CN105475130B - A kind of red cone isolated culture plant strain regeneration method - Google Patents

Abstract

The invention belongs to the technical field of plant tissue culture, and discloses a plant regeneration method for in vitro cultured red cones. The method is through explant treatment, basic medium design, different hormone types and concentration level ratio optimization, using the seeds of the red cone tree as explants, through adventitious bud induction, bud proliferation culture and rooting culture, to form a complete plant . Developed a substrate suitable for transplanting rooted seedlings of Red Cone tissue culture and management techniques for transplanted seedlings. This invention makes up for the gap in tissue culture and rapid propagation using the seeds of the red cone tree as explants, and provides a stable and efficient regeneration method for the isolated cultured plants of the red cone. The red cone seedlings are strong and uniform. Red cone is a precious timber tree species. At present, the afforestation area is large, and high-quality seedlings are scarce in the market. The implementation of this technology can provide technical support and good seed guarantee for the rapid promotion of excellent germplasm of red cone and improve the economic benefits of red cone plantation. It has broad application prospects.

Description

A kind of red cone in vitro culture plant regeneration method

technical field

The invention belongs to the technical field of plant tissue culture, and in particular relates to a plant regeneration method for in vitro cultured red cones.

Background technique

Castanopsis hystrix A.DC belongs to the genus Castanopsis of Fagaceae. The adult tree height can reach 30m, and the diameter at breast height is more than 1m. It is an important native broad-leaved precious timber and multi-purpose ecological public welfare forest tree species in South China. Red cones are naturally distributed in east longitude 95°20′～118°00′ and north latitude 18°30′～25°00′. The main production areas are concentrated in Guangdong, Guangxi, and southern Fujian, and its surrounding areas are distributed in Hainan, southern Yunnan, and southeastern Guizhou. And Hunan, Jiangxi and other provinces in the south.

Red cone has excellent characteristics such as fast growth, excellent material, wide adaptability and high benefit. Its trunk is straight, hard, red in color, beautiful in color and texture, strong in corrosion resistance, non-cracking, non-deformable, and easy to process. It is a high-quality and precious material for construction, shipbuilding, high-end furniture, wooden floors, and military supplies. , Sports equipment, etc. The seeds are rich in starch, which can be used for frying, feed and wine making. The seeds and shells are rich in tannin, which can be used to extract tannins. The red cone forest has strong germination ability and rapid growth of shoots. It can be harvested more than 10 times in one afforestation, and it can be profitable for hundreds of years if it is managed properly. Red Cone has dense branches and leaves, is more shade-tolerant, and has good mixed growth performance. It can be used as a timber and water conservation forest for pure forest planting, and can also be used for mixed forestry for reconstruction of residual forests and ecological public welfare forests. At present, many provinces in my country, such as Guangdong, Guangxi, and Fujian, have selected red cone as an excellent tree species with high economic value and great development prospects in the local area for promotion. The afforestation area is large and the promotion and application prospects are broad.

At present, most of the seedlings used in the cultivation of red cone plantation are raised by mixed seeds, which leads to large differentiation and unevenness of the individual seedlings and their afforestation stands, which in turn affects the afforestation quality, output and economic benefits of the plantation. Through the research of scientific and technological workers in the past ten years, a number of excellent single plants of Antonia chinensis have been selected and bred. However, due to the limited number of selected excellent plants, coupled with the young age and low seed setting rate, the harvested seeds are far from meeting the needs of afforestation. Preliminary studies have shown that no matter whether the red cone tree is propagated by grafting or cuttings, the reproductive rate is low, and there are different degrees of partial crown phenomenon, so the seedlings are difficult to use for production. Although there are reports on the tissue culture research on the budding branch of the excellent tree of the red cone, the age of the excellent tree is not indicated, which may be due to different genotypes. The reported tissue culture method is used for the tissue culture of the selected excellent tree in this project. There are problems such as serious pollution in induction culture, slow germination, and difficulty in establishing sterile lines; buds in multiplication culture are small and yellow, slow in growth, gradually withered and yellow, low in multiplication multiple and rooting rate, and are difficult to use in production.

