Method for tissue culture and rapid propagation of fig male tree germplasm material

Abstract

The invention discloses a tissue culture and rapid propagation method for a fig male tree germplasm material, and belongs to the technical field of plant tissue culture and rapid propagation. The specific method comprises the following steps: (1) obtaining and disinfecting an explant; (2) primary culture of buds; (3) subculture multiplication of buds; (3) rooting culture; and (4) hardening seedlings and transplanting. According to the method, the optimal culture medium for tissue culture and rapid propagation of the fig male tree is obtained by combining different hormones and adjusting different hormone concentration ratios, and the differentiation and proliferation of buds of the fig male tree and the growth and rooting rate of seedlings are remarkably improved. In the explant treatment, tebuconazole carbendazim, 75% alcohol and sodium hypochlorite are adopted for the first time, so that the use of a highly toxic reagent mercury bichloride is avoided; cefotaxime sodium is added into a primary culture medium, so that the pollution rate of explants is reduced; the method is simple to operate, time-saving, labor-saving, safe and environment-friendly, and improves the inoculation quality and efficiency. The method is not limited by seasons, enlarges the excellent male tree population of the figs in a short time, and lays a foundation for cultivating new germplasm of the figs.

Description

Technical Field

[0001] The invention relates to the field of seedling breeding of fig male tree germplasm materials, and in particular to a method for rapid tissue culture of fig male tree germplasm materials. Background Art

[0002] Fig belongs to the genus Ficus in the family Moraceae and is a perennial woody species with dioecious sexes. Domestic fig cultivars are parthenocarpic and can set fruit without pollination, and the probability of obtaining viable seeds is zero. Male fig trees provide pollen for fig hybridization, and innovative germplasm resources are created through hybridization and breeding. Due to the lack of male trees and their pollination media in China, there are no reports of fig hybrid breeding except for this team. After the applicant's team experimentally separated and sowed seeds of foreign figs, they obtained seedlings for the first time in China, and identified and selected some excellent male trees from them.

[0003] At present, domestic fig propagation mostly adopts cutting propagation, which is difficult to meet the current hybrid breeding needs due to the small source of materials, low reproduction rate, poor rooting, and labor-intensive and time-consuming. The tissue culture rapid propagation technology can eliminate the limitation of seasonal factors, is genetically stable, is an efficient way to retain the advantages of male fig trees, and can achieve rapid propagation of excellent male fig trees. The present invention realizes the year-round seedling cultivation of excellent male fig trees, significantly improves the reproduction coefficient, and lays the foundation for domestic autonomous hybrid breeding of figs.

[0004] Although there are reports in the prior art of patent literature similar to the rapid propagation of male fig trees through tissue culture, there are problems such as high infection rate and weak growth of tissue culture seedlings. In the process of in vitro rapid propagation of figs, the tissue culture regeneration systems of different fig varieties, or even different explants of the same variety, are not used together, and the pollution problem has not been effectively solved, and the pollution causes the tissue culture materials to fail to grow and develop normally. The prior art cannot be effectively applied to male fig trees and cannot achieve the desired effect. At present, if a method suitable for rapid propagation of male fig trees through tissue culture can be found, it will meet the urgent needs of fig breeding.

[0005] In summary, the problems existing in the prior art are: excellent male fig tree germplasm resources are scarce, and no reports on tissue culture rapid propagation technology of male fig tree germplasm materials have been found in China; the pollution problem of fig tissue culture rapid propagation has not been effectively solved, and the transplant survival rate is not high, which hinders domestic fig hybrid breeding. Summary of the invention

[0006] 1. Technical problems solved

[0007] The purpose of the present invention is to solve the defects of the above-mentioned prior art and provide a method for rapid propagation of fig male tree germplasm material tissue culture seedlings.

[0008] 2. Technical solution

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] (1) Collection and treatment of explants: The new shoots of the greenhouse are used as explants. After the explant materials are collected, the leaves are removed, the explants are cleaned in sequence, and the explants are trimmed into stem segments with axillary buds. They are treated to prevent browning and disinfected for later use.

[0011] (2) Primary culture: Cut the above stem segments into 0.5-1.0 cm pieces, inoculate them onto primary culture medium, place them under light for 16 h per day, light intensity of 2500 lx, and culture temperature of 26°C until adventitious buds are induced to form.

