阅读说明：本技术 一种建立金钗石斛遗传转化体系的方法 (Method for establishing dendrobium nobile genetic transformation system ) 是由 韩福贵 傅力 顾永华 杨如同 郑玉红 杨军 于 2019-04-15 设计创作，主要内容包括：本发明提供了一种建立金钗石斛遗传转化体系的方法,涉及遗传转化技术领域。本发明所述建立方法,包括以下步骤：将金钗石斛无菌苗接种到诱导培养基上,得愈伤组织；将所述愈伤组织接种于继代培养基上,得继代愈伤组织；将目的基因转入所述继代愈伤组织中,得侵染愈伤组织；将所述侵染愈伤组织接种于1/2MS的平板培养基上；将经过所述共培养的侵染愈伤组织接种于分化培养基上,得丛生芽；将所述丛生芽接种于壮芽培养基上,得壮芽；将所述壮芽接种于生根和壮苗共培养基上,得转基因金钗石斛。本发明所述体系基于组培快繁体系建立,为利用分子生物学手段对金钗石斛观赏性状和药用特性进行遗传改良奠定了技术基础。(The invention provides a method for establishing a dendrobium nobile genetic transformation system, and relates to the technical field of genetic transformation. The establishing method comprises the following steps: inoculating the dendrobium nobile sterile seedlings to an induction culture medium to obtain callus; inoculating the callus on a subculture medium to obtain a subculture callus; transferring a target gene into the subculture callus to obtain an infected callus; inoculating the infected callus on a plate culture medium of 1/2 MS; inoculating the infected callus subjected to co-culture on a differentiation medium to obtain cluster buds; inoculating the cluster buds to a strong bud culture medium to obtain strong buds; and inoculating the strong bud on a rooting and strong seedling co-culture medium to obtain the transgenic dendrobium nobile. The system is established based on a tissue culture and rapid propagation system, and lays a technical foundation for carrying out genetic improvement on the ornamental character and the medicinal characteristic of the dendrobium nobile by utilizing a molecular biological means.)

1. A transgenic method of dendrobium nobile lindl comprises the following steps: (1) inoculating the dendrobium nobile sterile seedlings to an induction culture medium for induction culture to obtain callus; the induction culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 25-35 g/L of sucrose, 6-9 g/L of agar, 1.3-1.5 mg/L of 6-BA and 0.15-0.25mg/L of NAA;

(2) inoculating the callus obtained in the step 1) on a subculture medium for subculture to obtain a subculture callus; the subculture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 24-35 g/L of sucrose, 6-9 g/L of agar, 0.8-1.2 mg/L of 6-BA and 0.05-0.15 mg/L of NAA;

(3) transferring a target gene into the subculture callus obtained in the step 2) to obtain an infected callus;

(4) inoculating the infection callus obtained in the step 3) on a solid culture medium of 1/2MS for co-culture;

(5) co-cultured in step 4)Inoculating the infected callus on a differentiation culture medium for differentiation culture to obtain cluster buds; the differentiation medium takes 1/2MS culture medium as a basic culture medium, and also comprises: 24-35 g/L of sucrose, 6-9 g/L of agar, 0.8-1.2 mg/L, NAA 0.05.05-0.15 mg/L of 6-BA, 0.8-1.5 mg/L of multi-walled carbon nano-tube and 100mg/L of antibiotic; the antibiotics comprise spectinomycin and timentin; the differential culture comprises dark culture and illumination culture which are sequentially carried out, wherein the dark culture time is 10-15 days; the illumination culture time is 20-25 d, and the illumination intensity of the illumination culture is 100-300 mu mol.m^-2·s^-1The illumination time of the illumination culture is 12-16 h/d;

(6) inoculating the cluster buds obtained in the step 5) on a bud-strengthening culture medium for bud-strengthening culture to obtain strong buds; the strong bud culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 35-45 g/L of sucrose, 6-9 g/L of agar, 0.3-0.8 mg/L of 6-BA, 0.1mg/L of NAA and 100mg/L of antibiotics; the antibiotics comprise spectinomycin and timentin;

(7) inoculating the strong bud on a rooting and strong seedling co-culture medium to carry out rooting and strong seedling co-culture to obtain the transgenic dendrobium nobile; the rooting and strong seedling co-culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 6-9 g/L of agar 35-45 g/L, 0.1-0.4 mg/L of 6-BA 1.5-3 mg/L, NAA 0.1 and 1.4-2.5 mg/L of IBA.

