CA2276003A1

CA2276003A1 - Method for rapid maturation and cultivation of ginseng plants regenerated from somatic embryo cultures

Info

Publication number: CA2276003A1
Application number: CA 2276003
Authority: CA
Inventors: Tei Sik Kyung; Ananchanok Tirajoh; Thomas Edward Tautorus; Christine Stewart; Samuel K. Foo; Carol Ann Bast; Zamir K. Punja; Susanna Mary Agnes Grimes
Original assignee: Individual
Current assignee: Phytogen Pharmaceuticals Inc
Priority date: 1998-06-22
Filing date: 1999-06-22
Publication date: 1999-12-22

Abstract

The present invention provides a method using in vitro tissue culture procedures to regenerate high frequency numbers of plantlets of North American ginseng (Panax quinquefolium L.) from mature leaf pieces and other explants. This method can be used for clonal propagation of the crop, especially of genetically superior lines. The method involves callus/tissue development, selection of embryogenic calli/tissue, propagation in a liquid suspension culture medium, plating and germination of embryos on semi-solid media, and development of plantlets. The plantlets may be acclimatized in soil and field planted.

Description

METHOD FOR RAPID MATURATION AND CULTIVATION OF GINSENG
PLANTS REGENERATED FROM SOMATIC EMBRYO CULTURES
FIELD OF THE INVENTION
The invention relates to embryogenic tissue culture of plant cells, plantlet regeneration from cultured cells and to maturation of the regenerated plantlets, and more particularly, to propagation of somatic embryo cultures from ginseng plants (genus Panax), regeneration of plantlets therefrom and acclimatization and maturation of the plantlets for field cultivation.
BACKGROUND OF THE INVENTION
Ginseng root represents an economically important source of metabolites commonly used in the pharmaceutical and food industries. It has been widely used in Asia for over 5000 years as a vitalizing and stimulating agent. The word "ginseng" is Chinese for "man-root," so named because the root is shaped like a human body.
Its main perceived benefits are as a general tonic for many ailments and as a body toner, giving the user a longer, healthier life. Ginseng is said to accomplish this by stimulating the brain, enhancing circulation, balancing the metabolism, decreasing blood sugar and cholesterol levels, and stimulating the glandular and hormonal systems (Proctor and Bailey 1987; Choi, 1988; Li, 1995; Shoyama et al., 1995). The active ingredients are a group of closely related chemicals called saponin glycosides or ginsenosides that are produced and stored in the plant.
There are at least six described Panax species (family Araliaceae), including P. quinquefolium L. (North American ginseng) and P. ginseng C.A.
Meyer (Chinese, Korean or Asian), which represent the predominant ginseng ~ plants grown commercially in North America and Asia, respectively. Although in the same genus, these species are very dissimilar. Whereas Asian ginseng is regarded as having a warming or stimulating effect, American ginseng is perceived as having a cooling or depressant effect (e.g., used for high blood pressure or diabetes). This may be attributed to differences in ginsenoside content between the two species (Ference, 1988).

Ginseng is a shade-loving, perennial aromatic herb with fleshy roots, and an annual stem bearing a whorl of palmate compound leaves (Thompson, 1987;
Choi, 1988; Li, 1995). A flowering plant may possess from 3 to 7 leaves in a whorl, with a peduncle terminated by a simple umbel at the center. The peduncle bears a terminal umbel with 10 to 80 flowers, according to the age of the plant and its growing condition (Choi, 1988). In general flowering of ginseng plants begins at the age of 3 years and lasts for 3 to 8 weeks depending upon the location. Two to 3 days after flowering, the embryo begins to develop, and small light green berries are formed 2 weeks later. The berries eventually turn red. It is general practice to collect the seeds once from 4-year old plants, and the fower buds are nipped off for better growth of the roots.
Despite there being a potentially high return for ginseng crops, conventional cultivation is an extreme high-input, long-term process, requiring intensive labor, (Oliver et al., 1990). One reason for this problem is that harvesting of ginseng does not occur until the end of a 4-year growing period. During this lengthy 1 S growth period it is plagued with many problems including susceptibility to high sunlight, heat stress, frost damage, strict nutritional and soil requirements, as well as weeds and diseases. According to Proctor and Bailey ( 1987), "the average yield of ginseng per acre in North America is not more than one-sixth to one-third of what might reasonably be expected, the shortage being caused almost entirely by the many diseases which attack the crop."
Another problem, is that currently all conventional ginseng cultivation is carried out using seed. It has been estimated that approximately 100 pounds are required for each acre of land. Unfortunately, ginseng undergoes a 3-year juvenile period before flowering and seed production occurs. A major problem with seed cultivation is that seeds must be collected from field grown plants and pre-treated (stratified) for about 15 months in the cold (4-6°C) before they can be planted in the fall for seedling emergence the following spring. Thus, in conventional seed cultivation, a 21-month period seed stratification and planting period is added to the three years required for the plant to mature for further seed production. There is therefore, a drastic shortage in seed supply and planting stock for growers making ginseng planting stock an important commodity.
Variation in vigour, pest and disease resistance, and levels of ginsenosides in the ginseng population is very common. Such a heterogenous population is due to mixture of seeds over the years, and uncontrolled pollination between plants. For crop improvement, it is highly desirable to cultivate only elite plants which would be fast growing, disease resistant, and contain high levels of selected ginsenosides. Elite plants may be produced through conventional breeding methods involving controlled hand pollination. Unfortunately, this is a very time-consuming process and would take many years to achieve for ginseng, which is a perennial crop with an extended juvenile phase. For some crops, an alternative approach is to screen and select superior plants and propagate them asexually (i.e., vegetatively) through rooted cuttings, grafting, etc. Unfortunately as yet, ginseng cannot be propagated through these traditional vegetative methods.
Cultivation by somatic embryogenesis represents an alternative method for propagating ginseng plants. Somatic embryogenesis is a process analogous to zygotic embryogenesis, but one in which a single cell or a small group of vegetative (i.e., somatic) cells are the precursors of the embryos (Ammirato, 1983).
Unlike organogenesis, therefore, somatic embryogenesis can recapitulate events in zygotic embryogenesis with the production of embryos having shoot and root apices (bipolarity). It has long been postulated (Haberlandt, 1902) that cells from any plant, given the appropriate stimuli and conditions, can be induced to regenerate plants. Since the development of somatic embryogenesis in carrot tissue cultures (Reinert, 1958, 1959; Steward et al., 1958), numerous angiosperm species have been regenerated in vitro by this method. However, at the present time understanding of the stimuli and conditions necessary for the induction and control of the somatic process is minimal.
The potential of somatic embryogensis as a method for rapid in vitro multiplication of plants and for production of artificial seeds has been emphasized previously (Parrott et al., 1991; Tautorus et al,. 1991 ). Furthermore, these methods are readily amenable to automation and mechanization. Theoretically, a culture initiated from a single explant can produce an unlimited number of embryos. Somatic embryogenesis therefore, offers significant advantages over other propagation methods, e.g., organogenesis, due to more rapid multiplication rates and lower costs per plantlet.
In addition, plants derived from somatic embryogenesis are less variable than those derived from organogenesis. Further, somatic embryogenesis has the potential to be more cost effective than seed propagation.
Successful propagation by somatic embryogenesis requires that somatic embryos should closely resemble their zygotic counterparts with the appropriate root, shoot, and coytledonary organs (Ammirato, 1983). Extraneous proliferations should be absent. They should be capable of growth into plants. Finally there should be no vascular connection with the mother tissue (Stolarz et al., 1991 ) as determined by histological sectioning. According to Haccius (1978), "the most distinctive characteristic of an embryo is its anatomically discrete (closed) radicular end."
Unfortunately, for too many reports, convincing documentation of the formation of well-developed true embryos capable of growth into plants is lacking. This is especially true for somatic embryos of ginseng plants. In addition, the distinction between structures derived by organogenesis and somatic embryogenesis can be sometimes be blurred. For these reasons, the field of somatic embryo culture for ginseng plants is in need of accurate histological information regarding the embryo development process to verify that somatic embryos have been produced. In addition, there is a need to characterize the type of germinants produced by somatic embryos and to provide methods of somatic embryo culture that produce the types of germinants most useful for regenerating plants .
In general a method of propagation using somatic embryo culture requires at least two steps. First, the somatic embryo culture must be established and maintained from an explant of plant tissue. This process involves sterilizing explant tissue to kill potential contaminants such as bacteria or fungus which may be present on the explant surface. The most commonly employed method involves immersion of the explant in a solution of bleach and a wetting agent such as Tween-20TM. The explants are then aseptically placed into petri dishes containing a special agar-solidified growth medium with phytohormones. Cultures are incubated in growth chambers under controlled temperature and lighting. After several weeks, an dedifferentiated cell mass is produced which is called callus. Under further incubation, or by subsequent transfer to modified media, a small percentage of callus may contain somatic embryos at various 5 stages of development. Callus tissue that produces somatic embryos is known as embryogenic callus.
Multiplication of embryos in an embryogenic culture may be carried out either by continued subculturing of embryogenic tissue on a semi-solid medium such an agar based medium, or by establishing suspensions in liquid medium, e.g., shake-flasks, bioreactors. In this latter state, cultures grow more rapidly than on agar solidified medium, generating large numbers of somatic embryos within several days (Tautorus and Dunstan, 1995). In addition, growth conditions and environmental factors can be carefully regulated and optimized. This reduces costs in terms of labor and presents opportunities for economical large-scale propagation of plants. For example, many millions of somatic cells can be maintained in an individual Erlenmeyer flask and sub-cultured by transferring a fraction of cells from one flask into another flask of fresh medium. A variety of methods have been used to generate embryogenic liquid suspension cultures depending upon species and laboratory. However, not all species or genotypes are amenable to liquid culture and methods for selecting a proper fraction of cells to transfer to fresh media so as to sustain propagation of the embryogenic cell culture are absent for many species. This is especially true for N. American ginseng which is known to be recalcitrant to liquid suspension culture techniques .
The second step in propagation by somatic embryo culture is embryo development, and plantlet regeneration. This step involves the transfer of a fraction of somatic embryos to regeneration conditions that involves optimization of several variables including media composition, phytohormones and culture conditions, all of which vary widely depending upon the particular species and type of culture.
The successful regeneration of plantlets from somatic embryo cultures is highly variable and differs widely across genotypes, species, and culture systems. A
variety of factors affect somatic embryogenesis and regeneration including, explant selection (e.g., age and type), culture conditions (e.g., media components, temperature, light), genotype, and biochemical/physiological influences. Numerous media and procedures have been utilized, but only some have been successful. For ginseng in particular, little is known about the specific conditions that effect the regeneration of plants from somatic embryos (i.e., conversion) which is of course essential to the final success of any cultivation system based on this process (Parrott et al., 1991). Many conditions effecting successful conversion may be effective with one species but ineffective for others. Unfortunately, the effect of the numerous variables that might impact the efficiency of conversion is rarely provided in the art. The majority of authors have only reported the successful recovery of plants, providing little or no information on conversion rates, methods of acclimatization or ultimate success in transplantation of regenerated plants to field conditions.
The use of somatic embryogenesis technology with ginseng has met with variable and incomplete success: There are few published reports of somatic embryogenesis in ginseng. (see Table l, below) In addition, they generally provide little information regarding specific methods and actual results (e.g., %
plantlet recovery).
Techniques for in vitro culture are significantly more amenable for Korean ginseng (Panax ginseng) than American ginseng. .Butenko et al. ( 1968) first induced somatic embryos from Korean ginseng root callus. Since then, somatic embryos of Korean ginseng have been induced from several types of explants including cotyledons (Choi, 1988), flower buds (Shoyama et al., 1988; Kishira et al., 1992), stem (Kishira et al., 1992), leaf (Cellarova et al., 1992; Kishira et al., 1992), zygotic embryos (Lee et al., 1990, 1991 ), and protoplasts (Arya et al., 1991 ). The most favorable explant in terms of induction appears to be flower buds in which approximately 80% of explants produced somatic embryos (Kishira et al., 1992).
Plantlet regeneration from embryogenic cultures of ginseng is still a major problem, and one of the least efficient steps is the acclimatization process.
Plantlets of Korean ginseng have been regenerated in vitro but it appears that none have been acclimatized in soil or field planted. There is only one recent report of ginseng plantlets being transferred to soil but there is not indication that a mature plant was ultimately produced or that the process is amenable to large scale cultivation because the percentage of plants surviving, if any, is unknown. Furthermore, the process used propagation of the somatic embryo cultures on semi-solid media which is time and labor intensive (Ahn, 1996).
The most extensively used basal medium formulation for Panax has been that of Murashige and Skoog ( 1962) (MS) either at full or half strength salts. The phytohormones most commonly used for somatic embryo induction in P. ginseng have been 2,4-dichlorophenoxyacetic acid (2,4-D) with or without kinetin (Kin) or benzylaminopurine (BA). For embryo maturation and plantlet regeneration, the most commonly used phytohormones have been gibberellic acid (GA3) and BA, used together at 0.5 or 1 mg/L (Table 1 ). These hormones have been shown to stimulate shoot formation from embryoids (Chang and Hsing, 1980).
Activated carbon (i.e., charcoal) has been used in various tissue culture media and is believed to be beneficial due to its ability to adsorb various phenolic or toxic compounds and/or due to its ability to adsorb excess levels of plant growth regulators. It has also been reported to be beneficial for embryogenic liquid suspension cultures of conifers (Handley and Godbey, 1996). Chang and Hsing ( 1980) reported using charcoal at 0.5% in combination with 0.1 mg/L GA3 for embryo maturation in P. ginseng. However, embryos failed to develop.
In terms of liquid culture, Asaka et al. (1993a) cultured embryogenic tissue of P. ginseng in 3 liter bioreactors for ginsenoside production. The tissues grew 9-fold in 42 days, and the ginsenoside pattern resembled that of ginseng leaves.
However, the authors did not attempt to obtain plantlets by transfer of embryos to semi-solid media to induce embryo maturation and plantlet regeneration. Kishira et al.
( 1992) reported culturing twenty mature P. ginseng somatic embryos in MS
liquid medium containing 0.5 mg/L GA3 at 2 rpm. However, the liquid suspension was not intended as a means to multiply embryos, but rather to achieve rapid elongation of the root. Results showed that although roots grew rapidly as compared to semi-solid medium, the growth of shoots was inhibited resulting in cotyledon-like plantlets.

There are several patents which describe methods for culturing embryogenic Panax ginseng cells in liquid and/or semi-solid media. Several are for the purpose of extraction of ginsenosides from the cells.
Japanese Patent (Sakono; PI JP09220036 A2; AI JP96-28277) describes a method of culturing ginseng callus using high dissolved oxygen and controlled pH.
The method resulted in a 20-fold higher adventitious embryo induction rate than prior arts.
Japanese Patent (Matsumoto 1993; PI JP05244838 A2; AI JP92-83293) describes a method of culturing embryogenic callus of ginseng in Gamborg BS
liquid medium for several generations, centrifugation to obtain the calli, and again culture of the calli in an agar medium. According to the inventors, the method is useful for enhanced induction of adventitious embryos and reproduction of male sterile ginseng plants.
Japanese Patent (Kishoshi et al., 1991; PI JP03035739; AI JP89-171609) describes a tissue culture method for inducing shoot primorida from adventitious embryos to produce seedlings. Adventitious embryos are cultured in a rotary fermenter with artificial illumination to induce shoot primordia; and the stationary culture of the shoot primordia to produce seedlings.
Japanese Patent (Kishoshi et al., 1991; PI JP03035740; AI JP89-171610) describes a method for mass production of plant seedlings by culturing adventitious embryos or shoot primordia in liquid medium which inhibits the differentiation of stem and leaf but promotes the development of roots. Roots are formed after 1-3 months.
Japanese Patent (WPI Acc. No. 90-079051/11) describes a method of screening, whereby juvenile plants, including adventitious embryos of P.
ginseng, are suspended or dispersed in a solvent of specified specific wt. The selected plants are then collected and washed.
Japanese Patent (WPI Acc. No. 89-064888/09) describes a method for production of root-like tissues of Panax ginseng by culturing adventitious embryos in liquid medium at 0-100 rpm for 20 days to 2 months.

A Russian Patent to Getmanova et al., (WPI Acc. No. 94-356458/44) describes a ginseng micropropagation process that can be speeded up by removing individual flower buds from racemes that have been induced by cultivating dormant rhizome nodules on modified MS. The mature embryoids obtained from these buds are then grown on a variant of MS to obtain rooted plant regenerates.
In contrast to Asian ginseng, N. American ginseng (P. quinquefolium) is extremely recalcitrant with regards to tissue culture and somatic embryogenesis. This is similar to Pinus spp. being highly recalcitrant towards somatic embryogenesis as compared to Picea spp. (Tautorus et al., 1991 ). A species is recalcitrant to tissue culture techniques when it does not respond to treatments that would be expected to evoke a response based on what has been observed with related species. Therefore, techniques applicable for one species are not useful for a recalcitrant species like American ginseng.
There are only four reports of somatic embryogenesis of American ginseng. None of these reports have resulted in successful soil transfer. Wang (1990) obtained somatic embryos from root-derived callus of Panax quinquefolium.
However, only a few plantlets were produced and none acclimatized in soil. Zheng and Huang (1994a,b) induced somatic embryos of P. quinquefolium from callus for electron microscopy and marker protein studies. No plantlets were produced. In 1995, Tirajoh and Punja reported somatic embryogenesis from root, leaf, and epicotyl explants in American ginseng. Only shoots were regenerated. In addition, there was no histological data from any report to document the actual presence of somatic embryos.
Furthermore, the most potentially useful somatic embryo culture for large scale cultivation is a liquid suspension culture. There have been no reports of a successful initiation and propagation of embryogenic suspension cultures of Panax quinquefolium.
To summarize, plantlet regeneration and soil acclimatization from somatic embryos of ginseng is extremely difficult for all ginseng plants. This is particularly true with American ginseng. There are no reports of successful transfer and survival of plants derived from somatic embryos into soil for either species.
In addition, American ginseng somatic embryos have not been previously cultured in liquid medium for the purpose of multiplying numbers and obtaining plantlets.
The result of several studies are summarized below in Table 1.

