CN113711914B - Caragana microphylla in-vitro regeneration method taking cotyledonary node as explant - Google Patents

Abstract

The invention discloses a caragana microphylla efficient regeneration method taking cotyledonary nodes as explants. The method has the advantages that the cotyledonary node is used as the initial explant, a rapid and effective regeneration system is established through a direct organogenesis way, the induction and differentiation of callus are not needed, the culture process is simplified, the caragana microphylla regeneration plant can be obtained in a short time, the phenotype consistency of the plant of the tissue culture seedling cultured by the method is high, and the excellent characteristics of a parent body are stably maintained. By establishing an in vitro regeneration system taking cotyledonary nodes as an initial explant, a powerful technical guarantee is provided for the expansion and development of a caragana microphylla rapid propagation system and a genetic transformation system, and further transgenic breeding work represented by caragana microphylla can be smoothly carried out.

Description

Caragana microphylla in-vitro regeneration method taking cotyledonary node as explant

Technical Field

The invention relates to the technical field of plant regeneration and rapid propagation, in particular to a caragana microphylla regeneration method.

Background

Caragana korshinskiii Kom belongs to Caragana deciduous shrubs in leguminosae, is widely distributed in arid and semiarid regions in China, is an important ecological economic shrub, not only has the ecological effects of preventing wind and fixing sand and maintaining water and soil, but also has economic values of firewood, feed, pulping and plate making, medicine and the like (Wang Yangdong. Cloning and function identification of middle Caragana oleic dehydrogenase gene (CaFAD 2) [ D ]. Chinese forest industry science research institute, 2007 ]. The plant grows in the environment resisting the adverse circumstances for a long time, forms a survival mechanism adapting to the severe circumstances, has strong stress resistance (Niu Xiwu. Caragana microphylla biological characteristic research [ J ]. North China agro-chemical newspaper 1998, 13 (4): 122-129), and becomes a star species for researching the stress resistance mechanism.

At present, no relevant research reports about caragana korshinskii tissue culture are seen abroad, although some researches about caragana korshinskii and sibling plant tissue culture have been made at home, a stable and efficient regeneration system is lacked. Gao Mei, etc. uses Caragana microphylla epicotyl and hypocotyl as explants to induce adventitious buds to obtain regenerated plants (Gaohua, li Tianran. Relationship between organ differentiation and endogenous phytohormone content during isolated culture of several wild family Caragana Fabr plants [ J ]. University of inner Mongolia (Nature science edition), 1990 (03): 127-137+ 162); cizhong Ling et al induced callus with caragana microphylla cotyledon and hypocotyl as explant, but did not differentiate into adventitious buds (Cizhong Ling. Caragana caragana callus culture and differentiation [ J ]. Proc. Inner Mongolia Ling academy of academic, 1998,20 (12): 15-19); song Junshuang et al, using caragana microphylla aseptic seedling cotyledon and hypocotyl as explants, were subjected to direct adventitious bud induction, successfully inducing adventitious buds, but failed to induce adventitious buds via callus pathway (Song Junshuang, wang Zan, sun Guizhi, et al, tissue culture of caragana microphylla [ J ]. Mediterrata, 2007,15 (1): 66-66); li Guangdao et al, using caragana microphylla twig as explant to induce adventitious bud (Li Guangdao, bai Shengcai, zhang Xiuzhi, li Jun. Caragana microphylla tissue culture research [ J ] Gansu agroltechnology 2010 (11): 18-20.); 3238 Zxft 3238 uses caragana korshinskii stem as explant to initially establish a set of fast breeding bodies (3262 Zxft 3262. Caragana korshinskii tissue culture technique [ J ]. Agricultural system science and comprehensive research (4): 469-474).

In general, caragana microphylla ex vivo regeneration is still in the trial stage, and no effective regeneration system is found, which is a problem to be overcome all the time. Therefore, establishing a rapid, efficient and stable regeneration platform is very important for caragana microphylla to carry out transgenic breeding work.

Disclosure of Invention

In order to solve the bottleneck problem, the inventor of the invention overcomes the defects in the prior art, develops the research on the in vitro generation of caragana microphylla leaf node, and finally obtains the in vitro regeneration system of caragana microphylla leaf node.