2. The transgenic method according to claim 1, wherein the temperature of the induction culture in step (1) is 25 ± 1 ℃, and the light intensity of the induction culture is 100-300 μmol-m^-2·s^-1The illumination time is 14-20 h/d.

3. The transgenic method according to claim 1, wherein the subculture in the step (2) is dark culture, and the temperature of the dark culture is 25 ± 1 ℃.

4. The method for transferring gene according to claim 1, wherein the step (3) of transferring the target gene is mediated by Agrobacterium.

5. The transgenic method according to claim 1, wherein the co-culture in the step (4) is dark culture, and the co-culture time is 65-80 h.

6. The transgenic method according to claim 1, wherein the temperature for culturing the strong bud of step (6) is 25 ± 1 ℃, and the light intensity for culturing the strong bud is 100-300 μmol-m^-2·s^-1The illumination time is 14-20 h/d.

7. The transgenic method according to claim 1, wherein the temperature for the co-culture of the rooting and the strong seedling in the step (7) is 25 +/-1 ℃, and the illumination intensity for the strong bud culture is 100-300 μmol-m^-2·s^-1The illumination time is 14-20 h/d.

8. The transgenic method according to claim 1, characterized in that after the transgenic dendrobium nobile is obtained in step (7), the identification of transgenic plants is further included.

9. The transgenic method of claim 8, wherein the identification comprises chromogenic identification.

10. The transgenic method according to claim 9, wherein the identification shows that yellow plants are differentiated to indicate that the target gene is successfully integrated into the genome of dendrobium nobile.

Technical Field

The invention belongs to the technical field of genetic transformation, and particularly relates to a method for establishing a dendrobium nobile genetic transformation system.

Background

Dendrobium nobile of the family Dendrobium, also known as the root of Hovenia acerba, Dendrobium nobile, Pinna, Panerochleaf cudweed, Panerochloe, etc., are perennial herbs and are famous for their ancient hairpin shapes. The stem of the dendrobium nobile is tufted, the upper part of the dendrobium nobile is slightly flat and slightly bent and ascended, the height of the dendrobium nobile is 10-60 cm, and the thickness of the dendrobium nobile is 1.4 cm. The raceme is emitted from the part above the middle part of the old stem, the flower is large, the white color is with a light purple tip, sometimes the whole body is light purple red or the rest is white except 1 purple red plaque on the labial disc; the flower type is peculiar, the gray is elegant, and the plant is an excellent ornamental plant species.

Dendrobium nobile is one of the certified products of Dendrobium nobile in 2015 edition of Chinese pharmacopoeia. Cold in nature, bitter, bland, bitter in taste; enter stomach and kidney meridians. Dendrobium nobile is first reported in Shen nong Ben Cao Jing as a medicinal herb, and has a history of more than 2000 years to date. About 1700 years ago, the medical record of dendrobium is recorded in famous medical records published by the Gregorian era of 220-450, and the application history is very long.

However, the application of dendrobium nobile germplasm resources in China is still limited to introduction, domestication and cultivation of wild resources whether the dendrobium nobile germplasm resources are used as ornamental plants or medicinal plants. The improvement of ornamental characters or medicinal components and contents thereof is still in the exploration stage, and particularly, the genetic improvement of the ornamental characters or medicinal components by using a molecular biological means is not reported.