REPORTS OF SOMATIC EMBRYOGENESIS IN PANAX SPP.
Explant Source Basal PhytohormonesResponse Reference Mediums (pM)b a) P. ginseng root MS 2,4-D embryoids Butenko et al., root i) MS; i) 22.6 2,4-Dembryoids, Change &
+

ii) BS 9.3 Kin; ii) flowers Hsing, 1980a 4.5 2,4-D; iii) 4.4 BA + 2.9 GAS

root i) MS, i) 4.5 2,4-D;plantlets Chang &

ii) 1/2 ii) 4.4 BA (none in Hsing, 1980b MS + 2.9 soil) or BS GA3 root MS i) 4.5 2,4-D Plantlets Asaka et + 0.5 al., Kin; ii) no (none in 1993, Norm. soil) 1994a,b flower bud MS, 1/2 i) 4.5 2,4-D;plantlets Shoyama MS

ii) 10.8 NAA (vermiculite)et al., + 1988 11.1 BA; iii) 1.4 GA3 + 2.2 BA;

iv) 5.4 NAA

(rooting) zygotic embryo i) MS; i) 4.5 2,4-D plantlets Lee et al., ii) '/z +

MS 0.05 Kin; (none in 1990 ii) 4.4 soil) BA + 2.9 GA3 protoplasts (zygotici) MS; i) 4.5 2,4-D plantlets Arya et + al., embryo) ii) 1/2 0.05 Kin; (none in 1991 MS ii) 4.4 soil) BA + 2.9 GA3 leaf LS i) 10.7 NAA shoots Cellarova +

2.3 Kin; ii) et al., 1.46 1992 GA3 + 2.2 BA

root MS i) 2.26 2,4-Dembryoids Jiu, 1992 +

0.44 BA; ii) 4.52 BA

flower buds, i) MS; i) 4.5 2,4-D;plantlets Kishira stems, et al., leaves ii) MS-vermii) 2.2 BA (none in 1992 + 1.4 soil)' iculite GA3 Explant Source Basal PhytohormonesResponse Reference Mediums (pM)b zygotic embryo MSs i) 4.5 2,4-D plantlets Arya et + al., 0.05 Kin; (none in 1993 ii) 4.5 soil) 2,4-D; iii) 4.7 Kin (shoot);

iv) 2.9 GA3 (root) zygotic embryo MS no hormones somatic Choi & Soh, embryos 1994, 1996a,b;

Choi et al., 1997a,b zygotic embryo MS, 1/2 i) no horm.; Plantlets Benkrima MS ii) 9 2,4-D + 2.3 (none in et al., Kin; soil) 1995 iii) 2.9 GA3 + 4.4 BA; iv) no Norm.

zygotic embryo MS i) 5 2,4-D plantlets Ahn 1996 + 0.5 BA; ii) 0.5 (transferred to 2,4-D + 5 soil') BA;

111) 5 GAg seedling sectionsMS i) 5 2,4-D; plant-like Ahn et al., ii) 0.3 NAA + 1 BA; structures 1996 iii) 5 GA3 b) P. japonicus flower bud i) MS; i) 4.5 2,4-D plantlets Fujoika + 5.4 ii) 1 /2 NAA; ii) 10.8(none in et al., MS soil) 1986;

NAA + 44.4 Shoyama BA;

iii) 2.9 GA3 et al., + 4.4 1995 BA; iv) 5.4 NAA

(rooting) c) P. notoginseng flower bud, petiolei) MS ii) i) 4.5 2,4-D;plantlets Shoyama MS ii) 1.4 GA3 (only in et al., + 2.2 1997 BA; iii) 5.4 vermiculite)'2 NAA

d) P. quinquefolium root MS i) 9.0 2,4-D plantlets Wang, 1990 + 4.7 Kin; ii) 9.0 (none in soil) dicamba; iii) 0.5 NAA + 2.5 IBA

Not reported MS 9.0 2,4-D none Zheng &

Huang, 1994a,b Explant Source Basal Phytohormones Response Reference Mediums (~M)b root MS i) 9.0 dicambashoots Tirajoh + &

5.0 Kin; ii) Punja, 9.0 1995 dicamba; iii) 4.4 BA + 2.8 GA3 leaf MS i) 10 NAA + shoots Tirajoh 9.0 &

2,4-D; ii) Punja, 4.4 1995 BA + 2.8 GAS

epicotyl MS i) 9.0 dicambashoots Tirajoh + &

5 Kin; ii) Punja, 4.4 1995 BA + 2.8 GAS

° Note: some of the basal media nave been mod~hed, refer to particular reference for details.
b Where indicated, different phythormone combinations were used for initiation of embryogenic tissue, embryo development, or plant regeneration.
Authors did not indicate which original explant was used for plantlet production.
Z No data reported as to how many plantlets survived transfer in soil or %
acclimatization.
Use of somatic embryo culture techniques for plant cultivation is especially useful when combined with techniques for the genetic transformation of plants. Advances in biotechnology and genetic engineering have enabled the introduction and expression of a desired gene in a recipient plant (Lal and Lal, 1993).
Through the use of genetic technologies, plants have been engineered to express gene sequences that are not normally or naturally present in the native plant, or to exhibit altered expression of naturally occurring genes (Parrott et al., 1991;
Potrykus, 1991).
Plants produced through the use of recombinant techniques are known as transgenic plants. The genetic transformation of plants has been achieved with selectable marker genes, such as antibiotic resistance genes, and more recently with genes encoding for agronomic traits. Characters such as resistance to viral diseases, insect pests, herbicides, and fungal infection have been transferred to various plants (Charest and Michel, 1991; Lal and Lal, 1993).
I S There are two approaches to gene transformation. Indirect gene transfer employs an intermediate organism, such as Agrobacterium tumefaciens or A.
rhizogenes, to transmit foreign DNA to the plant genome. Direct gene transfer can be accomplished using methods that deliver DNA directly to the plant cell without any intermediates.
Transgenic plants have been recovered using direct and indirect gene transfer methods.

The most advanced indirect technology for the genetic transformation of plants employs the phytopathogenic bacterium A. tumefaciens (which induces crown gall disease). The molecular basis of this disease is the transfer of plasmid DNA
(T-DNA or transfer DNA) to the plant cell genome and its subsequent expression, which induces abnormal proliferation of the transformed cell (Charest and Michel, 1991 ). Similarly, A. rhizogenes has also been used. Application of this technology to plants is limited by the host range of the bacteria.
Direct gene transformation methods overcome host range limitations inherent in using Agrobacterium species. These methods include:
electroporation of protoplasts, liposome-mediated delivery, polyethylene-glycol (PEG) delivery, microinjection, and microprojection. Except for microprojection, the majority of direct gene transfer methods rely on protoplasts from which plant regeneration must be achieved. Unfortunately, for many species, regeneration of plants from protoplasts is very difficult. Microprojection, is a technique in which microprojectiles, made by coating small tungsten or gold particles with DNA are accelerated with a particle gun to velocities that permit penetration of intact cells and tissues. Protoplasts are not required. This has been shown to be a very successful method of transformation for numerous plant species.
The transformation of ginseng tissue by indirect means has been reported, however there are no reports of successful acclimatization or maturation of plantlets regenerated from transformed ginseng. Roots of Panax ginseng have been successfully transformed with Agrobacterium rhizogenes (Inomata et al., 1993;
Inomata and Yokoyama, 1996). The transformed roots (i. e., hairy roots) show wide variation in their characteristics (e.g., ginsenoside production) between genotypes. In addition, Lee et al. (1995) reported successful transformation when cotyledonary explants of zygotic embryos of P. ginseng were cocultured with A. tumefaciens. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary explants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto regeneration media, somatic embryos developed into plantlets. Southern analysis confirmed that the ~3-glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants.
Tirajoh and Punja (1995) attempted to introduce a chitinase gene into American ginseng using Agrobacterium-mediated transformation. Coculture of A. tumefaciens with callused root explants resulted in putatively transformed calli.
However, verification of the presence of the foreign genes was not carried out.
The combination of genetic transformation methods with somatic embryogenesis offers enormous potential for transformation of important plant species.
The benefits of using embryogenic material for direct and indirect gene transfer has been emphasized by Parrott et al. (1991). In particular, the regeneration capability of embryogenic cells is a major advantage over other culture systems. This is particularly true when a liquid suspension culture system can be used to efficiently generate a high frequency of regenerated plants. Through somatic embryogenesis it should be possible to maximize the numbers of transformed cells from which regenerants can ultimately be produced.
Unfortunately, the development of an efficient liquid suspension culture system for propagating somatic embryo cultures of ginseng has not been provided.
Further, there is a need for efficient methods related to the techniques of plantlet regeneration, acclimatization and maturation of ginseng plants from somatic culture.
SUMMARY OF THE INVENTION
Therefore, an objective of the present invention is to provide methods for embryogenic cell culture, embryo maturation, plantlet regeneration, soil acclimatization, and rapid maturation of ginseng plants. A further objective is to provide mature ginseng plants capable of field planting where the ginseng plants have been regenerated from an embryogenic culture. Another objective is to provide a method for producing a germinant from an embryogenic culture of a plant, preferably a ginseng plant, where the germinant is a root-enhanced germinant capable of rapid development into a plantlet.
To these ends, one aspect of this invention provides a method of cultivating a ginseng plant that includes the steps of regenerating a plantlet from an embryogenic cell culture and maturing the plantlet into a mature plant, wherein the maturing includes acclimatizing the plantlet for field growth. Another aspect provides a mature ginseng plant derived from an embryogenic cell culture of a ginseng plant.
Preferred embodiments of the mature ginseng plant and of the method for cultivating ginseng plants include plants selected from Panax quinquefolium, P. ginseng, P. notoginseng, P. japonicus, hybrids of ginseng plants and transgenic ginseng plants.
More preferred embodiments include ginseng plants of an American variety, most preferably P. quinquefolium.
Another aspect of this invention is a method of regenerating a plantlet from an embryogenic cell culture of a plant that includes the steps of selecting globular embryos from the embryogenic cell culture and transferring the fraction of globular embryos to a germination media selected to induce the globular embryos to develop into a root enhanced germinant. A related aspect of this invention provides a root enhanced germinant derived from an embryogenic cell culture of any plant. A root-enhanced germinant is a germinant of a globular embryo that develops into a plantlet without passing through a conspicuous heart shaped or torpedo shaped embryo stage.
Root-enhanced germinants are capable of rapid development into a plantlet and of simultaneously developing root and shoot structures on a single media. In one embodiment of the method of regenerating plantlets through root-enhanced germinants, at least 3 root-enhanced germinants are produced per gram (Fresh wt.) of globular embryos plated. In a preferred embodiment, at least 50 root-enhanced germinants are produced per gram (Fresh wt.) of globular embryos plated.. In a more preferred embodiment, the ginseng plant is selected from Panax quinquefolium, P.
ginseng, P. notoginseng, P. japonicus, hybrids of ginseng plants and transgenic ginseng plants.
More preferred embodiments include ginseng plants of an American variety, most preferably P. quinquefolium.
Another embodiment of the method of regenerating a plantlet through root-enhanced germinants includes a step of transferring the fraction of globular embryos to a first media that lacks an active amount of phytohormones. Other embodiments further include transfer of the root-enhanced germinant to a second media selected to promote maturation of the root-enhanced germinant into a plantlet wherein the second media contains an active amount of phytohormones. These embodiments include a step of transferring globular embryos to the first media for a first period of growth to develop root-enhanced germinants, then transferring the root-enhanced germinants to the second media for a second period of growth. A preferred embodiment of this method provides that phytohormones contained in the second media are selected to simultaneously promote root and shoot growth. In a more preferred embodiment, the phytohormones contained in the second media include both NAA and GA3 where the NAA is present at a concentration of about 1 - 12 ~M, and the GA3 is present at a concentration of about 0.5 - 6.0 pM. In a most preferred embodiment, NAA is present at a concentration of about 4 - 8 pM, the GA3 is present at a concentration of about 2 - 4 pM and a ratio of NAA to GA3 concentration is about 2 to 4 fold.
Preferred embodiments of the root-enhanced germinant of this invention include root-enhanced germinant derived from a ginseng plant selected from Panax quinquefolium, P. ginseng, P. notoginseng, P. japonicus, hybrids of ginseng plants and transgenic ginseng plants. In more preferred embodiments, the root-enhanced germinant is derived from ginseng plants of an American variety, most preferably P.
quinquefolium.
Still another aspect of this invention provides a method of propagating a culture of somatic embryos of a ginseng plant including the steps of establishing a first somatic embryo culture from an explant of the ginseng plant, growing the first somatic embryo culture under conditions that promote production of somatic embryo bearing centers, selecting a fraction of globular embryos produced from the somatic embryo bearing centers, transferring the fraction of globular embryos to a growth media to establish a second somatic embryo culture, continuing growth of the second somatic embryo culture under conditions that promote production of additional somatic embryo bearing centers, and repeating these steps for a maintenance period. In another embodiment, the fraction of globular embryos is greater than a combined fraction of other somatic embryos consisting of heart-shaped embryos and torpedo shaped embryos. A preferred embodiment of this method provides culture propagation for a maintenance period of at least 2 years. A more preferred embodiment provides culture propagation for a maintenance period of at least 4 years. A most preferred embodiment provides culture propagation for a maintenance period that is indefinitely sustainable.
Further embodiments of the embryo culture propagating method of this invention include embodiments where at least one of the first somatic embryo culture and the second somatic embryo culture is a liquid suspension culture. In a preferred embodiment, both the first and the second somatic embryo cultures are liquid suspension cultures. In other embodiments, at least one of the first and the second somatic embryo cultures is maintained on semi-solid media. Another embodiment of the embryo culture propagating method includes selecting the first fraction of globular embryos by selecting an uppermost layer of a cell mass formed by allowing the suspension culture to settle to the bottom of a vessel by gravity.
Another aspect of this invention provides a method of acclimatizing regenerated ginseng plantlets for field growth. This method includes the steps of transferring the regenerated plantlet from a growth media to an aseptic soil, exposing the transferred plantlet in a sterile environment to a first condition that provides a first humidity and first light exposure for a first period of time, and changing from the first condition to a second condition over a second period of time wherein the second condition provides a second humidity lower than the first humidity and a second light exposure greater than the first light exposure. In one embodiment a total acclimatizing period including the first period and the second period is 3-7 months.
A further aspect of this invention provides a method of rapidly maturing ginseng plantlets regenerated from an embryogenic cell culture. Embodiments of this method include the steps of acclimatizing regenerated plantlets for field growth, senescing the acclimatized plantlets for an initial outgrowth period, and treating the senesced plantlets with a phytohormone composition for a flushing period to produce a mature ginseng plant is produced in a shortened maturation period. In a preferred embodiment, the shortened maturation period is less than 40 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least about 60 months age where the age of the seed grown plant is calculated from commencement of 1g seed stratification. In a more preferred embodiment, the shortened maturation period is less than 36 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least 60 months age. Another preferred embodiment of this rapid maturation method includes field growth of the matured ginseng plant. In another aspect, preferred embodiments include maturing ginseng plants selected from Panax quinguefolium, P. ginseng, P. notoginseng, P. japonicus, hybrids of ginseng plants and transgenic ginseng plants. More preferred embodiments include ginseng plants of an American variety, most preferably P. quinquefolium.
Still another aspect of this invention provides methods that include combinations of one or more steps of the separate methods of this invention.
In one embodiment, the method of cultivating a ginseng plants includes at least one step selected from the method of propagating somatic embryo cultures derived from a ginseng plant, the method of regenerating a plantlet by producing root-enhanced germinants, the method of acclimatizing regenerated ginseng plantlets and the method of maturing regenerated ginseng plants. In a preferred embodiment, the method of cultivating a ginseng plant includes at least two steps of the separate methods. In a more preferred embodiment, the method of cultivating a ginseng plant includes at least steps of the separate methods. In a most preferred embodiment, the method of cultivating a ginseng plant includes all of the separate methods provided by this invention.
These and other aspects of this invention will be evident upon reference to the attached drawings and following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates an overall protocol for one embodiment of a method for somatic embryogenesis, plantlet regeneration and rapid maturation of ginseng plantlets.
Figure 2 illustrates plant material and procedures provided in the invention. a) Shows an embryogenic tissue of P. quinquefolium derived from leaf tissue. Note the cotyledonary like embryo; (b) Shows an embryogenic cell culture of P. quinquefolium in liquid suspension; (c) Shows globular somatic embryos of P. quinquefolium from liquid suspension plated onto PL-1 media; (d) Shows an early heart-shaped somatic embryo of P. quinquefolium obtained from a liquid suspension plated onto a germination media (PL-1 media); (e) Shows a late heart-shaped somatic embryo derived from liquid suspension (7 days from plating); (fJ Shows a torpedo shaped somatic embryo derived from liquid suspension (14 days from plating);
(g) Shows a root-enhanced germinant with little cotyledon development; (h) Shows a root-enhanced germinant with cotyledon formation; (i) Shows early developing plantlets of P. quinquefolium regenerated from liquid suspension culture and plated on a post germination media containing activated charcoal (PG-3); (j) Shows a P. quinquefolium plantlet cultured in jar with activated charcoal media (PG-3); (k) Shows a Suncap used with a Magenta bottle for acclimatization of plantlets; ( 1 ) Shows a fully acclimatized P. quinquefolium plant derived from a somatic embryo culture and matured for field planting.
Figure 3 illustrates ordinary stages of zygotic embryo development in a dicot plant. (a) Shows a late globular embryo; (b) Shows an early heart-stage embryo;
(c) Shows a late-heart stage embryo; (d) Shows a torpedo stage embryo; (e) Shows an ntermediate stage embryo; (f) Shows a cotyledonary stage embryo; and (g) Shows a mature zygotic embryo. Modified from Raghaven ( 1976).
Figure 4 shows stages of embryo development from somatic embryo cultures of American ginseng as revealed by histological staining. (a) Globular;
(b) Heart-shaped; (c) Torpedo-shaped; (d) A root-enhanced germinant; (e) Cotyledonary with apical meristem; and (f) Another root-enhanced germinant. All photographs are of thin sections stained with safranin -fast green. Scale bar: 0.1 mm for (a);
0.2 mm for ( b) to (d) and 0.5 mm for (f).
Figure 5 illustrates a yield of root-enhanced germinants from a suspension culture of American ginseng (line 1 ) cultured on a bench-top shaker at 60 rpm in low light.

Figure 6 illustrates a yield of root-enhanced germinants from a suspension culture of American ginseng (line 1 ) cultured on a floor shaker at 120 rpm in low light.
Figure 7 illustrates a yield of root- germinants from a suspension culture 5 of American ginseng (line 1 ) cultured on a floor shaker at 120 rpm in the dark.
Figure 8 illustrates an effect of GA3 concentration on root-enhanced germinant shoot length.
Figure 9 illustrates an effect of media type on REG fresh weight.
Figure 10 illustrates an effect of media type on the frequency of REGs 10 with shoot length <5 mm.
Figure 11 illustrates an effect of media type on the frequency of REGs with a root length <10 mm.
Figure 12 shows a time line comparing the rapid maturation provided by a cultivation method of this invention to ordinary seed cultivation of American ginseng.
15 Figure 13 shows interdependency of root growth on charcoal on both block and % charcoal in PG-3 media.
Figure 14 illustrates root growth dependency on % charcoal within a block.
DETAILED DESCRIPTION OF THE INVENTION
20 This invention provides methods related to cultivating ginseng plants derived from embryogenic cell cultures. Several distinct methods are provided that may be used alone or in combination to provide alternative embodiments for the practice of this invention. The distinct methods herein provided herein include: 1 ) establishing and propagating an embryogenic cell culture derived from a ginseng plant;
2) regenerating of plantlets and production of root-enhanced germinants from the embryogenic cell culture; 3) acclimatization of plantlets derived from the embryogenic cell culture; and 4) rapid maturating of regenerated plantlets for field growth. Figure 1 illustrates a time line and conditions that represent one embodiment of a protocol that combines methods of the present invention.