As an important and precious afforestation tree species in my country, red cone has a huge demand for improved seedlings, but vegetative reproduction is difficult, and the contradiction between supply and demand of seedlings is prominent. This method uses the seeds of the selected red cone tree as explants to conduct tissue culture and rapid propagation technology research. It is an important way for the fine tree species of red cone to realize large-scale seedling cultivation and then realize industrial development. It has important research and development practical meaning of production.

There is no report about the tissue culture technology using the seeds of the eucalyptus as explants.

Using the seeds of the red cone tree with fast growth, excellent dry shape and wood properties, and strong stress resistance as explants, through explant treatment, basic medium design, and optimization of hormone types, concentrations and ratios, the establishment of Its in vitro culture plant regeneration technology achieves the goals of low pollution rate of explant induced culture, short time, many adventitious buds, fast and robust proliferative bud elongation, high value-added rate and rooting rate, and has developed a suitable tissue culture plant Substrate for transplanting roots and seedlings and management techniques for transplanted seedlings. The promotion through large-scale seedling breeding will provide technical support for the propagation of high-quality seedlings required for the cultivation of red cone plantations in my country, and is of great significance for improving the economic benefits of red cone plantations.

Contents of the invention

In order to overcome the shortcomings and deficiencies of the prior art, and to fill the technical gap of directly using the seeds of the red cone tree as explants for tissue culture and rapid propagation, the purpose of the present invention is to provide a method for the regeneration of red cone cultured plants in vitro. The method has the characteristics of fast induction of adventitious buds, many adventitious buds, high multiplication rate, thick buds and fast elongation, high rooting rate, developed and robust root system of rooted seedlings, and high transplanting survival rate.

The object of the present invention is achieved through the following technical solutions:

A method for plant regeneration of red cone in vitro culture, comprising the following steps:

(1) Design DX basal medium according to the physiological state of the seeds and the nutritional requirements of in vitro culture. The formula of the DX basal medium is as follows: 720mg/L NH ₄ NO ₃ , 480mg/L KNO ₃ , 600mg/L Ca(NO ₃ ) ₂ 4H ₂ O, 370mg/LMgSO ₄ 7H ₂ O, 170mg/L KH ₂ PO ₄ , 100mg/L KCl, 22.3mg/L MnSO ₄ 4H ₂ O, 8.6mg/L ZnSO ₄ 7H ₂ O, 6.2mg/LH ₃ BO ₃ , 0.83mg/L KI, 0.25mg/L Na ₂ MoO ₄ 2H ₂ O, 0.25mg/L CuSO ₄ 5H ₂ O, 0.05mg/LNiSO ₄ .6H ₂ O, 0.025mg/L CoCl ₂ , 27.8mg/L FeSO ₄ ·7H ₂ O, 37.3mg/L Na ₂ ·EDTA·H ₂ O, 120mg/LMyo-Insitol (inositol), 2.0mg/L Glycine ( glycine), 0.4mg/L Thiamine HCl (vitamin B1), 0.2mg/L Nicotinic acid (niacin), 0.2mg/L Pyridoxine HCl (vitamin B6); its pH value is 5.8;

(2) Explant preparation and processing;

(3) explant surface disinfection;

(4) Bud induction: insert the sterilized seeds into the prepared induction differentiation medium for cultivation; the induction differentiation medium is to add 6-BA0.4～1.0mg/L, KT 0.2～0.5mg/L, NAA0.02～0.2mg/L, sucrose 25～40g/L and carrageenan 8g/L, its pH value is 5.8～6.0;

(5) Proliferation culture: Cut out the buds with a length greater than 2 cm after induction for 25-30 days and transfer them to the proliferation medium for proliferation culture; proliferate and culture for 20-30 days, differentiate to form new bud clusters, and remove young shoots with a height of 3 cm or more Branches are cut into 1.0-1.5cm stem segments and transferred to new proliferation medium, and the multiplication factor is 3-4.2 times; after repeated subculture, proliferation, cultivation and expansion; the proliferation medium is added 6 -BA 0.5～2.0mg/L, KT 0.2～0.5mg/L, NAA0.05～0.2mg/L, sucrose 25～40g/L and carrageenan 8g/L, its pH value is 5.8～6.0;

