Allium fistulosum root callus induction and tissue culture regeneration method

Abstract

The invention discloses a green Chinese onion root callus induction and tissue culture regeneration method. The method comprises the following steps: (1) preparing aseptic seedlings: culturing to obtain the aseptic seedlings; 2) callus induction: inoculating the root system of the aseptic seedling to an induction culture medium; (3) differentiation of the callus: inoculating the callus onto a differential culture medium; 4) rooting of the regenerated seedlings: inoculating the callus with the adventitious buds to a rooting culture medium; 5) seedling hardening and transplanting: culturing the rapid propagation rooting seedlings under illumination, and transplanting after the root system is developed. The method has the beneficial effects that tissue culture and in-vitro regeneration research are carried out aiming at the root system of the green Chinese onion, and an efficient and stable green Chinese onion root system plant regeneration system is established through a series of research; the establishment of the system is of great significance to the subsequent research on the genetic transformation of the green Chinese onion by using the agrobacterium rhizogenes, and lays a foundation for the genetic improvement and molecular breeding of the green Chinese onion.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for inducing and regenerating root callus of green onion. Background Art

[0002] At present, onion breeding mainly relies on traditional breeding methods, which are accompanied by the disadvantages of long breeding cycles, slow breeding progress, and serious restrictions on improving the economic benefits of onion cultivation. With the development of biotechnology, the application potential of cell engineering technology in onion breeding is gradually being developed. Cell engineering technologies, such as tissue culture, gene editing, and molecular marker-assisted selection, can accelerate the breeding process, improve breeding efficiency, and help improve the agronomic traits of onions, such as yield, quality, disease resistance, and stress resistance.

[0003] Plant tissue culture technology can achieve efficient reproduction in a short time and keep the genetic characteristics of plants unchanged. It is an economical and effective method for germplasm preservation. Most plants can be rapidly propagated through tissue culture. Previous researchers have conducted tissue culture-related research on some Allium plants and made certain progress. For example, Tursunnai Dilixiati et al. used garlic bulbs as explants and found that the best induction effect was achieved with the medium of MS+NAA (0.5mg / L)+2,4-D (1.25mg / L). Mou Bin et al. used the stem tip of tillering onion bulbs as the experimental material. The results showed that MS+6-BA (0.4mg / L)+NAA (0.1mg / L) was the best hormone ratio for tillering onion induction. Gao Xingying explored the efficiency of different parts of the leek embryonic root tip as explants in callus induction differentiation and conducted a statistical analysis. The regeneration rate was the highest in the medium of MS+BA (1mg / L)+NAA (0.5mg / L)+KT (0.5mg / L). However, in vitro regeneration systems have been established only on a few Allium species, and different Allium species may have different selection of explants and optimal culture medium hormone ratios due to their different physiological characteristics and endogenous hormone levels. Therefore, more specific research is needed on the in vitro regeneration of green onions through tissue culture. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for inducing and regenerating root callus of green onion, which is achieved through the following technical solutions.

[0005] A method for inducing and regenerating root callus of green onion, comprising the following steps:

[0006] 1) Preparation of sterile seedlings:

[0007] Inoculating green onion seeds onto a sterile culture medium to obtain sterile seedlings;

[0008] 2) Callus induction:

[0009] Inoculate the root system of the sterile seedlings onto the induction medium to obtain callus tissue;

[0010] 3) Differentiation of callus:

[0011] Inoculating the callus onto a differentiation medium to allow the callus to differentiate into adventitious buds;

[0012] 4) Rooting of regenerated seedlings:

[0013] Inoculating the callus tissue that generates adventitious buds onto a rooting medium to allow it to take root and obtain fast-propagating rooted seedlings;

[0014] 5) Hardening and transplanting:

[0015] The fast-propagating rooting seedlings are cultured under light, and after the root system is well developed, they are taken out together with the rooting medium, and the rooting medium is rinsed with clean water and then transplanted to obtain complete plants.

