CN102293153A - A high-efficiency genetic transformation method of rubber tree based on secondary somatic embryogenesis - Google Patents

Abstract

The invention discloses a high-efficiency genetic transformation method of rubber tree based on secondary somatic embryogenesis, which is characterized in that it comprises the acquisition of a genetic transformation receptor-somatic embryoid body, the genetic transformation of a somatic embryoid body, and the proliferation of a transformed embryoid body , Plant regeneration of transformed embryoid body and transplanting process of transformed plant. The invention is simple in operation and high in efficiency, and effectively avoids the repeated, low-efficiency, and heavy physical labor of traditional genetic transformation in the continuous collection of inflorescences, disinfection, and callus induction, and the influence of environmental factors such as weather and seasons on the acquisition of genetic transformation receptors. At the same time, it makes full use of the characteristics that secondary somatic embryogenesis can efficiently obtain embryoid bodies and somatic embryos can proliferate cyclically, overcomes the defects of low efficiency of transforming embryoid bodies and transforming plants by traditional methods, and greatly improves the genetic transformation efficiency of rubber trees. Transgenic breeding of rubber trees and functional verification of genes play an important role in technical support.

Description

A high-efficiency genetic transformation method of rubber tree based on secondary somatic embryogenesis

technical field

The invention belongs to the field of biological technology, and in particular relates to a high-efficiency genetic transformation method for rubber trees based on secondary somatic embryogenesis.

Background technique

Natural rubber has the incomparable elasticity, ductility and thermal conductivity of synthetic rubber, and is an important industrial raw material, mainly derived from Hevea brasiliensis Muell.Arg. It takes 20 to 30 years to breed a new rubber tree variety through conventional hybrid breeding. Transgenic technology is a very effective way to shorten the breeding cycle of rubber trees and speed up the cultivation of new varieties.

Currently, there are two types of recipients used for Agrobacterium hevea transformation: anther embryogenic callus and long-term subcultured friable embryogenic callus.

The anther embryogenic callus has the following defects as the transformation recipient: the material is limited by the season, the stamen stripping is labor-intensive and time-consuming, and the anther regeneration ability is poor, so it cannot meet the transformation requirements of the four seasons, and it is difficult to obtain enough transgenic plants. Increasing demand for scientific research and production.

Long-term subculture of fragile embryogenic callus as a transformation recipient has the following defects: it takes more than half a year to obtain fragile embryogenic callus, the proportion of calli with rapid proliferation and strong embryogenicity is very low, and the induction and screening time Long; Fragile embryogenic callus regenerates deformed plants, and its growth and yield are not as good as old bud grafting trees, lacking application prospects.

Therefore, the introduction of a new receptor system is crucial to the establishment of an efficient genetic transformation system for rubber trees. Hua Yuwei et al. (2010) established a rubber tree secondary somatic embryogenesis system, which consists of three processes: 1) induction of primary somatic embryos: callus induced by anthers, inner integuments, roots or other explants, Then induce primary somatic embryogenesis; 2) induction of secondary somatic embryos: the primary embryo or secondary embryo is an explant, which is cut into an embryo block of 0.2-0.5×0.2-0.5cm, and then the callus is induced, Secondary somatic embryogenesis is induced again, and this process can be repeated; 3) Plant regeneration: select mature cotyledon-shaped embryoid bodies and transfer them to the emergence medium to induce plantlet regeneration. Using this system, a skilled worker can produce 8,000 self-rooted clones a year. The establishment of this system provides a new way for the establishment of a high-efficiency genetic transformation system for rubber trees. Embryoid bodies have obvious advantages as transformation recipients: 1. There are materials all year round, which can meet the needs of transformation at any time; 2. Save the time and labor for stripping off stamens or inducing and screening stable proliferation of fragile embryogenic callus; 3 times Somatic embryogenesis has a strong regeneration ability, and each worker can produce 8,000 tissue cultured seedlings per year, which can fully meet the needs of scientific research and production; 4. The root system of the regenerated plants is well developed and the growth is vigorous; 5. The number of explants required for transformation is small. It saves the time for transforming and recovering culture and clamping callus, and greatly improves the transforming speed.

