CN113711914A

CN113711914A - Caragana microphylla in-vitro regeneration method taking cotyledonary node as explant

Info

Publication number: CN113711914A
Application number: CN202110982317.XA
Authority: CN
Inventors: 李国婧; 朱木兰; 刘槟; 齐力旺; 王瑞刚; 郑珂媛
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-11-30
Anticipated expiration: 2041-08-25
Also published as: CN113711914B

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 korshinskii (Caragana korshinski Kom) belongs to Caragana deciduous shrubs in the family of 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 keeping water and soil, but also has economic values of firewood, feed, pulping and board making, medicine and the like (Wangyang middle Caragana oleic acid dehydrogenase gene (CaFAD2) cloning and function identification [ D ]. China forestry scientific 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 environment, has strong stress resistance (Niuxiang noon. Caragana microphylla biological characteristic research [ J ]. North China agricultural science, 1998, 13 (4): 122-.

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. Dahurian rose and the like use Caragana microphylla epicotyl and hypocotyl as explants to induce adventitious buds to obtain regenerated plants (Dahurian rose, plum nature, relationship between organ differentiation and endogenous phytohormone content in the process of in vitro culture of several wild species of the genus Caragana Fabr of the family Leguminosae [ J ]. university of inner Mongolia (Nature science edition), 1990(03): 127-; cizhong Ling et al induced callus with caragana microphylla cotyledon and hypocotyl as explants, which differentiated embryogenic cell mass but did not differentiate adventitious shoots (Cizhong Ling. Caragana caragana callus culture and differentiation [ J ]. Proc. inner Mongolia Lin academy 1998,20(12): 15-19); the method comprises the following steps of (1) performing direct adventitious bud generation by taking caragana korshinskii aseptic seedling cotyledons and hypocotyls as explants by Songjunshuang and the like, successfully inducing adventitious buds, but failing to successfully induce adventitious buds through a callus approach (Songjunshuang, Wangzang, Sunzui Zhi, and the like, tissue culture of caragana korshinskii [ J ] Tu, 2007,15(1): 66-66); plum light path and the like use caragana korshinskii twigs as explants to induce adventitious buds (plum light path, white talent, Zhangzhi, Lijun. caragana tissue culture research [ J ] Gansu agricultural science and technology, 2010(11): 18-20.); shaoyingling uses caragana microphylla stem as explant to initially establish a set of rapid propagation (Shaoyingling. caragana microphylla tissue culture technique [ J ] agricultural system science and comprehensive research (4): 469-.

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. Cleaning the seeds with detergent for 3min outside a super clean bench in a disinfection mode, wrapping the seeds with 75% recovered alcohol cotton to wipe the seeds, passing the seeds in the super clean bench with 75% recovered alcohol for one time, disinfecting the seeds with 75% alcohol for 2min, finally disinfecting the seeds with 10% sodium hypochlorite for 20min, cleaning the seeds with sterile water for 5-6 times, placing the disinfected seeds on sterile filter paper, and sucking water for later use;

(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 of 1.0-4.0mg/L, NAA, and culturing under light for 7-25d, preferably 10-20d, and most preferably 14-16 d; 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 0.1-1mg/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 and is 0.03-0.1mg/L, the clump buds are found to have obvious extension tendency after being cultured for 25-30d, and preferably, the original culture medium is continuously cultured for 20-25d without transferring subculture, so that a large amount of extended bud seedlings 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 0.5-1mg/L, and the culture time under the light is 30-40 d.

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 ℃, the illumination intensity is 2000-3000lx, and the illumination time is 12 h/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 taken 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: 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 1 cm.

FIG. 6: adventitious bud induction was initiated at bar 1 cm.

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

FIG. 8: adventitious buds were cultured by elongation at bar of 1 cm.

FIG. 9: adventitious buds were cultured by elongation at bar 2 cm.

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

FIG. 11: rooting adventitious buds at bar 2 cm.

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

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 full-grain caragana microphylla seeds without insect eyes and black spots, cleaning the caragana microphylla seeds for 3min by using a detergent, wrapping and wiping the caragana microphylla seeds with 75% recovered alcohol cotton, passing the recycled alcohol cotton once in an ultraclean bench, sterilizing the caragana microphylla seeds for 2min by using clean 75% alcohol, finally sterilizing the caragana microphylla seeds for 20min by using 10% sodium hypochlorite, cleaning the caragana microphylla seeds for 5-6 times by using sterile water, absorbing the moisture on the surface of the material by using sterile filter paper, inoculating the material into an MS basal medium, and culturing the material 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 ℃, the illumination intensity is 2000-.

