WO2019048382A1

WO2019048382A1 - Method for production of sugarcane seedlings

Info

Publication number: WO2019048382A1
Application number: PCT/EP2018/073621
Authority: WO
Inventors: Juliano Francisco BARELA; Marco David Molina Risco; Paulo Cesar Idalgo DONADONI
Original assignee: Bayer Aktiengesellschaft
Priority date: 2017-09-06
Filing date: 2018-09-03
Publication date: 2019-03-14
Also published as: MX2020002498A; CO2020002617A2; BR112020004341A2; AU2018329159A1; AR112999A1

Abstract

The invention relates to methods for production of sugarcane seedlings comprising treatment with growth regulators, a single pruning step and treatment with phytohormones.

Description

Method for production of sugarcane seedlings

Sugarcane (Saccharum sp.) crops are of great importance in the world socioeconomic scenario, both with regards to income generation and with regards to the production of currency. In recent years due to increased demand for bioenergy, areas cultivated with sugarcane are increasing all over the world, as well as investments in factories to produce mainly alcohol and electricity.

Sugarcane is an important food and bioenergy crop and a significant component of the economy of many countries in the tropics and sub-tropics. The value of sugar cane is based primarily on its high productivity, efficient use of agricultural input like water, fertilizer, labour and it is locally convertible to high value products, like sugar, molasses, ethanol and energy. Sugarcane is produced in nearly 100 countries. Although the area of sugarcane cultivation takes place on 1.5% of total world cropland only, the worldwide production of sugarcane harvested total biomass in terms of wet weight is the highest of all crop plants. Sugarcane is an allogamous (out-crossing), perennial plant, probably originating in the regions of Indonesia and New Guinea, belonging to the Poaceae family, sub-tribe Saccharinae tribe Andropogoneae, genus Saccharum. Its current cultivars are interspecific hybrids, with the species Saccharum officinarum, Saccharum spontaneum, Saccharum sinense, Saccharum barberi, Saccharum robustum and Saccharum edule, all participating in the set-up of the genotypes of cultivated species/varieties. The true seed of sugarcane is a caryopsis-type fruit. The caryopsis is described as a simple, dry, indehiscent, uniseminate fruit, the seed coat of which grows together with the pericarp along its entire length (2009, Regras para analise de sementes, Ministerio da Agricultura, Pecuaria e Abastecimento, Brasilia). The seeds are oval, brown or yellowish and about 1.5 mm long.

Sugarcane reproduces sexually. However, seed production depends on specific environmental characteristics, e.g. the need of constant conditions of elevated temperature and humidity for germination. Furthermore, cultivated sugarcane species exhibit high complexity of their genome structure. Cultivated sugarcane species are derived from interspecific hybridization resulting in highly polyploid and aneuploid genomes. The genomes of cultivated sugarcane species exhibit a high level of heterozygosity. (Moore et al. and Zhang et al. in Sugarcane: Physiology, Biochemistry and Functional Biology, Ed.: Moore and Botha, Wiley 2014, Chapter 1 and 23, respectively)

The highly heterozygous, complex genome and the outcrossing behaviour of sugarcane prevent generation of genetically and phenotypically uniform sugarcane plants from seeds. Environmental conditions influence even and complete seed germination. For maintenance of varieties, amplification and cultivation sugarcane ever was and still is propagated vegetatively. In conventional sugarcane production systems crops are established by sprouting of vegetative buds. Sugarcane crops are conventionally established from stalk pieces (setts). Crops established from setts are called plant cane to differentiate it from ratoon cane. Ratoon crops are grown from the part of the plant remaining in the ground after harvest (stool) (Bonnett in Sugarcane: Physiology, Biochemistry and Functional Biology, Ed.: Moore and Botha, Wiley 2014, Chapter 3).

Ethephon ((2-Chloroethyl)phosphonic acid) is used in sugarcane crops as a ripener, inducing maturation of stalks and desiccation of leaves. Silva et al. (2007, Bragantia 66, 545-552) discloses a stimulating effect of ethephon applied as ripener on tillering of the subsequent sugarcane ratoon crop of up to six months after application. But anyway, ssuccessively grown ratoon crops show yearly accumulating yield losses starting already in the first year. Successive re-growing ratoon crops in one field therefore are possible only for a few generations. Longer use of ratoon crops would lead to economically inacceptable yield depreciation. From time to time new crop generations have therefore to be established in a given field by replacing the ratoon crops with plant crops. The conventional method for re-establishment of new sugarcane crops from setts has some long known disadvantages.

For establishing sugarcane crops in fields, setts are placed into furrows end to end and covered with soil. The stalk pieces used as setts commonly comprise more than one, often three, sometimes more dormant axillary buds. Each bud of a single sett has the potential to produce a new sugarcane plant. However germination of buds is not consistent in terms of germinating number and germination time. Therefore, sugarcane plants grown in a field established from setts are not equally distributed. The field shows areas with higher densities and those with lower densities of plants. This leads to competition of plants for sunlight and nutrients in field areas of high plant density and a waste of available sunlight and nutrients in areas with low plant density with the consequence that the total yield potential of sugarcane plants in a given field is not fully exploited.

Stolf et al. (1981, In: CONGRESSONACIONAL DA STAB, 2, Rio de Janeiro. Anais... 3/4, 443-456) and Serafim et al. (2013, STAB, Piracicaba 31, 22-25) have shown that the average germination rate was 50% for manual planting, and 35% for mechanical planting when using conventional setts. This low germination rate results in formation of a non-uniform crop stand with low-density of primary tillers (mother tillers), causing a drop in productivity of sugarcane fields, even in the first crop cycle, if there is no replanting operation. Replanting, however, does incur further costs for labour and seeding material.

Sugarcane setts are conventionally produced from commercially grown crops. However, seeding material should be produced in nurseries or fields separated from sugarcane crops for enabling production of disease free planting material. Transportation of large bulks of sugarcane planting material from its production site to the fields generates significant costs in sugarcane plantings. Furthermore, sugarcane nurseries require a substantial amount of land, care, labour and investment in crop protection products.

Around 9 to 12 tons of sugarcane stalks per hectare is used in manual planting, whereas mechanized planting requires 18 to 24 tons per hectare. The planted material is buried in the soil, and cannot be used for the production of valuables like alcohol, sugar, electricity (Stolf and Lee, 1990, Alcool & Acucar, Sao Paulo, 10(53), 20-25). In another experiment 6-8 tons per hectare of seed stalks, containing three buds, were needed for establishing commercial crops. Planting material costs for sugarcane range from 22 to 25 % of the total production cost, and thus constitute a major part of expenditure in sugarcane cultivation (Srivastava et al., 1981, In: CONGRES SONACIONAL DA STAB, 2, Rio de Janeiro. Anais... 3/4, 443-456).

Therefore, over time different approaches for improving production of sugarcane planting material have been followed.

Tissue culture micropropagation technology has been developed for large scale propagation of sugarcane planting material (Snyman et al., 2011, Plant Cell Rep. 47, 324-249). Mishra et al. (2014, Sugar Tech 16(3), 255-263) disclose methods for in vitro multiplication (micropropagation) of sugarcane plants comprising excising the youngest bud from the top of a sugarcane plant as explant, transfer of the bud into tissue culture, applying growth regulators for bud elongation, applying hormones for proliferation (tillering) of elongated buds, transfer of tillers to hormone containing rooting media and hardening of rooted tillers in a glasshouse. Promotion of lateral shoot formation (tillering) by ethylene induction through ethrel (2-chloroethyl phosphonic acid) at lower but not at high concentrations in sugar cane in vitro cultures is demonstrated.

However, micropropagation technologies still are far too expensive in production of planting material for establishment of commercial crops (Bonnett in Sugarcane: Physiology, Biochemistry and Functional Biology, Ed.: Moore and Botha, Wiley 2014, Chapter 3).

Approaches for production of sugarcane planting material without involvement of in vitro and/or tissue culture have also been followed.

One approach uses tillers and is called the seblang or sprouting method. Tillering and areal branching in sugarcane depend on and can be induced by external factors, in particular growth conditions like fertilization and irrigation. In the sprouting method sugarcane plants are grown in light and fertile soil and tillering is promoted by establishing wide spacing between plants, shallow planting, frequent watering and ample fertilization. After root development tillers including roots are severed from mother plants and planted separately, allowed to tiller again followed by repeating severing and separately planting the tillers (Van Dillewijn, 1952, Botany of sugar-cane. Waltham, Mass. U. S. A. Chronica Botanica, 371).