There are three reported methods of propagation of rubber tree transgenic materials: 1. Transformation of fragile embryogenic callus to induce somatic embryogenesis and plant regeneration; Budding propagation. The proliferated callus in method 1 was induced somatic embryogenesis by suspension culture, the vitrification phenomenon was serious, and the regenerated transgenic plants were deformed, the growth vigor and yield were not as good as the control, and the application prospect was not optimistic; methods 2 and 3 were based on anther callus Tissues are used as transformation recipients. The main defect of this receptor is the low induction rate of resistant embryoid bodies and the low proportion of normal resistant embryoid bodies. Therefore, most of the transgenic embryoid bodies cannot regenerate plants. Micropropagation and budding only expand the transgenic plants, but do not really solve the technical bottleneck of low induction rate of resistant embryoid bodies and low regeneration frequency. The rubber tree secondary somatic embryogenesis is a method in which the embryoid body is used as an explant, and through cyclic somatic embryogenesis, the cyclic proliferation from embryo to embryo is realized, and the somatic embryo plant proliferation is finally realized. This regeneration system has a high induction rate of embryoid bodies and a high proportion of normal embryos, and is completely different from the previous three proliferation methods. It is to multiply the obtained resistant embryos, induce enough normal embryoid bodies to emerge again, and try to ensure that each transgenic All the embryoids can emerge, completely solving the problems of low embryoid induction rate and low emergence rate in the anther regeneration system.

The invention uses the secondary somatic embryogenesis system as the acceptor system to establish a high-efficiency rubber tree genetic transformation method different from the reported ones.

Contents of the invention

The purpose of the present invention is to provide a high-efficiency genetic transformation method of rubber tree based on secondary somatic embryogenesis, which is based on secondary somatic embryogenesis, and the somatic embryoid body is the Agrobacterium infection receptor. By inducing secondary somatic embryogenesis, the high-efficiency Obtaining transformed embryoid bodies and transformed plants provides an efficient genetic transformation method for transgenic breeding of rubber trees and transgenic verification of functional genes.

The technical scheme that the present invention adopts for solving its technical problem is:

A high-efficiency genetic transformation method for rubber tree based on secondary somatic embryogenesis, which is characterized in that it includes the acquisition of genetic transformation receptor-somatic embryoid bodies, the genetic transformation of somatic embryoid bodies, the proliferation of transformed embryoid bodies, and the transformation of embryoid bodies. The plant regeneration of shape body and the transplanting five steps of transformation plant:

(a) Acquisition of genetically transformed recipient-somatic embryoid bodies: select robust and vigorous embryoid bodies and separate them from the original culture medium as transformed recipients;

(b) Genetic transformation of somatic embryoid body: the process includes infection, co-cultivation, recovery culture, callus induction and differentiation process;

Infection: OD ₆₀₀ =0.1-0.6 Agrobacterium infection for 3-10min, put sterile filter paper to blot the bacterial solution;

Co-cultivation: transfer the infected embryoid bodies to the callus induction medium supplemented with 1-50mg/L silver nitrate, the pH value is 5.2, and co-cultivate at 20-28°C for 3-6 days;

Recovery culture: transfer the co-cultured embryoid bodies to a callus induction medium supplemented with 1-50 mg/L silver nitrate and antibiotics that inhibit the growth of Agrobacterium, and culture in the dark at 23-28°C for 3-10 days;

Callus induction: Cut the embryoid body after recovery culture into 0.2-0.5×0.2-0.5cm embryo blocks, and then transfer to the culture medium that has added 1-50 mg/L silver nitrate, antibiotics that inhibit the growth of Agrobacterium and corresponding screening agents. On the callus induction medium, its pH value is 5.8, and cultured in the dark at 23-28°C for 15-40 days;