[0016] Preferably, the step 1) includes the following sub-steps:

[0017] 11) Using 1 / 2MS as the basic medium, add 3.5 g / L agar, 15 g / L sucrose, pH 5.6-5.8, and sterilize at 121°C for 15 min to obtain a sterile medium;

[0018] 12) Selecting green onion seeds with full grains, soaking the green onion seeds in 10% sodium hypochlorite, and placing them in a magnetic stirrer at 200 rpm for 4 hours to break the seed coat surface;

[0019] 13) The green onion seeds with broken seed coats were soaked in alcohol for 3 minutes, then rinsed twice with sterile water, added with 100% sodium hypochlorite and soaked for 20 minutes, and finally rinsed six times with sterile water, and the moisture on the seed surface was removed with sterile filter paper;

[0020] 14) Inoculate the seeds onto a sterile culture medium in a 100 ml tissue culture bottle, with 20 seeds inoculated into one bottle. Incubate in the dark at 24-26°C until the seeds turn white, then light culture. Incubate under 3000-5000 Lx light for 7-10 days to obtain sterile seedlings.

[0021] Preferably, in the step 2), the root system of the sterile seedlings is divided into the upper part, the middle part and the lower part, and each part is cut into 1-2 cm; multiple induction culture media are set, and 10 portions of the root system of the upper, middle and lower sterile seedlings are inoculated on each induction culture medium respectively; the dark culture condition is maintained for 40 days, and the initial formation of light yellow callus is observed, and the dark culture is continued for 7 days, the growth state of the callus is observed, and the callus induction rate is calculated to determine the influence of different parts of the root system on the callus formation.

[0022] Preferably, in step 2), MS is used as the basic culture medium, and different ratios of 2,4-D, 6-BA and KT are added to prepare an induction medium, the root part of the sterile seedlings is inoculated on the induction medium, the callus induction rate is counted, and the optimal hormone ratio of the induction medium is determined.

[0023] Preferably, in the step 3), MS is used as the basic culture medium, and NAA, 6-BA and KT with different hormone ratios are added to prepare a differentiation medium. The callus tissue is inoculated on the differentiation medium and cultured at 25°C, a light intensity of 2000 lx, with 16 hours of light and 8 hours of darkness every 24 hours. The differentiation rate is counted after 30 days.

[0024] Preferably, in step 4), 1 / 2MS solid culture medium is used as the rooting medium.

[0025] The beneficial effect of the present invention is that tissue culture and in vitro regeneration research is carried out on the root system of green onions. Through this series of research, an efficient and stable green onion root plant regeneration system is established. The establishment of this system is of great significance for the subsequent research on genetic transformation of green onions using Agrobacterium rhizogenes, and it will lay the foundation for genetic improvement and molecular breeding of green onions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the description of the specific implementation methods will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 : Cultivation diagram of sterile green onion seedlings;

[0028] Figure 2 : The morphology of callus induced from different parts of the root system of sterile seedlings;

[0029] Figure 3 : The process of root regeneration of green onion. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] A method for inducing and regenerating root callus of green onion, comprising the following steps:

[0032] 1) Preparation of sterile seedlings:

[0033] The green onion seeds are inoculated into a sterile culture medium to cultivate sterile seedlings.

[0034] Step 1) includes the following sub-steps:

[0035] 11) Using 1 / 2MS as the basic medium, add 3.5 g / L agar, 15 g / L sucrose, pH 5.6-5.8, and sterilize at 121°C for 15 min to obtain a sterile medium;

[0036] 12) Selecting green onion seeds with full grains, soaking the green onion seeds in 10% sodium hypochlorite, and placing them in a magnetic stirrer at 200 rpm for 4 hours to break the seed coat surface;

[0037] 13) The green onion seeds with broken seed coats were soaked in alcohol for 3 minutes, then rinsed twice with sterile water, added with 100% sodium hypochlorite and soaked for 20 minutes, and finally rinsed six times with sterile water, and the moisture on the seed surface was removed with sterile filter paper;

[0038] 14) Inoculate the seeds onto a sterile culture medium in a 100 ml tissue culture bottle, with 20 seeds inoculated into one bottle. Incubate in the dark at 24-26°C until the seeds turn white, then light culture. Incubate under 3000-5000 Lx light for 7-10 days to obtain sterile seedlings.