Disclosure of Invention

In view of the above, the invention aims to provide a method for establishing a dendrobium nobile genetic transformation system, which establishes a technical foundation for carrying out genetic improvement on the ornamental character and medicinal characteristic of dendrobium nobile by utilizing a molecular biological means based on the establishment of a regeneration system.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a transgenic method of dendrobium nobile lindl, which comprises the following steps: (1) inoculating the dendrobium nobile sterile seedlings to an induction culture medium for induction culture to obtain callus; the induction culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 25-35 g/L of sucrose, 6-9 g/L of agar, 1.3-1.5 mg/L of 6-BA and 0.15-0.25mg/L of NAA;

(2) inoculating the callus obtained in the step 1) on a subculture medium for subculture to obtain a subculture callus; the subculture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 24-35 g/L of sucrose, 6-9 g/L of agar, 0.8-1.2 mg/L of 6-BA and 0.05-0.15 mg/L of NAA;

(3) transferring a target gene into the subculture callus obtained in the step 2) to obtain an infected callus;

(4) inoculating the infection callus obtained in the step 3) on a solid culture medium of 1/2MS for co-culture;

(5) inoculating the infection callus co-cultured in the step 4) on a differentiation medium for differentiation culture to obtain cluster buds; the differentiation medium takes 1/2MS culture medium as a basic culture medium, and also comprises: 24-35 g/L of sucrose, 6-9 g/L of agar, 0.8-1.2 mg/L, NAA 0.05.05-0.15 mg/L of 6-BA, 0.8-1.5 mg/L of multi-walled carbon nano-tube and 100mg/L of antibiotic; the antibiotics comprise spectinomycin and timentin; the differential culture comprises dark culture and illumination culture which are sequentially carried out, wherein the dark culture time is 10-15 days; the illumination culture time is 20-25 d, and the illumination intensity of the illumination culture is 100-300 mu mol.m^-2·s^-1The illumination time of the illumination culture is 12-16 h/d;

(6) inoculating the cluster buds obtained in the step 5) on a bud-strengthening culture medium for bud-strengthening culture to obtain strong buds; the strong bud culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 35-45 g/L of sucrose, 6-9 g/L of agar, 0.3-0.8 mg/L of 6-BA, 0.1mg/L of NAA and 100mg/L of antibiotics; the antibiotics comprise spectinomycin and timentin;

(7) inoculating the strong bud on a rooting and strong seedling co-culture medium to carry out rooting and strong seedling co-culture to obtain the transgenic dendrobium nobile; the rooting and strong seedling co-culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 6-9 g/L of agar 35-45 g/L, 0.1-0.4 mg/L of 6-BA 1.5-3 mg/L, NAA 0.1 and 1.4-2.5 mg/L of IBA.

Preferably, the culture temperature of the induction culture in step (1) is 25 + -1 deg.CThe light intensity of the induction culture is 100-300 [ mu ] mol.m^-2·s^-1The illumination time is 14-20 h/d.

Preferably, the subculture in the step (2) is dark culture, and the temperature of the dark culture is 25 +/-1 ℃.

Preferably, the target gene transfer in step (3) adopts an agrobacterium-mediated method.

Preferably, the co-culture in the step (4) is dark culture, and the co-culture time is 65-80 h.

Preferably, the culture temperature of the strong bud culture in the step (6) is 25 +/-1 ℃, and the illumination intensity of the strong bud culture is 100-300 mu mol.m^-2·s^-1The illumination time is 14-20 h/d.

Preferably, the culture temperature of the rooting and strong seedling co-culture in the step (7) is 25 +/-1 ℃, and the illumination intensity of the strong bud culture is 100-300 mu mol.m^-2·s^-1The illumination time is 14-20 h/d.

Preferably, after the transgenic dendrobium nobile is obtained in the step (7), the identification of transgenic plants is also included.

Preferably, said identification is an autonomous color method.