Differentiation: transfer the callused embryo blocks to the differentiation medium supplemented with antibiotics that inhibit the growth of Agrobacterium and corresponding screening agents; its pH value is 5.8, and it is cultured in the dark at 23-28°C for 30-70 days;

(c) Proliferation of transformed embryoid bodies: cut the embryoid bodies positive for GUS staining into 0.2-0.5×0.2-0.5 cm embryoids, repeat the callus induction and differentiation process described in step (b), but remove the callus The silver nitrate of the culture medium used in the induction process; this process can be recycled until the transformant obtains enough mature cotyledon-shaped embryoid bodies; after 1-3 cycles, the antibacterial antibiotics can be removed;

(d) Plant regeneration of transformed embryoid body: transfer mature cotyledon-shaped embryoid body to the emergence medium, its pH value is 5.8, 23-28 ° C light culture, 20-60 days for emergence;

(e) Transplanting of transformed plants: (1) Budding: use regenerated transformed plants as scions, seedlings or tissue cultured seedlings as rootstocks, and realize transplantation from the laboratory to the field through a series of processes of budding, unbinding, and bagging. Planting; (2) sand bed transplanting: wash the culture medium of the regenerated transformed plants and move them into the sand bed, control the temperature, humidity, and diseases, and gradually lift the shed after half a month, and the transformed plants will sprout new shoots and grow new roots in one month , and bagging after the leaves grow to a stable period.

The genetic transformation recipient used in the step (a) is an embryoid body obtained through somatic embryogenesis of explants such as anthers, inner integuments and roots, and secondary somatic embryogenesis of somatic embryos.

The callus induction medium of described step (b) is based on MS medium, and the potassium dihydrogen phosphate in MS medium, calcium chloride anhydrous, manganese sulfate tetrahydrate, magnesium sulfate heptahydrate The content is adjusted to potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, manganese sulfate tetrahydrate 10-40mg/L, magnesium sulfate heptahydrate 400-600mg/L, and add 2,4- D 0.5-3mg/L, KT 0.5-3mg/L, NAA 0.5-3mg/L, Phytagel 2-3g/L, sucrose 50-90g/L and coconut water 40-100ml;

The active ingredient of the differentiation medium of described step (b) is to take MS medium as basic medium, and potassium dihydrogen phosphate, calcium chloride anhydrous, manganese sulfate tetrahydrate, magnesium sulfate heptahydrate in MS medium The content is adjusted to potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, manganese sulfate tetrahydrate 10-40mg/L, magnesium sulfate heptahydrate 400-600mg/L, and add 6-BA 0.5-3mg/L, KT 0.5-3mg/L, NAA0.1-2mg/L, GA31-5mg/L, Phytagel 2-3g/L, sucrose 50-90g/L, activated carbon 0.5-3g/L and coconut 40-100ml of water.

The active ingredient of described step (d) emergence medium is to take MS medium as basic medium, and the potassium dihydrogen phosphate in MS medium, anhydrous calcium chloride, manganese sulfate tetrahydrate, magnesium sulfate heptahydrate The content is adjusted to potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, manganese sulfate tetrahydrate 10-40mg/L, magnesium sulfate heptahydrate 400-600mg/L, and KT 0.1-1.0 mg/L, 6A30.5-3mg/L, IAA0.5-4mg/L, Phytagel 2-4g/L, sucrose 30-70g/L, activated carbon 0.5-3g/L and coconut water 40-100ml.