(6) rooting culture: the tender stems exceeding 1.5cm in the proliferation medium are cut off, inserted in the rooting medium and cultivated, and the rooting rate reaches 80-90% after 20 days; the rooting medium is 1 /2DX basic medium is prepared by adding NAA 0.5～1.0mg/L, sucrose 15～20g/L and agar 6g/L, and its pH value is 5.8～6.0;

(7) Hardening and transplanting;

(8) Management of transplanted seedlings.

The DX basic medium in step (1) is sterilized at 121° C. for 15 to 20 minutes.

The preparation and treatment of the explants in step (2) is to peel off the shells of the seeds of the red cone tree in advance, peel off the inner and outer testa with a scalpel on the ultra-clean workbench, and pay attention to ensure the integrity of the embryo.

The surface disinfection of the explants in step (3) is to soak the treated seeds in alcohol with a concentration of 70 to 75% by volume for 20 to 30 seconds, rinse with sterile water for 1 to 2 times after pouring off the alcohol, and then add mass volume The percentage concentration is 0.05-0.1% mercury liter solution, soak for 1-2 minutes, shake constantly during this period, pour out the mercuric acid solution and rinse with sterile water for 5-6 times.

The culture in step (4) is carried out in a culture room with a temperature of 25±2°C, a light intensity of 30 μmol.m ^-2 .s ^-1 , and a light time of 8 h/d; after 10 days of culture, it starts from the cotyledon node Germination of axillary and adventitious buds.

The culturing in step (5) is carried out in a cultivating room with a temperature of 25±2°C, a light intensity of 60 μmol.m ^-2 .s ^-1 , and a light time of 12 h/d.

The culture in step (6) is carried out in a culture room with a temperature of 25±2°C, a light intensity of 80 μmol.m ^-2 .s ^-1 , and a light time of 12 h/d; the 1/2DX basic medium In order to halve the amount of a large number of elements contained in the DX basic medium, including NH ₄ NO ₃ , KNO ₃ , Ca(NO ₃ ) ₂ 4H ₂ O, MgSO ₄ 7H ₂ O, KH ₂ PO ₄ and KCl, The rest of the ingredients remain unchanged.

The seedling hardening and transplanting described in step (7) specifically follows the following steps: after rooting and culturing for 20 days, move the rooting bottle seedlings to a 60% shady greenhouse for seedling hardening for 7 to 10 days, and then set the cap of the tissue culture bottle from half-open to fully open. Seedlings for 1 day, then take the seedlings out of the culture bottle, wash the medium sticking to the roots, and transplant them into the seedling container filled with the substrate. The transplanting depth should be covered with soil just to cover the root system, and the substrate should be pressed tightly to stabilize the seedlings. The transplantation is complete. Afterwards, drench rooting water; the substrate is made of peat soil, perlite and coconut peat mixed in a volume ratio of 3:1:1.

The described transplanting seedling management of step (8) follows the steps below:

(1) In the first week after transplanting, use the cover film to keep moisture, and then uncover the film to keep the substrate moist, the relative air humidity is above 95%, the cultivation temperature is controlled at 15-30°C, and the awning is shaded with a 70% shading net;

(2) On the day after transplanting, use chlorothalonil or carbendazim solution with a mass volume percentage concentration of 0.1% to spray the seedlings, and then spray once every 10 days; 2‰ compound fertilizer, according to the growth status of the seedlings, spray once every 10 days;

(3) After transplanting, when the seedlings grow stably and grow new shoots and new roots, gradually increase the light intensity until the conventional management of full light is carried out, and the seedlings are used for afforestation when they are cultivated to a height of 20 cm.