[0012] (3) Proliferation culture: The adventitious buds grown to 1.0 cm in height were transferred to a proliferation medium for proliferation culture. The culture medium was placed under a light intensity of 3000 lx for 16 h per day and a culture temperature of 26°C. The buds were transferred every 30 days.

[0013] (4) Rooting culture: The above-mentioned clustered buds with a height of about 2.5 cm were cut and inoculated onto a rooting medium for rooting culture. The light intensity was 3000 lx for 16 h per day and the culture temperature was 26°C. The culture was continued until roots were formed.

[0014] (5) Hardening and transplanting: Remove the bottle caps of the rooted tissue culture seedlings about 4.0 to 6.0 cm in height and place them under natural light for 5 to 7 days. Then, take the tissue culture seedlings out of the culture bottles, wash off the root culture medium, plant them in a matrix mixed with peat and vermiculite (3:1) and transplant them into the field.

[0015] The primary culture medium is used for primary culture of fig male tree tissue culture seedlings, the proliferation culture medium is used for proliferation culture of fig male tree tissue culture seedlings, the rooting culture medium is used for rooting culture of tissue culture seedlings, and the transplanting matrix is ​​used for transplanting when the rooting of tissue culture seedlings meets the transplanting requirements.

[0016] Furthermore, in the method for rapid propagation of male fig tree tissue culture seedlings as described above, when collecting explants in (1), sunny days for more than 3 consecutive days should be selected. New buds are stem segments with buds at the top of branches that are healthy and free of diseases and insect pests.

[0017] Furthermore, in the method for rapid propagation of male fig tissue culture seedlings as described above, the cleaning method in (1) is: soaking in a dilute detergent solution for 10 minutes, rinsing with tap water, then soaking in a 5 mg / L pentazole·carbendazim solution for 2 hours, rinsing with tap water, and finally rinsing in running water for 1 hour.

[0018] Furthermore, in the method for rapid propagation of male fig tree tissue culture seedlings as described above, the method for pruning into stem segments with axillary buds in (1) is: cutting the new shoots into single axillary bud stem segments of about 1.0 to 1.5 cm.

[0019] Furthermore, in the method for rapid propagation of male fig tree tissue culture seedlings as described above, the (1) anti-browning treatment is: soaking the single axillary bud stem segment in a 1200 mg / L vitamin C solution at 4-8°C for 30 minutes.

[0020] Furthermore, in the method for rapid propagation of male fig tree tissue culture seedlings as described above, the (1) disinfection treatment is: sterilizing on a clean bench, surface sterilizing with 75% alcohol for 30 seconds, rinsing with sterile water for 3 times, soaking and shaking in 2% NaClO solution for 7 minutes, and rinsing with sterile water for 5 times.

[0021] Furthermore, in the method for rapid propagation of male fig tree tissue culture seedlings as described above, the primary culture medium in (2) is: MS+6-BA1.5mg / L+NAA0.2mg / L+sucrose 30g / L+agar 6g / L+cefotaxime sodium 200mg / L, with a pH of 5.8.

[0022] Furthermore, in the method for rapid propagation of male fig tree tissue culture seedlings as described above, the proliferation culture medium in (3) is: MS+6-BA0.5mg / L+NAA0.1mg / L+sugar 30g / L+agar 6g / L, with a pH of 5.8.

[0023] Furthermore, in the method for rapid propagation of male fig tree tissue culture seedlings as described above, the rooting medium of (4) is: 1 / 2MS+IBA1.0mg / L+sucrose 30g / L+agar 6g / L, with a pH of 6.0.

[0024] 3. Beneficial effects

[0025] Compared with the prior art, the present invention has the following beneficial effects: the present invention discloses a method for tissue culture and rapid propagation of male fig tree germplasm materials. The present invention uses vitamin C in the explant sterilization process to reduce the browning rate of male fig trees. The first use of penthazole, carbendazim and ceftriaxone sodium in primary culture reduces the contamination rate. Compared with the prior art, the use of the above scheme in combination with a suitable rapid propagation method can obtain a large amount of male fig tree germplasm materials in a relatively short period of time, opening up a new breeding path for male fig tree germplasm materials, accelerating the growth of male fig tree rooted tissue culture seedlings, improving the rooting rate and root length of male fig tree tissue culture seedlings, improving the survival rate of male fig tree tissue culture seedlings, and accelerating the breeding of excellent male fig trees, which has significant progress. DETAILED DESCRIPTION

[0026] 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.