The beneficial effects of the present invention are:

1. The transformation receptor of the present invention is different from the previous reports. The embryoid body has obvious advantages as a transformation receptor: a. Materials are available all year round, which can meet the transformation needs at any time; b. Elimination of stripping stamens or induction and screening The time and labor needed to stably proliferate fragile embryogenic callus; c. Somatic embryos have strong regeneration ability, and each worker can produce 8,000 tissue cultured seedlings per year, which can fully meet the needs of scientific research and production; d. Regenerate plant roots Well-developed and vigorous growth; e. The transformation requires a small number of explants, which saves the time for transformation and recovery culture to pick up the callus, and greatly improves the transformation speed.

2. The present invention adopts a transformant multiplication method that is different from previous reports. It is to multiply the obtained resistant embryos to induce enough normal embryoids to emerge again, to ensure that each transgenic embryoid can emerge as much as possible, and to completely solve the problem of anthers. The embryoid body induction rate of the regeneration system is low, and the seedling emergence rate is low.

3. The roots, stems and leaves of plants regenerated by secondary somatic embryogenesis are very strong, which is completely different from the thin plants regenerated by long-term subcultured callus, and the RITA culture method with the strongest regeneration ability of long-term subcultured callus will A certain number of vitrified seedlings appeared, but the plants regenerated from cotyledon-shaped embryoids through secondary somatic embryogenesis completely avoided this phenomenon. The transgenic plants are strong, which greatly improves the survival rate of transplanting, thereby further improving the transformation rate.

4. The regenerated transgenic plant after secondary somatic embryogenesis has a root system developed by the radicle. This root system is the same as the seedling root system and has a well-developed main root. It helps to improve the resistance of Hevea brasiliensis after planting in the field. wind capacity. The roots of the transgenic plants obtained by micropropagation are regenerated roots, which can develop into similar taproots after planting in the field, but the connection between the taproot and the stem is weak, which is not conducive to the wind resistance of Hevea brasiliensis.

Detailed ways

Example 1:

The present invention will be further described below by taking the infection of Agrobacterium tumefaciens EHA105 as an example. It should be noted that the preparation of engineering bacteria and the molecular detection of transformed plants are the same as the previous methods.

(a) Acquisition of genetic transformation recipient-somatic embryoid bodies: select robust and vigorous embryoid bodies differentiated through anthers, inner integuments, tree root culture, secondary somatic embryogenesis or other somatic embryogenesis pathways, and It was isolated from the original medium as the transformed recipient. If the culture time is too long, vitrified, albino and brown embryoid bodies are not suitable as transformation recipients.

(b) Genetic transformation of somatic embryoid bodies: Infection: Adjust the OD value of Agrobacterium to 0.5, and use the embryoid bodies selected by the method (a) as recipients to infect for 5 minutes, and dry the bacteria solution with sterile filter paper ; Co-cultivation: the co-cultivation temperature is 25 ° C, the co-culture medium is adjusted to 5.2 for the pH value, and the callus induction medium with 5 mg/L silver nitrate is added; recovery culture: the embryoid bodies after transformation are transferred to 5 mg/L silver nitrate Callus induction medium (pH5.8) of silver nitrate and 500mg/L temindin (ticarcillin:potassium clavulanate=15:1), recovery culture in dark room at 25°C for 10 days; callus induction: recovery culture The embryoid body after cutting into the embryo piece of 0.2 * 0.2cm, transfers to then added 5mg/L silver nitrate, the callus induction medium (PH5.8 ), cultured in the dark at 25°C for 20 days. Differentiation: Transfer the resistant calli to the differentiation medium (PH5.8) supplemented with 500 mg/L terminin and 50 mg/L kanamycin, and culture in the dark at 25°C for 50 days to obtain mature embryoid bodies. The callus induction medium is based on MS medium, and the contents of potassium dihydrogen phosphate, anhydrous calcium chloride, manganese sulfate tetrahydrate, and magnesium sulfate heptahydrate in the MS medium are adjusted to diphosphate Potassium hydrogen 350mg/L, calcium chloride anhydrous 200mg/L, manganese sulfate tetrahydrate 20mg/L, magnesium sulfate heptahydrate 400mg/L, add 2,4-D 2mg/L, KT 1mg/L, NAA 1.5mg /L, Phytagel 2g/L, sucrose 80g/L, and coconut water 100ml; Calcium chloride, manganese sulfate tetrahydrate, magnesium sulfate heptahydrate content adjusted to potassium dihydrogen phosphate 350mg/L, calcium chloride anhydrous 200mg/L, manganese sulfate tetrahydrate 20mg/L, magnesium sulfate heptahydrate 400mg/L, And add 6-BA 0.5mg/L, KT 1.0mg/L, NAA 0.1mg/L, GA32mg/L, Phytagel 2.2g/L, sucrose 50g/L, activated carbon 1.0g/L, coconut water 40ml.