[0039] like Figure 1 As shown, Figure 1 A in the middle is the seeds of sterile green onion seedlings. Figure 1 B in the middle is a sterile seedling cultured for 20 days.

[0040] 2) Callus induction:

[0041] The roots of sterile seedlings were inoculated onto induction medium to obtain callus tissue.

[0042] Figure 3 A: Different parts of the root system (upper, middle and lower) of sterile seedlings after 20 days of culture; B: Inoculation into induction medium; C: Callus formation after 47 days; D: Callus differentiation; E: Growth in rooting medium for 14 days; F: Complete plants grown in rooting medium; G: Plant transplantation.

[0043] In the step 2), the root system of the sterile seedlings was divided into the upper part, the middle part and the lower part, and each was cut into 1-2 cm; 10 roots of the upper part, the middle part and the lower part of the sterile seedlings were inoculated on the induction medium respectively; the dark culture condition was continued for 40 days, and the initial formation of light yellow callus was observed, and the dark culture was continued for 7 days, the growth state of the callus was observed, and the callus induction rate was calculated (Table 1) to determine the effect of different parts of the root system on callus formation.

[0044] Table 1 Callus formation efficiency in different parts of the root system

[0045]

[0046] Note: The cure rate is mean ± SD. Different letters after the data in the same column indicate significant differences (P<0.05).

[0047] As shown in Table 1, there are significant differences in callus induction rates in different parts of the root system, among which the induction rate in the lower part of the root system is the highest, reaching 39.58%, which is much higher than that in the upper and middle parts.

[0048] Figure 2 A in the middle shows the morphology of callus tissue induced in the lower part of the root system; Figure 2 Middle B shows the morphology of callus tissue induced in the upper and middle parts of the root system.

[0049] Depend on Figure 2 It can be seen that in the lower part of the root system, the induced callus tissue is mostly yellow, compact, granular, showing obvious embryonic characteristics and good growth condition ( Figure 2 A). In contrast, the callus induction rates in the upper and middle parts of the root system were lower, at 14.66% and 15.56%, respectively. These calli were light yellow, had loose structures, and lacked embryonic characteristics ( Figure 2 B). These results indicate that different root positions have significant effects on callus induction in explants, and the lower root system has an advantage in callus formation.

[0050] In order to explore the optimal hormone ratio for callus, MS was used as the basic culture medium, and different ratios of 2,4-D, 6-BA and KT were added to make the induction medium. The root part of the sterile seedlings was inoculated on the induction medium, and the callus induction rate was counted. The callus induction efficiency was statistically analyzed (Table 2).

[0051] Table 2 Callus formation efficiency of different hormone ratios

[0052]

[0053]

[0054] Note: The cure rate is mean ± SD. Different letters after the data in the same column indicate significant differences (P<0.05).

[0055] Analysis of Table 2 shows that, under the condition of single hormone addition, KT failed to induce callus formation, while the induction efficiency of 6-BA was relatively low. In contrast, 2,4-D showed a higher induction efficiency, and the induction rates of different concentrations of 2,4-D all reached more than 30%, and when the concentration was 1 mg / L, the callus induction rate was as high as 86.67%, and then the induction rate decreased with the increase of 2,4-D concentration. In the case of adding two hormones, when the concentration of 2,4-D was 1 mg / L, the callus induction rate decreased with the increase of 6-BA concentration, and the induction rate of 2,4-D (1 mg / L) + 6-BA (1 mg / L) reached 76.67%, which was higher than other concentration ratios of the two hormones. When all three hormones were added, the induction rate of 2,4-D (1.5 mg / L) + 6-BA (3.5 mg / L) + KT (0.5 mg / L) was 73.33%, and the induction rates of the other hormone ratios did not exceed 70%. Therefore, the most suitable hormone to be added to the callus induction medium in this experiment is 2,4-D, with a concentration of 1 mg / L.