Preferably, when the dyeing identification is carried out, the bud differentiated from the callus develops into a yellow differentiated plant, which indicates that the target gene is successfully integrated into the plant genome.

The invention provides a transgenic method of dendrobium nobile lindl, which comprises the following steps: (1) inoculating the dendrobium nobile sterile seedlings to an induction culture medium for induction culture to obtain callus; (2) inoculating the callus on a subculture medium for subculture to obtain a subculture callus; (3) transferring GUS gene into the subculture callus to obtain infection callus; (4) inoculating the infection callus on a plate culture medium of 1/2MS for co-culture; (5) inoculating the infection callus subjected to co-culture on a differentiation medium for differentiation culture to obtain cluster buds; (6) inoculating the cluster buds on a bud-strengthening culture medium to carry out bud-strengthening culture to obtain strong buds; (7) and inoculating the strong bud on a rooting and strong seedling co-culture medium to carry out rooting and strong seedling co-culture, thereby obtaining the transgenic dendrobium nobile. The establishment method is based on the regeneration system of the dendrobium nobile lindl, establishes the dendrobium nobile lindl genetic transformation system, and lays a technical foundation for genetic improvement of the dendrobium nobile lindl from the molecular biology level.

Detailed Description

The invention provides a transgenic method of dendrobium nobile lindl, which comprises the following steps: (1) inoculating the dendrobium nobile sterile seedlings to an induction culture medium for induction culture to obtain callus; the induction culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 25-35 g/L of sucrose, 6-9 g/L of agar, 1.3-1.5 mg/L of 6-BA and 0.15-0.25mg/L of NAA;

(2) inoculating the callus obtained in the step 1) on a subculture medium for subculture to obtain a subculture callus; the subculture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 24-35 g/L of sucrose, 6-9 g/L of agar, 0.8-1.2 mg/L of 6-BA and 0.05-0.15 mg/L of NAA;

(3) transferring a target gene into the subculture callus obtained in the step 2) to obtain an infected callus;

(4) inoculating the infection callus obtained in the step 3) on a solid culture medium of 1/2MS for co-culture;

(5) inoculating the infection callus co-cultured in the step 4) on a differentiation medium for differentiation culture to obtain cluster buds; the differentiation medium takes 1/2MS culture medium as a basic culture medium, and also comprises: 24-35 g/L of sucrose, 6-9 g/L of agar, 0.8-1.2 mg/L, NAA 0.05.05-0.15 mg/L of 6-BA, 0.8-1.5 mg/L of multi-walled carbon nano-tube and 100mg/L of antibiotic; the antibiotics comprise spectinomycin and timentin; the differential culture comprises dark culture and illumination culture which are sequentially carried out, wherein the dark culture time is 10-15 days; the illumination culture time is 20-25 d, and the illumination intensity of the illumination culture is 100-300 mu mol.m^-2·s^-1The illumination time of the illumination culture is 12-16 h/d;

(6) inoculating the cluster buds obtained in the step 5) on a bud-strengthening culture medium for bud-strengthening culture to obtain strong buds; the strong bud culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 35-45 g/L of sucrose, 6-9 g/L of agar, 0.3-0.8 mg/L of 6-BA, 0.1mg/L of NAA and 100mg/L of antibiotics; the antibiotics comprise spectinomycin and timentin;

(7) inoculating the strong bud on a rooting and strong seedling co-culture medium to carry out rooting and strong seedling co-culture to obtain the transgenic dendrobium nobile; the rooting and strong seedling co-culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 6-9 g/L of agar 35-45 g/L, 0.1-0.4 mg/L of 6-BA 1.5-3 mg/L, NAA 0.1 and 1.4-2.5 mg/L of IBA.