(c) Proliferation of transformed embryoid body: Cut the positive embryoid body of GUS staining into 0.2 × 0.2 cm embryo blocks, repeat the callus induction and differentiation process described in (b), but remove the medium used in the callus induction process silver nitrate. This process can be repeated until the transformants obtain enough mature cotyledon-shaped embryoid bodies. After 1-3 cycles, the antibacterial antibiotics can be removed.

(d) Plant regeneration of transformed embryoid bodies: transfer the mature cotyledon-shaped embryoid bodies to the emergence medium (PH5.8), culture under light at 25° C., and seedlings emerge in 20-60 days. The effective components of the emergence medium are MS medium as the basic medium, and the content of potassium dihydrogen phosphate, anhydrous calcium chloride, manganese sulfate tetrahydrate, and magnesium sulfate heptahydrate in the MS medium is adjusted to dihydrogen phosphate Potassium 350mg/L, anhydrous calcium chloride 200mg/L, manganese sulfate tetrahydrate 20mg/L, magnesium sulfate heptahydrate 400mg/L, and add KT 0.5mg/L, GA32mg/L, IAA 0.5mg/L, Phytagel 2g /L, sucrose 50g/L, activated carbon 0.5g/L, coconut water 100ml.

(e) Transplanting of transformed plants: (1) Budding: use regenerated transformed plants as scions, seedlings or tissue cultured seedlings as rootstocks, and realize transplantation from the laboratory to the field through a series of processes of budding, unbinding, and bagging. Planting; (2) sand bed transplanting: wash the culture medium of the regenerated transformed plants and move them into the sand bed, control the temperature, humidity, and diseases, and gradually lift the shed after half a month, and the transformed plants will sprout new shoots and grow new roots in one month , and bagging after the leaves grow to a stable period.

Example 2:

The present invention will be further described below by taking the infection of another Agrobacterium tumefaciens LBA4404 as an example. Similarly, the preparation of engineering bacteria and the molecular detection of transformed plants are no different from the previous methods.

(a) Acquisition of genetic transformation recipient-somatic embryoid bodies: select robust and vigorous embryoid bodies differentiated through anthers, inner integuments, tree root culture, secondary somatic embryogenesis or other somatic embryogenesis pathways, and It was isolated from the original medium as the transformed recipient. If the culture time is too long, vitrified, albino and brown embryoid bodies are not suitable as transformation recipients.