[0056] 3) Differentiation of callus:

[0057] The callus tissue is inoculated onto differentiation medium to allow the callus tissue to differentiate into adventitious buds.

[0058] Using MS as the basic medium, NAA, 6-BA and KT with different hormone ratios were added to make differentiation medium. The callus was inoculated on the differentiation medium and cultured at 25°C, 2000lx light intensity, 16h of light and 8h of darkness every 24h. After 30d, adventitious buds were generated ( Figure 3 (D) , statistical differentiation rate, as shown in Table 3.

[0059] Table 3 Callus differentiation efficiency of different hormone ratios

[0060]

[0061]

[0062] Note: Differentiation rate is mean ± SD; Different letters after the data in the same column indicate significant differences (P<0.05).

[0063] As shown in Table 3, when one hormone is added, the differentiation rate of 6-BA is lower than that of KT. When two hormones are added, the differentiation efficiency is better (30%) when 6-BA (2.0 mg / L) + KT (1.0 mg / L) and 6-BA (0.5 mg / L) + KT (2.0 mg / L), which is higher than other hormone ratios. And after adding NAA, the differentiation efficiency increases to a certain extent, so these two groups of hormone concentrations are repeated on the basis of adding NAA. After repeated multiple times, it is found that the combination of adding NAA (0.1 mg / L) + 6-BA (2.0 mg / L) + KT (1.0 mg / L) is the best ratio to promote callus differentiation. The differentiation rate under this hormone ratio reaches 34.63%, showing a higher efficiency.

[0064] 4) Rooting of regenerated seedlings:

[0065] The callus tissue that produces adventitious buds is inoculated onto a rooting medium to allow it to take root and obtain rapidly propagated rooted seedlings.

[0066] 1 / 2MS solid medium was used as rooting medium.

[0067] The callus tissue that produces adventitious buds was inoculated onto the rooting medium, and 1 / 2MS solid medium was used as the rooting medium. After cultivation, roots began to form around the adventitious buds ( Figure 3 Afterwards, more roots gradually sprouted from the lower end of the adventitious buds in the area in contact with the culture medium, and these roots rapidly extended, with a rooting efficiency exceeding 90% ( Figure 3 Middle F).

[0068] 5) Hardening and transplanting:

[0069] The fast-propagating rooting seedlings are cultured under light, and after the root system is well developed, they are taken out together with the rooting medium, and the rooting medium is rinsed with clean water and then transplanted to obtain complete plants.

[0070] in conclusion:

[0071] The callus rates in different parts of the root system vary greatly. The callus rate in the lower part of the root system is as high as 39.58%, and the callus formation is fast and more uniform, while the callus induction rate in the middle and upper parts of the root system is only about 15%. Therefore, the lower part of the root system of sterile onion seedlings was selected for callus induction.

[0072] In the induction medium, 2,4-D showed a high induction efficiency. The induction rates of different concentrations of 2,4-D were all above 30%. When the concentration was 1 mg / L, the callus induction rate was as high as 86.67%. Then, as the concentration of 2,4-D increased, the induction rate decreased. Therefore, the most suitable hormone to be added to the callus induction medium in this experiment was 2,4-D at a concentration of 1 mg / L.