In the establishing method, the dendrobium nobile sterile seedlings are inoculated to an induction culture medium for induction culture to obtain callus; the induction culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 25-35 g/L of sucrose, 6-9 g/L of agar, 1.3-1.5 mg/L of 6-BA and 0.15-0.25mg/L of NAA. The source of the dendrobium nobile sterile seedlings is not specially limited, and the dendrobium nobile sterile seedlings can be obtained by utilizing the conventional commercially available sterile seedlings in the field. The induction culture medium comprises sucrose, and the concentration of the sucrose is preferably 25-34 g/L, more preferably 28-32 g/L, and most preferably 30 g/L.

The induction culture medium comprises agar, and the concentration of the agar is preferably 6.5-8.5 g/L, more preferably 7-8 g/L, and most preferably 7.5 g/L.

The induction culture medium comprises 6-BA, and the concentration of the 6-BA is 1.3-1.5 mg/L, preferably 1.4 mg/L. The 6-BA can play a role in inducing the formation of the callus.

The induction culture medium comprises NAA, and the concentration of the NAA is 0.15-0.25mg/L, preferably 0.2 mg/L. The NAA can play a role in promoting the proliferation of the callus. In the present invention, the source of each component in the induction medium is not particularly limited, and a conventional reagent in the art may be used.

The dendrobium nobile lindl aseptic seedlings are subjected to induction culture on the induction culture medium, and the temperature of the induction culture is preferably 25 +/-1 ℃. The illumination intensity of the induction culture is preferably 100-300 mu mol.m^-2·s^-1More preferably 200 to 280. mu. mol/m^-2·s^-1Most preferably 250. mu. mol. m^-2·s^-1. The illumination time of the induction culture is preferably 14-20 h/d, more preferably 15-18 h/d, and most preferably 16 h/d. In the invention, after 3 weeks of induction culture by the induction culture medium, green protrusions appear around the rhizoid, which are the germination of the callus, and the green protrusions are seen to expand to form green callus after 4 weeks.

After obtaining the callus, inoculating the callus on a subculture medium for subculture to obtain a subculture callus; the subculture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 24-35 g/L of sucrose, 6-9 g/L of agar, 0.8-1.2 mg/L of 6-BA and 0.05-0.15 mg/L of NAA. The subculture medium comprises sucrose, and the concentration of the sucrose is preferably 25-34 g/L, more preferably 28-32 g/L, and most preferably 30 g/L.

The subculture medium comprises agar, and the concentration of the agar is preferably 6.5-8.5 g/L, more preferably 7-8 g/L, and most preferably 7.5 g/L.

The subculture medium comprises 6-BA, and the concentration of the 6-BA is preferably 0.8-1.2 mg/L, and more preferably 1.0 mg/L. The 6-BA can promote the differentiation of the callus.

The subculture medium comprises NAA, and the concentration of the NAA is preferably 0.06-0.13 mg/L, more preferably 0.08-0.12 mg/L, and most preferably 0.1 mg/L. The NAA can play a role in promoting callus differentiation. In the present invention, the source of each component in the subculture medium is not particularly limited, and a conventional reagent in the art may be used. The invention carries out subculture on the subculture medium, wherein the subculture is preferably dark culture, and the temperature of the dark culture is preferably 25 +/-1 ℃.

In the process of subculture, the method preferably further comprises the step of performing spectinomycin resistance screening on the dendrobium nobile callus, and preferably comprises the following steps: inoculating the dendrobium nobile callus into the subculture medium added with spectinomycin with different concentrations, wherein the inoculation amount of each concentration is not less than 10 callus, counting the survival rate of the callus every 5 days, and stopping counting after 30 days. The concentrations of spectinomycin in the invention are respectively 50, 100, 150, 200 and 250 mg/L. The results show that: 100mg/L is the spectinomycin lethal concentration of the dendrobium nobile callus.

After the subculture callus is obtained, the target gene is transferred into the subculture callus to obtain the infection callus. The method for transferring the yellow gene is not particularly limited, and an agrobacterium-mediated method is preferably adopted, and specifically comprises the following steps: constructing a vector and transforming agrobacterium; the source of the target gene is not particularly limited in the present invention.