This invention is generally applicable to all Panax species, wild or cultivated, including but not limited to the following varieties: Panax quinquefolium, P. ginseng, P. notoginseng, and P. japonicus. In addition, the current invention is applicable to interspecies hybrids of Panax as well as transgenic ginseng plants. As used herein, the term ginseng plant refers to any and all varieties and genotypes of the genus Panax.
1. Initiation and Establishing of Embryo~enic Cell Cultures In the culture establishing step, explant material is collected from a ginseng plant and placed into in vitro culture to initiate somatic embryo production and establish a first somatic embryo culture. This first somatic embryo culture is then grown under conditions that promote production of somatic embryos and somatic embryo bearing centers. As used herein an explant is any tissue or cells obtained from a plant. This includes, but is not limited to, seed, leaves, flower buds, and roots. Plants at any stage of growth may used to obtain explants. A preferred source of explant material is leaf tissue of young seedlings, leaf tissue of mature plants, or flower buds of mature plants, however, explants from of other tissue can be used as well.
Alternatively, plant cultures grown in vitro or in the greenhouse may also be used as a source of the explant.
Once the explant has been obtained it is surface-sterilized, placed in a liquid or on a semi-solid culture medium and incubated in growth rooms at about 22-26°C to initiate callus formation and somatic embryo production. As used herein, semi-solid media is media comprised of an aqueous based gel typically formed with agars, agaroses, starches or other gelling agents often used in plant cell culture such as Phytogel~ or GelRite~. (Sigma Chemical Company, St. Louis, Mo.). Culture media may be comprised of any one of a variety of formulations for promoting tissue growth.
A common basal medium used to culture plant tissue is MS salts (Murashige and Skoog, 1962) which provides a formulation of macro and micro nutrient salts needed for most plant growth in vitro. The Panax cultures of the present invention use MS
based media at either half strength or full strength however, the invention is not limited by the source or strength of media used. MS media is usually supplemented with a carbon source, typically sucrose at about 3% (w/v). Nitrogen supplements such as casein may also be used where appropriate for the source of the explant to be cultured.
Other supplements and typical amounts often added to MS media are 100 mg/liter myo-inositol, 0.1 mg/liter thiamine-HCI, 0.5 mg/liter nicotinic acid, 0.5 mg/liter pyridoxine-HC1, 2 mg/liter glycine, and Gamborg's vitamins as recomended (Gamborg et al., 1968). The pH of the media adjusted to about 5.8 with 1 N NaOH or HCl as needed.
The production of somatic embryo cultures from an explant usually requires the presence of phytohormones. The most commonly used phytohormones for somatic embryo induction in P. ginseng has been 2,4-dichlorophenoxyacetic acid (2,4-D).with or without kinetin (Kin) or benzylaminopurine (BA). Other combinations of phytohormones may also be used according to the type and source of explant.
It is preferred that a combination of phytohormones be selected that is optimized in amount and ratio for somatic embryo induction from the particular explant source being used.
For explants of Panax plants, phytohormones and amounts include, but are not limited to: 2,4-D (4.5, 9.0); napthalene acetic acid (NAA) (5.0, 10.0, 15.0, 20.0) with either kinetin (Kin) (2.5, 5.0) or 2,4-D (9.0); 2,4-D (9.0) with Kin (5.0); dicamba alone (range of 4.5-13.5), and dicamba (4.5, 9.0, 13.5) with Kin (5.0, 10.0) (concentrations are in pM).
As mentioned earlier, somatic embryo culture of P. quinquefolium has been problematic. However, this invention provides embodiments for preferred combinations of phytohormones that are useful for somatic embryogenesis for this species. For explants of seedling leaves, a combination of NAA ( 10.0 ~M) and 2,4-D
(9.0 p.M) gives a high rate of somatic embryo formation (about 30% in 2 months and 40% in 3 months). For epicotyl seedling explants, somatic embryo formation is more recalcitrant, however, a combination of diacmaba (9.0 pM) and Kinetin (5.0 ~M) will induce formation of somatic embryos at a rate of about 2% in 9 months. (See EXAMPLE 3). For explants of mature leaves, a variety of combinations of dciamba/Kin and NAA/2,4-D can produce healthy embryogenic callus. A preferred combination is NAA/2,4-D (10.0/9.0 ~M) which produces somatic embryos at a rate of about 30% in 5 months. (See Example 3). For explants of mature root tissue, a preferred combination is to initiate callus induction on dicambalKin (4.5/10.0 or 9.0/5.0) and then transfer callus tissue to dicamba alone at about 9.0 ~M
which produces a rate of somatic embryo induction of about 16% in 3 months. (See Example 3). Similarly, a two step process is preferred for explants of flower buds where callus is initially formed with 2,4-D/Kin (9.0/4.7) and somatic embryos are induced after transfer to NAA/2,4-D (2.5/2.3) which produces a somatic embryo formation rate of about 36%
in 5 months. This combination is effective with flower buds from Asian as well as American ginseng. (See Example 4).
Various conditions may be employed to initiate somatic embyro production from the explant. The length of time an explant needs to be cultured before somatic embryogenesis is induced will vary depending on the source of the explant, culture conditions, and frequency of transfer. Other conditions that may be varied to optimize somatic embryo production include but are not limited to media formulation, light exposure, temperature, culture vessel and protocols for scheduled transfer to one a fresh or different media. Figure 1 illustrates one embodiment of conditions selected for somatic embryo production from American ginseng. One skilled in the art will readily vary culture conditions to optimize both cell proliferation and embryo production for the particular plant by using the information provided by the description and Examples of this invention.
Once an initial somatic embryo culture has been obtained from an explant the initial culture is used to establish a first embryogenic cell culture that can be further maintained and propagated for continued production of somatic embryos.
As used in herein, a somatic embryo culture or embryogenic cell culture refer to any culture derived from an explant that multiplies and produces embryos that can be matured into a plantlet. It includes cultures derived from both somatic and zygotic source.
The terms "establishing" and "established" refer to the process of starting the first embryogenic cell culture from the initial culture obtained from an explant and growing the first culture for a period of time sufficient to provide a cell mass that can be used to continue propagation of the embryogenie cell culture. Callus and callus culture refer generally to any cultured tissue derived from an explant and includes but is not limited to embryogenic cell cultures.
In a preferred embodiment, the first embryogenic cell culture is established in a liquid media although cultures can may also be established on semi-solid media after initiating a somatic embryo culture. Establishing the first embryogenic cell culture in a liquid media entails taking a sample of the initial explant culture and transferring the sample to fresh media that promotes continued growth of somatic embryos. In a preferred protocol about 2 -3 grams of tissue from the initial somatic culture is transferred to about 20 ml of liquid media to start the first embryogenic cell culture. Cultures may take 1-2 months to become established.
After a liquid suspension culture is established it may serve as the first somatic embryo culture for the propagating method of this invention. The liquid media is usually comprised of the same components and phytohormones contained the semi-solid media used for the initial explant but without the gelling agent. It is preferred that the liquid media be contained in a vessel that can be agitated such as an Erlemenyer flask as illustrated in Figure 2b. The liquid media should fill about 1/6 to 1/ 4th the volume of the flask to permit sufficient aeration of the culture. The flask can be agitated by placement on a rotary shaker, typically at speed of about 30 to about 120 rpm.
A speed of about 60 rpm is preferred for initial establishing. Light conditions may vary from darkness to low light without significantly altering characteristics of the culture Growth in darkness is preferred to suppress unwanted differentiation of somatic embryos. The liquid media is replenished about once every week by removing a portion of the used media and adding an equal portion of fresh media. Typically , 18- 20 ml of media is replenished.
The culture is routinely examined for cellular multiplication and somatic embryo production. Somatic embryo production can be determined by microscopic examination or histological staining of a sample of tissue. An illustration of somatic embryos produced in liquid culture is shown in Figures 2 - 4. Figure 2 shows somatic embryos produced by this invention, Figure 3 shows representative samples of dicot embryo stages, and Figure 4 shows histological staining of somatic embryos produced by this invention After a period of about 3 weeks the culture mass will have increased and will contain embryos of various stages including globular (Figures 2(d), 3(a), 4(a), heart shaped, Figures 2(f), 3(b-c), 4(b), torpedo shaped, Figures 2(f), 3 d-e, 4 d), with or without early cotyledonary development, Figures 2(f), 3(d-e), 4(d-e). After about five 5 weeks this first embryogenic cell culture can be divided and a fraction of somatic embryos transferred to fresh media to form a second embryogenic cell culture.
Alternatively, the fraction of somatic embryos can be transferred back to semi-solid media to from the second embryogenic cell culture or the whole of the first embryogenic cell culture can be used to inoculate a larger amount of liquid media to 10 form the second embryogenic cell culture. A fraction of somatic embryos is usually about 1-5 ml of packed cells obtained from the first embryogenic culture. At this point the first embryogenic cell culture may be considered established An established . embryogenic cell culture produced by the method described here may contain some large clusters of cells that appear to have more active 15 growth centers than other clusters. . It is suggested that these centers generate further production of somatic embryo's and hence are designated herein as somatic embryo bearing centers (SEBC). SEBCs continuously give rise to smaller globular somatic embryos. The production of SEBCs and the production of a fraction of globular embryos is an important characteristic of an established embryogenic cell culture. As 20 will be shown hereafter, long term propagation of an embryogenic cell culture is a that stems from the production of a large fraction of globular embryos from SEBCs.
In preferred embodiments, the fraction of globular embryos produced is at least 10% of the embryos produced by an established cell culture. In a more preferred embodiment, the culture produces a fraction of globular embryos greater than a fraction comprised of 25 both heart shaped and torpedo shaped embryos.
In some cases globular embryos present in an established embryogenic cell culture will undergo some maturation/differentiation in liquid to form other stages.
The longer suspension are incubated, the further differentiation of the culture may occur. When a culture becomes over differentiated it may either fail to grow or fail to produce a sufficient quantity of somatic embryos to be continuously propagated. The rapidity of this depends on culture conditions and the particular genotype.
However, in the practice of this invention, the culture line may be maintained continuously undifferentiated ensuring the presence of SEBCs by routinely transferring a fraction of a culture that is enriched with globular embryos to fresh liquid medium.
(Example 6).
The above methods allow an embryogenic cell culture to be established from a variety of sources. The sources include different explant tissues and different varieties of ginseng plants. The occurrence of somatic embryogenesis in Panax sp. is affected by several factors, including source of explants, age of explants, and type of growth regulator combinations supplemented to the basal medium. The shortest period for somatic embryo formation was 2 months from seedling-derived leaf explants plated onto MS medium with NAA/2,4-D (10.0/9.0 p,M). The highest frequency of somatic embryo formation was observed with seedling leaf explants (40%) within a 3 month period. The age of leaf explants, i.e., from juvenile (seedling) or mature leaf, influenced the response to the growth regulators tested.
2. Propagation And Maintenance Of Embryogenic Cell Cultures Once an embryogenic cell culture is established, it can be continuously propagated either as a liquid suspension culture or on a semi-solid medium.
Propagation of embryogenic cell culture includes the steps of growing the first somatic embryo culture established as described, under conditions as previously described that that promote production of SEBC, then selecting a fraction of globular embryos contained in the first somatic embryo culture, and transferring the fraction of globular embryos to a fresh media to establish a second somatic embryo and continuing growth of the second somatic embryo culture under conditions that promote production of additional somatic embryo bearing centers. Propagation is accomplished by repeating the growing, selecting, transferring and growth steps of a maintenance period.
In one embodiment, both the first somatic embryo culture and the second somatic embryo culture are liquid suspension cultures. In another embodiment, at least one of the first somatic embryo culture and the second somatic embryo culture is a liquid suspension culture. The other culture may be maintained on a semi-solid media.