[0073] In the differentiation medium, NAA, as an auxin analog, can significantly accelerate the differentiation rate of callus and improve the differentiation rate, while 6-BA and KT, as cytokinins, mainly promote bud formation and cell division. Different hormone ratios will affect the differentiation direction and efficiency of callus. The present invention compares the culture medium with different concentration ratios of these three hormones, and the results show that the ratio of NAA (0.1 mg / L) + 6-BA (2.0 mg / L) + KT (1.0 mg / L) is most suitable for the differentiation of callus, and the differentiation rate is 34.63%.

[0074] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for inducing callus and tissue culture regeneration of green onion roots, characterized in that: The method comprises the following steps: 1) Preparation of sterile seedlings: Inoculating green onion seeds onto a sterile culture medium to obtain sterile seedlings; 2) Callus induction: Inoculate the root system of the sterile seedlings onto the induction medium to obtain callus tissue; 3) Differentiation of callus: Inoculating the callus onto a differentiation medium to allow the callus to differentiate into adventitious buds; 4) Rooting of regenerated seedlings: Inoculating the callus tissue that generates adventitious buds onto a rooting medium to allow it to take root and obtain fast-propagating rooted seedlings; 5) Hardening and transplanting: The rapidly propagated rooting seedlings are cultured under light, and after the root system is well developed, they are taken out together with the rooting medium, and the rooting medium is rinsed with clean water and then transplanted to obtain complete plants.

2. The method for inducing callus and regenerating green onion roots according to claim 1, characterized in that: The step 1) includes the following sub-steps: 11) Using 1 / 2MS as the basic medium, add 3.5 g / L agar, 15 g / L sucrose, pH 5.6-5.8, and sterilize at 121°C for 15 min to obtain a sterile medium; 12) Selecting green onion seeds with full grains, soaking the green onion seeds in 10% sodium hypochlorite, and placing them in a magnetic stirrer at 200 rpm for 4 hours to break the seed coat surface; 13) The green onion seeds with broken seed coats were soaked in alcohol for 3 minutes, then rinsed twice with sterile water, added with 100% sodium hypochlorite and soaked for 20 minutes, and finally rinsed six times with sterile water, and the moisture on the seed surface was removed with sterile filter paper; 14) Inoculate the seeds onto a sterile culture medium in a 100 ml tissue culture bottle, with 20 seeds inoculated into one bottle. Incubate in the dark at 24-26°C until the seeds turn white, then light culture. Incubate under 3000-5000 Lx light for 7-10 days to obtain sterile seedlings.

3. The method for inducing callus and regenerating green onion roots according to claim 1, characterized in that: In the step 2), the root system of the sterile seedlings is divided into the upper part, the middle part and the lower part, and each part is cut into 1-2 cm; multiple induction culture media are set, and 10 portions of the root system of the upper part, the middle part and the lower part of the sterile seedlings are inoculated on each induction culture medium; the dark culture condition is maintained for 40 days, and the initial formation of light yellow callus is observed, and the dark culture is continued for 7 days, the growth state of the callus is observed, and the callus induction rate is calculated to determine the influence of different parts of the root system on the callus formation.

4. A method for inducing callus and tissue culture regeneration of green onion roots according to claim 3, characterized in that: In the step 2), MS is used as the basic culture medium, and different ratios of 2,4-D, 6-BA and KT are added to prepare an induction medium. The root part of the sterile seedling is inoculated on the induction medium, the callus induction rate is counted, and the optimal hormone ratio of the induction medium is determined.

5. A method for inducing callus and regenerating green onion roots according to claim 4, characterized in that: In the step 3), MS is used as the basic culture medium, and NAA, 6-BA and KT with different hormone ratios are added to prepare a differentiation medium. The callus tissue is inoculated on the differentiation medium and cultured at 25° C., a light intensity of 2000 lx, with 16 hours of light and 8 hours of darkness every 24 hours. The differentiation rate is counted after 30 days.

6. A method for inducing callus and regenerating green onion roots according to claim 5, characterized in that: In the step 4), 1 / 2MS solid culture medium is used as the rooting medium.

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