Compared with the prior art, the present invention has the following advantages and beneficial effects: adopting the method of the present invention to induce buds of red cone explants is fast; the induction rate is as high as 100%; the multiplication coefficient is large, reaching 4.2; The buds elongate and grow fast, and the buds are as high as 4-5cm after 30 days of cultivation; the rooting rate is high, the root system is developed, up to 90%, and the average root number is 3.4 roots/plant; the transplanting survival rate is high, up to 90%. Through large-scale seedling breeding, it will provide technical support for the rapid promotion of fine germplasm of red cone in my country, and provide high-quality seedling guarantee for improving the economic benefits of red cone plantation, which is of great significance.

Description of drawings

Fig. 1 is the bud induction of aseptic material of Youshu seeds.

Fig. 2 is a bud cluster of induced bud formation.

Fig. 3 is the proliferating shoots in the proliferating medium.

Fig. 4 is the root system of rooted seedlings in the rooting medium.

Fig. 5 is rooted seedling transplanting.

Detailed ways

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

The DX basic medium was designed according to the physiological state of the seeds and the nutritional requirements of in vitro culture. The formula of the DX basic medium involved in the following examples is as follows: 720mg/L NH ₄ NO ₃ , 480mg/L KNO ₃ , 600mg/L Ca(NO ₃ ) ₂ 4H ₂ O, 370mg/L MgSO ₄ 7H ₂ O, 170mg/L KH ₂ PO ₄ , 100mg/L KCl, 22.3mg/L MnSO ₄ 4H ₂ O, 8.6mg /L ZnSO ₄ ·7H ₂ O, 6.2mg/LH ₃ BO ₃ , 0.83mg/L KI, 0.25mg/L Na ₂ MoO ₄ ·2H ₂ O, 0.25mg/L CuSO ₄ ·5H ₂ O, 0.05mg/L L NiSO ₄ .6H ₂ O, 0.025mg/L CoCl ₂ , 27.8mg/L FeSO ₄ ·7H ₂ O, 37.3mg/L Na ₂ ·EDTA·H ₂ O, 120mg/L Myo-Insitol (inositol), 2.0mg/L Glycine (glycine), 0.4mg/L Thiamine·HCl (vitamin B1), 0.2mg/L Nicotinic·acid (niacin), 0.2mg/LPyridoxine·HCl (vitamin B6); its pH value is 5.8.

The 1/2DX basal medium is to halve the amount of the macroelements (NH4NO3, KNO3, Ca(NO3)2 4H2O, MgSO4 7H2O, KH2PO4, KCl) contained in the DX basal medium, and the remaining ingredients remain unchanged .

Embodiment one Adopt the following steps to realize the in vitro culture plant regeneration of red cone:

(1) Induction of adventitious buds from seeds

Preparation and treatment of explants: Peel off the shell of the selected excellent tree seeds in advance with branch shears, and peel off the inner and outer seed coats with a scalpel on the ultra-clean workbench, paying attention to ensure the integrity of the embryo.

Surface disinfection of explants: first scrub the seeds with sterile water on the ultra-clean workbench, then use alcohol with a volume percentage concentration of 75%, soak for 20s, then rinse with sterile water once, and then add mass volume percentage Concentration of 0.05% mercuric chloride solution, soak for 2 minutes, shake constantly during this time, drain the mercuric liter solution and rinse with sterile water 6 times. Blot the water droplets on the surface of the sterile material with sterile filter paper for later use.

Bud induction: Inoculate the sterilized embryos into induction medium DX+0.8mg/L 6-BA+0.2mg/L KT+0.02mg/L NAA+sucrose 30g/L+carrageenan 8g/L (hormones purchased from Sigma- Aldrich Company), each bottle was inoculated with one explant. After 2 to 3 days, the embryos began to germinate. One week later, the epicotyl reached 0.2 cm, and 20 days later, many small buds germinated (Fig. 1, Fig. 2), and the induction rate was 100%.