The invention provides a caragana microphylla in vitro regeneration method taking cotyledonary nodes as explants, which comprises the following steps:

(1) Preparation of sterile seedlings: inoculating sterilized caragana korshinskii seeds into MS culture medium for culture. Preferably, the specific operation of the step (1): selecting caragana microphylla seeds with full grains, no insect eyes and no black spots, sterilizing and inoculating to MS culture medium. Wherein, the disinfection mode is that the cleaning liquid is used for cleaning for 3min outside a super clean bench, 75% recovered alcohol cotton is wrapped to wipe seeds, the seeds enter the super clean bench and are disinfected for 2min by 75% recovered alcohol, and are finally disinfected by 10% sodium hypochlorite for 20min, and are cleaned for 5-6 times by sterile water, and the disinfected seeds are placed on sterile filter paper and are dried for standby;

(2) Adventitious bud induction culture

There are two ways for adventitious bud induction, the first is to induce adventitious bud through callus, the time consumption is long, and it is possible to generate mutation; the second is that the explant directly induces adventitious buds without a callus induction way, the multiplication of the way is fast, the seedling formation is fast, and the excellent characteristics of the parent can be stably maintained, so the way is taken as the main way for caragana microphylla seedling breeding.

Selecting the sterile seedlings cultured for 10-20 days in the step (1), performing plane cutting at a position 2mm below the cotyledon joint by using a sterile blade, and removing a hypocotyl area and a true leaf part to obtain a cotyledon node explant. Inoculating the cotyledon node to an adventitious bud induction culture medium which is an MS basal culture medium, adding 0.5-6.0mg/L of 6-Benzylaminopurine (BA) and 0.05-0.60mg/L of naphthylacetic acid (NAA), preferably adding 0.10-0.40mg/L of BA into 1.0-4.0 mg/L, NAA, and culturing under light for 7-25d, preferably 10-20d, and most preferably 14-16d; preferably, after the adventitious bud induction of the plant is started, subculture is carried out for about 2 times.

(3) Adventitious bud elongation culture

Inducing cluster buds to be elongated in the step (2), inoculating the cluster buds to an adventitious bud elongation induction culture medium, wherein the adventitious bud elongation culture medium takes the MS culture medium in the step (2) as a basic culture medium, and 0.1-1 mg/L of 6-BA and 0.01-0.10mg/L of NAA are added; preferably, the addition amount of BA is 0.3-1.0mg/L, NAA is 0.03-0.1mg/L, and the multiple buds are found to have obvious extension tendency after being cultured for 25-30 days, and preferably, the original culture medium is continuously cultured for 20-25 days without transferring subculture, so that a large number of extended buds can be obtained.

Further, the method also comprises the following steps, namely (4) rooting culture: dividing the cluster buds subjected to subculture elongation in the step (3) into individual plants, inoculating the individual plants into a rooting medium, and performing rooting induction. Preferably, the rooting medium MS culture medium is used as a basic culture medium, 0.1-1.5mg/L of NAA (N-acetylneuraminidase) is added, preferably 0.5-1 mg/L, and the culture time under the light is 30-40d.

Preferably, sucrose 30g/L, inositol 0.1g/L and agar powder 5.5g/L are added to the culture medium used in (1) to (4), and the pH value of the culture medium is adjusted to 5.8. The culture conditions are 20-25 deg.C, illumination intensity is 2000-3000lx, and illumination time is 12h/d.

Further, the method also comprises the following steps of (5) hardening off the seedlings: and (4) selecting bottle seedlings with strong growth for hardening seedlings from the rooted regenerated seedlings obtained in the step (4). The seedling exercising method comprises the following steps: opening the culture bottle cap, injecting 0.5-1cm clear water, standing at room temperature under natural illumination for 2-3 days, taking out bottle seedling, cleaning culture medium attached to the base, and preparing for transplanting.

Still further, the method also comprises the following steps, namely (7) transplanting: transplanting the regenerated seedlings after hardening, preferably, the transplanting matrix is peat soil: perlite: vermiculite =3:1:1, soaking the matrix in water to completely wet the matrix before transplanting, bagging and moisturizing after transplanting, spraying the surface of the plant by using a spray can when the humidity is insufficient, removing the bag after the seedling grows sturdy, and maintaining normally.