This permits continued propagation in liquid media and provides a supply of somatic embryos on semi-solid media that can be used as a back up to establish additional cultures in a liquid media or used for other purposes such as genetic transformation. In another embodiment, both the first and the second cultures are maintained on semi-solid media. However, propagation of embryogenic cell cultures on semi-solid media is an extremely labor intensive process and requires many petri dishes to bulk up an individual culture line for large scale embryo multiplication and development.
Further, semi-solid media may cause inconsistencies resulting from variance in the type and purity of agar or other gelling agent used. (Dunstan et al., 1995).
Therefore, in the most preferred embodiment of the propagation method, both the first and the second somatic embryo culture are liquid suspension cultures. In addition to ease, another advantage of propagation by liquid suspension culture is that with proper selection of cells, cultures can maintain production of embryogenic cells for an indefinitely sustainable period. This is in contrast with prior liquid suspension cultures of ginseng that either fail to grow or fail to continue production of somatic embryos after a short period. A further advantage is that liquid suspension cultures can be rapidly scaled to larger sizes for the production of very large numbers of embryogenic cells.
i) Propagation on semi-solid media Ginseng embryogenic cultures may be propagated on a semi-solid medium (same as described for initiating somatic embryo development) by subculturing masses of embryogenic tissue present in the first somatic embryo culture every weeks to a second, fresh semi-solid medium. Somatic embryos appear to grow from somatic embryo bearing centers present in the first somatic embryo culture.
Embryogenic cells are carefully picked off each individual clump of tissue and then aseptically transferred to a new petri dish containing the second medium using tweezers or a similar tool. The environmental conditions for culture maintenance are the same as for culture initiation (e.g., 22-26°C/dark). Ginseng embryogenic cell cultures maintained on semi-solid media in this manner have been successfully propagated for more than 4 years of continuous culture without showing a decline in growth rate or in the production of somatic embryos.
ii) Propagation as liquid suspension cultures Liquid cultures of ginseng somatic embryos maybe propagated in a variety of different vessels, including shake-flasks as described above and bioreactors.
As used herein, a bioreactor is a vessel having a volume of at least 1 liter or larger, used to culture animal, plant, or microbial cells under controlled conditions of aeration, agitation, and potentially other environmental parameters such as temperature.
Bioreactors provide many advantages for the growth of plant cultures compared to shake-flasks, including increased working volume, maintenance of a nearly homogenous culture, and control of the media and physical environment for optimum growth. The inoculum for establishing a liquid suspension culture is rapidly growing embryogenic tissue from another previously established somatic embryo culture.
In preferred embodiments, embryogenic tissue is selected that contains a fraction of globular embryos. When the first somatic embryo culture has been maintained on semi-solid media the fraction of globular embryos is transferred to a second liquid media contained in Erlenmeyer flasks as described above for establishing an embryogenic cell culture. Alternatively, Delong baffle flasks may be used as the baffle ridges on the insides of each shake-flask help to both break up the tissue and to maintain the tissue in suspension early in the establishment phase; eventually flasks without baffles are used routinely. The liquid maintenance medium is usually, but not necessarily comprised of the same components as the semi-solid medium on which the embryogenic tissue was cultured but without the gelling agent.
The most preferred embodiment of the propagating method is when the both the first somatic embryo culture and the second somatic suspension cultures are liquid suspension cultures. When the first somatic embryo is culture is propagated in liquid suspension the culture should be dense and particulate prior to selecting a fraction for transfer to a second media. Ideally, agitation conditions should be chosen so the particulate mass of cells present in the culture will contain both large clusters of about 1 to 5 mm having SEBCs and smaller clusters of less than 2 mm comprised of somatic embryos. An agitation rate of between 60 to 120 rpm on a gyratory shaker is usually preferred. This will vary depending on the cell type, the physical characteristics of the culture and growth conditions. Cultures grown in the dark will tend to have more SEBCs and can be agitated at a higher rate of speed to produce more of the smaller clusters containing globular embryos than cultures grown in the light.
In the most preferred embodiment of propagation by liquid suspension culture, the fraction of globular embryos transferred to the second media is selected to be enriched with globular embryos. In this method, the culture vessel is allowed to settle without agitation so as to allow the mass of cells to pack to the bottom of the vessel by gravity. This can easily be accomplished by tilting a flask at about 45°. The majority of globular embryos tend to form a layer that floats just above the main cell mass. A small portion of packed cells from this upper layer are selected with a pipette and transferred to the second media to form the second somatic embryo culture.
'The amount of the cells transferred depends on the amount of media in the first and the second cultures. As a rough estimate, transfer of about 1-2 ml of packed cells from a first culture containing 50 ml of media is sufficient to propagate a second 50 ml second culture. (See Example 6).
Globular embryos may be transferred weekly from the suspension cultures and either used to inoculate further liquid suspensions or plated onto semi-solid media to maintain the culture line or plated for plant regeneration as described hereafter under Somatic Embryo Maturation and Plantlet Regeneration. The constant transfer of only globular embryos to fresh liquid medium maintains the line at an early embryogenesis stage permitting an embryogenic culture to be maintained indefinitely.
Cultures have been maintained in this manner for more than two years while retaining their embryogenic capacity. This is a unique method for propagation of Panax somatic embryos, especially for P. quinquefolium.
The exact conditions for the propagating method of this invention will vary with culture type and variety. Most variations will relate to the rate of growth, amount of cells selected, frequency of transfer cells and intended use. In addition, light requirements, temperature, agitation and nutrients may differ depending on the source explant used to establish the suspension culture. Examples 5 - 9 illustrate test procedures that can be used to optimize some of these variables.
5 3. Production of Root-enhanced ~,erminants (REGs) from Somatic Embryo Cultures: Embryo Maturation and Plantlet Development Once a culture producing somatic embryos is obtained, the embryos can be matured, isolated and germinated to form plantlets. Somatic embryos can be matured from either liquid suspension or from solid agar plates. One embodiment of 10 this invention provides for plantlets produced from somatic embryos of ginseng that undergo ordinary development stages in vitro. In a preferred embodiment, however, plantlets are produced from embryos that develop into a novel germinant designated herein as a "root-enhanced germinant".
A root-enhanced germinant (REG) refers to a germinant derived from 15 globular somatic embryos of a plant, having a bipolar structure, with a small extended root and an apical initial. Early cotyledons may or may not be present. REGs derived from ginseng plants are illustrated in Figure 2(g-h) and Figure 4(d-f). There is no prior description of a germinant having the characteristics of REGs for any plant.
One characteristic of a REG is that rather than following standard zygotic embryo 20 developmental stages, i.e., globular, heart-shaped, torpedo, cotyledonary, etc. REGs appear to develop from globular embryos without passing through a conspicuous heart shaped or torpedo shaped embryo stage fewer intervening stages and yet still develop into a plantlet. The standard embryo stages are illustrated in Figure 3. In the case of REGs, either the earlier stages are bypassed (e.g., heart), or they are much more rapid.
25 One useful characteristic of REGs is that they directly develop into plantlets more rapidly than standard germinants. Another useful characteristic of REGs is that regenerated plantlets matured therefrom can develop both root and shoot structures with fewer treatments of phytohormones than conventional techniques used in plantlet regeneration. Therefore, REGs can be matured from embryogenic cell cultures on a media containing phytohormones that simultaneously promote both root and shoot development, thereby providing for rapid maturation of the plantlet.
Development of REGs from a ginseng globular embryo culture and regeneration of plantlets matured therefrom are illustrated in Figures 2i -21.
This invention provides characteristics of REGs that can be produced from a somatic embryo culture of any plant. Although the Examples provided here pertain to REGs prepared from ginseng plants, the description of REGs and of methods for their production will enable one of ordinary skill in the art to readily prepare REGs from somatic embryo cultures of any plant.
Therefore, one embodiment of this invention provides a method of regenerating a plantlet from an embryogenic cell culture of any plant through use of an REG intermediate. The method includes the steps of: selecting globular embryos from the embryogenic cell culture and transferring the fraction of globular embryos to a germination media selected to induce the globular embryos to develop into root-enhanced germinants. In the practice of this method, REGs can be derived from either liquid suspension cultures or from cultures maintained on semi-solid media. As will be apparent from this description and Examples that follow, REGs derived from globular embryos contained in liquid suspension cultures are preferred over those derived from cultures maintained on semi-solid media.
Embryo maturation into REGs and regeneration of plantlets begins by selection and transfer of globular embryos obtained from an embryogenic cell culture onto a plating media. Unlike conventional plantlet regeneration techniques, the plating media used for the production of REGs is a media that lacks an active amount of phytohormones. As used herein, an active amount of phytohormones is an amount that will elicit a developmental response in a plant tissue. In a preferred embodiment, the plating media does not contain any phytohormones, the effect on the production of REGs is more properly attributed to the absence of an active amount of phytohormones because REGs will likely be produced in the presence of low amounts of phytohormones. The plating media may be a semi-solid or liquid media but semi-solid media is preferred A typical plating media contains MS salts at half to full strength, a carbon source such as sucrose, vitamins (e.g. Gamborg vitamins and thiamine-HCL) and activated charcoal. See Examples 7 -10. The selected globular embryos are first washed with a phytohormone free media before transfer to the plating media.
It is preferable to separate globular embryos transferred onto semi-solid germination media in order to facilitate growth and identification of germinant characteristics. Typically about O.Sg fresh weight of globular embryos are arranged on a standard petri plate. Plates containing transferred embryos are incubated under a low light and for a photoperiod selected to promote development of REGs. For embryos obtained from ginseng cultures, the low light used was about 2.7-18.8 pmol m-ZS-1, and the photoperiod was 16 hr. The incubation temperature is room temperature. (22-26°C).
After about 1- 3 weeks of growth on the plating medium the embryos are examined under a microscope with or without histological staining to score for the presence of REGs.
Histological analyses of ginseng somatic embryo development produced by this invention are shown in Figure 4. Similar histological analysis may be performed for somatic embryo development from any plant. Developing embryos from semi-solid medium and from suspension cultures may be classified into globular, torpedo, heart, cotyledonary, and REG according to the their appearance as determined by histology or light microscopy. For histological staining, embryos were fixed in formalin-acetic acid, dehydrated through an ethanol series, embedded in paraffin and sectioned at 10 ~M. Sections were fixed onto microscope slides and stained with safranin-fast green.
Figure 4 shows various stages of embryo development from somatic embryo cultures of American ginseng as revealed by histological staining. Figure shows various stages of embryo development as revealed by light microscopy alone. These figures clearly demonstrate the true production of ordinary somatic embryos and REGs through the practice of this invention.
Variables such as the type of embryogenic cell culture, media and other conditions of maintenance can effect the production of REGs. These variables can be optimized by varying conditions and selecting conditions that produce the largest amount of REGs after transfer to the plating media. As explained below and in the Examples provided conditions can be found where at least 3 REGs are produced per gram (fresh wt.) of globular embryos plated. In more preferred methods at least SO
REGs are produced per gram (fresh wt.) of globular embryos plated.
i) REGs produced from somatic embryos propagated as liquid suspension cultures In embodiments using liquid suspension cultures, REGs are obtained by selecting a fraction of globular embryos from the liquid suspension as provided above for culture propagation. These globular embryos are plated onto the semi-solid germination media and scored for REG development as described. REG production from liquid suspension cultures is affected by the speed of agitation and exposure to light. Figure 5 shows that cultures exposed to low light and a low agitation rate of about 60 rpm produce a moderate and variable number of REGs in a cyclical pattern that persists for several months. Figure 6 shows that cultures exposed to low light and a high agitation rate of about 120 rpm produce a large amount of REGs However, REG
production rapidly declines in a cyclical manner over time because these cultures tend to produce a higher amount of embryos that develop through the ordinary stages of embryo development. Figure 7 shows that cultures propagated in the dark at a high rate of agitation continually produce moderately high number of REGs in a cyclical manner that is less variable and more persistent over time. It is therefore preferred that REG
production from ginseng plants be accomplished by liquid suspension cultures propagated in the dark and at a moderate to high agitation rate of 60 -120 rpm. These results indicate that culture conditions of the suspension culture have a dramatic effect on REG production and should be optimized for each plant variety and genotype used.
Plantlets are regenerated from REGs and other germinants of somatic embryos by transfer of the somatic embryos to a germination media that contains an active amount of phytohormones. As mentioned in the "Background of the Invention", for ginseng embryo maturation and plantlet regeneration, the most commonly used phytohormones have been GA3 and BA, used together at 0.5 or 1 mg/L. These hormones have been shown to promote shoot formation from ginseng embryoids (Chang and Hsing, 1980). Generally, following treatment to promote shoot formation, a second step is needed to promote root formation, i.e., transfer to media containing a rooting auxin, e.g., IBA (indole-butyric acid) or NAA. It is essentially a two or three step organ induction process.
With the root-enhanced germinants provided by this invention a new protocol may be used because both an early developed root and shoot are already present. Therefore, REGs may be matured into plants much more quickly, using a maturation protocol that avoids a separate period of growth on a media required to promote rooting in addition to another period of growth on a media required to promote shooting. This allows a novel combination of phytohormones to be used as a second media in plantlet regeneration that will simultaneously promote both shoot formation and increase root growth.
In a preferred embodiment, the germination media contains a combination of GA3 and NAA. The amount of NAA and GAS can be optimized by varying the concentrations and ratios of each. The amounts of GA3 and NAA that will be optimal is expected to vary for the induction of REGs from differing cultures and from differing varieties or species of plant. In some embodiments, NAA
concentrations of between 1- 12 ~M and GA3 concentrations of about 0.5 - 6 ~M
should be optimal. In other embodiments, the NAA concentration should be about 4 - 8 ~M
and the GA3 concentration should be about 2-4 ~M. In most other embodiments, it is expected that a ratio of NAA to GA3 concentrations should be about 2 to 4 fold. In the embodiment illustrated in Figure 8, GA3 was optimal at 2.6 ~M when NAA was set at 5.4 ~M. Other combinations of root and shoot promoting hormones might be used to accomplish similar same results for different plant varieties.
REGs are transferred to the germination media after about 1 -3 weeks of development on the plating media lacking phytohormones. REGs are maintained on the germination media for a period sufficient to cause maturation of a plantlet having both shoots and roots. This period may be from 1-5 weeks but is often within 1 -2 weeks.
Root and shoot growth may be scored in a number of ways including frequency of germinants developing both tissue and the size of tissues developed (See Examples 8-16). Exposure to light during growth on the germination media also promotes maturation of root and shoot tissue. A typical light exposure of 2 to 5 pmol m-2s-~ with a 16 hour photoperiod is sufficient, but light exposures can be varied to optimize maturation of plantlets from particular germinant and plant varieties. For maturation of REGs obtained from a suspension culture of American ginseng, light exposures of up to S 18.8 pmol m-2s-~ significantly enhanced root and shoot development over a lower light exposure of about 2.7 pmol m Zs.-~ (See Example 10). In a similar manner, sucrose and salt content of the germination media can be varied to achieve optimal results. For American ginseng, a media containing half strength MS salts and about 3%
sucrose was significantly better than media having higher and lower amounts of each of these 10 components. (See Example 11 ).
After growth on the germination medium the germinants are transferred to a third, post-germination media that again lacks phytohormones in order to promote maturation into a plantlet. In a preferred embodiment, REGs are germinated for about 1 week on the germination media before transfer to the post germination media.
As with 15 the other media of this invention, alternative formulations of the post-germination media will work so long as the media promotes plantlet maturation. In a preferred embodiment, the post germination media was comprised of half strength MS
salts, full strength Gamborg vitamins, 3% sucrose and 1 % charcoal. Charcoal at 1 % was found to enhance media characteristic for a variety media including the plating media, the 20 germination media, the post germination media and for other media described hereafter.
(See, e.g. Example 12) The presence of charcoal is particularly important in the germination and post germination media because root development from REGs is impaired in media lacking charcoal. (See Example 15) Therefore, charcoal should be evaluated in any embodiment of this invention that seeks to optimize media 25 composition for other plant varieties.
ii) Maturation of plantlets produced from somatic embryos propagated on semi-solid media In embodiments where somatic embryos are propagated on a semi-solid media rather than liquid suspension cultures REGs are produced at a lower frequency than REGs produced form suspension cultures. Globular embryos capable of maturing into REGs tend to be present as a smaller fraction on semi-solid media than in liquid suspension cultures thereby requiring a more labor intensive process to search for REGs produced on semi-solid media. When REGs are found, they can be matured and regenerated into plantlets as with the method described for liquid suspension cultures.
However, a more preferred embodiment for cultures maintained on semi-solid media is to transfer all globular embryos to a germination media that contains an active amount of phytohormones that promotes ordinary embryo stage development.
In preferred embodiments, the phytohormones contained in the germination media include, but are not limited to, one or more of BA, GAS NAA, BA, IBA and/or 2,4-D. The preferred media base is half strength or full strength MS media with sucrose and vitamins and charcoal. In one embodiment, the germination medium further includes a combination of BA/GA3 at 4.4/2.9 pM. More preferred embodiments include a combination of NAA/2,4-D at 0.5/4.5 ~M or 2.5/2.25 pM. Other preferred embodiments include a combination of GA3/ NAA; at 2.6 /5.4 ~M or GA3 /IBA at 2.6/4.9 ~M. Less preferred embodiments include a combination of BAP/GA~ at 4.4/2.9 ~M. The maturation of globular embryos on media containing these phytohormones closely follows the angiosperm zygotic embryo developmental pathway illustrated in Figure 3. The effect of phytohormone combinations is evaluated by first transferring embryos to the germination media for a period of 1-3 weeks then transferring the germinants to a post-germination media containing no phytohormones for about 3 weeks to score for shoot, leaf and root development.
For somatic embryos obtained from both Asian and American cultures maintained on semi-solid media, the most rapid germination was observed when the germination media contained NAA/2,4-D (5.0/4.5 pM). (See Example 13). For plantlet recovery from somatic embryos of Korean ginseng, the combination of BAP/GA3 at 4.4/2.9 pM promoted shoot but not root development. This abnormality is one of the characteristics of precocious germination. These plantlets could be further developed in some cases, by transfer to a rooting medium containing an auxin such as NAA at about 5.0 pM, however, none of these plantlets were successfully acclimatized by the acclimatization method provided below as another aspect of this invention.
For American ginseng cultures maintained on semi-solid media, germination on a media containing combinations of GA3 with either NAA or IBA
was also effective. The combination containing NAA produces roots and a 22%
enhancement in shoot development over controls lacking GA3 whereas the combination containing IBA produced about a 17% enhancement in shoot development. Neither combination significantly effected root development. See Example 16.
After germination for a period of 1-3 weeks on the germination media, germinants having developed root and shoot structures are transferred to a post germination media for plantlet regeneration as is this case for plantlet regeneration through REG germination. Plantlets are grown in a covered and sterile growth container such as a jar as shown in Figure 2(j) or a Magenta bottle shown in Figure 2(k).
The humidity inside the container is at least about 95% RH and the plantlets are grown with a 16 hr photoperiod of low light exposure of about 18.8 pmol m z s.-' The regeneration period on the post germination media is 1 -3 months.
4. Soil Acclimatization and Rapid Maturation of Plantlets into Mature Plants Another aspect of this invention is a method for acclimatization of ginseng plantlets regenerated from embryogenic cell cultures. As mentioned in the Background of the Invention, acclimatization of regenerated ginseng plantlets is one of the major difficulties in cultivation by embryogenic cell culture. In particular, there are no methods demonstrating acclimatization of regenerated ginseng plants for field growth that can be reliably reproduced for large scale cultivation. This is because roots and leaves formed in vitro are not adapted to the relatively harsh conditions experienced in the field. A drop in relative humidity of nearly 100% in vitro to as low as 35% in the field has been the most difficult factor although infection by plant pathogens naturally present in the field is also a problem. In the method provided by this invention, plantlets regenerated from embryogenic cell cultures can be reliably acclimatized using steps amenable to large scale cultivation.

The method of acclimatizing regenerated ginseng plantlets for field growth is essentially a method for gradually adjusting the plantlet's exposure to ordinary soil, reduced humidity, and increased light as would be encountered in the field. The method includes the steps of transferring the regenerated plantlet from a growth media to an aseptic soil, exposing the transferred plantlet in a sterile environment to a first condition that provides a first humidity and first light exposure for a first period of time, changing from the first condition to a second condition over a second period of time where the second condition includes a second humidity lower than the first humidity and a second light exposure greater than the first light exposure.
As used hereafter, the first condition may be called high humidity/low light and the second condition may be called low humidity/high light. In using this method, plantlets can be acclimatized for field growth in 3 - 5 months from the time they are fist transferred to the sterile soil. The transfer to aseptic soil permits hardening of the plantlets to resist pathogens while the gradual change in humidity and light permit the plantlets to adopt to the light and humidity conditions likely to be encountered in the field.
In vitro ginseng plantlets should be selected for acclimatization in soil by the following criteria: Plantlets should have a true leaf/leaves, an actively growing root, be of reasonable size, (about 1 cm or larger) non-vitrified and not callused.
Normally, plantlets regenerated from liquid suspension cultures are ready for acclimatization approximately 4 months after an embryo has been transferred from the liquid suspension culture to the germination media.
Several embodiments of the method of acclimatizing plantlets relate to the period required for complete acclimatization. It is expected that the period required will vary according to the type and source of plantlet being acclimatized. In one embodiment, the first period of high humidity/low light is 1-4 weeks and the second period of low humidity/high light is at least 2 months. In another embodiment, the first period is 1-4 weeks and the second period is 2-4 months. In another embodiment the first period is 2-3 weeks and the second period is at least 2 months. In a preferred embodiment, the first period is about 3 weeks and the second period is about 4 months.

In another preferred embodiment, a sum of the first period and the second period is 4 -5 months. When used with a regenerated plantlet grown in media for about 4 months, this last embodiment provides for acclimatized plantlets to be ready for field growth within 8 - 9 months from the time an embryo is transferred to the germination media to commence plantlet regeneration.
Other embodiments of the method of acclimatizing plantlets relate to the humidity and light conditions of the first and second periods. In a preferred embodiment, the first humidity is about 95% and the second humidity is between about 30%-65% RH. In another embodiment, the first humidity is greater than about 60% RH
and the second humidity is greater than about 30% RH. With regard to the first light exposure, in one embodiment, the first light exposure is less than about 60 pmol m-Z s'.
In a preferred embodiment, the first light exposure is gradually raised during the first period from about 18.8 to about 60 pmol m Z s-'. With regard to the second light exposure, in one embodiment, the second light exposure is between about 55-120 ~mol m'' s'. In a preferred embodiment, the second light exposure is gradually raised from about 55 to 120 pmol m 2 s' over the second period of time.
An easy way to adjust the humidity and light is by growing the covered plantlets in a container having a vented lid and placing the container in a growth chamber that allows light to be regulated. When the vents are covered the plantlet is exposed to a high humidity of about 95% RH. When the vents are uncovered the humidity will be lowered to about 65-75%. Later the lid can be perforated to further lower the humidity. Finally the lid can be removed to expose the plantlet to ambient humidity. During the entire acclimatization period the light can be adjusted as needed by setting the output and photoperiod of the growth chamber. Acclimatization of ginseng plantlets ordinarily uses a photoperiod of 16 hr.
The following protocol describes an embodiment of the acclimatization method that has been used to successfully acclimatized hundreds of American ginseng plantlets: Small drainable pots are filled with a porous soil mixture such as a 1:1 peat:
sand mixture and placed into an autoclavable vessel, such as a glass jar or Magenta~
bottle. A layer of sand in the vessel bottom provides drainage and pot stability. The soil is pre-wetted with water, and the sealed vessels sterilized by autoclaving.
Afterwards, the plastic vessel lids are replaced by covers that permit light entry and venting but maintain a sterile environment. In this protocol, Suncap~ covers (Sigma Chemical Co., St. Louis, Missouri, USA) were used and the vents temporarily covered 5 with tape. A Suncap~ cover is shown in Figure 2(k).
A plantlet regenerated from an embryogenic culture of P. quinquefolium of about 4 months of growth in a post germination medium was aseptically transferred to the sterile soil. Sterile conditions of growth are maintained for the first few weeks to months of growth in the soil. After transfer to the soil the vessels are placed in a growth 10 chamber with a photoperiod set to 16 hr and a light output of about 18.8 -28 ~,mol m Zs.'~ Plantlets are exposed to this first condition for a period of about 2 weeks during which time they overcome transplant shock and root growth resumes. During this time the plantlets are exposed to a first humidity of about 95%RH. After about three weeks of growth under this first condition the light output is adjusted to about 55 ~mol m-ZS'~, 15 and the Suncap covers are perforated to adjust the humidity to a second level of about 65-75%RH. Plantlets are grown under this second condition for about one month after which time the light exposure is adjusted to about 80 ~mol m-2s.-~ Soil moisture content is carefully monitored after this point, as the plants are still quite vulnerable to any stress. After about 3 - 4 month of growth under the second condition the Suncap 20 covers are removed completely and the second light exposure is increased to about 118 ~mol m Zs.-~ At this point the plantlets are capable of tolerating a humidity as low as about 30% RH and capable of transplantation for growth in the field.
If desired, plantlets can be successfully transplanted to the field after an acclimatization period of 3-5 months without further maturation steps being taken. The 25 above method of acclimatizing regenerated plantlets is one distinct embodiment of this invention. However, a more preferred embodiment provides a method of rapidly maturing regenerated ginseng plantlets that includes the acclimatization process as part of a maturation method and provides for field planting a matured plant 12 - 13 months after acclimatization is complete.