(2) Cluster bud proliferation

Cut the induced axillary buds into 1.0-1.5 cm stem segments and transfer them to the proliferation medium. The formula of the proliferation medium is DX+6-BA 0.5mg/L+KT 0.2mg/L+NAA 0.05mg/L+sucrose 30g/L+carrageenan 8g/L. After 4 weeks, single buds form buds The buds elongate and grow quickly, the multiplication coefficient reaches 4.2, and the effective buds (bud height≧2cm) reach 3.32 per cluster (Figure 3).

(3) Rooting culture

Select robust effective buds and transfer them to the rooting induction medium of 1/2DX+NAA 0.5mg/L+sucrose 15g/L+agar 6g/L for culture. After 15 days, the induction rate of adventitious roots is 85%, and the average root number is 3.4 /strain (Figure 4).

(4) Transplanting management of tissue culture seedlings

According to the volume ratio of 3:1:1, mix the peat soil, perlite and coir peat evenly, put it into a container bag, and disinfect it with a solution of potassium permanganate with a mass volume percentage concentration of 3‰ for later use; the environmental conditions for transplanting are temperature controlled at 25°C, 70% shading, and a greenhouse with a relative air humidity of more than 95%; move the rooted bottle seedlings that have been cultivated for 20 days to a 60% shady greenhouse to adapt to the external environment for 7 days, and change the cap of the tissue culture bottle from half open to full Open the seedlings for 1 day, then carefully take out the group culture seedlings, wash the medium sticking to the roots, transplant them into container bags, plant one seedling in each bag, and transplant the soil to a depth just enough to cover the root system to stabilize the seedlings, and compact the substrate After the transplanting is completed, water the fixed root water thoroughly; use the cover film to moisturize the first week after transplanting, and then uncover the film to keep the substrate moist; on the day after transplanting, use chlorothalonil (or multibacterial Spirit) solution to spray the seedlings, and then spray once every 10 days to prevent the growth of germs; 15 days after transplanting, start to spray the compound fertilizer with a mass volume percentage concentration of 2‰, and spray once every 10 days according to the growth status of the seedlings After the seedlings grow stably and grow new shoots and new roots, gradually increase the light intensity until the conventional management of full light is carried out. When the seedlings are cultivated to a height of 20 cm, they can be used for afforestation. The transplanting survival rate was higher than 90% (Fig. 5).

Embodiment two Adopt the following steps to realize the in vitro culture plant regeneration of red cone:

(1) Induction of adventitious buds from seeds

Preparation and treatment of explants: Peel off the shell of the selected excellent tree seeds in advance with branch shears, and peel off the inner and outer seed coats with a scalpel on the ultra-clean workbench, paying attention to ensure the integrity of the embryo.

Disinfection of the surface of the explants: first scrub the seeds with sterile water on the ultra-clean workbench, then use alcohol with a volume percentage concentration of 72%, soak for 30s, then rinse with sterile water twice, and then add the mass volume percentage Concentration of 0.1% mercuric chloride solution, soak for 1min, shake continuously during this period, drain the mercuric chloride solution and rinse with sterile water 5 times. Blot the water droplets on the surface of the sterile material with sterile filter paper for later use.

Bud induction: inoculate the sterilized embryos into induction medium DX+0.4mg/L 6-BA+0.2mg/L KT+0.1mg/L NAA+sucrose 25g/L+carrageenan 8g/L (hormones purchased from Sigma- Aldrich Company), each bottle was inoculated with one explant. After 8-10 days, the embryos began to germinate. After half a month, the epicotyls reached 0.2 cm. After 30 days, many small buds germinated, and the induction rate was 85%.

(2) Cluster bud proliferation

Cut the induced axillary buds into 1.0-1.5 cm stem segments and transfer them to the proliferation medium. Proliferation medium formula is DX+6-BA 1.0mg/L+KT 0.2mg/L+NAA 0.1mg/L+sucrose 25g/L+carrageenan 8g/L in the proliferation medium, after 4 weeks, single buds form buds The buds elongate and grow quickly, the multiplication coefficient reaches 3, and the effective buds (bud height≧2cm) reach 2.82 per cluster.