One further approach is known as the rayungan method. Rayungan is an Indonesian term, referring to the development of a single sprout (primary shoot) from lateral (axillary) buds of a sugarcane stalk, after pruning the apex of the mother plant. Primary lateral shoots are excised form the mother plant and planted (Sugiyarta & Winarsih, 2009, Sugar Tech 11(1), 22-27).

Compared to the rayungan method the stratified rayungan method produces several shoots arising on a single secondary bud due to decapitation of the primary (lateral first order) shoots. Primary and secondary shoots are removed from the stalk together with parts of the node to which the bud is attached for producing un-rooted seedlings. The number of shoots produced in the stratified rayungan method depends on position of the bud on the stalk, viability depends on number of leave blades on the shoot and for rooting shoots with less than four leaves should be treated with rooting stimulants like indole- acetic acid (Sugiyarta & Winarsih, 2009, Sugar Tech 11(1), 22-27).

Keethipala et al. (2001, Sugar Tech 3(3), 106-108) discloses a method including decapitation of sugarcane mother plants, followed by decapitation of primary lateral shoots, followed by decapitation of secondary lateral shoots, followed by decapitation of tertiary shoots, separation of lateral shoots from the mother plants and subsequent transfer into polythene bags for rooting. Planting material production takes 15 - 16 months including growing of mother plants for about 8 months. One mother stalk is said to produce up to 40 lateral shoots when decapitation is performed at least three times. JP 08280244 discloses the production of sugarcane seedlings from axillary buds. Sugarcane mother plants are decapitated inducing formation of primary lateral shoots. The primary lateral shoots are decapitated again for inducing production of secondary lateral shoots. The secondary lateral shoots can be excised from the plants for producing seedlings. However, for preparation of a sufficient number of lateral shoots, decapitation of second followed by decapitation of tertiary and even higher order lateral shoots is preferred in efficient seedling multiplication. At least two but preferably four or even more times decapitation is required.

JP 2003 204716 and JP 2000 135025 disclose production of sugarcane planting material by decapitation of a stalk at a height of about 120 cm of a fully grown (approximately 3 meters high) sugarcane mother plant. Primary shoots developed from axillary buds are grown until seven leaves are present and cut off again at the base of the primary shoot. Secondary shoots are cut off again at the same stage in the same way as primary shoots. Tertiary or even higher order shoots produced after further cut off of the next lower order shoots are excised from the mother plant stalk and transferred to a nursery for rooting before transplanting into a field. The so called single eye method relies on the fact that a small volume of node tissue together with a single root primordium adhering to a bud is sufficient to develop into a sugarcane seedling, although seedlings established from minimum tissue appear thin (Ramaiah et al., 1977, Proc Inter Soc Sugar Cane Technol, 1509-1513). Based thereon, several methods have been described for the production of sugarcane seedlings which rely either on setts comprising a single node or even on so called bud chips comprising a bud excised along with only a portion of the respective sugarcane stalk node.

One example, of the so called single eye method used in some regions for propagating sugarcane comprises producing mother shoots having at least four areal internodes on the stalks and then cut the stalks into pieces comprising one node (eye). The cut surfaces are dipped into hot paraffin, stalk pieces are germinated in special containers and young plants are hardened before transplanted into the field (Van Dillewijn, 1952, Botany of sugar-cane. Waltham, Mass. U. S. A. Chronica Botanica, 371).

WO 2012/140177 discloses a method for production of a sugarcane seedling comprising cutting mother stalks into pieces comprising at least one node (including one bud), placing those pieces into a humid medium for growing shoots from the buds, cutting the top part of the shoots emerged from buds above the apical meristem, cutting off the top part of the newly emerged (primary) shoots from buds above the apical meristem, optionally repeating the last step, cutting off lateral shoots obtained, planting those shoots into growth medium and growing seedlings from the shoots. As exemplified, the production of seedlings from cutting the mother stalks into pieces until planting took at least 105 days.

A series of patent applications using single bud (eye) setts have been published. All these applications rely on the use of stalk pieces (setts) from sugarcane plants comprising one single node (including a bud) and directly planting of those single node setts into the field for raising sugarcane crops. The amount of sugarcane plants for establishing a sugarcane crop and the tonnage of seeding material can be reduced by using stalk pieces having a single node compared to conventional methods using three bud setts. Based on the just described single bud sett method, WO 2009/000398 emphasizes cold storage (below 15°C) of the single bud setts before planting into the field. WO 2009/000399 emphasizes that the single bud setts can be treated or coated with growth promoting compounds including pesticides, sugars, fertilizers or nutrients/micronutrients before directly planting into the field. WO 2009/000400 emphasizes that the single bud setts can be treated in a way aiming at retaining the moisture of the stalk pieces (including encapsulating into paraffin) before directly planting into the field. WO 2009 000401 emphasizes that the single bud setts can be treated with crop protection chemicals including fungicides, insecticides, herbicides or plant activators before directly planting into the field. WO 2009 000402 emphasizes that the single bud setts can be treated with a coating (e.g. paraffin) to retain moisture of the stalk pieces and a crop protection chemical (including fungicides, insecticides, herbicides or plant activators) or a compound having growth promoting activity before directly planting into the field. The bud chip technology uses even smaller parts of a sugarcane plant for raising a seedling compared to the single bud sett method. Bud chips are produced from sugarcane stalks by excising axillary buds together with surrounding stalk material but without totally severing the stalk. Bud chips have the advantage of being even smaller than setts used conventionally in sugarcane planting and that a single stalk can be used for producing more bud chips compared to two to three bud settings, which reduces the area needed for growing seed cane and volume of planting material to be transported to fields. In addition, the remaining part of the stalk used for excising the bud chip can be processed into valuable products, like sugar, alcohol, electricity.

Ravindra et al., 2013 (Sugar Tech 15(1), 27-35) discloses comparison of mechanical planting of sugarcane seedlings produced from bud chips with manual planting of such seedlings. Yield and juice quality of sugarcane planted by both methods were comparable, wherein costs were higher for manual planting.

Ramaiah et al., 1977 (Proc Inter Soc Sugar Cane Technol, 1509-1513) discloses comparison of sugarcane crops established by using seedlings produced from bud chips with sugarcane crops established by conventional three bud sett planting. A fungicide treatment of the bud chips before transfer into growth medium was included into the method. No significant differences in yield were seen between the crops established by both methods but the cost of the bud chip method was significantly lower. This was mainly due to far less material from mother plants had to be used in the bud chip method (200 - 300 kg per hectare) compared to the conventional planting method (8.000 kg per hectare).

Jain et al. (2010, Sugar Tech 12(1), 67-69) discloses that treatment of bud chips with Ethephon (2- chloroethylphosphonic acid) or CaCh has positive effects on sprouting frequency of bud chips, root number and weight and shoot weight of plantlets obtained from bud chips. Also disclosed is a previous finding that Ethephon and CaCh promote sprouting of sugarcane setts and underground buds (tillers). Loganandhan et al. (2013, Sugar Tech 15(1), 98-102) summarizes main results previously obtained in planting sugarcane crops from bud chips and compares sugarcane planting from bud chip produced nursery sugarcane seedlings with conventional sett planting. In the bud chip method, fungicide treatment of the chips before transfer into growth medium is recommended. Fields planted with nursery seedlings produced from bud chips revealed higher tiller numbers, higher millable stalk numbers and higher yields. Optimum parameters concerning age and size of the bud, cultivation medium and tray size for raising seedlings from bud chips are presented. Furthermore, a wider spacing between seedlings transplanted into the field is proposed for increasing yield.

A further known approach is the so called polybag method developed originally by Silva (1975, In: Seminario Copersucar da agroindustria Acucareira, 3. Aguas de Lindoia, Anais... Aguas de Lindoia: COPERSUCAR, 211-214) for control of ratoon stunting disease (infection by Leifsonia xyli subsp. Xyli). The determining aspect of this method is that each sugarcane seedling or settling is included in a containment (plastic bags or tubes) and planted into the field together with the containment. Sugarcane seedlings for the polybag method may be obtained by the (stratified) rayungan method (Keethipala et al., 2001, Sugar Tech 3(3), 106-108) or the single bud (eye) method (Landell et al., 2013, Ribeirao Preto: Instituto Agronomico de Campinas, 16 (IAC. Documentos, 109).