Based on the current situation of caragana korshinskii tissue culture, researches show that the cotyledonary node of caragana korshinskii is used as an explant, and a high-efficiency and stable regeneration system is finally and successfully researched through a direct organ generation way by selecting a culture medium, selecting different hormone combinations and using the hormone combinations with different concentrations and proportions. Particularly, cotyledonary nodes are used as explants, adventitious bud in vitro regeneration is directly carried out without a callus approach, and no report is found on caragana or other species of caragana. The method of the invention can cultivate the tissue culture seedling plants with high phenotype consistency and stably keeping the excellent characteristics of the parent. By establishing an in vitro regeneration system taking cotyledonary nodes as an initial explant, a powerful technical guarantee is provided for the expansion and development of a caragana rapid propagation system and a genetic transformation system, and further the caragana regeneration and agrobacterium-mediated transformation research can be applied to smoothly develop transgenic breeding work represented by caragana.

Drawings

FIG. 1: different explants induced an adventitious bud formation state.

FIG. 2 is a schematic diagram: the hypocotyl region induces an adventitious bud formation state.

FIG. 3: explants were grown in different media.

FIG. 4: different hormone combinations induce growth states on adventitious buds.

FIG. 5: sterile explant material, bar =1cm.

FIG. 6: adventitious bud induction initiation, bar =1cm.

FIG. 7: adventitious bud induction culture, bar =1cm.

FIG. 8: adventitious buds were grown and cultured with bar =1cm.

FIG. 9: adventitious buds were grown and cultured with bar =2cm.

FIG. 10: and (5) carrying out subculture on old piles, wherein the bar =2cm.

FIG. 11: rooting of adventitious buds, bar =2cm.

FIG. 12: and 4 weeks after transplanting the regeneration seedlings into the soil, wherein the bar =2cm.

Detailed Description

The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.

Example one

(1) Preparation of explant Material

Selecting caragana microphylla seeds with full grains, no insect eyes and no black spots, cleaning for 3min by using detergent, wrapping and wiping seed coats by using 75% recovered alcohol cotton, then operating in an ultra-clean bench, filtering by using recovered alcohol, sterilizing for 2min by using clean 75% alcohol, finally sterilizing for 20min by using 10% sodium hypochlorite, cleaning for 5-6 times by using sterile water, sucking the moisture on the surface of the material by using sterile filter paper, inoculating the material into an MS (Mass spectrometer) basic medium, and culturing for about 20d to obtain the sterile material with dark green cotyledons. Wherein, 30g/L of sucrose, 0.1g/L of inositol and 5.5g/L of agar powder are added into the MS culture medium, and the pH value of the culture medium is adjusted to 5.8; the culture conditions are 25 + -2 deg.C, illumination intensity is 2000-3000lx, and illumination is 14h/d.

(2) And (4) performing adventitious bud induction culture.

a. Selection of explants pre-experiment: the selection of the explant is very important for the in vitro rapid propagation of the plant, and when the in vitro culture is carried out, the hypocotyl, cotyledon, root, stem, leaf and the like of the seedling are frequently cut and taken as the explant to carry out the in vitro culture. The inventor finds out in earlier researches that the hypocotyl has stronger differentiation capability and can successfully induce adventitious buds (A in figure 1); while the roots and leaves induced callus, but differentiation of adventitious buds at the later stage was difficult (B and C in FIG. 1); the stem segment can induce adventitious buds, but the number of adventitious buds is small (D in FIG. 1); cotyledon response status was less obvious and there was little change (E in fig. 1), so the embryonic axis was selected as recipient material for caragana regeneration and further genetic transformation. Because the embryonic axis has the differentiation capability, the two parts of the embryonic axis, namely the cotyledonary node area and the hypocotyl area, are further distinguished through further analysis, and are respectively subjected to preliminary experiments to study the performance of the embryonic axis in regeneration. The results of preliminary experiments show that adventitious buds cannot be induced from hypocotyls (hypocotyl regions), only callus is formed, the adventitious buds cannot be successfully differentiated in later culture (figure 2), and a regeneration system of the hypocotyls needs further research; and the cotyledonary node as explant can produce adventitious bud well. Therefore, the use of further differentiated cotyledonary nodes is expected to increase the efficiency of induction and regeneration relative to the direct use of the hypocotyl as explant. Therefore, the invention adopts the cotyledonary node as the initial explant to carry out further research and exploration, and establishes a high-efficiency in vitro regeneration system with the cotyledonary node as the initial explant.

b. Selection of basal medium preliminary experiment: the results of preliminary experiments on three basic media, namely MS, B5 and White media, show that the caragana microphylla cotyledons node has different growth states on the same hormone concentration and different media, as shown in FIG. 3. As the culture time increases, the growth state of the adventitious buds induced on the MS culture medium is the best (shown in the left picture of figure 3), the explants in the B5 and WPM culture media have the phenomenon of browning and death, wherein the explants have certain activity on the B5 culture medium (shown in the middle picture of figure 3), and the adventitious buds mostly become yellow seedlings on the WPM culture medium (shown in the right picture of figure 3). Therefore, the MS culture medium is selected as a basic culture medium for in vitro regeneration of caragana microphylla leaf segments.