As used herein, the term "rapidly maturing" means to provide a shortened period of time for development of a plantlet so that it obtains characteristics of a plant that ordinarily requires a longer period of time to develop when the plant is cultivated by seed. One characteristic includes the outgrowth of a first bud.
This ordinarily requires an 18 month period of seed stratification plus a first season of growth for a seed grown plant when the time is calculated from commencement of seed stratification. Another characteristic is outgrowth of a second bud that ordinarily requires a second season of growth that follows after the first season and a period of natural dormancy. This second season of growth is ordinarily completed at 33 months as calculated from commencement of seed stratification. Still other characteristics include the development of root and leaf structures to a size where the plant is suitable for commercial harvest. This ordinarily requires about 60 months for American ginseng The development of root and leaf structures is correlated with commercially useful characteristics such as the production of seeds and ginsenosides. Accordingly, as used herein, a mature or a matured ginseng plant refers to a plant that has at least acquired the characteristics of a seed cultivated plant in its first season of field growth.
The method of rapidly maturing ginseng plantlets includes acclimatizing regenerated plantlets for field growth, senescing the acclimatized plantlets for an initial outgrowth period, and treating the senesced plantlets with a phytohormone composition for a flushing period. This process produces a ginseng plant that is matured in a shortened maturation period of time relative to a seed cultivated plant.
In one embodiment, the senescing comprises full outgrowth of a first leaf bud under a condition of controlled humidity and light. Outgrowth and senescing ordinarily occurs for a period of 3-7 months and usually is complete within 3-5 months.
In still another embodiment, the treating includes periodic application of the phytohormone composition to a secondary leaf bud of the plantlet. One embodiment of this last method includes treating with the phytohormones for a flushing period that lasts for 1-5 months. Another embodiment provides that the phytohormone composition used for flushing is comprised of GA3.

Other embodiments of the rapid maturation period relate to the shortened maturation period in relationship to the characteristics obtained. In one embodiment, the shortened maturation period is less than 40 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least about 48 months age where the age of the seed grown plant is calculated from commencement of seed stratification. In a preferred embodiment the shortened maturation period is less than 40 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least about 60 months age where the age of the seed grown plant is calculated from commencement of seed stratification. In a most preferred embodiment, the shortened maturation period is less than 36 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least about 60 months age where the age of the seed grown plant is calculated from commencement of seed stratification.
The time at which plants are transplanted to the field is about 12 - 13 months after the transfer of the plantlets to sterile soil which is about 18 months after the somatic embryo was first plated on media for regeneration of a plantlet.
Therefore, the method of rapid maturation produces a field growing ginseng plant in about months that has characteristics that would ordinarily take about 45 - 48 months for a seed grown plant to acquire. This benefit of rapid maturation applies to the remaining seasons of field growth needed to further mature the plant for commercial harvest.
Hence, commercially mature plants may be harvested after about 35 months from the time of embryo maturation rather than the typical 60 month period required for ordinary seed cultivation. A comparison of the rapid maturation method of this invention to ordinary seed cultivation is shown in Figure 12.
The following describes one protocol that embodies the method for rapid maturation of plantlets. Plantlets regenerated from P. quinquefolium cultures are acclimatized for about 3 weeks in the manner described above. About 3 weeks after transfer of the plantlets to sterile soil, fertilization is provided by Hoagland's solution (Hoagland and Snyder, 1933) diluted to a conductivity of 500 microSiemens which is applied regularly during active growth. A first outgrowth of a primary leaf bud develops on the maturing plantlet during the first period of growth under the Suncap, at 95% RH. This primary bud corresponds to a first season bud for a seed cultivated plant grown in the field. Suncap covers are perforated at about the same time, marking the end of the first high humidity period of the acclimatization method. After 3-5 months in soil, just about the time that the Suncaps are to be removed, the primary leaf bud begins to senesce and a secondary leaf bud begins to develop. This secondary bud corresponds to a bud that ordinarily develops in the second season of growth for a seed grown plant.
The secondary bud is treated by a single application of GA3 at about 5 -ppm to break the natural dormancy that ordinarily occurs after a first season of 10 outgrowth. This causes flushing of the next prong of the secondary bud within several weeks. Breaking the dormancy by flushing the bud with GA3 causes a continual growth cycle that eliminates about five months off an ordinary season of ginseng growth. The second growth season occurs for 4-6 months in a growth chamber until natural senescence occurs. At this point the matured plantlets are treated to about 100 days of cold exposure at 0 - 5 °C to satisfy the plant's need for dormancy.
The end of the cold treatment occurs about 18 months after an embryo was plated on germination media. At this point the fully matured plants is transplanted to the field. For transplantation, plants are carefully removed from the pots so that the potting mix remained attached to roots and transplanted to a prepared field.
After about three months of field growth the plant has acquired the characteristics of a 45 month, third season plant cultivated by ordinary seed cultivation Figure 12 illustrates a time comparison of ordinary seed cultivation and the rapid maturation method of this invention.
To date the matured plants of this invention have survived field transplantation and appear phenotypically normal relative to standard seed cultivated Panax quinquefolium seedlings. The above methodology has resulted in the successful acclimatization of over 200 ginseng plantlets over a 4 month period with over 80%
survival.

5. Trans~enic Plants The above described methods can also be used to propagate plants which are variants of the explant source, e.g., transgenic plants. As used herein, a "variant" is defined as any plant or tissue which contains a genetic alteration not present in the plant form from which the explant is derived from. Transgenic plants of ginseng may be produced through the introduction of exogenously supplied DNA. Embryogenic tissue, mature embryos, REGs, and plantlets may all be used for transformation methods. As described in the Background of the Invention, numerous techniques have been developed to introduce DNA into plant cell cells including ginseng. Such methods are particularly compatible with the methods of this invention as virtually all plant transformation methods currently rely on culturing embryogenic cells at some point in the transformation procedure. Using Agrobacterium transformation with the practice of this invention provides a novel method for ginseng transformation as illustrated by the following procedure:.
A. tumefaciens disarmed strain EHA 105 containing the plasmid p1779C
was used to transform callus tissue derived from root explants of ginseng plants. The plasmid contains both the neomycin phosphotransferase-II (NPT-II) and cucumber acidic chitinase gene within the T-DNA borders. Initially, the bacteria were grown overnight in 10 ml of Luria broth medium ( 10 g/L trypton, S g/L yeast extract, 5 g/L
NaCI, pH 5.4) containing 20 mg/L gentamycin at 29C. The next day, 1 ml of the overnight bacterial solution was resuspended in 25 ml Minimal Medium (MM) containing 20 mg/L gentamycin. Then, the bacterial suspension was spun at 2000 rpm for 10 minutes and the pellet was resuspended in MS medium (pH 5.4) to a final density of about 1 Og bacterial cells/ml. Prior to transformation, acetosyringone ( 100mM) was added.
Tissue infection was done by immersing the ginseng callus in the bacterial solution for 5 minutes, followed by rinsing with MS medium and finally blotting dry using sterile filter paper. The explants were placed on a semi-solid callus induction medium (MS with dicamba (9.0 ~M)) and kinetin (5.0 ~M) which is the same as medium used to propagate the embryogenic tissue, and the plate was incubated at 27°C in the dark for 2 days of cocultivation. Explants were then transferred to a selective medium (same as callus induction medium) but supplemented with 50 mg/L
kanamycin and 500 mg/L carbenicillin. Positive controls (root explants with no 5 infection placed onto callus initiation medium only) as well as negative controls (root explants with no infection but placed onto selective medium) were included in each test.
In addition to root explants, leaf explants were cocultivated as above and transferred to selective MS medium with NAA/2,4-D (5.0/4.5 pM) and 50 mg/L
kanamycin. All explants are incubated in the dark 24-26°C and are transferred to fresh 10 selective media at to initiate growth of somatic embryos.
Transformed tissue were identified by continued growth and enlargement on the selective medium while control explants plated on selective medium without prior infection (negative control) failed to grow in the presence of kanamycin. This indicates the presence of transformed tissue. Tissue that continues growth on the 15 selective medium is transferred to fresh selective media on a monthly basis until a sufficient cell mass is generated to establish an embryogenic cell culture according to the method described in this invention.
Using direct gene transfer by bombardment of globular embryos and SEBCs is another preferred way of using the methods of this invention for ginseng 20 transformation. A fraction of an embryogenic cell culture of American ginseng that contains SEBCs and globular embryos is transferred to semi-solid media and placed in a Biolistics particle gun chamber under vacuum. It is then bombarded with gold particles coated with vector DNA containing the gene for (3-glucuronidase (gus) to be used as a reporter gene and the NPT II gene to be used as a selectable marker.
25 Following bombardment, embryos are transferred to a fresh semi-solid media ordinarily used for embryogenic cell propagation as described above, i.
e., half strength MS supplemented with 3% sucrose, and pNAA/2,4-D at 5.0/4.5 ~M and 1%
charcoal. Embryos are incubated in the absence of kanamycin for 24 -48 hours to allow recovery and thereafter transferred to the same media supplemented with 25 mg/L
30 kanamycin. At the same time a sub-sample of embryos were assayed for gus activity.

The tissues following bombardment had dark areas that corresponded to wounded regions, but grew on MS medium. When stained with the conventional chemicals to illustrate gus activity, the characteristic blue color indicative of gus expression indicates that transient expression has been achieved. The embryos plated S on selective media are transferred weekly to fresh selective media. After about 3 weeks, embryo tissue that continues growth and shows a bright, soft and friable appearance on selective media is separated from darkening necrotic tissue. This separated tissue is used to establish a first embryogenic cell culture as previously described except that kanamycin is present at 50 mg/L in all growth media. After about 2-5 months a new embryogenic cell culture is established that can propagated, regenerated and matured by the methods of this invention. During the course of establishing and propagating the embryogenic culture, samples can be removed and stained for gus activity as a way to confirm transformation.
The following Examples demonstrate materials and methods used in the practice of this invention, and are offered by way of illustration, not limitation.
EXAMPLES

ESTABLISHING A SOMATIC EMBRYO CULTURE
2O FROM ROOT EXPLANTS OF AMERICAN GINSENG.
American ginseng plants, ranging from 3- to 5- years old, and grown under field conditions, were uprooted, placed in plastic bags with moistened paper towels, and transported to the laboratory. The taproot was washed in tap water and surface-sterilized in 70% ethanol for 7 min and then in a 40% solution of commercial bleach (containing 6.25% NaOCI) for 10 min, followed by 3-5 rinses in sterile distilled water. The epidermal tissues were discarded and pith sections (0.4-0.6 cmz) were removed from the root and plated in petri dishes (100 x 15 mm) containing approximately 25 ml of basal medium and the various growth regulator combinations described below.

The basal medium used was MS (Murashige and Skoog, 1962) supplemented with 100 mg/liter myo-inositol, 0.1 mg/liter thiamine-HCI, 0.5 mg/liter nicotinic acid, 0.5 mg/liter pyridoxine-HCI, 2 mg/liter glycine, 30 g/liter sucrose and 9 g/liter tissue culture agar. All chemicals used were tissue culture grade (Sigma Chemical Co., St. Louis, MO). The pH was adjusted to 5.8 with 1 N NaOH or HC1 prior to autoclaving. The following growth regulator combinations were evaluated for callus development and somatic embryo formation (concentrations are in pM):
2,4-D
(4.5, 9.0); napthalene acetic acid (NAA) (5.0, 10.0, 15.0, 20.0) with either kinetin (Kin) (2.5, 5.0) or 2,4-D (9.0); 2,4-D (9.0) with Kin (5.0); dicamba alone (range of 4.5-13.5), and dicamba (4.5, 9.0, 13.5) with Kin (5.0, 10.0). Petri dishes with explants were sealed with Parafilm~ (American Can Co.) and incubated in the dark at 22-26°C.
An average of 9-10 replicate petri dishes, each with 7 to 10 explants, were used, and the experiments were repeated at least twice over a 4-year period.
After 3-4 months of incubation, embryogenic calluses from MS medium with dicamba/Kin (4.5/10.0, 9.0/5.0) were transferred to a second medium (embryo induction medium). The combinations of growth regulators tested to promote somatic embryo development (in ~M) were: dicamba (4.5, 9.0, 13.5) with Kin (5.0, 10.0);
dicamba (4.5, 6.8, 9.0); NAA (2.5, 5.0) with Kin (4.6, 9.3); NAA/2,4-D
(1.25/2.25, 2.5/4.5); and NAA (5.4, 10.8) with N6-isopentenyladenine (2iP) (4.9, 9.8). All dishes were incubated in the dark. The percentage of calluses with globular somatic embryos was determined after 3 months.
Pith sections began to develop callus of a pale yellow color within 2-3 wk of plating, which was generally compact in appearance. Callus growth and appearance was optimal on dicambalkin (4.5/10.0, 9.0/5.0), dicamba (9.0) and 2,4-D/kin (9.0/5.0), whereas callus growth on all of the other growth regulators tested was less satisfactory. During the first three months on callus induction media, somatic embryos were observed only with the growth regulators dicamba/kin (4.5/10.0, 9.0/5.0).
When these calli were transferred to various embryo induction medium and incubated for three additional months, 15.6% of the calluses from callus induction medium with dicamba/kin (9.0/5.0), transferred to dicamba (9.0), formed somatic embryos, a few of which germinated to produce shoots. No somatic embryos developed on calluses transferred to any of the other growth regulator combinations.

ESTABLISHING A SOMATIC EMBRYO CULTURE FROM MATURE LEAF
S EXPLANTS OF AMERICAN GINSENG
Mature leaves from 3, 4 and S year-old American ginseng were obtained from field grown plants. Mature leaves have an advantage over seedling leaves in that they are larger and the prolonged seed germination step is avoided. Material was collected during June to August, placed in plastic bags with moistened paper towels, and transported in a cooler. The leaves were dipped in 70% ethanol for 2S sec and then in a 10% solution of bleach for 10 min and subsequently rinsed 3-S times with sterile distilled water. Leaf segments (0.2-0.4 cm2) were plated onto MS medium containing the growth regulator combinations described previously (Example 1 ).
Callus first developed at the edges of the leaf explants, generally near a 1 S midrib or vein. Callus appearance was soft and friable. Among the growth regulators, dicamba/Kin (4.S/5.0, 9.0/5.0, 13.5/5Ø 4.S/10.0 pM) and NAA (5.0, 10.0, 15.0, 20.0 pM) with 2,4-D (9.0 pM) produced healthy callus. Somatic embryos were first observed from leaf explants cultured on MS with NAA/2,4-D at S months (Figure 2a).
They initially appeared as small yellowish-white globular masses, which sometimes continued to develop into heart-shaped embryos. Somatic embryos developed at a frequency of 30% on NAA/2,4-D (10.0/9.0 p,M) and 7.2% on NAA/2,4-D (S.0/9.0 ~M).
Using leaf explants, both callus initiation and embryo formation/development were achieved on the same growth regulators (NAA/2,4-D) eliminating the need for successive transfers onto different media.

ESTABLISHING A SOMATIC EMBRYO CULTURE FROM LEAF

Dehusked American ginseng seeds which had been previously stratified over a 9 to 12-month period with a fluctuating cool:warm:cool temperature regime were immersed in 70% ethanol for 5 min and then in a 20% solution of Javex for 10 min and rinsed three times in sterile water. The seeds were placed on MS medium supplemented with 6-benzylaminopurine (BA) at 4.4 pM and gibberellic acid (GA3) at 2.9 pM.
All dishes were incubated at 4°C under low light (intensity of 7.0 pmol Zs') with a 16 hr/day photoperiod. When the seeds began to germinate, they were transferred to ambient temperatures of 22 to 26°C with the same light conditions. The seedlings were transferred to fresh medium until the first true leaves expanded (5-8 months from initial plating). Leaf segments (0.2-0.4 cmZ) and epicotyl segments (0.2-0.3 cm long) from these in vitro-derived seedlings were plated onto MS medium containing the growth regulator combinations described previously (Example 1 ).
Callus development from leaf explants was observed within 2 weeks and generally formed at the cut edges of the explant and was soft and friable in appearance.
Among all of the growth regulators evaluated, the combination of NAA (10.0 pM) and 2,4-D (9.0 pM) gave the highest somatic embryo formation. After 2 months of incubation, the percentage of embryo formation was 30%, while after 3 months, the frequency of somatic embryo production increased to 40%. By transferring the calluses to fresh medium at monthly intervals, the capacity to form somatic embryos was retained even after 3 years of culture.
Epicotyl-derived callus was soft and friable and similar in appearance to that of seedling-derived leaf explants. Among the growth regulator combinations, only calluses on dicamba (9.0 ~M) and kinetin (5.0 ~M) formed somatic embryos after months of culture, at a frequency of 2%.

ESTABLISHING A SOMATIC EMBRYO CULTURE
FROM FLOWER BUDS OF AMERICAN AND KOREAN GINSENG
Flower buds from 3- to 4- year-old American ginseng, and 6- to 7-5 year-old Korean ginseng were obtained from field grown plants. Material was collected during spring, placed in plastic bags with moistened paper towels, and transported in a cooler. The flower buds were washed for approximately 1 hour and then surface-sterilized by dipping in 70% ethanol for 25 sec and then in a 10%
solution of bleach for 20 min (+2 drops Tween-20) and subsequently rinsed 3-5 times with sterile 10 distilled water. Flower buds were then aseptically inoculated onto petri dishes containing MS salts with Gamborg's vitamins (Gamborg et al., 1968) and 100 mg/L
casein hydrolysate, 9 pM 2,4-D and 4.7 ~M Kin, 30 g/L sucrose and 6 g/L agar.
Material was subcultured to fresh media every 6-8 weeks.
After 1-2 months culture in the dark, callus was observed on 100% of the 15 cultures. Callus cultures were then transferred to MS salts and vitamins with 2.5 pM
NAA, 2.3 pM 2,4-D, 30 g/L sucrose and 7 g/L agar. Within 5 months post-inoculation, embryogenic callus were observed in 36% and 33%, respectively, of the American and Korean cultures respectively.

SOMATIC EMBRYOS AND SOMATIC EMBRYO BEARING CENTERS
FROM EMBRYOGENIC TISSUE OF GINSENG
For initiation of suspensions, embryogenic tissue of American ginseng initially derived from leaf material was used (Line 1). A first somatic embryo culture 25 established on MS agar plates was carefully separated into smaller tissue sections prior to inoculation. Approximately 2-Sg fresh weight of embryogenic tissue was selected from agar plates and inoculated into a 125 ml Erlenmeyer flask containing a second media comprised of 20 ml of MS basal liquid medium with 3% sucrose, 2.5 ~M
NAA and 2.3 p.M 2,4-D, pH 5.8 (suspension medium; same as solid). Flasks were capped with aluminum foil closures. Cultures were incubated at 60 rpm on a gyrotary shaker under low light. After 2 weeks, spent medium was removed and 20 ml of fresh MS media added. Fresh medium was then added at weekly intervals.
Within 3 weeks incubation, cultures were observed to be producing numerous globular embryos and the culture had increased in biomass. The culture was now considered to be established. After 5 weeks, the entire culture was selected and transferred to a 250 ml Erlenmeyer flask containing 50 ml of the same medium.
The flask was then agitated at 115 rpm in the dark on a gyrotary shaker. At weekly intervals spent medium was removed and 50 ml of fresh medium added. Repeating growth of fraction transferred back to semi-solid media, selection of embryogenic tissue, and transfer of a fraction containing globular embryos back to liquid media permitted an embryogenic cell culture to be routinely propagated in this manner.
Further liquid suspensions were prepared by, i) pipetting ( 10 ml wide-mouth pipette) 10-20 ml of embryogenic cells from the 250 ml Erlenmeyer flask into another 250 ml Erlenmeyer flask containing 50 ml of the same medium; or ii) aseptically transferring by spatula approximately 1/3 of the fresh weight of embryogenic cells from the 250 ml Erlenmeyer flask into another 250 ml Erlenmeyer flask containing 50 ml of the same medium. Cultures were quickly bulked up into numerous flasks using this methodology. Suspensions were also successfully established in 500 ml and 1 L Erlenmeyer flasks containing 150 ml and 300 ml of medium respectively.
Upon microscopic examination, suspensions were composed of a mixture of globular, heart-shaped, Figure 2(c-e), and early cotyledonary somatic embryos. The majority of embryos were globular. In addition, larger embryogenic clusters were present which may be referred to as somatic embryo bearing centers (SEBC's). It is suggested that the SEBC's continuously give rise to the smaller globular somatic embryos. The globular embryos then undergo some maturation/differentiation in liquid to form other stages. As suspensions continue to become incubated, further differentiation of the culture may occur. The rapidity of this depends on culture conditions and the particular genotype. However, in the practice of this invention, the culture line may be maintained continuously undifferentiated by routinely transferring only the globular embryos to fresh liquid medium (Example 6).
The above methodology was repeated successfully a minimum of 12 different times with genotypes initially derived from leaf, flower bud, and root.