Landell et al. (2013, Ribeirao Preto: Instituto Agronomico de Campinas, 16 (IAC. Documentos, 109) discloses a method for obtaining sugarcane settlings from single eye (one node) setts. 5 to 10 month old stalks are cut into discs of approximately 3 cm height, wherein each disc comprises a bud, primordial roots and reserve tissue. The stalk discs are treated with crop protection chemicals, in particular fungicides and heat (for supressing growth of pests) before they are transferred into a substrate and placed in a greenhouse (sprouting nursery). After 12 days, the sprouted (germinated) discs are placed into containers (plastic sacks or tubes) and transferred to another greenhouse (growth nursery). After a period of 60 days, the containers including the settlings can be transplanted to the field. Mohanty et al. (2015, Sugar Tech 17(2), 116-120) disclose production of sugarcane seedlings from bud chips excised from healthy sugarcane plants by allowing germination of buds in containments (cones) before transplanting into the field (SSI technology). Number, length and perimeter of millable canes, average cane weight and cane yield were significantly higher in sugarcane fields planted by SSI technology compared to those planted by the conventional setts (each comprising three buds). From above discussion it follows, that known methods for production of sugarcane planting material each has its advantages and disadvantages.

Although still used in many sugarcane growing countries and areas conventional methods using two, three or more bud setts for planting sugarcane crops needs a large amount of material which on one side does reduce the harvest material which can be converted into valuable products and on the other side requires a huge amount of planting material tonnage to be transported to the fields. The highest costs in sugarcane planting arise from production of respective planting material.

Tissue culture micropropagation is far too expensive for commercial production of sugarcane planting material and thus is non-competitive to conventional planting methods.

Because of reduced sett size compared to conventional methods the single bud (eye) method does save seed material and costs for transport of sugarcane planting material to the field. Single bud (eye) setts directly planted into the field however do show a decreased germination rate compared to planting three or two bud setts leading to reduction in yield per hectare (Darpana & Patel, 2014, The Biosacn 9(1), 55- Bud chips reduce the amount of material to be taken from the mother plant and thus allow more material from the mother plant to be harvested and converted into high value products. However, Shanthy & Ramanjaneyulu (2014, Indian Res. J. Ext. Edu 14(3), 93) disclose that raising sugarcane seedlings from bud chips have problems even when practiced in a nursery: Bud chips should be taken from younger plants (6-8 months); even slight damage of the buds has to be avoided; high quality soil is required for growing the seedlings; severing of bud chips from the sugarcane stalks is labours.

The rayungan method requires a large amount of space, because only primary lateral shoots are used as planting material (Sugiyarta & Winarsih, 2009, Sugar Tech 11(1), 22-27). The use of secondary and higher order lateral shoots in the stratified rayungan method requires less space as the rayungan method but several pruning (decapitation) steps are needed. After each pruning, time is required until the next higher order lateral shoots emerge. Thus, the stratified rayungan method is time and labour intensive.

Using sugarcane crops as mother plants for producing sugarcane seeding material is known to bear the high risk of producing infected seeding material. Thus, it is well accepted that sugarcane for seedcane production should be grown separately from sugarcane crops. Raising seedlings in a nursery further contributes to raising disease free sugarcane planting material and saving water during the seedling growing phase. Using pre-grown seedlings for raising sugarcane crops allows establishing a well- defined planting scheme leading to a regular crop stand and thus an increase in yield of harvested and millable stalks.

It is therefore still some need for further improvement in sugarcane seed material production. The object of the present invention therefore is the provision of a method for production of sugarcane seeding material eligible for establishing sugarcane crops in a time, cost and labour efficient way.

A first embodiment of the invention therefore concerns a method for producing a sugar cane seedling comprising the steps of a) treating sugarcane plants with an ethylene releasing plant growth regulator b) further growing the sugarcane plants sprayed according to step a) c) pruning the top parts of the main stalks and the top parts of the newly produced lateral shoots of the sugarcane plants grown according to step b) d) further growing the pruned sugarcane plants obtained in step c) e) removing lateral shoots from the plants obtained in step d) f) treating the lateral shoots obtained according to step e) with an auxin g) transferring the lateral shoots treated according to step f) into a growth substrate and grow lateral shoots in a conditioned climate h) obtaining (rooted) sugarcane seedlings from the lateral shoots grown according to step g).

The term "sugarcane" as used herein shall have the current common meaning in the art, comprising the species Saccharum spontaneum L., Saccharum sinense (Roxb) Jesw., Saccharum barberi Jesw., Saccharum officinarum L., Saccharum robustum Brandes & Jeswiet ex Grassl. and Saccharum edule Hassk. Saccharum spontaneum and Saccharum robustum are wild species. Saccharum sinense, Saccharum barberi, Sacchatum officinarum and Saccharum edule are commonly known and also understood herein to represent "cultivated sugarcane species" or "cultivated sugarcane plants". (Moore et al. in Sugarcane: Physiology, Biochemistry and Functional Biology, Ed.: Moore and Botha, Wiley 2014, Chapter 1).

"Sugarcane seedling" as used herein is understood to be a young sugarcane plant having leaves, a stalk (culm, shaft) and roots. Settlings represent a specific embodiment of a sugarcane seedling.

"Settling" shall be understood herein to mean a sett, which comprises root(s) and shoot(s) including leaves.

"Sett" shall be understood herein to mean stalk pieces or billets comprising one or more nodes including lateral (axillary) buds.

The age of a sugarcane plant is determined by calculating the elapsed time between the actual date and the start date. The age can be calculated in common terms of time measurement, like days, months, years, but preferably is measured herein in months. In the art several systems have been established for growing sugarcane plants or sugarcane crops. When seedlings are planted, the start date is the day of transfer of the seedling into normal growing conditions or transplanting the seedling into the field. When ratoon sugarcane plants are grown, the start date is the date at which tillers become visible on the stool. When setts are used for planting sugarcane plants, the start date is the date at which plantlets emerge from the soil.

"Ratoon" is to be understood herein as a plant or crop which is grown from the stool (Roots and rhizomes, remained under the soil)

"Stool" is commonly understood in the art and also to be understood herein to be the part of a sugarcane plant which is left in the ground after harvest including underground roots and rhizomes. "Tiller" as used herein has the common meaning in the art and shall be understood to mean shoots which arise from underground (below soil surface) buds of the main stalk (culm, shaft). The sugarcane plants in step a) of the method for producing a sugarcane seedling according to the invention can be any sugarcane plant, including sugarcane plants of a commercial variety, or sugarcane plants of varieties which shall be propagated including new varieties to be propagated for commercialisation. Thus, the method for producing a sugarcane seedling according to the invention can be used for production of sugarcane seeding material of already existing commercial varieties or it can be used for multiplication of (new) varieties. The sugarcane plants in step a) of the method for producing a sugarcane seedling according to the invention may have been grown from any suitable material like single or multiple bud setts, settlings, ratoons, bud chips, seedlings, wherein the seedlings can have been obtained from any suitable material, including in vitro or tissue culture grown or propagated material. "Bud chip" is commonly understood in the art and also shall be understood herein to mean a short stalk part which comprises a part of a node including a single lateral (axillary) bud.

In respect with step a) of the method for producing a sugarcane seedling according to the invention, the sugarcane plants have the age of between 4 months to 14 months, preferably between 5 months to 12 months, more preferably between 5 months to 10 months, further more preferably between 5 months to 9 months, even more preferably between 5 months to 8 months, particular preferably between 5 months to 7 months and most preferably between 6 to 7 months when treated with an ethylene releasing growth regulator.

"Ethylene releasing plant growth regulator" is to be understood herein to mean a chemical substance inducing the production of ethylene in plant tissue and/or inducing the release of ethylene from plant tissue.

It is known in the art, that ethylene itself, when applied to plants stimulates its own production in plant tissues and release of ethylene from these tissues (Woodward & Barrel, 2005, Annals of Botany 95, 707- 735; E. F. George et al. (eds.), 2008, Plant Propagation by Tissue Culture 3^rd Edition, Springer, 227- 281). Therefore, ethylene itself is understood herein to represent an ethylene releasing plant growth regulator.

Further ethylene plant growth regulators are known in the art and comprise substances which stimulate the production of ethylene in plants and its subsequent release to the surrounding environment. Such substances comprise the substances 1-aminocyclopropane-l-carboxylic acid (ACC), methionine natural auxins and synthetic auxins. Substances releasing ethylene from plants are also known in the art. The most common substance which is taken up into plants, subsequently broken down in the plant followed by releasing ethylene is 2- chloroethylphosphonic acid (CAS-Number: 16672-87-0) also known under the common name "Ethephon" (E. F. George et al. (eds.), 2008, Plant Propagation by Tissue Culture 3^r Edition, Springer, 227-281).