c. Preliminary experiments for hormone combination selection

The results of preliminary experiments show that the caragana microphylla leaf nodes have different growth states in different phytohormone combinations. Based on the research of Gaoya, li Tianran, the concentration of exogenous cytokinin required in the Caragana tissue culture is much greater than that of auxin (Gaoya, li Tianran. The relationship between organ differentiation and endogenous phytohormone content in the process of in vitro culture of several wild family Caragana Fabr plants [ J ]. University of inner Mongolia proceedings (Nature science edition), 1990 (03): 127-137+ 162), so the combination of high-concentration cytokinin and low-concentration is adopted for preliminary experiments. The caragana microphylla leaf node is taken as an explant, MS is taken as a basic medium, cytokinin (4 mg/L) and auxin (0.4 mg/L) are added at the same concentration, and as a result, the following results are found: in the combination of 6-BA + NAA, the explant can induce about 3 adventitious buds (A in figure 4); in medium supplemented with 6-BA +2,4-D, explants expanded, basal calluses, occasionally producing 1 shoot (B in FIG. 4); in the TDZ + NAA combination, the base of the explant generates a large amount of callus, the growth of adventitious buds is inhibited, seedlings turn yellow, and the base callus is browned (C in figure 4) along with the increase of culture time; in the case of the TDZ +2,4-D combination, the explants produced callus, which died upon further culture, and it was difficult to induce adventitious shoots (D in FIG. 4). Therefore, the combination of cytokinin (6-BA) and auxin (NAA) used in the present invention was used for further study of adventitious bud induction culture.

d. Adventitious bud Induction experiment

Selecting the sterile material obtained in the step (1), namely selecting the sterile material (shown in figure 2) with cotyledon to be opened, texture and dark green color, performing flat cutting at a position 2mm below the cotyledon joint by using a scalpel, and removing a hypocotyl region and a true leaf part to obtain the caragana microphylla leaf node explant.

The study of adventitious bud induction culture was performed by inoculating cotyledonary nodes in MS basal medium supplemented with different concentrations of treated 6-BA (0, 1, 2, 3, 4 mg/L) and NAA (0, 0.1, 0.2, 0.3 mg/L), 3 replicates per treatment, 10 explants per replicate. Wherein, 30g/L of sucrose, 0.1g/L of inositol and 5.5g/L of agar powder are added into the MS basal medium, and the pH value of the medium is adjusted to be 5.8; the culture conditions are 25 + -2 deg.C, illumination intensity is 2000-3000lx, and illumination is 14h/d. After 15 days of culture, the cotyledon node expanded and the growth vigor was good (fig. 6), and after 1 time of subculture, an adventitious bud cluster was formed (fig. 7), and the adventitious bud induction rate was counted (table 1).

The experimental results show that: as can be seen from the table 1, when caragana microphylla leaf nodes are inoculated to the medium without adding 6-BA and NAA, the explant has no response, occasionally induces 1-2 small buds, and experimental observation shows that the bud seedlings can not grow in the later stage and can be yellowed and withered; the optimal hormone ratio for inducing adventitious buds of cotyledonary nodes is 3 mg/L6-BA +0.3mg/L NAA, the adventitious bud induction rate is about 73%, when certain level of NAA is unchanged, the adventitious bud induction rate shows a trend that the adventitious bud induction rate increases and then decreases along with the increase of 6-BA concentration, when the 6-BA concentration is 3mg/L, the adventitious bud induction rate is the largest, and when the 6-BA concentration is 4mg/L, the induction rate begins to decrease, which indicates that the higher-concentration cytokinin can inhibit the adventitious buds. When 6-BA is not changed at a certain level, the adventitious bud induction rate does not show obvious change along with the change of the NAA concentration.

Based on the above results, 3mg/L of 6-BA +0.3mg/L of NAA is shown to be the optimum concentration most suitable for adventitious bud induction of cotyledon node.

In addition, experimental observations have shown that callus formation occurs at the base of explants as the time for explant culture increases. Through subculture, a part of callus is cut off during subculture, so that the explant has a new wound, and a small amount of callus increases the absorption surface in contact with a culture medium, so that nutrition can be better absorbed.