PROPAGATION OF LIQUID SUSPENSION CULTURES CONTAINING
GLOBULAR EMBRYOS AND SEBCS OF GINSENG
Suspensions of American ginseng (Line 1 ) were established and maintained in 250 ml Erlenmeyer flasks as described in Example 5. The globular embryos were separated from the rest of the culture by simply inverting the Erlenmeyer flask at approximately 45 degrees and allowing the suspension to settle by gravity. The majority of globular embryos tend to float to just above the main cellular biomass where they form a layer. This fraction of globular embryos was selected and aseptically removed as a layer with a pipette. In this manner, approximately 1-2 ml of packed cell volume of globular embryos was transferred from the 250 ml Erlenmeyer flask containing 50 ml medium to a 50 ml Erlenmeyer flask containing 5 ml liquid medium (same medium as in Example 1 ). The culture was incubated on a gyrotary shaker at 60 rpm under low light. For 3 weeks, spent medium was removed and 10 ml of fresh medium added each week. After 4 weeks, the entire culture was transferred to a 125 ml Erlenmeyer flask containing 20 ml liquid medium. At this time the culture was observed to be growing as seen by increased biomass.
Propagation of the culture was continued as described in Example 1.

PRODUCTION OF REGS FROM LIQUID SUSPENSION CULTURES:
EFFECT OF SHAKE-FLASK CULTURE CONDITIONS ON REG PRODUCTION
American ginseng Line 1 was initially derived from leaf tissue of field-grown plants. In order to compare various culture treatments, embryogenic suspensions of Line 1 were maintained either on a lab bench-top shaker (60 rpm) under low light, or on a floor shaker (120 rpm) in low light or total darkness. All treatments were carried out in 250 ml Erlenmeyer flasks with 50 ml of suspension medium.
Every week, globular embryos were aseptically removed from each flask (approx. 0.5 g fresh wt/flask) and collected into a 250 ml Millipore Sterifil system with 220 ~m nitex cloth.
Globular embryos were then washed with a minimum of 200 ml liquid 1/2 MS + 3%
sucrose (no hormones). The nitex cloth filter with globular embryos was removed and placed onto a sterile filter paper to remove excess moisture. The nitex cloth filter was then carefully transferred onto a plating media (PL-lmedia: MS basal with 1/2 salts and full vitamins; 0.7% agar; 3% sucrose, 1% activated charcoal, pH 5.75) in petri dishes (100 x 15 mm plates). The filter was inverted onto PL-1 plates and the back of the filter lightly pressed to allow globular embryos to stick to the media. Approximately 0.3 g to 0.8 g fresh wt. of globular embryos were distributed per plate.
Using a stereomicroscope the globular embryos were aseptically separated so that they do not stick together. Plates were wrapped with Parafilm~, or Saran Wrap~, and incubated at 22-26°C under 3.5-5.5 ~molrri 2S-~, l6hr photoperiod.
After 1-3 weeks incubation on PL-1, globular embryos were shown to develop either one of two ways. A small proportion (approximately 10%) progressed through stages identical to that of angiosperm zygotic embryogenesis, i.e., heart-shaped, torpedo, etc. Figures 2(d- f). However, a larger portion of the globular embryos matured into REGs. Figures 2(g-h). In routine practice, at least 3 REGs were produced per gram of globular embryos plated. Often at least 50 REGs were produced per gram of globular embryos.
Figures 5-7 show the numbers of REGs produced from each of the treatments over approximately 3-4 months. As can be seen, numbers of REGs from each treatment were produced in a cyclical manner. High numbers of REGs would be produced one week, usually followed by a lower number the next week. The highest number of REGs from line 1 (1477), were produced from suspensions grown at 120 rpm in low light on the floor shaker (Figure 7). However, these cultures were also shown to differentiate the fastest into root/callus masses resulting in low numbers of globular embryos and hence REGs. Within 3 months cultures grown at 120 rpm in the light were producing less than 100 REGs. For comparison, cultures grown in darkness at 120 rpm showed the most consistent production of REGs over the time period (Figure 7). These cultures did not decline in numbers over the time period.
Furthermore, cultures grown at 60 rpm on the lab bench-top shaker differentiated the slowest.

MATURATION OF REGS AND PLANTLET DEVELOPMENT
Globular embryos from American ginseng Line 1 were collected and plated onto the first PL-1 media as described in Example 7. Plates were wrapped with Parafilm or Saran Wrap and incubated at 22-26°C, 3.5-5.5 pmolm-2S-I, l6hr photoperiod. After 1-3 weeks incubation, embryos were shown to develop into REGs.
Root-enhanced germinants were paired as for size and level of cotyledon development, suspension flask of origin, and length of time on the plating media (PL-I
media), with one of each being placed on a second media. For the purpose of this study the composition of the second media was varied to include or omit phytohormones 'The second media lacking phytohormones (GM-0 media) was comprised of GM base media (MS basal with 1/2 salts and full-strength vitamins, 0.7% agar; 3% sucrose; no charcoal pH 5.75). The second media containing phytohormones (GM-1) was GM supplemented with GA3 (2.6 ~M) and NAA (5.4 PM) REGs were cultured for one week at 22-26°C, 3.5-5.5 ~molrri 2S I, 16 hr photoperiod. After one week on the second media the REGs were transferred to a post-germination media (PG-I) (MS basal with 1/2 salts and full vitamins, 0.9% agar; 1 % sucrose; 1 % charcoal, pH 5.75) media. Cultures were incubated under a 16 hr photoperiod for about 40 days.
This study consisted of 11 pairs of plates with the number of REGs per plate ranging from 11 to 28, depending upon their availability at the time of plating on GM media. Shoot/leaf length and root length were measured after 40 days. The shoot and root lengths were measured and placed in one of 3 classes: <Smm; 5 to lOmm, and >lOmm (Table 2). Statistical analysis was based on the percentage of REGs having SS
shoot/root length >lOmm. The percentage data was normal. A t-test of paired sample means was done on the percent greater than l Omm for both shoot and root.

PERCENTAGE OF ROOT-ENHANCED GERMINANTS HAVING BOTH SHOOT AND ROOT

GM-0 GM-1 P(T<=t) two 90% CI
tail Shoot 0.3% 6.6% 0.02 +/-2.73 Root 3S% 43% 0.08 +/-6.88 GM-O:no hormones; GM-I GA3 (2.6 ~M) and NAA (5.4 ~M).
These results demonstrate that treatment of REGs with GA3 and NAA
improved the frequency of REGs developing into plantlets having shoot and root lengths greater than 10 mm, relative to media without either hormone.

In this study, the optimum concentration of GA3 for embryo/root-1 S enhanced germinant maturation and development was investigated. Globular embryos from American ginseng Line 1 were collected and plated onto the first PL-1 media as described in Example 7. Plates were wrapped with Parafilm and incubated at 22-26°C
under low light. After 1-3 weeks incubation, embryos were shown to develop into REGs.
REGs from Line 1 were transferred from the first PL-1 media to a second GM media with varying concentrations of GA3: 0.0, 1.3, 2.6, 3.9, 5.2, and 7.8, pM respectively. NAA was used at S.4 ~M for each treatment. REGs were cultured for one week at 22-26°C, 3.S-S.S ~molm-2S-I, l6hr photoperiod. After one week on the GM
media, the REGs were transferred to PG-1 media as in Example 8. After S weeks, the 2S shoot length of each REG was measured, and assigned to one of 3 classes: <S
mm, S to 10 mm, and > 10 mm.

This study was designed as a completely randomized design, with 4 replications, of 40 REGs per treatment. Where suspension plating dates differed within a rep, the number of REGs used from each culture remained constant over all treatments within a rep. Statistical analysis was carried out on the percentage of REGs >
10 mm in overall shoot length. The data expressed as percentage of REGs >10 mm, was normal, and therefore did not require transformation prior to analysis.
Analysis of the data indicated a significant difference among treatment means, at a=0.004 (Table 3). Graphing the data, averaged over the four reps (Figure 9) indicates that maximum shoot development occurred between 2.6 and 3.9 pM GA3.
Exposure of the REGs to GA3 did not prohibit leaf expansion. Therefore, a preferred embodiment of plantlet regeneration through REG production includes the use of a second media comprised of NAA at about 5 pM and GA3 at about 2. to 4 ~M
wherein a ratio of NAA to GA3 is about 1.5 to 4 fold.

ANOVA OF PERCENTAGE OF ROOT-ENHANCED GERMINANTS WITH GREATER THAN

df MS F

Treatment5 27.13 5.12**

Error 18 5.29 Total 23 j J

** F 5/18; a=0.004=5.12.

INFLUENCE OF LIGHT LEVEL ON POST-GERMINATION
SHOOT GROWTH FROM REGS
This study was carried out in order to determine the influence of increased light on shoot growth. Globular embryos from American ginseng Line 1 were collected and plated onto the first media (PL-1 media) as described in Example 7.
REGs were transferred from PL-1 media to the second media (GM-1 media) for one week, 14 to 37 days after plating from suspension. Both the suspension platings and the REGs on GM-1 were cultured under 2.7 to 4 ~molrri 2S I cool white fluorescent lights for a 16 hr photoperiod at 22-24°C.
Prior to transfer to the post germination media (PG media) REGs were paired for similar growth form (20 pairs/rep) and then one of each was placed on PG
media for 5 weeks. Each was then assigned without bias to be cultured under a light intensity of either 2.7 or 18.8 Pmol m 2s-I' Twenty pairs of REGs were assessed for maximum shoot growth. A total of twenty replicates were carried out. The frequency of REGs having shoots greater than l Omm was evaluated after 5 weeks. The data was analyzed via a paired t-test.
Results showed that increasing the light intensity during the first PG
phase increased the frequency of REGs with shoot length >lOmm by four fold (Table 4). The 95% confidence interval was + 3,3%.

INFLUENCE OF LIGHT INTENSITY ON SHOOT GROWTH
Light Intensity (~molm-zsec-')% Root-enhanced germinants with Shoots>10 mm 2.7 2.2 18.8 9.0 EFFECT OF SUCROSE CONCENTRATION AND BASAL SALT STRENGTH
ON POST GERMINATION SHOOT GROWTH OF REGS
This study was done in order to evaluate the effect of sucrose and salt concentration on plantlet growth, and to define the relationship between growth and the two factors.
Globular embryos from American ginseng Line 1 were collected and plated onto the first media (PL-1 media) as described in Example 7. REGs were transferred from PL-1 media to the second media (GM1 media) for one week, 14 to 37 days after plating from suspension. Both the suspension platings and the REGs on GMl were cultured under 2.7 to 4 ~molrri 2S-1 cool white fluorescent lights for a 16 hr photoperiod at 22-26°C.
The REGs were grouped according to morphological similarity and plating date, and then REGs in each group were equally distributed to one of 8 treatments such that there were 25 REGs/ treatment. The REGs were grown under the previous culture conditions, with light intensity increased to 18.8 ~molm-2S-~. The 8 treatments were a 2x4 factorial: basal (MS) salts at 1/2 and full strength;
and 4 levels of sucrose: l, 3, 5, and 7% arranged in a Randomized Complete Block Design with blocks (replicates) in total. After 5 weeks the REGs were classed for shoot length (<Smm, 1 to 10 mm, >lOmm), root length (<lOmm, 10- 15 mm and >l5mm), and fresh weight/ 25 REGs. Data from the lowest growth classes (shoot<5 mm and root<lOmm) were chosen for analysis, as this would best demonstrate, which treatments impaired REG growth relative to the others.
The Anova (Tables 5-7) analysis was consistent over the 3 traits assessed: the 2 factors, MS salt, and sucrose concentration were independent and significant. The lack of significance for the interaction between MS salt and sucrose concentration allowed them to be evaluated independently. Graphing of the treatment means (Figures 8-10) supports that 1/2 MS yielded larger REGs (g/25 REGs), with both longer roots (fewer having root length<10 mm) and larger shoots (fewer with shoot length <Smm). All three traits exhibited higher order relationships in response to sucrose concentration: with the highest order being cubic in the case of fresh weight and quadratic for both shoot and root length. Although root length increases with sucrose concentration, higher sucrose concentrations compromise both embryo mass accumulation and shoot growth. It was evident that the variation in REG
response, differed between blocks, in regards to embryo fresh weight, shoot length, and root length.

ANOVA EFFECT OF MEDIA (MS SALT AND SUCROSE CONCENTRATION) ON
ROOT-ENHANCED GERMINANT FRESH WEIGHT (G/ 2S REGS) SS df MS F

Treatment 0.6513 (7) 0.0930 9.541 **

MS 0.0973 1 0.0973 9.974 3% Sucrose 0.5257 3 0.1753 17.974 **

Linear 0.0411 1 0.0411 4.212 Quadratic 0.3992 1 0.3992 40.939 **

Cubic 0.0855 1 0.0855 8.770 **

MSxSucrose 0.0282 3 0.0094 0.966 ns Block 0.7144 13 0.0549 5.636 **

Error 0.8873 91 0.0097 Total I 2.2531 111 ~

F0.01, 13/91:2.33; F0.01, 7/91:2.84: F0.01, 3/91:4.01; F0.01, 1/91:6.93;
F0.05, 1/91:3.95 TART F F
ANOVA EFFECT OF MEDIA (MS SALT AND SUCROSE CONCENTRATION) ON THE
FREQUENCY OF ROOT-ENHANCED GERMINANTS WITH SHOOT LENGTH<SMM
SS df MS F

Treatment 0.4648 (7) 0.0664 9.21 MS 0.1819 1 0.1819 25.26 **

3% Sucrose 0.2249 3 0.07496 10.40 Linear 0.0032 1 0.0032 0.44 ns Quadratic 0.2096 1 0.2096 29.09 Cubic 0.012 1 0.0121 1.68 ns MSxSucrose 0.0579 3 0.0192 2.68 ns Block 0.8037 13 0.0618 8.58 **

Error 0.6556 91 0.0072 Total 1.924 111 F0.01, 13/91: 2.33; F0.01, 7/91:2.84: F0.01, 3/91: 4.01; F0.01, 1/91:6.93;
F0.05, 1/91:3.95.

ANOVA EFFECT OF MEDIA (MS SALT AND SUCROSE CONCENTRATION) ON THE
FREQUENCY OF ROOT-ENHANCED GERMINANTS WITH ROOT LENGTH <1 OMM
SS df MS F
I Treatment ~ 3.616 ~ (7) I 0.5166 ~ 38.39 **

SS df MS F

MS 1.506 1 1.0506 111.96 **

3% Sucrose 2.045 3 0.6817 50.66 **

Linear 1.694 1 1.6942 125.91 **

Quadratic 0.348 1 0.3481 25.87 **

Cubic 0.0027 1 0.0027 0.20 ns MSxSucrose 0.0645 3 0.0215 1.60 ns Block 1.223 13 0.0941 6.99 **

Error 1.224 91 0.0135 Total 6.063 111 F0.01, 13/91: 2.33; F0.01, 7/91:2.84: F0.01, 3/91:4.01; F0.01, 1/91: 6.93;
F0.05, 1/91:3.95.

TABLE OF MEANS
MS 1/2 trength Full S Strength Means Sucrose1% 3io 5% 7% 1% 3% 5% 7 95%
CI

Fresh g/ 25 0.41 0.610.5 0.50 0.350.520.50 0.410.018 Wt. Root Shoot %<5mm 78 72 77 84 94 80 82 89 1.6 length Root %<lOmm 72 47 37 32 90 68 59 63 1.1 length In conclusion, both the graphs and the table of means (Table 8) support that optimum growth during the first month of maturation is supported by media containing 1/2 MS basal salts and 3% sucrose.

1 O EFFECT OF CHARCOAL, PHYTOHORMONES, AND BASAL MEDIA STRENGTH
ON EMBRYO MATURATION OF PLANTLETS FROM SOMATIC EMBRYOS
MAINTAINED ON SEMI-SOLID MEDIA
Charcoal at 1 % (w/v) was added to each of the following germination media: MS medium with BA/GA3 (4.4/2.9 ~tM) or NAA/2,4-D (0.5/4.5 pM, 2.5/2.25 15 ~M). MS salts were tested at either full strength or half strength salts.
Somatic embryos originating from seedling and mature leaf explants and maintained as embryogenic tissue on agar plates were used. Somatic embryos were placed on the above media, 20 embryos per dish, and with 8 replicate dishes of each medium.
All dishes were incubated at ambient temperatures of 22-26°C with low light intensity. The frequency of embryo germination was rated weekly, for up to 8 weeks (Table 9).
Upon transfer of globular embryos propagated and maintained on semi-solid media containing NAA/2,4-D onto a germination media containing half strength MS medium with activated charcoal, heart shaped and cotyledonary-stage embryos were observed. Transfer of embryogenic masses gave rise to various stages of embryo development in clusters. Plantlets developed from embryos after 6 weeks, with well-developed roots and shoots. The developmental stages of embryos of ginseng were found to be comparable to the stages of somatic embryo development in other plants, such as carrot.
The most rapid germination was observed on a germination media comprised of half strength MS salts with charcoal and on NAA/2,4-D (5.0/4.5 ~M) with charcoal. The addition of charcoal was observed to significantly improve embryo germination in all media except that containing BA/GA3 (Table 9). The addition of charcoal likely resulted in the absorption of phenolic compounds and other chemicals that may have inhibited embryo development.
For plantlet recovery from somatic embryos of Korean ginseng, the combination of BAP/GAj at 4.4/2.9 pM as suggested by several authors (see Table 1), was found to promote shoot but not root development in the present study. This abnormality is one of the characteristics of precocious germination. None of the plants produced on this media were successfully acclimatized. The highest percentage of plantlet recovery was observed on half strength MS salts with charcoal, and on NAA/2,4-D (5/4.5 ~M) with charcoal.