Ethephon has the following formula:

Ethephon is commercially available e.g. from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Munich, Germany). Ethephon is the active compound of various agricultural products like Ethrel® (Bayer S/A, Rua Domingos Jorge, 1.100 - CEP: 04779-900 - Sao Paulo/SP).

The term "ethephon" as used herein shall not only comprise the acid form but also salts thereof. Preferred salts in connection with the present invention are alkali metal, alkaline earth metal or ammonium salts of ethephon. Concerning the alkali metal salts of ethephon, sodium or potassium salts are preferred herein. Concerning alkaline earth metal salts of ethephon, magnesium or calcium salts are preferred herein.

The term "auxin" is understood herein to comprise natural and synthetic auxins.

"Natural auxins" are those which are produced by plants and do comprise the substances indole-3-acetic acid (IAA), 2-phenylacetic acid (PAA), 4-chloroindole-3-acetic acid (4-Cl-IAA), Indole-3-butyric acid (IBA) (Sauer et al., 2013, J. Exp. Botany 64(9), 2565-2577).

"Synthetic auxins" are those not naturally produced in plants and comprise alp ha-nap tylacetic acid (NAA), 3,6-dichloroanisic acid (dicamba), 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5- trichlorophenoxyacetic acid (2,4,5-T), 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4-amino-3,5,6- trichloropicolinic acid (picloram), 2-chloro-3(2,3-dichlorophenyl) propionitrile (CDPPN), para- chlorophenoxyacetic acid (PCPA), fceta-naphfhyloxyacetic acid (NOA), 2,4-dichlorophenylselenoacetic acid, 3-(benzo[b]selenienyl) acetic acid (E. F. George et al. (eds.), 2008, Plant Propagation by Tissue Culture 3^rd Edition, Springer, 175-204), 2-chloro-3-(3-chloro-2-methylphenyl) propionitrile (CCMPPN), 2-chloro-3-(2,3-dichlorophenyl) butyronitrile (CDPBN) (Nemeth, 1981, Scientia Horticulturae 14, 253- 259). Indole-3-propionic acid (IP A) acts on plants as an auxin, is naturally produced by bacteria present in the human gastrointestinal tract, but not by plants. It is therefore understood herein to be a synthetic auxin.

In respect with step a) of the method for producing a sugarcane seedling according to the invention the ethylene releasing plant growth regulator can be any substance which results in ethylene release from plant tissue to the surrounding environment. Preferably the ethylene releasing plant growth regulator in step a) of the method for producing a sugarcane seedling according to the invention is selected from the group consisting of 1-aminocyclopropane-l-carboxylic acid (ACC), natural or synthetic auxins, or ethephon.

In this respect, preferred natural auxins to be used in step a) of the method for producing a sugarcane seedling according to the invention are indole-3-acetic acid (IAA), 2-phenylacetic acid (PAA), 4- chloroindole-3-acetic acid (4-Cl-IAA), Indole-3-butyric acid (IBA), more preferably IAA, IBA or PAA, most preferably IAA or IBA.

Preferred synthetic auxins to be used in step a) of the method for producing a sugarcane seedling according to the invention are NAA (a//?¾a-naphtylacetic acid), dicamba (3,6-dichloroanisic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5 trichlorophenoxyacetic acid), MCPA (2-methyl-4- chlorophenoxyacetic acid), picloram (4-amino-3,5,6-trichloropicolinic acid), CDPPN (2-chloro-3(2,3- dichlorophenyl) propionitrile), PCPA (para-chlorophenoxyacetic acid), NOA (fceto-naphthyloxyacetic acid), 2,4-dichlorophenylselenoacetic acid, 3-(benzo[b]selenienyl) acetic acid, more preferred are NAA, dicamba, 2,4-D, 2,4,5-T, MCPA or picloram, even more preferred are NAA, 2,4-D, dicamba, MCPA or picloram most preferred are 2,4-D, dicamba, MCPA or picloram. In the most preferred embodiment of the invention, the ethylene releasing plant growth regulator in respect with step a) of the method for producing a sugarcane seedling according to the invention is ethephon or a salt thereof.

Treating plants with auxins normally shows a detrimental effect on plant growth, as is demonstrated by the fact that a number of synthetic auxins (e.g. 2,4-D, dicamba, picloram) are used as active ingredients in herbicides or are used as pre-harvest desiccants, leading to plant leaves drying out. The plant growth regulator ethephon is also commonly used in sub-tropical regions for inducing sugarcane desiccation. It was therefore surprising that auxins and growth regulators when applied to sugarcane plants do induce production of lateral shoots instead of arresting plant growth when commonly used.

Treating with an ethylene releasing plant growth regulator in respect with step a) of the method for producing a sugarcane seedling according to the invention means applying an ethylene releasing plant growth regulator by any means which brings the sugarcane plant directly in contact with an ethylene releasing plant growth regulator. Treatment therefore includes applying gaseous substances like ethylene by means of aeration, solid substances in form of powders or in form of solutions, emulsions or dispersions. Solutions, emulsions or dispersions can be applied by any suitable means to the sugarcane plants, preferably they are applied by spraying the sugarcane plants in step a) of the method for producing a sugarcane seedling according to the invention with a respective solution, emulsion or dispersion. Dicamba, 2,4-D, MCPA or picloram are commercially available as readily formulated herbicides. Ethephon is also commercially available as readily formulated growth regulator (e.g. Ethrel®, Bayer S/A). Respective formulations can be used for preparing appropriately diluted solutions or where applicable emulsions or dispersions, for treating the sugarcane plants in step a) of the method for producing a sugarcane seedling according to the invention. In a specifically preferred embodiment of the invention in step a) of the method according to the invention for producing a sugarcane seedling a solution comprising ethephon or a salt thereof is sprayed onto the sugarcane plant.

Spraying can be performed by any suitable means, including hand spraying with hand sprayer devices of any kind, machine spraying by using the respective devices like tank sprayer, automatic sprayers etc. The person skilled in the art knows the different spraying methods and devices. The method and type of spraying, spraying equipment or device is not decisive and can be chosen by the skilled person according to the given circumstances.

In a preferred embodiment of the invention the sugarcane plants in step a) of the method for producing a sugarcane seedling according to the invention is sprayed with a solution comprising ethephon in an amount of 14.5 gram per litre (g/1) to 36.0 g/1, more preferred in an amount of 18.5 g/1 to 33.0 g/1, even more preferred in an amount of 22.0 g/1 to 29.5 g/1, further more preferred in an amount of 24.5 g/1 to 27.5 g/1 and most preferred in an amount of 25.5 g/1 to 26.5 g/1. The amounts in g/1 are calculated on the dry weight basis of active ingredient (ethephon) present in 1 litre of final (spraying) solution.

When using the commercially available products Ethrel® 720 (Bayer S/A) or Ethephon 720 (e.g. Kenso agacare, Bulimba, Qld 4171, Australia or Cheminova, North Ryde, NSW 2113, Australia) the sugarcane plants in step a) of the method for producing a sugarcane seedling according to the invention is sprayed with a solution comprising Ethrel® 720 or Ethephon 720 in an amount of 20.0 millilitre per litre (ml/1) to 50.0 ml/1, more preferred in an amount of 26.0 ml/1 to 46.0 ml/1, even more preferred in an amount of 31.0 g/1 to 41.0 ml/1, further more preferred in an amount of 34.0 ml/1 to 38.0 ml/1 and most preferred in an amount of 35 ml/1 to 37 ml/1.

In another preferred embodiment of the invention the ethylene releasing plant growth regulator in respect with step a) of the method for producing a sugarcane seedling according to the invention is applied, preferably sprayed over the canopy of the sugarcane plants, preferably the ethylene releasing plant growth regulator is applied, preferably sprayed only over the canopy of the sugarcane plants.