(3) Rooting culture

Select robust effective buds and transfer them to the rooting induction medium of 1/2DX+NAA 0.7mg/L+sucrose 20g/L+agar 6g/L for culture. After 15 days, the induction rate of adventitious roots is 80%, and the average root number is 2.5 / strain.

(4) Transplanting management of tissue culture seedlings

According to the volume ratio of 3:1:1, mix the peat soil, perlite and coir peat evenly, put it into a container bag, and disinfect it with a solution of potassium permanganate with a mass volume percentage concentration of 3‰ for later use; the environmental conditions for transplanting are temperature controlled at 15°C, 70% shading, and a greenhouse with a relative air humidity of more than 95%; move the rooted bottle seedlings that have been cultivated for 20 days to a 60% shady greenhouse to adapt to the external environment for 8 days, and change the cap of the tissue culture bottle from half open to full Open the seedlings for 1 day, then carefully take out the group culture seedlings, wash the medium sticking to the roots, transplant them into container bags, plant one seedling in each bag, and transplant the soil to a depth just enough to cover the root system to stabilize the seedlings, and compact the substrate After the transplanting is completed, water the fixed root water thoroughly; use the cover film to moisturize the first week after transplanting, and then uncover the film to keep the substrate moist; on the day after transplanting, use chlorothalonil (or multibacterial Ling) solution to spray the seedlings, and then spray once every 10 days to prevent the growth of germs; 15 days after transplanting, start to spray the compound fertilizer with a mass volume percentage concentration of 1‰, and spray once every 10 days according to the growth status of the seedlings After the seedlings grow stably and grow new shoots and new roots, gradually increase the light intensity until the conventional management of full light is carried out. When the seedlings are cultivated to a height of 20 cm, they can be used for afforestation. The transplanting survival rate was higher than 82% (Fig. 5).

Embodiment three Adopt the following steps to realize the in vitro culture plant regeneration of red cone:

(1) Induction of adventitious buds from seeds

Preparation and treatment of explants: Peel off the shell of the selected excellent tree seeds in advance with branch shears, and peel off the inner and outer seed coats with a scalpel on the ultra-clean workbench, paying attention to ensure the integrity of the embryo.

Disinfection of the explant surface: scrub the seeds with sterile water on the ultra-clean workbench, then use alcohol with a volume percentage concentration of 70%, soak for 30s, and then rinse with sterile water once, and then add the mass volume percentage Concentration of 0.1% mercuric chloride solution, soak for 2 minutes, shake continuously during this period, pour out the mercuric liter solution and rinse with sterile water for 6 times. Blot the water droplets on the surface of the sterile material with sterile filter paper for later use.

Bud induction: Inoculate the sterilized embryos into induction medium DX+1.0mg/L 6-BA+0.5mg/L KT+0.2mg/L NAA+sucrose 40g/L+carrageenan 8g/L (hormones purchased from Sigma- Aldrich Company), each bottle was inoculated with one explant. After 6-7 days, the embryos began to germinate. After 10 days, the epicotyl reached 0.2 cm. After 30 days, many small buds germinated, and the induction rate was 90%.

(2) Cluster bud proliferation

Cut the induced axillary buds into 1.0-1.5 cm stem segments and transfer them to the proliferation medium. Proliferation medium formula is DX+6-BA 2.0mg/L+KT 0.5mg/L+NAA 0.2mg/L+sucrose 40g/L+carrageenan 8g/L in the proliferation medium, after 4 weeks, single buds form buds The buds elongate and grow quickly, the multiplication coefficient reaches 3.5, and the effective buds (bud height≧2cm) reach 2.52 per cluster.

(3) Rooting culture

Select healthy and effective buds and transfer them to the rooting induction medium of 1/2DX+NAA 1.0mg/L+sucrose 15g/L+agar 6g/L for culture. After 15 days, the induction rate of adventitious roots is 82%, and the average number of roots is 3 / strain.