GERMINATION OF GINSENG SOMATIC EMBRYOS UPON TRANSFER TO DIFFERENT MEDIA
WITH AND WITHOUT ACTIVATED CHARCOAL
Charcoal Medium Present Germination (%) at various times (weeks) BAP/GA3 No 0 15.9 31.6 58.6 69.1 80.Sb (4.4/2.9 Yes 6.5 20.5 38.4 51.8 65.5 7l.lc ~M) NAA/2,4-D No 0 9.5 11.9 15.6 21.8 35.6e (5.0/4.5 Yes 20.5 54.6 71.4 89.3 100.0 100.0a PM) NAA/2,4-D No 0 10.2 15.3 39 47.1 Sl.Sd (2.5/2.25 Yes 3.5 20.4 28.7 41.5 58.6 69.8c ~tM) Half strengthNo 5.8 22.5 35 58.5 76 82.Sb MS salts Yes 25.0 56.8 76.4 94.2 100.0 100.0a Full-strengthNo 0 12.5 28.4 41.9 63.5 71.3c MS salts Yes I 4.2 22.7 48.5 61.0 71.8 84.Sb I I

Treatments were compared for significant differences at week 12. Means in a column followed by the same letter are not significantly different (Tukey HSD test, P=0.05) Note: Somatic embryos were derived from embryogenic tissue cultured on solid agar plates.

EFFECT OF GA3, AND NAA OR IBA ON EMBRYO MATURATION

The purpose of this study was to evaluate and compare the effect of combinations of auxin at various stages (torpedo, early cotyledon, enlarged cotyledon) on embryo development. Further, both a shoot promoting phytohormone (GA3) and a root promoting phytohormone (NAA or IBA) were selected to be used together and at the same time in the germination media.
American ginseng Line 2 was initially derived from flower buds of field-grown plants. Embryogenic tissue of Line 2 had been maintained on solid maintenance media (MS + 2.5 PM NAA / 2.25 ~tM 2,4-D + 3% sucrose + 0.7% agar) for at least 1 year. The various stages of embryos were isolated from embryogenic tissue using a stereomicroscope and inoculated onto 1/2 MS with no hormones, with 2.6 ~M GA3 / 5.4 ~M NAA; or 1/2 MS with 2.6 ~M GA3 / 4.9 ~M IBA. Each of the treatments received 20 embryos of each stage at four per plate. The treatment was carried out for 1 week, then embryos were transferred to 1/2 MS + 1% activated charcoal with no hormones. After 3 weeks incubation, embryos were scored for shoot differentiation, the presence of both petiole and leaf, and root formation.
Experiments were carried out 2 times. The experiment was designed and analyzed as a split plot, with hormone being the main plot and embryo type being the sub plot, with a=0.10.
Table 10 indicates a significant difference among the means in shoot development over the three different media. Table 11 shows that treatment with GA3 / NAA enhanced shoot development over all types of embryos at 22%, compared with GA3 / IBA 17%, and the control, 0%. Neither the embryo type nor the interaction between embryo type and hormone compliment were significant in this study.
Table 12 indicates that IBA and NAA in conjunction with GA3 did not significantly (a=0.10) effect root development. However embryo type was significant (a=0.025) in terms of root production. Sixty-eight percent of torpedo embryos produced roots whereas early cotyledonary embryos and enlarged cotyledonary embryos produced 52% and 35%, respectively (Table 13).

2O ANALYSIS OF SHOOT DIFFERENTIATION IN GINSENG (LINE 2) SOMATIC EMBRYOS AT
THREE DIFFERENT STAGES OF DEVELOPMENT IN RESPONSE TO NO HORMONES, GA3 + NAA oR GAS + IBA' df SS MS F

hormone 2 0.1183 0.0592 9.35*

rep 1 0.0175 0.0175 2.77 ns hormone x rep 2 0.0127 0.0063 embryo 2 0.0047 0.0024 0.016 ns embryo x hormone4 0.0152 0.0038 0.263 ns split error 6 0.0867 0.0144 ' data was transformed (In (+1 )) prior to analysis.
* F~,2 0. I o=9.00 PERCENTAGE OF EMBRYOS THAT DEVELOPED SHOOTS (PETIOLE/LEAF) Torpedo Early CotyledonaryEnlarged CotyledonaryMean Control 0 0 0 0 GA3 + IBA 17 15 17 17 GA3 + NAA 17 32 15 22 S ANALYSIS OF ROOT FORMATION IN GINSENG (LINE 2~ SOMATIC EMBRYOS AT THREE
DIFFERENT STAGES OF DEVELOPMENT IN RESPONSE TO NO HORMONES, GA~3 + NAA oR GA; + IBA' df SS MS F

hormone 2 0.2617 0.1308 1.33 ns rep 1 0.0464 0.0464 0.472 ns main plot error hormone2 0.1963 0.0981 x rep embryo 2 0.1306 0.0653 9.19*

embryo x hormone 4 0.0429 0.0107 1.51 ns split plot error 6 0.0454 0.0071 ' data was transformed (In(+1)) prior to analysis.
* F2,6 0.10=3.46; F2,6 0.25=7.26 PERCENTAGE OF EMBRYOS THAT DEVELOPED ROOTS
Torpedo Early CotyledonaryEnlarged Cotyledonary Control 42 25 25 GAS + IBA 65 47 40 GA3 + NAA 97 82 40 Mean 68 52 35 The purpose of this study was to determine the maximum number of embryos per gram of embryogenic tissue on solid agar.

American ginseng Line 2 was initially derived from flower buds of field-grown plants. Embryogenic tissue of Line 2 had been maintained on solid maintenance media ( MS Medium with 0.47 mg/L NAA + 0.50 mg/L 2,4-D + 3%
sucrose, + 0.7% agar) for at least 1 year. Embryogenic tissue of Line 2 (0.57+p,p2g 5 fresh weight) was placed onto fresh P3A media and cultured in the dark for 4 weeks (22-26C). After 4 weeks, embryos ranging from globular to early cotyledonary, were enumerated and placed onto the plating media (PL-1 media) for 13 days. The embryogenic tissue was also weighed after removing the embryos. Developing embryos were then transferred to a germination media (GM-1 media) for 1 week.
The 10 number of secondary embryos was enumerated at this time. The embryos were then transferred to a post germination media (PG-3 media). The number of REGs was recorded at this time.
The experiment was repeated using the same embryogenic tissue from which the embryos were removed. The embryogenic tissue was transferred to fresh 15 P3A media and data was collected as per the previous experiment. The experiment was carried out with 12 replicates.

YIELD EVALUATION OF EMBRYOS PRODUCED FROM EMBRYOGENIC TISSUE
First Month 95% Second Month 95%
CI CI

Growth Rate ~a~ 121 _+ 21 --No. Embryos/g tissue 31.5 _+ 9.5 40.7 _+ 14.1 No. Embryos/g tissue 23.8 _+ 8.1 32.2 _+ 11.9 No. Embryos/g tissue 22.6 _+ 7.4 18.1 _+ 8.6 PG-3b Secondary Embryo GM-1 8.6 _+ 8.2 8.9 _+ 6.2 REGs PG-3 2.1 + 2.8 10.1 + 5.6 a % Growth Rate = (embryogenic tissue mass (g)4 Weeks - embryogenic tissue mass (g);~;,;ay~embryogenic tissue mass (g);~;~;a~) x 100.
b yields are based on initial mass of Line 2 inoculated in each experiment.
25 Results are shown in Table 14. Embryogenic tissue was shown to double in mass over 4 weeks of culture, as shown by the growth rate. Highest numbers of embryos were produced at time of transfer to PL-1. The embryo yields declined over the 3 phases of the culture process, primarily due to recallusing and impaired development. The percentage of secondary embryos (<10%), and REGs (<11%), were low in the semi-solid culture system, irrespective of the initial mass of tissue cultured.
The majority of embryos produced in the semi-solid culture system were primary embryos and their development paralleled the morphologies typical to zygotic embryo development. The liquid culture system reverses these trends in that it relies on secondary embryo production with the majority of embryos going through REG
development. These results illustrate that suspension cultures produce a higher number of REGS as compared to solid culture and that somatic embryos from semi-solid media develop in a pathway more closely resembling zygotic embryogenesis.
The above methodology for embryo maturation and plantlet regeneration from cultures maintained on semi-solid method was successful with embryogenic material irrespective of the original explant source, e.g., root, leaf, flower bud or zygote.

EFFECT OF CHARCOAL ON REG DEVELOPMENT ANDGROWTH OF THE ROOT
The effect of charcoal on REG root development and growth during the PL-1 and PG-3 phases of the culture process were evaluated. This study was restricted to American ginseng Line 1.
Design and Analysis The experiment was designed as a split-split plot Randomized Complete Block Design with the PL-1 being the whole plot and the PG-3 phase as the split factor (Table 15). The suspensions were randomly assigned to either PL-1 media with or without charcoal ( 1 %, 0%). 40 REG's, either 17 or 22 days old were randomly assigned from the PL-1 plates to the PG-3 treatments.
Both PL-l and PG-3 were considered fixed factors. The root length of each REG was measured after 6 weeks on the appropriate PG3 media. The 160 REG's or replicates within each block ( 80/PL sub plot and 40/PG 3subplot) was considered a random factor.

S MEDIA
Media Phases Charcoal 1 PL-1 1% 0%

0% 0% 0% 0%

3 PG-3 1 % 0% 1 % 0%

treatment (PL-1; 1 (1;1)2 (1;0)3 (0;1)4 (0;0) PG-3) Blocking By Suspension Plating Date The blocks were formed by using globular embryos/REG's from 3 sequential suspension plating dates (from the same batch) , with 2 samplings from each plating date, one at day 17 the other at day 22. Due to contamination in block 6, the data analysis was based on 5 blocks. Block was considered a fixed factor.

BLOCKING
Suspension Plating # Block Days on PL-1 Date 24/2a 3 6 22 a contamination resulted in loss of data, block #6 not used The SS interactions with the random factor (rep) were pooled if found to not be different by F statistic at a=0.05.
D.~.~r l=..,....sl., To summarize the Anova (Table 17) charcoal is not required during the early phases of embryo development (PL-1); charcoal is required for root growth to occur during the PG phase; and the presence or absence of charcoal in the first phase does not influence the second phase. A general observation was that the REG's on PG-3 without charcoal were short, thick, brittle and brown, where as those in charcoal were elongated, white , flexible and thin.
There was no significant differences in root length among block means, or days on PL-1. Contrasting suspension plating dates, (Feb. 10 v Feb. 17)-was significant. However, this difference was minimal: Feb. 10 plantlet roots were l.OScm and those from Feb. 17 were 0.93 cm. The days on PL addresses differences in embryo ages at the time of plating on their development rate, whereas plating date addresses the differences in embryo quality over time. This difference of 0.08cm is considered negligible.
The interaction of Block with PG reflects that REG growth was not consistently predictable: block 2 REG's compared to the other blocks produced both the longest roots on 1 % charcoal and the shortest roots on 0% charcoal (Figures 13, 14).
Respective to quality there was no readily observable , or tabulated, difference between the embryo's/REG's in this block compared to other.
Evaluation of root tip quality, more rounded and dense appearance versus thin and filmy, was not feasible at the time. Ignoring the block effect, larger roots were obtained by growing REG's in PG-3 media with 1 % charcoal (Figure 14) with mean being 1.29 ~ 0.07 and in the absence of charcoal 0.67 ~ 0.07 cm. Additionally the appearance of the roots cultured in the absence of charcoal during the PG phase suggests that these roots were not viable, appearing to deteriorate over time.

ANOVA OF THE EFFECT OF CHARCOAL ON REG ROOT GROWTH
SS MS F

PL 1 0.002 0.002 0.01 ns rep 39 8.117 0.208 PL x rep 39 9.154 0.235 PG 1 77.501 77.501 350.01 *

PLxPG 1 0.794 0.794 3.58 ns pooled error (a) 78 17.271 0.221 (B)lock 4 1.4171 0.354 1.64 ns Contrast: plating date ( 1.089 1.089 5.04*
10/2 vs 17/2 1 ) Contrast: days on PL ( 0.277 0.277 1.28 ns ) B x PL 4 0.282 0.071 0.33 ns B x PG 4 2.889 0.722 3.35 B x PL x PG 4 0.92_6 0.231 1.07 ns pooled error (a) 624 134.725 0.216 **F0.01 1,39 :7.33; F0.01,1,78: 6.97; F0.01 4,624 :3.35; F0.01,1,624: 6.68 *F 0.05 1,39 : 4.09; F 0.05 1,78: 3.96; F 0.05 4,624:2.39; F 0.05 1,624: 3.85 ns: not significant :a: MS error terms were pooled if found to not differ by F' 0.05.
From the foregoing, it will be appreciated that, although specific embodiments of this invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.
REFERENCES CITED
Gupta, PK; Pullman; GS ( 1990). Method for reproducing coniferous plants by somatic embryogenesis. (to Weyerhaeuser Company). USA 04957866, 09 Mar 1989.
Handley,LW; Godbey,AP (1996): Embryogenic coniferous liquid suspension cultures. (to Westvaco Corporation) Application: USA 5,491,090, 13 Feb 1996. USA Patent.

Wann,SR; Goldner,WR (1994): Induction of somatic embryogenesis in Taxus, and the production of taxane-ring containing alkaloids therefrom. (to Union Camp Corporation) Application: US 5,310,672, OS August 1993., issued 10 May 1994.
Ahn,IO ( 1996): Regeneration of ginseng (Panax ginseng C.A. Meyer) 5 through the maturation process of somatic embryos. J. Korean Soc. Hort.
Sci.37, 777-780.
Ahn,lO; Van Le,B; Tran Thanh Van,K (1996): Direct somatic embryogenesis through thin cell layer culture in Panax ginseng. Plant Cell Tissue Organ Cult. 45, 237.
10 Ammirato,PV (1983): Embryogenesis. In: Handbook of Plant Cell Culture. Vol. 1. (Eds: Evans,DA; Sharp,WR; Ammirato,PV; Yamada,Y) Macmillan Publishing Co, New York, NY, 82-123.
Arya,S; Arya,ID; Eriksson,T (1993): Rapid multiplication of adventitious somatic embryos of Panax ginseng. Plant Cell Tissue Organ Cult.34, 15 157-162.
Arya,S; Liu,JR; Eriksson,T (1991): Plant regeneration from protoplasts of Panax ginseng (C.A. Meyer) through somatic embryogenesis. Plant Cell Rep.
10, 277-281.
Asaka,I; Ichio,I; Hirotani,M: Asada,Y; Yoshikawa,T; Furnya,T (1994a):
20 Mass production of ginseng (Panax ginseng embryoids on media containing high concentrations of sugar. Planta Med. 60, 146-148.
Asaka,I; Ii,I; Hirotani,M; Asada,Y; Furuya,T (1993a): Production of ginsenoside saponins by culturing ginseng (Panax ginseng) embryogenic tissues in bioreactors. Biotech. Lett. 15, 1259- 1264.
25 Asaka,I; Ii,I; Hirotani,M; Asada,Y; Furuya,T (1994b): Ginsenoside contents of plantlets regenerated from Panax ginseng embryoids. Phytochem.36, 61-63.
Asaka,I; Ii,I; Yoshikawa,T; Hirotani,M; Furuya,T (1993b): Embryoid formation by high temperature treatment from multiple shoots of Panax ginseng.
Planta Med.59, 345-346.

Benkrima,L; Sun,LH; Sain,S; Zhu,J; Ma,YC; Kont,C;
Plaut-Carcasson,YY (1995): Adventitious somatic embryogenesis, plant regeneration and in vitro flowering of ginseng (Panax ginseng C.A. Meyer). Presented at the International Ginseng Conference, Vancouver, B.C. July 17-22, 1994.
Butenko,RG; Brushwitzky,IV; Stepyan,LI (1968): Organogenesis and somatic embryogenesis in the tissue culture of Panax ginseng C.A. Meyer. Bot.
Zh.
7,906-913.
Cellarova,E; Rychlova,M; Vranova,E (1992): Histological characterization of in vitro regenerated structures of Panax ginseng. Plant Cell Tissue Organ Cult.30, 165-170.
Chang,W; Hsing,Y (1980a): In vitro flowering of embryoids derived from mature root callus of ginseng (Panax ginseng. Nature 284, 341-342.
Chang,WC; Hsing,YI (1980b): Plant regeneration through somatic embryogenesis in root-derived callus of ginseng (Panux ginseng C.A. Meyer).
Theor.
Appl. Genet. 57, 133-135.
Charest, PJ; Michel, M-F ( 1991 ). Basics of plant genetic engineering and its potential applications to tree species. Information Report PI-X-104.
Petawawa National Forestry Institute, Forestry Canada. 48 pg.
Choi,KT ( 1988): Panax ginseng C.A. Meyer: Micropropagation and the in vitro production of saponins. In: Biotechnology in Agriculture and Forestry. Vol. 4.
(Ed: Bajaj,YPS) Springer-Verlag, Berlin, Heidelberg, 484-500.
Choi,YE; Kim,HS; Soh,WY; Yang,DC (1997a): Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid. Plant Cell Rep. 16, 738-744.
Choi,YE; Soh,WY (1994): Origin of somatic embryo induced from cotyledons of zygotic embryos at various developmental stages of ginseng. J.
Plant Biology 37, 365-370.
Choi,YE; Soh,WY (1996a): Effect of plumule and radicle on somatic embryogenesis in the culture of ginseng zygotic embryos. Plant Cell Tissue Organ Cult. 45, 137-143 Choi,YE; Soh,WY (1996b): The role of excision on somatic embryogenesis from mature ginseng zygotic embryos. Phytomorphology 46, 151-159.
Choi,YE; Yang,DC; Kim,HS; Choi,KT (1997b): Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination. Plant Cell Rep. 16, 841-846.
Dunstan,DI; Tautorus,TE; Thorpe,TA (1995): Somatic embryogenesis in woody plants. In: In Vitro Embryogenesis in Plants. (Ed: Thorpe,TA) Kluwer Academic Publishers, Dordrecht, The Netherlands, 473-539.
Fujioka,N; Kurisu,Y; Miyagawa,H; Kohda,H; Yamasaki,K; Shoyama,Y;
Nishioka,I (1986): Studies on the tissue culture of Panax japonicus(1).
Multiplication by somatic embryogenesis of flower bud and rhizome. Shoyak. Zasshi 40, 152-158.
Gamborg, O.L., Miller, RA., Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cultures. Exp. Cell Res. 50, 151-158.
' Haberlandt,G ( 1902): Kulturversuche mit isolierten Pflanzenzellen.
Sitzungsber.Math.-Naturwiss. Kl.Kais.Akad.Wiss.Wien. 111,69-92.
Haccius, B. (1978): Question of unicellular origin of non-zygotic embryos in callus culture. Phytomorp. 28:74-81.
Hoagland, DR, Snyder, WC (1933). Nutrition of strawberry plant under controlled conditions. Proc. Amer. Soc. Hort. Sci.30:288296.
Inomata,S; Yokoyama,M (1996): Genetic transformation of Panax ginseng for increased production of ginsenosides. In: Biotechnology in Agriculture and Forestry VII. Vol. #38. (Ed: Bajaj,YPS) SpringerVerlag, Berlin, 253-269.
Inomata,S; Yokoyama,M; Gozu,Y; Shimizu,T; Yanagi,M (1993):
Growth pattern and ginsenoside production of Agrobacterium-transformed Panax ginseng roots. Plant Cell Rep. 12, 681-686.
Jiu,SY (1992): Plant regeneration from adventitious root-derived calli of ginseng (Panax ginseng C.A. Meyer). J. Agric. Assoc. China New Ser., 41-48.
Kishira,H; Takada,M; Shoyama,Y (1992): Micropropagation of Panax ginseng C.A. Meyer by somatic embryos. Acta Hort.319, 197-202.