The method for constructing the vector and transforming the agrobacterium of the present invention is not particularly limited, and preferably comprises:

the vector construction preferably comprises:

(1) connecting: mu.L (20ng/L) of the target gene, 23011. mu.L of the cloning vector pCAMBIA, a total volume of 4. mu.L, and reaction at 25 ℃ for 15 min.

(2) And (3) transformation: taking escherichia coli DH5 alpha stored at-70 ℃, melting on ice, adding 4 mu L of ligation product when the escherichia coli DH5 alpha is in a semi-melting state, flicking and uniformly mixing, carrying out ice bath for 20-30 min, carrying out heat shock for 30s at 42 ℃, immediately placing on ice for 2min, adding 250 mu L of nonresistant LB liquid medium into an ultra-clean workbench, and carrying out shake culture at 200rpm and 37 ℃ for 1 h.

(3) Coating a plate: centrifuging, removing supernatant, collecting 100 μ L of bacterial liquid, uniformly spreading on a 50mg/L Km resistant plate, and culturing at 37 deg.C for 8-12 h.

(4) And (3) positive clone detection: and (3) selecting a white single colony, inoculating the white single colony in an LB liquid culture medium containing 50mg/LKm, shaking and culturing at 250rpm and 37 ℃ for 4h, detecting a positive clone by PCR (polymerase chain reaction), and sequencing and verifying.

The transformation of said agrobacterium preferably comprises:

(1) taking agrobacterium tumefaciens competent cells stored at the temperature of minus 80 ℃, placing the agrobacterium tumefaciens competent cells on a palm piece, allowing the palm piece to be partially melted, and inserting the palm piece into ice when the palm piece is in an ice-water mixed state;

(2) adding 0.01-1.0 μ g plasmid into 100 μ L competent cells, tapping the tube bottom with finger, mixing, and standing in ice, liquid nitrogen, 37 deg.C water bath and ice bath for 5 min;

(3) adding a sterile LB liquid culture medium, and performing shake culture at 28 ℃ for 2-3 h;

(4) centrifuging at 6000rpm for 1min to collect bacteria, taking 50 mu L of supernatant, slightly blowing and beating the heavy suspension bacteria block, coating the heavy suspension bacteria block on an LB (lysogeny broth) plate containing 50mg/L spectinomycin, and inversely placing the heavy suspension bacteria block on a shaking table at 28 ℃ for culturing for 2-3 d.

(5) And (3) positive clone detection:

white single colonies are picked and inoculated in LB liquid culture medium containing 50mg/L spectinomycin, the culture is carried out for 4h at 250rpm and 37 ℃, PCR detection positive clones are carried out by using primers, and sequencing verification is carried out.

The infection is preferably carried out by putting the dendrobium nobile callus into 1/2MS liquid culture medium containing transformed agrobacterium tumefaciens, and the infection time is preferably 20-30 min, more preferably 22-28 min, and most preferably 25 min. The infection process of the invention is preferably accompanied by shaking, which is beneficial to the invasion of the transformed agrobacterium into the callus.

After the infection callus is obtained, the infection callus is inoculated on a plate culture medium of 1/2MS for co-culture. The present invention preferably further comprises an infection solution adsorbing the surface of the infected callus before the inoculation, and the method of the present invention for the adsorption is not particularly limited, and is preferably performed by using sterile filter paper. The infected callus after adsorbing the infection liquid is inoculated on a plate containing 1/2MS culture medium for co-culture, the co-culture is preferably dark culture, the dark culture time is preferably 3d, and the dark culture temperature is preferably 25 +/-1 ℃.