(4) Transplanting management of tissue culture seedlings

According to the volume ratio of 3:1:1, mix the peat soil, perlite and coir peat evenly, put it into a container bag, and disinfect it with a solution of potassium permanganate with a mass volume percentage concentration of 3‰ for later use; the environmental conditions for transplanting are temperature controlled at 30°C, 70% shading, and a greenhouse with a relative air humidity above 95%; move the rooted bottle seedlings that have been cultivated for 20 days to a 60% shady greenhouse to adapt to the external environment for 10 days, and change the cap of the tissue culture bottle from half open to full Open the seedlings for 1 day, then carefully take out the group culture seedlings, wash the medium sticking to the roots, transplant them into container bags, plant one seedling in each bag, and transplant the soil to a depth just enough to cover the root system to stabilize the seedlings, and compact the substrate After the transplanting is completed, water the fixed root water thoroughly; use the cover film to moisturize the first week after transplanting, and then uncover the film to keep the substrate moist; on the day after transplanting, use chlorothalonil (or multibacterial Spirit) solution to spray the seedlings, and then spray once every 10 days to prevent the growth of germs; 15 days after transplanting, start to spray the compound fertilizer with a mass volume percentage concentration of 2‰, and spray once every 10 days according to the growth status of the seedlings After the seedlings grow stably and grow new shoots and new roots, gradually increase the light intensity until the conventional management of full light is carried out. When the seedlings are cultivated to a height of 20 cm, they can be used for afforestation. The transplanting survival rate was higher than 85% (Fig. 5).

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (7)