Lal, R; Lal, S (1993). Genetic Engineering of Plants for Crop Improvement. CRC Press Inc, Florida, 231, Pg.
Lee,HS; Kim,SW; Lee,K-W; Eriksson,T; Liu,JR (1995):
Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted B-glucuronidase gene in regenerants from isolated protoplasts.
Plant Cell Rep. 14, 545-549.
Lee,HS; Lee,K-W; Yang,SG; Liu,JR (1991): In vitro flowering of ginseng (Panax ginseng C.A. Meyer) zygotic embryos induced by growth regulators.
Plant Cell Physiol. 32, I 111-1113.
Lee,HS; Liu,JR; Yang,SG; Lee,YH (1990): In vitro flowering of plantlets regenerated from zygotic embryo-derived somatic embryos of ginseng.
Hortsci.25, 1652- 1654.
Li,TSC (1995): Asian and american ginseng - a review. HortTech. 5, 27-34.
IS Murashige,T; Skoog,F (1962): A revised medium for rapid growth and big-assays with tobacco tissue cultures. Physiol. Plant. I5, 473-498.
Oliver,A; Van Lierop,B; Buonassisi,A (Eds.) (1990): American ginseng culture in the arid climates of British Columbia. B.C. Ministry of Agriculture and Fisheries, Victoria,B.C. 37 pages.
Parrott,WA; Merkle,SA; Williams,EG (1991): Somatic embryogenesis:
potential for use in propagation and gene transfer systems. In: Advanced Methods in Plant Breeding and Biotechnology. (Ed: Murray,DR) C.A.B. International, Wallingford, I 58-200.
Potrykus, I ( 1991 ): Gene transer to plants: Assessment of published approaches and results. Ann. Rev. Plant Physiol. Plant Molec. Biol. 42, 205-222.
Proctor,JTA; Bailey,WG (1987): Ginseng:Industry, botany, and culture.
In: Horticultural Reviews. Vol. 9. (Ed: Janick,J) Van Nostrand Reinhold Company Ltd, New York, NY, 187-236.
Raghaven, V (1976): Experimental Embryogenesis in Vascular Plants.
Academic Press, London.

Reinert,J (1958): Morphogenese and ihre Kontrolle an Gewebekulturen aus Carotten. Naturwissen 45, 344-345.
Reinert,J (1959): Uber die Kontrolle der Morphogenese and die Induktion von Adventivembryonen an Gewebekulturen aus Karotten. Planta Med.53, 318-333.
Shoyama,Y; Kamura,K; Nishioka,I (1988): Somatic embryogenesis and clonal multiplication of Panax ginseng. Planta Med. 54, 155-156.
Shoyama,Y; Matsushita,H; Zhu,XX; Kishira,H ( 1995): Somatic embryogenesis in ginseng (Panax species). In: Biotechnology in Agriculture and Forestry. Vo1.31. (Ed: Bajaj,YPS) Springer-Verlag, Berlin, 343-356.
Shoyama,Y; Zhu,XX; Nakai,R; Shiraishi,S; Kohda,H (1997):
Micropropagation of Panax notoginseng by somatic embryogenesis and RAPD
analysis of regenerated plants. Plant Cell Rep. 16, 450-453.
Steward,FC; Mapes,MO; Mears,K (1958): Growth and organized development of cultured cells. II. Organization in cultures grown from freely suspended cells. Amer. J. Bot. 45, 705-708.
Tautorus, T.E., Dunstan, DI. (1995). Scale-up of embryogenic plant suspension cultures in bioreactors. In: Somatic Embryogenesis in Woody Plants.
Vol 1.
(ed. Jain, S, Gupta, P, Newton,R), 265-292.
Tautorus,TE; Fowke,LC; Dunstan,DI ( 1991 ): Somatic embryogenesis in conifers. Can. J. Bot. 69, 1873-1899.
Thompson,GA (1987): Botanical characteristics of ginseng. In: Herbs, Spices, and Medicinal Plants: Recent Advances in Botany, Horticulture, and Pharmacology. Vol. 2. (Eds: Craker,LE: Simon,JE) Oryx Press. Phoenix, AZ.
Tirajoh,A; Punja,ZK (1995): Tissue culture and Agrobacterium-mediated transformation of American ginseng (Panax quinquefolium L.). In: The Challenges of the 21st Century. (Eds: Bailey,WG; Whitehead,C;
Proctor,JTA; KyIe,JT) SFU, Burnaby, BC, 144-155.
Wang,AS (1990): Callus induction and plant regeneration of American ginseng. Hortsci.25, 571-572.

Zheng,X-f; Huang,B-q ( I 994a): Intranuclear and cytoplasmic inclusions in embryogenic embryoid cells of Panax quinquefolius. Acta Bot. Sin. 36, 73-74.
Zheng,X-f; Huang,B-q (1994b): Marker proteins for somatic embryogenesis in several plants. Acta Bot. Sin. 36, 175-180.

Claims

1. A method of cultivating a ginseng plant comprising the steps of:
regenerating a plantlet from an embryogenic cell culture; and maturing the plantlet into a mature plant by acclimatizing the plantlet for field growth.

2. The method of claim 1 wherein the embryogenic cell culture is a suspension culture maintained in a liquid media.

3. The method of claim 1 wherein the embryogenic cell culture is maintained on a semi-solid media.

4. The method of claim 1 wherein the embryogenic cell culture is a somatic embryo culture.

5. The method of claim 1 wherein the ginseng plant is selected from Panax quinquefolium, P. ginseng, P. notoginseng and P. japonicus.

6. The method of claim 1 wherein the ginseng plant is an American variety.

7. The method of claim 1 wherein the ginseng plant is P. quinquefolium.

8. The method of claim 1 wherein the ginseng plant is an Asian variety.

9. The method of claim 1 wherein the ginseng plant is P. ginseng.

10. The method of claim 1 wherein the ginseng plant is a hybrid variety.

11. The method of claim 1 wherein the ginseng plant is a transgenic plant.

12. A matured ginseng plant derived from an embryogenic cell culture of a ginseng plant.

13. The matured ginseng plant of claim 12 wherein the ginseng plant is selected from Panax quinquefolium, P. ginseng, P. notoginseng and P.
japonicus.

14. The matured ginseng plant of claim 12 wherein the ginseng plant is an American variety.

15. The matured ginseng plant of claim 12 wherein the ginseng plant is P.
quinquefolium.

16. The matured ginseng plant of claim 12 wherein the ginseng plant is an Asian variety.

17. The matured ginseng plant of claim 12 wherein the ginseng plant is P. ginseng.

18. The matured ginseng plant of claim 12 wherein the ginseng plant is a hybrid variety.

19. The matured ginseng plant of claim 12 wherein the ginseng plant is a transgenic plant.

20. A method of producing a root-enhanced germinant from an embryogenic cell culture of a plant comprising the steps of:
selecting globular embryos from the embryogenic cell culture; and transferring the globular embryos to a plating media selected to induce the globular embryos to develop into root-enhanced germinants wherein the root-enhanced germinant is a germinant that can mature into a plantlet without passing through a conspicuous heart shaped or torpedo shaped embryo stage.

21. The method of claim 20 wherein the embryogenic culture is a somatic embryo culture.

22. The method of claim 20 wherein at least 3 root-enhanced germinants are produced per gram of embryogenic cell culture.

23. The method of claim 20 wherein at least 50 root-enhanced germinants are produced per gram of embryogenic cell culture.

24. The method of claim 20 wherein the plating media contains less than an active amount of phytohormones.

25. The method of claim 20 wherein the germination media contains an active amount of phytohormones selected to simultaneously promote root and shoot growth.

26. The method of claim 20 wherein the plant is selected from Panax quinquefolium, P. ginseng, P. notoginseng and P. japonicus.

27. The method of claim 20 wherein the plant is an American ginseng variety.

28. The method of claim 20 wherein the plant is P. quinquefolium.

29. The method of claim 20 wherein the plant is an Asian ginseng variety.

30. The method of claim 20 wherein the plant is P. ginseng.

31. The method of claim 20 wherein the plant is a hybrid ginseng variety.

32. The method of claim 20 wherein the plant is a transgenic ginseng plant.

33. The method of claim 20 wherein the plant is an American ginseng and at least at least 3 root-enhanced germinants are produced per gram of embryogenic cell culture.

34. A method of regenerating a ginseng plantlet from an embryogenic cell culture comprising the steps of:
selecting somatic embryos from the embryogenic cell culture; and transferring the somatic embryos to a germination media selected to induce the somatic embryos to mature into a plantlet.

35. The method of claim 34 wherein the germination media contains charcoal.

36. The method of claim 34 wherein the somatic embryos are transferred to a plating media to develop a root-enhanced germinant prior to being transferred to the germination media.

37. The method of claim 36 wherein the plating media contains charcoal.

38. The method of claim 34 wherein the plantlet is further transferred to post germination media selected to promote growth of the plantlet.

39. The method claims 38 wherein the post germination media contains charcoal.

40. The method of claim 34 wherein the embryogenic cell culture is a liquid suspension culture.

41. The method of claim 34 wherein the plantlet is a P. Quinquefolium plantlet.

42. The method of claim 34 wherein the phytohormones contained in the germination media are selected to simultaneously promote root and shoot growth.

43. The method of claim 34 wherein the phytohormones contained in the germination media are comprised of NAA at a concentration of about 1-12 µM
and GA3 at concentration of about 0.5-6.0 µM.

44. The method of claim 34 wherein the phytohormones contained in the second media are comprised of NAA at a concentration of about 4 - 8 µM, and GA3 at a concentration of about 2 - 4 µM and a ratio of NAA to GA3 is concentrations is about 2 to 4 fold.

45. The method of claim 34 wherein the NAA concentration is about 4.0 to 6.0 µM and the GA3 concentration is about 2.0 to 3.0 µM.

46. The method of claim 1 wherein the regenerating of a plantlet from an embryogenic cell culture is done according to the method of claim 20.

47. A root-enhanced germinant of a plant wherein the root-enhanced germinant comprises a germinant derived from a somatic embryo that develops into a plantlet without having a conspicuous heart shaped or torpedo shaped embryo stage.

48. The root-enhanced germinant of claim 47 wherein the plant is selected from Panax quinquefolium, P. ginseng, P. notoginseng and P. japonicus.

49. The root-enhanced germinant of claim 47 wherein the plant is an American ginseng variety.

50. The root-enhanced germinant of claim 47 wherein the plant is P.
quinquefolium.

51. The root-enhanced germinant of claim 47 wherein the plant is an Asian ginseng variety.

52. The root-enhanced germinant of claim 47 wherein the plant is P. ginseng.

53. The root-enhanced germinant of claim 47 wherein the plant is a hybrid ginseng variety.

54. The root-enhanced germinant of claim 47 wherein the plant is a transgenic ginseng plant.

55. A method of propagating a culture of somatic embryos of a ginseng plant comprising the steps of:
establishing a first somatic embryo culture from an explant of the ginseng plant;
growing the first somatic embryo culture under conditions that promote production of somatic embryo bearing centers;
selecting a fraction of globular embryos produced from the somatic embryo bearing centers;
transferring the fraction of globular embryos to a growth media to establish a second somatic embryo culture;
continuing growth of the second somatic embryo culture under conditions that promote production of additional somatic embryo bearing centers; and repeating the growing, selecting, transferring and continuing growth steps for a maintenance period.

56. The method of claim 55 wherein the maintenance period is at least 2 years.

57. The method of claim 55 wherein the maintenance period is at least 4 years.

58. The method of claim 55 wherein the maintenance period is indefinitely sustainable.

59. The method of claim 55 wherein the fraction of globular embryos is greater than a combined fraction of other somatic embryos consisting of heart-shaped embryos and torpedo shaped embryos.

60. The method of claim 55 wherein at least one of the first somatic embryo culture and the second somatic embryo culture is a liquid suspension culture.

61. The method of claim 55 wherein the first somatic embryo culture and the second somatic embryo culture are each liquid suspension cultures.

62. The method of claim 55 wherein the first somatic embryo culture and the second somatic embryo culture are each maintained on semisolid media.

63. The method of claim 55 wherein at least one of the first somatic embryo culture and the second somatic embryo culture is a liquid suspension culture and selecting the first fraction of globular embryos includes selecting an uppermost layer of a cell mass formed by allowing the suspension culture to settle to the bottom of a vessel by gravity.

64. The method of claim 55 wherein the explant is obtained from a plant tissue selected from root, shoot, epicotyl, leaf and flower bud.

65. The method of claim 55 wherein the explant is obtained from a leaf.

66. The method of claim 55 wherein the explant is obtained from a flower bud.

67. The method of claim 55 wherein the ginseng plant is selected from Panax quinquefolium, P. ginseng, P. notoginseng and P. japonicus.

68. The method of claim 55 wherein the ginseng plant is an American ginseng variety.

69. The method of claim 55 wherein the ginseng plant is P. quinquefolium.

70. The method of claim SS wherein the ginseng plant is an Asian ginseng variety.

71. The method of claim 55 wherein the ginseng plant is P. ginseng.

72. The method of claim 55 wherein the ginseng plant is a hybrid ginseng variety.

73. The method of claim 55 wherein the ginseng plant is a transgenic ginseng plant.

74. The method of claim 1 wherein the embryogenic cell culture is propagated according to the method of claim 55.

75. The method of claim 1 wherein the embryogenic cell culture is a liquid suspension culture propagated according to the method of claim 63.

76. The method of claim 20 wherein the embryogenic cell culture is propagated according to the method of claim 55.

77. The method of claim 20 wherein the embryogenic cell culture is a liquid suspension culture propagated according to the method of claim 63.

78. A method of acclimatizing regenerated ginseng plantlets for field growth comprising the steps of:
transferring the regenerated plantlet from a growth media to an aseptic soil;
exposing the transferred plantlet in a sterile environment to a first condition that provides a first humidity and first light exposure for a first period of time;
changing from the first condition to a second condition over a second period of time wherein the second condition provides a second humidity lower than the first humidity and a second light exposure greater than the first light exposure.

79. The method of claim 78 wherein the first period is 1-4 weeks and the second period is at least 2 months.

80. The method of claim 78 wherein the first period is 1-4 weeks and the second period is 2-4 months.

81. The method of claim 78 wherein the first period is 2-3 weeks and the second period is at least 2 months.

82. The method of claim 78 wherein the first period is 2-3 weeks and the second period is about 2-4 months.

83. The method of claim 78 wherein a sum of the first period and the second period is 3 - 5 months.

84. The method of claim 78 wherein the first humidity is greater than about 60% RH and the second humidity is greater than about 30% RH.

85. The method of claim 78 wherein the first humidity is about 95% and the second humidity is between about 30%-65% RH.

86. The method of claim 78 wherein the first light exposure is less than about 60 µmol m-2 s-1.

87. The method of claim 78 wherein the first light exposure is raised during the first period from about 15 to about 60 µmol m-2 s-1.

88. The method of claim 78 wherein the second light exposure is between about 55-120 µmol m-2 s-1.

89. The method of claim 78 wherein the second light exposure is raised from about 55 to about 120 µmol m-2 s-1 over the second period of time.

90. A method of rapidly maturing ginseng plantlets regenerated from an embryogenic cell culture comprising the steps of:
acclimatizing regenerated plantlets for field growth;
senescing the acclimatized plantlets for an initial outgrowth period; and treating the senesced plantlets with a phytohormone composition for a flushing period to produce a matured ginseng plant in a shortened maturation period.

91. The method of claim 90 wherein the ginseng plantlet is selected from Panax quinquefolium, P. ginseng, P. notoginseng and P. japonicus.

92. The method of claim 90 wherein the ginseng plantlet is an American ginseng variety.

93. The method of claim 90 wherein the ginseng plantlet is P.
quinquefolium.

94. The method of claim 90 wherein the ginseng plantlet is an Asian ginseng variety.

95. The method of claim 90 wherein the ginseng plantlet is P. ginseng.

96. The method of claim 90 wherein the ginseng plantlet is a hybrid ginseng variety.

97. The method of claim 90 wherein the ginseng plantlet is a transgenic ginseng plantlet.

98. The method of claim 90 wherein the senescing comprises full outgrowth of a primary leaf bud under a condition of controlled humidity and light.

99. The method of claim 90 wherein the initial outgrowth period is 3-7 months.

100. The method of claim 90 wherein the initial outgrowth period is 3-5 months.

101. The method of claim 90 wherein the treating includes periodic application of the phytohormone composition to a secondary leaf bud of the plantlet.

102. The method of claim 90 wherein the flushing period is 1-5 months.

103. The method of claim 90 wherein the phytohormone composition is comprised of GA3.

104. The method of claim 90, further including a step of cold treating the matured ginseng plant for about 100 days at 0-5 °C after treating the plantlet with the phytohormone composition.

105. The method of claim 90 wherein the shortened maturation period is less than 40 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least about 60 months age where the age of the seed grown plant is calculated from commencement of seed stratification.

106. The method of claim 90 wherein the shortened maturation period is less than 34 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least about 48 months age where the age of the seed grown plant is calculated from commencement of seed stratification.

107. The method of claim 90 wherein the shortened maturation period is less than 34 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least 60 months age where the age of the seed grown plant is calculated from commencement of seed stratification.

108. The method of claim 90 wherein the shortened maturation period is less than 12 months and the mature ginseng plant has characteristics of a seed grown ginseng plant of at least about 33 months age where the age of the seed grown plant is calculated from commencement of seed stratification.

109. The method of claim 90, further including a step of field growth of the mature ginseng plant.

110. The method of claim 1 wherein the maturing is accomplished by the method of claim 90.

111. A method of cultivating a ginseng plants comprising at least two steps selected from the steps of propagating a somatic embryo culture derived from a ginseng plant according to the method of claim 55, regenerating a plantlet from a somatic embryo culture according to the method of claim 20, acclimatizing a plantlet regenerated from a somatic embryo culture according to the method of claim 78 and rapidly maturing a regenerated plantlet into a mature plant according to the method of claim 90.

112. A method of cultivating a ginseng plants comprising at least three steps selected from the steps of propagating a somatic embryo culture derived from a ginseng plant according to the method of claim 55, regenerating a plantlet from a somatic embryo culture according to the method of claim 20, acclimatizing a plantlet regenerated from a somatic embryo culture according to the method of claim 78 and rapidly maturing a regenerated plantlet into a mature plant according to the method of claim 90.

113. A method of cultivating a ginseng plants comprising the steps of propagating a somatic embryo culture derived from a ginseng plant according to the method of claim 55;
regenerating a plantlet from a somatic embryo culture according to the method of claim 20;
acclimatizing a plantlet regenerated from a somatic embryo culture according to the method of claim 78; and rapidly maturing a regenerated plantlet into a mature plant according to the method of claim 90.