(b) Genetic transformation of somatic embryoid bodies: Infection: adjust the OD value of Agrobacterium to 0.3, and use the embryoid bodies selected by the method (a) as recipients to infect for 5 minutes, and dry the bacteria solution with sterile filter paper ; Co-cultivation: the co-cultivation temperature is 20 ° C, the co-culture medium is adjusted to a pH value of 5.2, and the callus induction medium is added with 10 mg/L silver nitrate; recovery culture: the embryoid body after transformation is transferred to the culture medium added with 10 mg/L silver nitrate Callus induction medium (pH5.8) of silver nitrate and 300mg/L Temindin (ticarcillin: potassium clavulanate = 15:1), recovery culture in darkroom at 25°C for 5 days; callus induction: recovery culture After the embryoid body is cut into 0.2 * 0.2cm embryo block, then transfers to the callus induction medium (PH5.8) that has added 5mg/L silver nitrate, 300mg/L teminding and 5mg/L hygromycin , cultured in the dark at 25°C for 20 days. Differentiation: transfer the resistant callus to the differentiation medium (pH5.8) supplemented with 300 mg/L temindin and 5 mg/L hygromycin, and culture in the dark at 25°C for 60 days to obtain mature embryoid bodies. The callus induction medium is based on MS medium, and the contents of potassium dihydrogen phosphate, anhydrous calcium chloride, manganese sulfate tetrahydrate, and magnesium sulfate heptahydrate in the MS medium are adjusted to diphosphate Potassium hydrogen 500mg/L, anhydrous calcium chloride 400mg/L, manganese sulfate tetrahydrate 20mg/L, magnesium sulfate heptahydrate 400mg/L, and add 2,4-D 1mg/L, KT1mg/L, NAA 1mg/L , Phytagel 2g/L, sucrose 60g/L, coconut water 40ml; The active ingredient of described differentiation medium is to take MS medium as basic medium, and potassium dihydrogen phosphate in MS medium, anhydrous chlorinated The contents of calcium, manganese sulfate tetrahydrate and magnesium sulfate heptahydrate are adjusted to potassium dihydrogen phosphate 500mg/L, calcium chloride anhydrous 400mg/L, manganese sulfate tetrahydrate 20mg/L, magnesium sulfate heptahydrate 400mg/L, and add 6-BA1.0mg/L, KT 1.0mg/L, NAA 0.2mg/L, GA3 3mg/L, Phytagel 2.2g/L, sucrose 70g/L, activated carbon 1.0g/L, coconut water 40ml.

(c) Proliferation of transformed embryoid body: Cut the positive embryoid body of GUS staining into 0.2 × 0.2 cm embryo blocks, repeat the callus induction and differentiation process described in (b), but remove the medium used in the callus induction process silver nitrate. This process can be repeated until the transformants obtain enough mature cotyledon-shaped embryoid bodies. After 1-3 cycles, the antibacterial antibiotics can be removed.

(d) Plant regeneration of transformed embryoid bodies: transfer the mature cotyledon-shaped embryoid bodies to the emergence medium (PH5.8), culture under light at 25° C., and seedlings emerge in 20-60 days. The active ingredients of the emergence medium are MS medium as the basic medium, and the content of potassium dihydrogen phosphate, anhydrous calcium chloride, manganese sulfate tetrahydrate, and magnesium sulfate heptahydrate in the MS medium is adjusted to dihydrogen phosphate Potassium 500mg/L, anhydrous calcium chloride 400mg/L, manganese sulfate tetrahydrate 20mg/L, magnesium sulfate heptahydrate 400mg/L, and add KT 0.7mg/L, GA3 2.5mg/L, IAA 0.3mg/L , Phytagel 2.2g/L, sucrose 50g/L, activated carbon 0.5g/L, coconut water 50ml.

(e) Transplanting of transformed plants: (1) Budding: use regenerated transformed plants as scions, seedlings or tissue cultured seedlings as rootstocks, and realize transplantation from the laboratory to the field through a series of processes of budding, unbinding, and bagging. Planting; (2) sand bed transplanting: wash the culture medium of the regenerated transformed plants and move them into the sand bed, control the temperature, humidity, and diseases, and gradually lift the shed after half a month, and the transformed plants will sprout new shoots and grow new roots in one month , and bagging after the leaves grow to a stable period.