"Canopy" shall be understood in connection with the present invention as the top leaves of a sugarcane plant or stand forming a continuous layer of leaves. After the sugarcane plants have been treated with an ethylene releasing plant growth regulator, new lateral shoots will be produced on the mother stalks of the treated plants when further grown according to step b) of the method for producing a sugarcane seedling according to the invention. It was surprisingly found that treatment of sugarcane plants with specific amounts of ethephon induced production of lateral shoots. Furthermore, it was found that the number of shoots significantly increased after application of ethephon to sugarcane plants compared to the rayungan method, wherein lateral shoot production is induced by pruning of the main sugarcane stalk. 2 to 3 times more lateral shoots could be produced on a single plant when using ethephon, compared with the traditional rayungan method. Thus, use of ethephon does reduce space needed for lateral shoot production, because less mother plants are used for producing a specific amount of lateral shoots. Furthermore, the production of lateral shoots upon ethephon treatment was found to be more uniform, compared to traditional methods. Therefore, the lateral shoots produced on the mother stalk all are in a comparable, similar developmental stage. Further treatments needed for sugarcane seedling production thus can be performed at one point in time for all plants which saves time compered to different groups of plants having to be treated differently. This saves labour and time and consequently costs. Furthermore, a more uniform lateral shoot production enables improvement of precise planning of the seedling production process. When e.g. it is known that a pre-determined amount of seedlings is required at a defined point in time, it can be easily defined when to start with seedling production, when performing which further steps and when the required amount of new seedlings will be available. Thus, the method disclosed herein does enable precise planning of seedling production and thus allows just in time delivery of seedlings to or for the farmer.

"Stalk" is commonly understood and shall have the meaning herein to refer to the millable stem of a sugarcane plant formed of repeating phytomeric units, each containing a node and an internode.

"Stalk" is used in the art as well as herein synonymously and interchangeably with the terms "shaft" or "culm".

"Shoot" shall mean herein to be a young stalk arising from a bud.

"Lateral shoot" is commonly and shall herein be understood to mean all shoots which arise at the location of a bud located on areal (above ground) parts of a stalk or shoot. The generic term "lateral shoot" encompasses primary, secondary tertiary and all higher order shoots arising at the location of a bud located on areal (above ground) parts of the stalk.

"Lateral shoot" is used in the art as well as herein synonymously and interchangeably with the term "axillary shoot". "Primary lateral shoot" is commonly and shall herein be understood to refer to only those shoots directly arising from a bud located on the areal parts of the stalk.

"Secondary lateral shoot" is commonly and shall herein be understood to refer to only those shoots directly arising from a bud located on primary lateral shoot. Likewise, higher order (tertiary, quaternary etc.) lateral shoots are commonly and shall herein be understood to refer to only those shoots directly arising from a bud located on a shoot of the previous lower order (secondary, tertiary etc., respectively) lateral shoot.

"Mother stalk" shall be understood herein to refer to the stalk from which planting material is derived or excised (severed). Growing of the sugarcane plants concerning to step b) of the method for producing a sugarcane seedling according to the invention can be done as conventionally known. The plants can be kept under the conditions, they have been grown before. A skilled person well knows the conditions under which sugarcane is to be grown.

In respect with step b) of the method for producing a sugarcane seedling according to the invention the sugarcane plants are grown until lateral shoots have been produced, preferably the sugarcane plants are grown for another 14 days to 21 days after spraying, more preferably for another 16 days to 19 days after spraying, further more preferably for 17 days to 18 days after spraying, even more preferably the sugarcane plants are grown for another 14 days to 16 days after spraying.

The pruning in step c) of the method for producing a sugarcane seedling according to the invention can be done by conventionally known methods. Pruning can be done by handwork using any suitable tool or pruning can be done by using suitable machines. Preferably the top of the mains stalks and the top of the newly produced lateral shoots in step c) of the method for producing a sugarcane seedling according to the invention are cut at a position which removes the apical meristem from the stalk. The newly produced lateral shoots to be cut in step c) of the method for producing a sugarcane seedling according to the invention can comprise primary and secondary lateral shoots.

"Apical meristem" as used herein has the meaning common in the art and is understood to be the growing point at the top (tip) of the stalk or shoot which is responsible for vertical growth of the stalk or shoot, respectively.

Growing the sugarcane plants after pruning will lead to the production of secondary or higher order lateral shoots from the primary or lower order lateral shoots. Growing of the sugarcane plants concerning to step d) of the method for producing a sugarcane seedling according to the invention can be done as conventionally known. The plants can be kept under the conditions, they have been grown before. A skilled person well knows the conditions under which sugarcane is to be grown.

In respect with step d) of the method for producing a sugarcane seedling according to the invention the sugarcane plants are grown until secondary or even higher order lateral shoots have been produced, preferably the sugarcane plants are grown for another 20 days to 23 days after pruning, more preferably, the sugarcane plants are grown for another 18 days to 21 days after pruning, even more preferably the sugarcane plants are grown for another 16 days to 19 days after pruning.

In step e) of the method for producing a sugarcane seedling according to the invention the newly formed lateral shoots are removed from the mother stalk. The lateral shoots to be removed from the mother stalk can be primary or secondary lateral shoots, preferably lateral shoots to be removed from the mother stalk are secondary lateral shoots. Lateral shoots can be removed from the mother stalks by severing the lateral shoots using knifes or using specific tools, like sprout extractor scissors.

Optionally the lateral shoots removed in step e) of the method for producing a sugarcane seedling according to the invention are kept at reduced temperature after removal from the mother stalk. This optional part of step e) is in particular used, when the lateral shoots are to be stored for some time before treatment with an auxin according to step f) is performed. If the lateral shoots are kept under low temperature, preferably lateral shoots are immediately transferred to a low temperature environment after removal from the mother stalk. In this respect, immediately transferring the lateral shoots to reduced temperature preferably shall be understood to mean transferring the lateral shoots to an environment of reduced temperature less than 25 minutes, more preferably less than 20 minutes, further more preferably less than 15 minutes, even more preferably less than 10 minutes, most preferably less than 5 minutes after removal from the mother stalk.

Reduced temperatures in the optional part of step e) of the method for producing a sugarcane seedling according to the invention preferably means a temperature which is significantly below the temperature of the environment from which the lateral shoots are obtained, preferably, the reduced temperature has a value of between 12°C and 18°C, more preferably of between 13°C and 17°C, further more preferably of 14°C and 16°C.

In one preferred embodiment of the invention lateral shoots in the optional part of step e) of the method for producing a sugarcane seedling according to the invention are kept at the reduced temperature at most for 30 hours, more preferably for at most for 24 hours, further preferably for at most for 18 hours, even more preferably for at most for 12 hours, most preferably for at most for 6 hours. The reduced temperature environment in the optional part of step e) of the method for producing a sugarcane seedling according to the invention can be established by any means suitable for a person skilled in the art. The environment can be e.g. cooled boxes, colling bags, refrigerators etc.

When lateral shoots were kept under reduced temperature according to the optional part of step e) of the method for producing a sugarcane seedling according to the invention the lateral shoots are treated with an auxin immediately after having been removed from the reduced temperature environment and subsequently transferred into a growth substrate.

In case the optional step of step e) of the method for producing a sugarcane seedling according to the invention is not performed, the treatment with an auxin according to step f) of the method for producing a sugarcane seedling according to the invention is performed shortly after removal of the lateral shoots from the plants, preferably the treatment with an auxin is performed between 0 minutes (min) and 180 min, more preferably between 0 min and 150 min, further more preferably between 0 min and 120 min, even more preferably between 0 min and 90 min, even further more preferably between 0 min and 60 min, most preferably between 0 min and 30 min after removal of the lateral shoots from the plants. Preferably only the bottom parts of the lateral shoots are treated with an auxin in step f) of the method for producing a sugarcane seedling according to the invention. Preferably, 2 centimetre (cm) to 10 cm , more preferably 2 cm to 8 cm, even more preferably 2 cm to 6 cm and most preferably 2 cm to 5 cm of the bottom part of the lateral shoot is dipped into an auxin containing solution in step f) of the method for producing a sugarcane seedling according to the invention. Treatment with an auxin in step f) of the method for producing a sugarcane seedling according to the invention can occur by any suitable means, e.g. the the bottom part ofthe lateral shoots can be sprayed or brushed with or dipped into an auxin containing solution.