1. a red cone in vitro culture plant regeneration method is characterized in that comprising the following steps: (1) Design DX basal medium according to the physiological state of the seeds and the nutritional requirements of in vitro culture. The formula of the DX basal medium is as follows: 720mg/L NH ₄ NO ₃ , 480mg/L KNO ₃ , 600mg/L Ca(NO ₃ ) ₂ 4H ₂ O, 370mg/LMgSO ₄ 7H ₂ O, 170mg/L KH ₂ PO ₄ , 100mg/L KCl, 22.3mg/L MnSO ₄ 4H ₂ O, 8.6mg/L ZnSO ₄ 7H ₂ O, 6.2mg/LH ₃ BO ₃ , 0.83mg/L KI, 0.25mg/L Na ₂ MoO ₄ 2H ₂ O, 0.25mg/L CuSO ₄ 5H ₂ O, 0.05mg/LNiSO ₄ 6H ₂ O, 0.025mg/L CoCl ₂ , 27.8mg/L FeSO ₄ ·7H ₂ O, 37.3mg/L Na ₂ ·EDTA·H ₂ O, 120mg/L Myo-Insitol, 2.0mg/L Glycine, 0.4mg/L L Thiamine·HCl, 0.2mg/L Nicotinic·acid, 0.2mg/L Pyridoxine·HCl; its pH value is 5.8; (2) Preparation and treatment of explants: Peel off the shells of the seeds of the red cone tree in advance, peel off the inner and outer seed coats with a scalpel on the ultra-clean workbench, and pay attention to ensure the integrity of the embryos; (3) Surface disinfection of explants: Soak the treated seeds in alcohol with a concentration of 70 to 75% by volume for 20 to 30 seconds, pour off the alcohol and rinse with sterile water for 1 to 2 times, then add 0.05% by volume ～0.1% mercuric acid solution, soak for 1～2min, shake constantly during this period, pour off the mercuric solution and rinse with sterile water for 5～6 times; (4) Bud induction: Insert the sterilized seeds into the prepared induction differentiation medium for cultivation; the induction differentiation medium is to add 6-BA 0.4～1.0mg/L, KT 0.2 ~0.5mg/L, NAA 0.02~0.2mg/L, sucrose 25~40g/L and carrageenan 8g/L, its pH value is 5.8~6.0; (5) Proliferation culture: cut off the buds with a length greater than 2 cm after induction for 25 to 30 days and transfer them to the proliferation medium for proliferation culture; proliferate and culture for 20 to 30 days, differentiate to form new bud clusters, and young buds with a height ≥ 3 cm Branches are cut into 1.0-1.5cm stem segments and transferred to new proliferation medium, and the multiplication factor is 3-4.2 times; after repeated subculture, proliferation, cultivation and expansion; the proliferation medium is added 6 -BA 0.5～2.0mg/L, KT 0.2～0.5mg/L, NAA 0.05～0.2mg/L, sucrose 25～40g/L and carrageenan 8g/L, its pH value is 5.8～6.0; (6) rooting culture: the tender stems exceeding 1.5cm in the proliferation medium are cut off, inserted in the rooting medium and cultivated, and the rooting rate reaches 80-90% after 20 days; the rooting medium is 1 /2DX basic medium is prepared by adding NAA0.5～1.0mg/L, sucrose 15～20g/L and agar 6g/L, and its pH value is 5.8～6.0; the 1/2DX basic medium is DX The amount of a large number of elements contained in the basic medium, including NH ₄ NO ₃ , KNO ₃ , Ca(NO ₃ ) ₂ 4H ₂ O, MgSO ₄ 7H ₂ O, KH ₂ PO ₄ and KCl, was halved, and the remaining ingredients were unchanged. Change; (7) Hardening and transplanting; (8) Management of transplanted seedlings. 2 . A method for plant regeneration in vitro cultured red cone according to claim 1 , characterized in that: the DX basal medium in step (1) is sterilized at 121° C. for 15 to 20 minutes. 3 . 3. A method for regeneration of red cone cultured plants in vitro according to claim 1, characterized in that: the cultivation in step (4) is carried out at a temperature of 25±2°C and a light intensity of 30 μmol.m ⁻² .s ^-1 , cultured in a culture room with a light time of 8h/d; after 10 days of culture, axillary buds and adventitious buds began to germinate from the cotyledon nodes. 4. A method for regeneration of red cone cultured plants in vitro according to claim 1, characterized in that: the cultivation in step (5) is carried out at a temperature of 25±2°C and a light intensity of 60 μmol.m ⁻² .s ^-1 , cultured in a culture room with a light time of 12h/d. 5. A method for regeneration of red cone cultured plants in vitro according to claim 1, characterized in that: the cultivation in step (6) is carried out at a temperature of 25±2°C and a light intensity of 80 μmol.m ⁻² .s ^-1 , cultured in a culture room with a light time of 12h/d. 6. a kind of red cone in vitro culture plant regeneration method according to claim 1 is characterized in that: the described seedling hardening transplanting of step (7) is specifically according to the following steps: after rooting and cultivating 20d, rooting bottle seedling is moved to Harden the seedlings in a 60% shade greenhouse for 7 to 10 days, then turn the cap of the tissue culture bottle from half-open to fully open for 1 day, then take the seedlings out of the culture bottle, wash the medium sticking to the roots, and transplant them into the filled matrix In a seedling container, the depth of transplantation should be covered with soil just to cover the root system, and the substrate should be compacted to stabilize the seedlings. After the transplantation is completed, drench the root water; the substrate is made of peat soil, perlite and coconut bran at a ratio of 3:1:1. The volume ratio is mixed. 7. a kind of red cone in vitro culture plant regeneration method according to claim 1, is characterized in that: the described transplanting seedling management of step (8) is according to the following steps: (1) In the first week after transplanting, use the cover film to keep moisture, and then uncover the film to keep the substrate moist, the relative air humidity is above 95%, the cultivation temperature is controlled at 15-30°C, and the awning is shaded with a 70% shading net; (2) On the day after transplanting, use chlorothalonil or carbendazim solution with a mass volume percentage concentration of 0.1% to spray the seedlings, and then spray once every 10 days; 2‰ compound fertilizer, according to the growth status of the seedlings, spray once every 10 days; (3) After transplanting, when the seedlings grow stably and grow new shoots and new roots, gradually increase the light intensity until the conventional management of full light is carried out, and the seedlings are used for afforestation when they are cultivated to a height of 20 cm.