Claims (4)

1. bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo is characterized in that: comprise the acquisition of genetic transformation acceptor-somatic cell embryoid, the genetic transformation of somatic cell embryoid, the propagation that transforms embryoid, the plant regeneration that transforms embryoid and five steps of transplanting of transformed plant:

(a) acquisition of genetic transformation acceptor-somatic cell embryoid: select stalwartness, embryoid that vitality is vigorous, it is separated as transformation receptor from original medium;

(b) genetic transformation of somatic cell embryoid: its process comprises and infects, cultivates altogether, recovers cultivation, callus of induce and atomization;

Infect: OD ₆₀₀=0.1-0.6 Agrobacterium is infected 3-10min, puts aseptic filter paper and blots bacterium liquid;

Cultivate altogether: the embryoid after infecting changes on the callus of induce medium that adds the 1-50mg/L silver nitrate, and its pH value is 5.2, cultivates altogether 3-6 days for 20-28 ℃;

Recover to cultivate: the embryoid after cultivating altogether changes over to and adds the 1-50mg/L silver nitrate and suppress Agrobacterium and grow on the antibiotic callus of induce medium, and its pH value is 5.8,23-28 ℃ of dark the cultivation 3-10 days;

Callus of induce: the embryoid after will recovering to cultivate is cut into the embryo piece of 0.2-0.5 * 0.2-0.5cm, change over to then and added the 1-50mg/L silver nitrate, on the antibiotic of inhibition Agrobacterium growth and the callus of induce medium of corresponding selective agent, its pH value is 5.8,23-28 ℃ of dark the cultivation 15-40 days;

Differentiation: change the embryo piece of callusization over to the antibiotic of interpolation inhibition Agrobacterium growth and the differential medium of corresponding selective agent, its pH value is 5.8,23-28 ℃ of dark the cultivation 30-70 days;

(c) transform the propagation of embryoid: the embryoid of GUS stained positive is cut into the embryo piece of 0.2-0.5 * 0.2-0.5cm, the described callus of induce of repeating step (b), atomization, but remove the silver nitrate of the used medium of callus of induce; This process is capable of circulation, obtains enough ripe cotyledon shape embryoids until transformant; After circulation 1-3 time, antibacterial antibiotic can be removed;

(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the medium of emerging, its pH value is 5.8, and 23-28 ℃ of illumination cultivation emerged in 20-60 days;

(e) transplanting of transformed plant: (1) bud grafting: as scion, seedling or tissue cultivating seedling be as stock with the transformed plant of regeneration, by bud grafting, separate and tie up, pack a series of processes and realize transplanting from the laboratory to the land for growing field crops; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned medium, control temperature, humidity, disease are lifted canopy gradually behind the two weeks, and transformed plant was extracted sprouting out, grown new root in one month, treated to pack after blade grows to stationary phase.

2. a kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo according to claim 1 is characterized in that: the employed genetic transformation acceptor of described step (a) is the embryoid that acquisition takes place by the secondary body embryo of flower pesticide, inner integument and tree root explant and body embryo thereof.

3. a kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo according to claim 1, it is characterized in that: the callus of induce medium of described step (b) is to be basal medium with the MS medium, and with the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, the content furnishing of epsom salt is potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L, and add 2,4-D 0.5-3mg/L, KT 0.5-3mg/L, NAA 0.5-3mg/L, Phytagel 2-3g/L, sucrose 50-90g/L and Sucus Cocois 40-100ml; The active ingredient of described differential medium is for being minimal medium with the MS medium, and the content of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt is adjusted into potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L, and add 6-BA 0.5-3mg/L, KT 0.5-3mg/L, NAA 0.1-2mg/L, GA31-5mg/L, Phytagel 2-3g/L, sucrose 50-90g/L, activated carbon 0.5-3g/L and Sucus Cocois 40-100ml.

4. a kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo according to claim 1, it is characterized in that: described step (d) is emerged the active ingredient of medium for being minimal medium with the MS medium, and with the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, the content of epsom salt is adjusted into potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L, and add KT 0.1-1.0mg/L, GA3 0.5-3mg/L, IAA 0.5-4mg/L, Phytagel 2-4g/L, sucrose 30-70g/L, activated carbon 0.5-3g/L and Sucus Cocois 40-100ml.