TABLE 1 Effect of different hormone combinations on explant Induction

(3) And (5) elongation culture of the adventitious bud.

Transferring the adventitious bud clusters obtained in the step (2) to an elongation culture medium. Based on the result of the adventitious bud induction experiment, the combination of the low-concentration 6-BA and the NAA hormone is found to induce a small number of adventitious buds, but the adventitious buds are well extended in the later period, and the bud seedling growth state is good. The adventitious bud elongation culture medium takes MS as a basic culture medium, and is added with two common phytohormones of 6-BA (0, 0.3, 0.7 and 1 mg/L) and NAA (0, 0.03, 0.07 and 0.1 mg/L) at low concentration for different concentration combinations, each treatment is carried out for 3 times, and each treatment is carried out for 6 explants, so as to carry out the study on the adventitious bud elongation. Adding 30g/L of sucrose, 0.1g/L of inositol and 5.5g/L of agar powder into an MS basal medium, and adjusting the pH value of the medium to 5.8; the culture conditions are 25 + -2 deg.C, illumination intensity is 2000-3000lx, and illumination is 14h/d. After 30 days of culture, the adventitious bud clusters tended to significantly elongate (FIG. 8), and after 20 days of continued culture, a large number of adventitious buds extended (FIG. 9), and the adventitious bud elongation was counted (Table 2).

The experimental results show that: the induced adventitious bud basically has no extension tendency on the bud seedling on a culture medium without adding any hormone, the plant is short, and the phenomenon of yellow and withered seedling can occur. However, under the condition of high concentration hormone proportion, the color of the plant is dark green, the phenomenon of water stain is caused, a large number of callus clusters are basically generated, under the condition of low concentration hormone proportion, each explant only has about 3 seedlings which are obviously elongated, when the concentration of 6-BA is 0.7mg/L and the NAA is 0.07mg/L, the influence on the elongation of the adventitious bud is most obvious, the elongation of the adventitious bud is 84%, and therefore, the optimal hormone combination for the elongation of the adventitious bud is 6-BA 0.7mg/L + NAA 0.07mg/L.

TABLE 2 Effect of different hormone combinations on adventitious bud elongation

Cutting bud seedlings with the extension being more than or equal to 3cm (figure 6), inoculating the bud seedlings to a rooting culture medium for inducing rooting, and transferring the residual bud seedlings with the adventitious bud clusters to subculture to obtain a large amount of adventitious bud seedlings (figure 10).

(4) Rooting culture

Dividing the bud seedling (figure 9) with the length of more than 3cm obtained in the step (3) into single plants, cutting one extending bud, inoculating the bud to a rooting culture medium containing NAA and IBA with certain concentration, and performing rooting induction. The result shows that the induced root side roots are less, the roots are more enlarged and the transplanting survival rate is lower on the IBA rooting culture medium. Therefore, NAA is selected for rooting induction. In order to determine the influence of the basic culture medium on the sprouts, the MS is selected as the basic culture medium in the preliminary experiment that the sprouts can not grow due to lack of nutrition in the overground part of the sprouts cultured on the 1/2MS culture medium, and the overground part of the plants can normally grow on the MS culture medium. NAA (0.5, 1.0, 2.0 mg/L) was added at different concentrations as shown in Table 3, thereby confirming the adventitious bud-inducing rooting formulation. Wherein the culture medium contains 30g/L of sucrose and 5.5g/L of agar powder, and the pH value of the culture medium is adjusted to 5.8; the culture conditions are 25 + -2 deg.C, illumination intensity is 2000-3000lx, and illumination is 14h/d.

The results show that: the NAA with a certain concentration can promote the growth of adventitious buds to root, the rooting rate of the adventitious buds can be improved to a certain extent by increasing the concentration of the NAA within a certain range, when the concentration of the NAA is higher, the callus is firstly formed at the base part of the stem section, the root is directly induced from the callus, and the plant is not easy to survive by using the rooting seedling to acclimatize. And determining that 1mg/L NAA is the best hormone for induction culture of adventitious bud rooting. The growth of shoots of about 3cm was observed after 15 days of cultivation, and FIG. 11 shows the growth state of adventitious shoot roots induced for 50 days.