The auxin in step f) of the method for producing a sugarcane seedling according to the invention can be a natural auxin or a synthetic auxin. In one embodiment of the invention the auxin in step f) of the method for producing a sugarcane seedling according to the invention is selected from the group consisting of indole-3-acetic acid (IAA), 2-phenylacetic acid (PAA), 4-chloroindole-3-acetic acid (4-Cl-IAA), indole-3-butyric acid (IBA), alpha- naphtylacetic acid (NAA), fceto-naphthyloxyacetic acid (NOA), 2,4-dichlorophenylselenoacetic acid, 3- (benzo[b]selenienyl) acetic, indole-3-propionic acid (IPA), 2-chloro-3-(3-chloro-2-methylphenyl) propionitrile (CCMPPN), 2-chloro-3-(2,3-dichlorophenyl) propionitrile (CDPPN), 2-chloro-3-(2,3- dichlorophenyl) butyronitrile (CDPBN). In a preferred embodiment of the invention the auxin in step f) of the method for producing a sugarcane seedling according to the invention is selected from the group consisting of IAA, PAA, 4-Cl-IAA, IBA, NAA, NOA, IPA or CDPPN, more preferably the auxin in step f) of the method for producing a sugarcane seedling according to the invention is selected from the group consisting of IAA, IBA, NAA, IPA, further more preferably the auxin in step f) of the method for producing a sugarcane seedling according to the invention is selected from the group consisting of IAA, IBA or NAA, even more preferably the auxin in step f) of the method for producing a sugarcane seedling according to the invention is selected from the group consisting of IBA or NAA, most preferably the auxin in step f) of the method for producing a sugarcane seedling according to the invention is IBA.

In one embodiment of the invention the auxin in step f) of the method for producing a sugarcane seedling according to the invention the lateral shoots are treated with an auxin solution, wherein the concentration of auxin in the solution is between 0.5 gram per litre (g/1) to 2.7 g/1, preferably between 0.7 g/1 to 2.5 g/1, more preferably between 0.9 g/1 to 2.3 g/1, further more preferably betweenl.l g/1 to 2.1 g/1, further more preferably between 1.3 g/1 to 1.9 g/1, most preferably between 1.5 g/1 to 1.7 g/1.

In a specific embodiment of the invention the auxin in step f) of the method for producing a sugarcane seedling according to the invention the lateral shoots are treated with an auxin solution, wherein the auxin is IBA and the concentration of IBA in the solution is between 0.5 gram per litre (g/1) to 2.7 g/1, preferably between 0.7 g/1 to 2.5 g/1, more preferably between 0.9 g/1 to 2.3 g/1, further more preferably betweenl.l g/1 to 2.1 g/1, further more preferably between 1.3 g/1 to 1.9 g/1, most preferably between 1.5 g/1 to 1.7 g/1.

The lateral shoots according to step f) of the method for producing a sugarcane seedling according to the invention are transferred to a growth substrate after treatment with an auxin. Growing allows production of roots by the lateral shoots. Preferably the growth substrate is present in a container. The container may be selected from any suitable container, like pots, buckets, trays, bags or trays with separations and the like. The containers can be made of synthetic and/or natural material, including biodegradable material. Pots, buckets, or trays with separations, allowing the growth of a single lateral shoot in a single growth area are preferred.

The growth substrate can be any substrate eligible for growing sugarcane. Such sugarcane eligible substrates are well known to a person skilled in the art. Preferably the substrate comprises vermiculite and/or coconut fibre, more preferably vermiculite and coconut fibre in a ratio of 1:5 to 5: 1, further more preferably vermiculite and coconut fibre in a ratio of 1 :3 to 3: 1, even more preferably vermiculite and coconut fibre in a ratio of 1 :2 to 2: 1 and most preferably vermiculite and coconut fibre in a ratio of 1 : 1.τ

After transfer to a growth substrate the lateral shoots, or, as applicable, the containers comprising the lateral shoots are grown in a conditioned climate. The environment for establishing a conditioned climate according to step g) of the method for producing a sugarcane seedling according to the invention is not decisive, as long as at least it allows growing of the sugarcane shoots under uniform humidity and/or temperature conditions. It is preferred to use an environment for growing the sugarcane shoots which is equipped with technical means for controlling humidity and/or temperature. More preferably the conditioned climate environment is a greenhouse, further more preferably a greenhouse equipped with air condition and/or humidity control. Concerning step g) of the method for producing a sugarcane seedling according to the invention the climate is in average preferably conditioned to a temperature between 24°C and 36°C, more preferably to a temperature between 26°C and 34°C, further more preferably to a temperature between 28°C and 32°C, even more preferably to a temperature between 29°C and 31°C and most preferably to a temperature of about 30°C. The relative humidity of the conditioned climate according to step g) of the method for producing a sugarcane seedling according to the invention is preferably conditioned to 70% to 98%, more preferably to 75% to 98%, further more preferably to 77% to 98% even more preferably to 80% to 98%, particular preferably to 80% to 95%, most preferably to 80% to 90%.

The lateral shoots are grown in step g) of the method for producing a sugarcane seedling according to the invention until roots have been sufficiently developed by the shoots. Preferably the lateral shoots are grown under conditioned climate in step g) of the method for producing a sugarcane seedling according to the invention for at least 5 days, more preferably for at least 7 days, further more preferably for at least 10 days.

The lateral shoots are grown under conditioned climate in step g) of the method for producing a sugarcane seedling according to the invention preferably for at most 15 days, more preferably for at most 13 days, further more preferably for at most 12 days, even more preferably for at most 11 days, most preferably for at most 10 days.

Preferably the lateral shoots are grown under conditioned climate in step g) of the method for producing a sugarcane seedling according to the invention for 5 days to 15 days, more preferably for 5 days to 13 days, further more preferably for 5 days to 12 days, even more preferably for 5 days to 11 days, most preferably for 5 days to 10 days.

In the method for producing a sugarcane seedling according to the invention after the lateral shoots grown according to step g) have developed roots, seedlings are obtained according to step i). The seedlings can be grown into sugarcane plants in any suitable and desirable way. The seedlings can e.g. be grown in containers in any suitable or desirable environment or they can be transferred to fields. The seedlings can be grown for further amplification purposes by e.g. simply repeating the method for producing a sugarcane seedling according to the invention or they can be transferred to a field for establishing a sugarcane crop and/or for production of further seedcane. Another embodiment of the present invention concerns a method for producing a sugarcane seedling according to the invention comprising a further step j) which consists of growing the seedling obtained in step i) in an acclimation environment.

Growing the seedlings obtained by the method according to the invention for production of a sugarcane seedling in an acclimation environment is in particular recommendable to be performed for seedlings intended to be transferred to the natural environment, including fields, for further growing sugarcane plants.

The term "acclimation environment" as used herein shall be understood to be an environment which allows the sugarcane seedling (stepwise) adaptation to another environment, in particular adaptation to different and more volatile humidity and/or temperature.

Acclimation environments and the setup of respective conditions for sugarcane seedlings are known to a person skilled in the art.

Acclimation is done with the progressive modification of temperature and humidity, until it equals or is similar to the conditions of temperature and humidity of the environment of the region into which the sugarcane seedlings are intended to be transferred. Conditions can be chosen according to the common knowledge of a person skilled in the art and depend on the given environment into which the sugarcane seedlings are intended to be transferred.

Preferably the seedlings obtained by a method according to the invention for production of a sugarcane seedling are grown according to additional step j) in an acclimation environment for 16 days to 28 days, more preferably for 18 days to 26 days, further more preferably for 18 days to 24 days, even more preferably for 19 days to 22 days, most preferably for about 21 days.

Key advantages of the method for producing a sugarcane seedling according to the invention disclosed herein can be summarised as follows.

More than 95% of the lateral shoots produced on the main stalk developed into viable seedlings. The high reproduction rate reduces the amount of main stalks and thus the space needed for production of a defined number of seedlings. It also reduces costs labour and space in establishing seedlings because a low number of lateral shoots will have to be discarded after the growth phase for root development. Uniform lateral shoot development after ethephon treatment is to be made partly accountable for the high survival rate after the rooting growth phase. Compared to known methods for sugarcane seedling production, the multiplication rate, which defines the number of sugarcane seedlings producible from a single sugarcane stalk or tiller, is increased. In the so-called conventional poly bag method, using (single bud/eye) settlings germinated in a growth substrate containing bag, the multiplication rate is about 1:20. The conventional bud chip method has a multiplication rate of about 1 :40. For the method for producing a sugarcane seedling according to the invention multiplication rates of 1:200 to 1:350 were observed. Thus, compared to conventional methods, far less sugarcane plants have to be cultivated for producing the same number of seedlings when using the method for producing a sugarcane seedling according to the invention disclosed herein. This saves space for sugarcane plant cultivation and resources, like water, fertiliser, crop protection agents, work labour etc.