After the co-culture is finished, inoculating the infection callus subjected to the co-culture on a differentiation medium for differentiation culture to obtain cluster buds; the differentiation medium takes 1/2MS culture medium as a basic culture medium, and also comprises: 24-35 g/L of sucrose, 6-9 g/L of agar, 0.8-1.2 mg/L, NAA 0.05.05-0.15 mg/L of 6-BA, 0.8-1.5 mg/L of multi-walled carbon nano-tube and 100mg/L of antibiotic; the antibiotics comprise spectinomycin and timentin; the differential culture comprises dark culture and illumination culture which are sequentially carried out, wherein the dark culture time is 10-15 days; the light cultureThe culturing time is 20-25 d, and the illumination intensity of the illumination culture is 100-300 mu mol.m^-2·s^-1And the illumination time of the illumination culture is 12-16 h/d. The differentiation medium comprises sucrose, and the concentration of the sucrose is preferably 25-34 g/L, more preferably 28-32 g/L, and most preferably 30 g/L.

The differentiation medium comprises agar, and the concentration of the agar is preferably 6.5-8.5 g/L, more preferably 7-8 g/L, and most preferably 7.5 g/L.

The differentiation medium comprises 6-BA, and the concentration of the 6-BA is preferably 0.9-1.1 mg/L, and more preferably 1.0 mg/L. The 6-BA can play a role in promoting callus differentiation.

The differentiation medium comprises NAA, and the concentration of the NAA is preferably 0.06-0.14 mg/L, more preferably 0.08-0.12 mg/L, and most preferably 0.1 mg/L. The NAA can play a role in promoting callus differentiation.

The differentiation medium comprises multi-walled carbon nanotubes, and the concentration of the multi-walled carbon nanotubes is preferably 0.85-1.4 mg/L, more preferably 0.9-1.2 mg/L, and most preferably 1 mg/L. The multi-walled carbon nano-tube can promote the absorption of water and nutrients so as to promote the differentiation of cells, and simultaneously can inhibit the growth and the propagation of agrobacterium tumefaciens. In the present invention, the sources of the components in the differentiation medium are not particularly limited, and conventional reagents in the art may be used.

The differential culture is carried out on the differential culture medium, the differential culture comprises dark culture and illumination culture which are carried out in sequence, the dark culture time is 10-15 d, and the temperature of the dark culture is preferably 25 +/-1 ℃; according to the invention, after the callus begins to differentiate, the callus is transferred to illumination culture, and the illumination culture time is preferably 20-25 d. The illumination intensity of the illumination culture is preferably 100-300 mu mol.m^-2·s^-1More preferably 200 to 280. mu. mol/m^-2·s^-1Most preferably 250. mu. mol. m^-2·s^-1. The illumination time of the illumination culture is preferably 12-16 h/d, and more preferably 16 h/d. The present invention can be subcultured every 20 days after the dark culture. The invention can achieve the effect of promoting callus differentiation by first darkening and then shining.

After cluster buds are obtained, the cluster buds are inoculated on a bud-strengthening culture medium for bud-strengthening culture to obtain strong buds; the strong bud culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 35-45 g/L of sucrose, 6-9 g/L of agar, 0.3-0.8 mg/L of 6-BA, 0.1mg/L of NAA and 100mg/L of antibiotics; the antibiotics include spectinomycin and timentin. The strong bud culture medium comprises sucrose, and the concentration of the sucrose is preferably 36-44 g/L, more preferably 38-42 g/L, and most preferably 40 g/L.

The strong bud culture medium comprises agar, and the concentration of the agar is preferably 6.5-8.5 g/L, more preferably 7-8 g/L, and most preferably 7.5 g/L.

The bud-strengthening culture medium comprises 6-BA, and the concentration of the 6-BA is preferably 0.4-0.6 mg/L, and more preferably 0.5 mg/L. The 6-BA can play a role in the growth of seedlings.

The bud-strengthening culture medium comprises NAA, and the concentration of the NAA is preferably 0.06-0.14 mg/L, more preferably 0.08-0.12 mg/L, and most preferably 0.1 mg/L. The NAA can play a role in promoting the growth of seedlings.