TABLE 3 Effect of hormones on inducing adventitious bud rooting

(6) Hardening off and transplanting

And (5) hardening the rooted and strong bottle seedlings in the step (5), wherein the hardening method comprises the following steps: opening the culture bottle cap, injecting 0.5-1cm clear water, standing at room temperature under natural illumination for 2-3 days, taking out bottle seedling, cleaning culture medium attached to the base, and preparing for transplanting. The transplanting matrix is peat soil: perlite: vermiculite =3:1:1, soaking the substrate in water to completely wet the substrate before transplanting, bagging and moisturizing after transplanting, spraying the substrate on the surface of a plant by using a spray can when the humidity is insufficient, removing the bag after the seedling grows sturdy, and normally maintaining (figure 12). And after 1 month, the survival rate is counted and is over 97 percent.

Claims (9)

1. A caragana microphylla ex vivo regeneration method taking cotyledonary node as explant comprises the following steps:

(1) Preparation of sterile seedlings: inoculating the sterilized caragana korshinskii seeds into an MS basal culture medium, 30g/L of cane sugar, 0.1g/L of inositol and 5.5g/L of agar powder for culture;

(2) Adventitious bud induction culture: selecting the sterile seedlings cultured in the step (1) and 10-20d, performing flat cutting at a position 2mm below the cotyledon joint by using a sterile blade, and removing a hypocotyl area and a true leaf part to obtain a cotyledon node explant; inoculating cotyledon nodes to an adventitious bud induction culture medium, wherein the adventitious bud induction culture medium is MS basal culture medium, 30g/L of cane sugar, 0.1g/L of inositol, 5.5g/L of agar powder, 1.0-4.0 mg/L of BA, 0.10-0.30 mg/L of NAA, culturing under light for 7-25d, and performing subculture for 1-3 times after the induction of the adventitious bud is started;

(3) Adventitious bud elongation culture: inducing cluster buds to be elongated in the step (2), inoculating the cluster buds to an adventitious bud elongation induction culture medium, wherein the adventitious bud elongation induction culture medium is MS basal medium, 30g/L of sucrose, 0.1g/L of inositol, 5.5g/L of agar powder, 0.7-1.0 mg/L of BA, 0.03-0.1mg/L of NAA, and culturing 25-30d to find that the cluster buds have obvious elongation tendency; continuously culturing 20-25d in the original culture medium to obtain a large amount of elongated buds;

(4) Rooting culture: dividing the cluster buds subjected to subculture elongation in the step (3) into individual plants, inoculating the individual plants into a rooting medium, and performing rooting induction.

2. The method according to claim 1, wherein the medium used in (1) to (3) has a pH of 5.8; the culture conditions are 20-25 deg.C, illumination intensity is 2000-3000lx, and illumination time is 12h/d.

3. The method according to claim 1, wherein the sterilization mode is specifically operated as follows: cleaning with detergent for 3min, wrapping and wiping seeds with 75% recovered alcohol cotton, passing through 75% recovered alcohol after entering a super clean bench, sterilizing with 75% alcohol for 2min, sterilizing with 10% sodium hypochlorite for 20min, cleaning with sterile water for 5~6 times, placing the sterilized seeds on sterile filter paper, and sucking water for use.

4. The method of claim 1, wherein the specific operation of step (1): caragana korshinskii seeds with full grains, no insect eyes and no black spots are selected, sterilized and inoculated into MS basal culture medium, 30g/L of cane sugar, 0.1g/L of inositol, 5.5g/L of agar powder.

5. The method of claim 1, wherein the culturing time under light in step (2) is 10-20 d.

6. The method of claim 1, wherein in step (4), the rooting medium is MS medium + sucrose 30g/L + agar powder 5.5g/L + NAA 1-2mg/L, and is cultured under light at 30-40d.

7. The method of claim 1, further comprising the step of (5) exercising the seedling: and (5) selecting bottle seedlings with strong growth for hardening seedlings from the rooted regenerated seedlings obtained in the step (5).

8. The method of claim 7, wherein the seedling exercising method comprises: opening the culture bottle cap, injecting 0.5-1cm clear water, standing at room temperature under natural illumination for 2-3 days, taking out bottle seedling, cleaning culture medium attached to the base, and preparing for transplanting.

9. The method of claim 7, further comprising the step of (6) transplanting: transplanting the regenerated seedlings after hardening, wherein the transplanting matrix is peat soil: perlite: vermiculite =3:1:1, soaking the matrix in water to completely wet the matrix before transplanting, bagging and moisturizing after transplanting, spraying the surface of the plant by using a spray can when the humidity is insufficient, removing the bag after the seedling grows sturdy, and maintaining normally.

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