Compared to conventional methods for producing sugarcane seedlings, the amount of time needed for growing the sugarcane shoots in a conditioned climate is reduced when using the method for producing a sugarcane seedling according to the invention described herein. In the so-called conventional poly bag method e.g. at least 60 days are required for lateral shoot production from bud chips under conditioned climate. In the method for producing a sugarcane seedling according to the invention disclosed herein a time frame of around 28 days is required for growth under conditioned climate for root production and acclimation. At most up to 36 days but generally less time is needed for growth under conditioned climate in the method for producing a sugarcane seedling according to the invention disclosed herein. This is a significant reduction of time for growth under conditioned climate needed in the seedling production process which reduces input costs for establishing the required climate.

With the method for producing a sugarcane seedling according to the invention disclosed herein not only more sugarcane seedlings per stalk (space) but also seedlings of higher uniformity can be produced, compared to conventional methods for sugarcane seedling production. This was mainly due to the effect that the treatment with an ethylene releasing growth regulator did not impact viability of axillary buds but did allow lateral shoot production over a wide range of nodes from the base to the top of the sugarcane stalk.

In addition, lateral shoots developed much more uniformly after treatment with an ethylene releasing growth promoter, when compared to conventional methods for producing sugarcane seedlings. Higher uniformity of lateral shoots increases the number of viable sugarcane seedlings which can be produced from those shoots.

Conventional methods for producing a sugarcane seedling comparable to the method disclosed herein require a number of consecutive pruning operations. The use of an ethylene releasing growth promoter in the method for production of a sugarcane seedling according to the invention reduces the number of necessary pruning operations to a single one. This again saves time and labour in seedling production.

Sugarcane seedlings produced by a method for producing a sugarcane seedling according to the invention can be transplanted into the field e.g. for establishing a (commercial) sugarcane crop or for propagation of a desired variety, a desired breeding line or a desired parent of a hybrid. A further embodiment of the invention therefore concerns a method for establishing a sugarcane crop comprising the steps of a) obtaining or producing seedlings according to a method for producing a sugarcane seedling according to the invention b) transplant the seedlings according to step a) into a field.

In connection with the present invention the term "sugarcane crop" has the common meaning and is to be understood herein to be any specific sugarcane plant or variety which is or is intended to be grown or cultivated in a scale for profit or subsistence and which after growing is harvested for food, clothing, livestock, fodder, biofuel, medicine, or other uses by humans. In particular the term "sugarcane crop" has the meaning herein to be any specific sugarcane plant or variety which is or is intended to be grown or cultivated in groups on a scale for profit and which after growing is harvested for providing sugar, ethanol, fodder and/or bagasse for production of electric power. By the term intended to be grown used in the current definition it shall be made explicitly clear that also any breeding or propagation material eligible for producing any specific sugarcane plant or variety envisaged to be grown or cultivated as crop is comprised by the term "sugarcane crop".

The term "sugarcane crop" as used herein is generic and comprises the terms "commercial sugarcane (crop)" and "cultivated sugarcane species".

Transplanting in step b) of the method for establishing a sugarcane crop according to the invention can be done with any suitable means known to a person skilled in the art. The transplanting of the seedlings can be performed by hand work or it can be done by machine planting as disclosed e.g. by Ravindra et al. (2013, Sugar Tech 15(1), 27-35).

A further embodiment of the invention concerns the use of a seedling obtained or obtainable by a method for production of a sugarcane seedling according to the invention for establishing a sugarcane crop. One advantage of establishing a sugarcane crop by transplanting sugarcane seedling obtained by a method for producing a sugarcane seedling according to the invention is that the sugarcane seedling can be planted into the field in an equal distribution. Spacing between rows and plants in a row therefore can be adapted as desired. It is known in the art that uniform spacing between plants increases yield in terms of number, length and weight of millable stalks in sugarcane crops (Mohanty et al., 2015, Sugar Tech 17(2), 116-120). Therefore, with the efficient seedling production method disclosed herein, the revenue of a farmer will be increased, because the sugarcane seedling production costs are reduced and at the same time the yield can be increased. Regular and more frequent substitution of sugarcane crops by planting new seedlings instead of growing numerous ratoon crops is also possible with the method for establishing a sugarcane crop according to the invention because the costs for the production of seedlings by the method for producing a sugarcane seedling according to the invention disclosed herein is reduced and the higher costs for replanting with seedlings may be over-compensated by the yield improvement due to the regular and circumstances adjusted planting, compared to re-growing ratoon crops.

Treatment of sugarcane with ethephon was shown to have several different effects. Induction of lateral shoots upon treatment of sugarcane plants with ethophon has not been described. It has in particular not been described that treatment of sugarcane canopy leaves with ethephon would increase production of lateral buds and uniformity in lateral shoot production. This effect of ethephon is first described herein.

A further embodiment of the invention therefore concerns the use of ethephon for inducing lateral shoot growth in sugarcane plants. A preferred embodiment of the invention is the use of a solution containing ethephon for spraying the canopy (leaves) of a sugarcane plant for inducing lateral shoot growth. Preferably the solution containing ethephon used for spraying the canopy (leaves) of a sugarcane plant for inducing lateral shoot growth contains ethephon in an amount of 14.5 gram per litre (g/1) to 36.0 g/1, more preferably in an amount of 18.5 g/1 to 33.0 g/1, even more preferably in an amount of 22.0 g/1 to 29.5 g/1, further more preferably in an amount of 24.5 g/1 to 27.5 g/1 and most preferably in an amount of 25.5 g/1 to 26.5 g/1. Preferably the solution containing ethephon used for spraying the canopy (leaves) of a sugarcane plant for inducing lateral shoot growth is sprayed onto the canopy (leaves) of a 4 months to 12 months, more preferably a 5 months to 11 months, further more preferably a 6 months to 10 months, even more preferably a 7 months to 9 months old sugarcane plant.

A further embodiment of the invention is the use of a seedling obtainable or obtained by a method for production of a sugarcane seedling according to the invention for establishing a (commercial) sugarcane crop. Another embodiment of the invention is the use of a seedling obtainable or obtained by a method for production of a sugarcane seedling according to the invention for establishing, growing harvesting and subsequently processing a sugarcane crop. Here processing preferably means milling. More preferably processing means milling, obtaining sugarcane juice and subsequently producing sucrose from the juice by crystallization or subsequently producing ethanol from the juice by means of fermentation. Description of the Figures

Fig. 1: Average number of internodes per stalk/tiller of sugarcane plants at 0 DAS (Days After

Spraying Ethrel® 720 or pruning, repectively) and at 14 DAS. Shows the results for the average number of buds (buttons) per stalk/tiller obtained for each of the different treatments as described in Example 2.

Dose response curve for the number of sprouts produced in response to the amount of Ethrel® 720 (ethephon) sprayed on sugarcane plants.

Picture for comparison of sugarcane plants having been treated with ethephon (pots 3 and 4) and sugarcane plants having been decapitated/pruned instead of ethephon treatment (pots 1 and 2).

Picture demonstrating severing lateral shoots form stalks/tillers.

Number of dead (non-rooting) lateral shoots after transfer of lateral shoots into a growth substrate in dependency from pre-treatment with different amounts of indole-butyric acid.

Fig. 7: Survival (rooting) rate of lateral shoots in relation to indole-butyric acid treatment, its concentration and regression curve calculated on basis of the data points obtained.

Examples

1. Internode production after treatment with ethephon vs. pruning

Sugarcane plants (variety RB92579) were grown in randomized blocks in a greenhouse. Treatments with ethephon (Ethrel® 720) were performed by spraying the canopy of six month old sugarcane plants according to Table 1. One set of plants was not sprayed with ethephon but instead the tops of the stalks were decapitated below the apical meristem. Each of the experiments was performed in in five independent replications.

The average number of internodes per stalk/tiller at day 0 (day at which ethephon treatment or pruning, respectively, was performed) and average number of internodes at day 14 (14 days after ethephon treatment or pruning, respectively, was performed) was determined. The results are shown in Table 1 and Fig. 1.

Table 1 : Number of internodes of sugarcane plants present at day 0, defined as the the point in time of pruning (Tl) or treatment with different concentrations of ethephon (T2 to T6) compared to the number of internodes 14 days after the respective treatments. The amount of ethephon in mg/1 were calculated on the manufacturers Ethrel® 720 label information according to which the product comprises 720 g ethephon per litre. The results were submitted for variance analysis, using the F test at 5% and the t significance test (p < 0.05).