The bud-strengthening culture medium also comprises antibiotics, and the sources of the components in the bud-strengthening culture medium are not particularly limited and can be obtained by using conventional reagents in the field.

The invention carries out strong bud culture in the strong bud culture medium, and the culture temperature of the culture is preferably 25 +/-1 ℃. The illumination intensity of the strong bud culture is preferably 100-300 mu mol.m^-2·s^-1More preferably 200 to 280. mu. mol/m^-2·s^-1Most preferably 250. mu. mol. m^-2·s^-1. The illumination time for the bud-strengthening culture is preferably 14-20 h/d, more preferably 15-18 h/d, and most preferably 16 h/d. The time for culturing the strong buds is preferably 25-40 d, more preferably 28-35 d, and most preferably 30 d.

After the strong buds are obtained, the strong buds are inoculated on a rooting and strong seedling co-culture medium to carry out rooting and strong seedling co-culture, and the transgenic dendrobium nobile is obtained; the rooting and strong seedling co-culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 35-45 g/L of sucrose, 6-9 g/L of agar, 1.5-3 mg/L, NAA 0.1.1-0.4 mg/L of 6-BA and 1.4-2.5 mg/L of IBA. The rooting and strong seedling co-culture medium comprises sucrose, and the concentration of the sucrose is preferably 36-44 g/L, more preferably 38-42 g/L, and most preferably 40 g/L.

The rooting and strong seedling co-culture medium comprises agar, and the concentration of the agar is preferably 6.5-8.5 g/L, more preferably 7-8 g/L, and most preferably 7.5 g/L.

The rooting and strong seedling co-culture medium comprises 6-BA, and the concentration of the 6-BA is preferably 1.6-2.8 mg/L, more preferably 1.8-2.5 mg/L, and most preferably 2 mg/L. The 6-BA can play a role in promoting the growth of seedlings.

The rooting and strong seedling co-culture medium comprises NAA, and the concentration of the NAA is preferably 0.15-0.3 mg/L, more preferably 0.18-0.25 mg/L, and most preferably 0.2 mg/L. The NAA can play a role in promoting the growth of seedlings.

The rooting and strong seedling co-culture medium comprises IBA, and the concentration of the IBA is preferably 1.5-2.4 mg/L, more preferably 1.8-2.2 mg/L, and most preferably 2 mg/L. The IBA can play a role in promoting the rooting of seedlings. The sources of the components in the rooting and strong seedling co-culture medium are not particularly limited, and conventional reagents in the field can be utilized.

The invention utilizes the rooting and strong seedling co-culture medium to carry out rooting and strong seedling co-culture, and the culture temperature of the rooting and strong seedling co-culture is preferably 25 +/-1 ℃. The illumination intensity of the rooting and strong seedling co-culture is preferably 100-300 mu mol.m^-2·s^-1More preferably 200 to 280. mu. mol/m^-2·s^-1Most preferably 250. mu. mol. m^-2·s^-1. The illumination time of the rooting and strong seedling co-culture is preferably 14-20 h/d, more preferably 15-18 h/d, and most preferably 16 h/d. The invention is used for rooting and strengthening seedlingsThe culture time is preferably 25-40 days, more preferably 28-35 days, and most preferably 30 days. In the invention, after rooting and strong seedling co-culture is carried out for 30 days, test-tube seedlings with stem diameter of 1-2 mm and 3-5 root systems can grow.

After the transgenic dendrobium nobile is obtained, the identification of transgenic plants is preferably included, and the identification preferably includes a self-color development method. The identification of the invention shows that the bud differentiated from the callus develops into a yellow plant, which indicates that the target gene is successfully integrated into the plant genome, and the plant is a transgenic plant. The method for sequencing and identifying is not particularly limited in the present invention, and the identification can be performed by using a conventional test method in the art.

The following will explain the establishment method of the dendrobium nobile genetic transformation system provided by the present invention in detail with reference to the examples, but they should not be construed as limiting the scope of the present invention.