(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: preliminary experiments were performed on three basal media, MS, B5 and White, and the results showed that caragana microphylla leaf nodes had 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 cotyledonary nodes have different growth states in different plant hormone combinations. Based on the natural study of Gaohai and Li, the exogenous cytokinin concentration required in the Caragana tissue culture is much higher than that of auxin (Gaohai, Li's natural. several wild species of the family Leguminosae, Caragana Fabr.) plants have a relationship between organ differentiation and endogenous phytohormone content during the in vitro culture [ J ]. university of inner Mongolia (Nature science edition), 1990(03): 127-. The caragana microphylla leaf node is taken as an explant, MS is taken as a basic medium, cytokinin (4mg/L) and auxin (0.4mg/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 the medium supplemented with 6-BA +2,4-D, the explants expanded and the bases callized, 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). Thus, the combinations of cytokinin (6-BA) and auxin (NAA) employed in the present invention were used for further studies 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, 4mg/L) and NAA (0, 0.1, 0.2, 0.3mg/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 5.8; the culture conditions are 25 +/-2 ℃, the illumination intensity is 2000-. 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 the caragana microphylla leaf node is inoculated to the caragana microphylla leaf node without adding 6-BA and NAA, the explant does not respond, 1-2 small buds are induced occasionally, and experimental observation shows that the bud seedlings do not grow in the later stage and become yellow 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 1mg/L) and NAA (0, 0.03, 0.07 and 0.1mg/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 ℃, the illumination intensity is 2000-. 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 a 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 obviously extend, when the concentration of 6-BA is 0.7mg/L and the level of NAA is 0.07mg/L, the influence on the elongation rate of the adventitious bud is most obvious, the elongation rate 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 0.07 mg/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 was selected for rooting induction. In order to determine the effect of the basal medium on the sprouts, it was previously found that the sprouts were cultured on 1/2MS medium, the aerial part of which did not grow due to lack of nutrients, while the aerial part of the plants could grow normally on the MS medium, so MS was selected as the basal medium. NAA (0.5, 1.0, 2.0mg/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 ℃, the illumination intensity is 2000-.

The results show that: the NAA with a certain concentration can promote the growth of adventitious buds to root, the concentration of the NAA is increased within a certain range to improve the rooting rate of the adventitious buds to a certain extent, when the concentration of the NAA is higher, the base part of a stem segment firstly forms callus, the root is directly induced from the callus, and the plant is not easy to survive by using the rooting seedling. And determining that 1mg/L NAA is the best hormone for adventitious bud rooting induction culture. 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 before transplanting to completely wet the substrate, 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

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 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, disinfecting and inoculating the caragana microphylla seeds to an MS culture medium;

(2) adventitious bud induction culture: selecting the sterile seedlings cultured for 10-20 days in the step (1), performing plane cutting at a position 2mm below a 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 of 1.0-4.0mg/L, NAA, and culturing under light for 7-25d, preferably 10-20d, and most preferably 14-16 d; preferably, after the adventitious bud of the plant is induced and started, subculturing for 1-3 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 adding 0.1-1mg/L of 6-BA and 0.01-0.10mg/L of NAA; preferably, the addition amount of BA is 0.3-1.0mg/L, NAA and is 0.03-0.1mg/L, and the clumpy buds have obvious extension tendency after being cultured for 25-30 days; preferably, the transfer subculture is not needed, and the original culture medium is continuously cultured for 20-25d to obtain a large amount of elongated sprouts;

(4) rooting culture: dividing the cluster buds elongated by the subculture in the step (3) into single plants, inoculating the single plants into a rooting culture 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 0.5-1mg/L, and the culture time under the light is 30-40 d.

2. The method according to claim 1, wherein the medium used in (1) to (4) is supplemented with sucrose (30 g/L), inositol (0.1 g/L), agar powder (5.5 g/L), and the pH of the medium is adjusted to 5.8; the culture conditions are 20-25 ℃, the illumination intensity is 2000-3000lx, and the illumination time is 12 h/d.

3. The method according to claim 1, wherein the sterilization mode is specifically operated as follows: cleaning with a detergent for 3min, wrapping and wiping seeds with 75% recovered alcohol cotton, putting the seeds into an ultra-clean bench, passing the seeds with 75% recovered alcohol for one time, disinfecting with 75% alcohol for 2min, finally disinfecting with 10% sodium hypochlorite for 20min, cleaning with sterile water for 5-6 times, placing the disinfected seeds on sterile filter paper, and sucking water for later use.

4. 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).

5. The method of claim 4, 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.

6. The method of claim 4, further comprising the step of (6) 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.