It is derivable from the data obtained, that pruning does prevent production of new internodes in sugarcane plants. Treatment with ethephon did not prevent production of new internodes in sugarcane plants. During the 14 day period after spraying the sugarcane canopy with etheophon in average two new internodes per stalk/tiller were produced, whereas the pruned sugarcane plants did not produce new internodes during this period. It can be concluded that pruning does arrest further internode development whereas ethephon does not. 2. Lateral shoot development after ethephon application followed by pruning vs. subsequent pruning

Sugarcane plants were grown and treated (pruning or ethephpon application) as described in Example 1.

Fourteen days after pruning or ethephon application, a simultaneous pruning of the top of the sugarcane stalks and lateral shoots was performed in treatments T2, T3, T4, T5 and T6. In treatment Tl, only pruning of the lateral shoots was performed at this point in time. At the end of the experimental period, the number of sugarcane tillers, internodes and lateral shoots in the different treatments was determined. The results were submitted for variance analysis using the F test at 5%. In cases of significant variations, the Fisher significance test (p < 0.05) was applied and regression analysis was performed in order to determine the best dose of ethephon to be used.

Fig. 2 shows the results obtained for the average number of lateral shoots per stalk/tiller in each of the different treatments. A positive effect of the application of Ethrel® 720 is seen on the production of sugarcane lateral shoots. The average of all of the treatments that used Ethrel® 720 was approximately 30 lateral shoots per stalk/tiller. For the conventional methods, solely using subsequent pruning treatments, the number of sugarcane lateral shoots was 13 in average.

The greatest production of lateral shoots was observed with application of 40 ml/1 of Ethrel® 720 (equals 28.80 g/1 ethephon). At this concentration in average 40 sugarcane lateral shoots per stalk/tiller were obtained. In control treatments using subsequent pruning without application of ethephon in average 13 lateral shoots per stalk/tiller were obtained. Examples comparing sugarcane plants treated with ethephon followed by pruning compared with sugarcane plants subsequently pruned but without previous ethephon treatment is shown in Fig 4.

With the results obtained for different of Ethrel® 720 concentrations sprayed on sugarcane plants versus production of lateral shoots per sugarcane stalk/tiller, a regression equation was established. The second degree polynomial model gave the equation: Y= -0.0149X² + 1.0792X + 13.272 (R²=0.7373)

The respective dose response curve is shown in Fig. 3.

This equation has an elevated correlation coefficient (r) of 86%, indicating that the equation represents sufficient correlation between the production of sugarcane lateral shoots with the application of different doses of Ethrel® 720. Using the above equation, it was calculated that the maximum production of approximately 35 lateral shoots per stalk/tiller was obtained when spraying the canopy of sugarcane plants with a solution comprising 36 ml/1 Ethrel® 720 (equating 25.92 gram ethephon per litre of spray solution).

3. Root development vs. auxin concentration Plants were grown and treated as described in Examples 1 and 2 according to T2 to T6 in Table 1. Lateral shoots were severed from the stalks/tillers as exemplified in Fig. 5 and the bottom end of the lateral shoots were submersed in an indole-butyric acid (IBA) containing solution. About 100 lateral shoots were submersed in each case in a solution comprising 0.0 gram per litre (g/1), 0.5 g/1, 1.0 g/1, 1.5 g/1 or 2.0 g/1 of indole-butyric acid (IBA). Right afterwards, the sugarcane buds were placed in trays containing substrate (1 : 1 of vermiculite and coconut fibre) and placed in an environment with a temperature of approximately 27 °C and a relative humidity of about 85%. After a 10 day period, the lateral shoots were evaluated. A count of dead plants was performed. Dead lateral shoots indicated that no rooting had occurred. Surviving lateral shoots did develop a root system and developed into a sugarcane seedling. The results were submitted for variance analysis at 5% probability and subsequent regression analysis for the comparison of mean values.

Results are shown in Fig. 6. Without the application of indole-butyric acid (0 g/1), increased death of lateral shoots was observed (23 seedlings). After application of 0.5 g/1; 1.0 g/1; 1.5 g/1 and 2.0 g/1 indole- butyric acid, 6, 7, 5 and 2, respectively, dead lateral shoots were obtained. This is a clear reduction in the death rate of the sugarcane lateral shoots compared to the lateral shoots which have not been treated with indole-butyric acid.

The percentage of viable plants (% of surviving plants) was calculated. With the results obtained a regression equation was established representing the correlation between concentration of indole-butyric acid versus rooting and survival of lateral shoots (Fig. 7).

The second degree polynomial model gave the equation: Y= -7.7143X² + 25.229X + 76.543 (R²=0.8376)

This equation has an elevated correlation coefficient (r) of 92%, indicating that the equation represents sufficient correlation between rooting and indole-butyric acid application. Using this model, it was found that maximum rooting of lateral shoots was obtained at a concentration of 1.6 g/1 of indole-butyric acid (approximately 97% of buttons produce roots and survive). Despite the optimal indole-butyric acid found to be optimal at 1.6 g/1 when using above equation, it can be seen from the data points presented in Fig. 7 that the lateral shoot survival rate is above 90% in each case when indole-butyric acid is used between 0.5 g/1 to 2.0 g/1. In contrast thereto without indole- butyric acid treatment, the survival rate of lateral shoots is below 75%. Thus, treatment of sugarcane lateral shoots with any concentration of indole-butyric acid between 0.5 g/1 to 2.0 g/1 significantly increases root formation and sugarcane seedling development.

Claims

1. A method for producing sugarcane seedlings characterized in that the method comprises the steps of a) treating sugarcane plants with an ethylene releasing plant growth regulator

b) further growing the sugarcane plants sprayed according to step a)

c) pruning the top parts of the main stalks and the top parts of the newly produced lateral shoots of the sugarcane plants grown according to step b)

d) further growing the pruned sugarcane plants obtained in step c)

e) removing lateral shoots from the plants obtained in step d)

f) treating the lateral shoots obtained according to step e) with an auxin

g) transferring the lateral shoots treated according to step f) into a growth substrate and grow lateral shoots in a conditioned climate

h) obtaining sugarcane seedlings from the lateral shoots grown according to step g).

2. The method according to claim 1 wherein in step a) the canopy of the sugarcane plants is treated with an ethylene releasing plant growth regulator.

3. The method according to any of claims 1 or 2 wherein the concentration of ethylene releasing plant growth regulator in step a) is between 14.5 g/1 and 36.0 g/1.

4. The method according to any one of claims 1 to 3 wherein the plants in step b) are grown for another 16 to 19 days after treatment with an ethylene releasing growth regulator.

5. The method according to one of claims 1 to 4, wherein the plants in step d) are grown for another 20 to 23 days after pruning.

6. The method according to any one of claims 1 to 5, wherein step e) comprises a further part consisting of keeping the removed lateral shoots at reduced temperature after removal from the mother stalk.

7. The method according to any one of claims 1 to 6 wherein the concentration of auxin in step e) is between 1.1 g/1 and 2.1 g/1.

8. The method according to any one of claims 1 to 7 wherein the auxin in step f) is selected from the group consisting of indole butyric acid (IBA), indole-3-acetic acid (IAA), 2-phenylacetic acid (PAA), 4-chloroindole-3-acetic acid (4-Cl-IAA), a//?¾a-naphtylacetic acid (NAA) or, beta- naph thy loxy acetic acid (NO A).

9. The method according to any one of claims 1 to 8 wherein the climate in step g) is conditioned to a temperature between 24°C to 36°C.

10. The method according to any one of claims 1 to 9 wherein the climate in step g) is conditioned to a relative humidity of 70% to 98%.

11. The method according to any one of claims 1 to 10 wherein the lateral shoots are grown under conditioned climate according step g) for at least 5 days.

12. The method according to any one of claims 1 to 11 wherein the method comprises a further step j) which consists of growing the seedling obtained in step i) in an acclimation environment.

13. The method according to claim 12 wherein in step j) the seedlings are grown in the acclimation area for 19- 25 days.

14. A method for establishing a sugarcane crop characterized in that the method comprises the steps of a) obtaining seedlings according to any of the methods of claims 1 to 13

b) transplanting the seedlings according to step a) to a field.

15. Use of a seedling obtainable by a method for production of a sugarcane seedling according to any of claims 1 to 13 for establishing a sugarcane crop.

16. Use of a seedling obtainable by a method for production of a sugarcane seedling according to any of claims 1 to 13 for establishing, growing, harvesting and subsequently processing a sugarcane crop.