[0027] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the initial generation of tissue culture seedlings provided by the embodiment of the present invention Figure 1 .

[0029] Figure 2 This is the initial generation of tissue culture seedlings provided by the embodiment of the present invention Figure 2 .

[0030] Figure 3 This is a diagram of the proliferation of tissue culture seedlings provided in an embodiment of the present invention.

[0031] Figure 4 The rooting situation of the tissue culture seedlings provided in the embodiment of the present invention Figure 1 .

[0032] Figure 5 The rooting situation of the tissue culture seedlings provided in the embodiment of the present invention Figure 2 .

[0033] Figure 6 This is the transplanting situation of tissue culture seedlings provided in the embodiment of the present invention Figure 1 .

[0034] Figure 7 This is the transplanting situation of tissue culture seedlings provided in the embodiment of the present invention Figure 2 .

[0035] Figure 8 This is the transplanting situation of tissue culture seedlings provided in the embodiment of the present invention Figure 3 .

[0036] Fig. 9 This is the transplanting situation of tissue culture seedlings provided in the embodiment of the present invention Figure 4 .

[0037] Specific implementation cases

[0038] The experimental methods and detection methods described in the examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0039] Some of the reagents involved in the following examples are 6-BA (6-benzyladenine), TDZ (thidiazuron), NAA (α-naphthylacetic acid), IBA (3-indolebutyric acid), etc.

[0040] Some of the instruments used in the following examples are a clean bench (HCB-1300V), an autoclave (LS-35HD), a culture container (7.5 cm in diameter, 320 mL), a constant temperature incubator (YKRD-500B4), and the like.

[0041] Example 1

[0042] This example provides a method for sterilizing male fig tree explants and a primary culture experiment.

[0043] (1) Collection and treatment of explants: The new shoots of the year in the greenhouse were used as explants. After collecting the explant materials, the leaves were removed and cut into single axillary bud stem segments of about 1.5-2 cm. They were soaked in a dilute detergent solution for 10 min, rinsed with tap water, and then soaked in a 5 mg / L pentamethylene · carbendazim solution for 2 h, rinsed with tap water, and finally rinsed with running water for 1 h. They were soaked in a 1200 mg / L vitamin C solution at 4-8 °C for 30 min. They were sterilized on a clean bench, sterilized on the surface with 75% alcohol for 30 s, rinsed with sterile water 3 times, soaked and shaken in a 2% NaClO solution for 7 min, rinsed with sterile water 5 times, and the excess water was filtered out. The treated buds were set aside for later use. An experiment was set up with cefotaxime sodium as the research object to study its effect on inhibiting tissue culture contamination of male fig trees. Four concentration levels were set: 0, 100, 200, and 300 mg / L. Three explants were inoculated in each bottle, and 30 bottles were inoculated in each treatment, which was repeated 3 times. After 15 days, the bud contamination rate was calculated, and the growth and differentiation of the buds were observed and recorded. The data processing method was as follows:

[0044] Contamination rate = (number of contaminated explants / total number of inoculated explants) × 100%.

[0045] Table 1 Antibacterial effect of ceftriaxone sodium on fig male tree tissue culture seedlings

[0046]

[0047]

[0048] As can be seen from Table 1, at 15 days of culture, the contamination rate of tissue culture seedlings in the medium without antibiotics was as high as 94.07%, and the contamination rate of tissue culture seedlings in the medium with cefotaxime sodium added decreased. The antibacterial effects of different concentrations of cefotaxime sodium on fig male tree tissue culture seedlings were different. The contamination rate of tissue culture seedlings decreased with the increase of cefotaxime sodium concentration. Both 200mg / L and 300mg / L cefotaxime sodium can achieve the effect of inhibiting the contamination rate, and the difference between the two treatments is not significant, but the leaf state of 300mg / L cefotaxime sodium tissue culture seedlings is not good, and the leaves are yellowing. The results show that 200mg / L cefotaxime sodium has the best antibacterial effect.

[0049] Example 2

[0050] This embodiment provides an experiment on the effect of different concentrations of cytokinins on bud induction of fig male trees. Single factor experiments were set up with cytokinins 6-BA and TDZ as research objects to study their effects on bud induction culture. Six concentration levels of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mg / L were set respectively. Three explants were inoculated in each bottle, 30 bottles were inoculated in each treatment, and repeated 3 times. After inoculation, they were placed in the tissue culture room for culture. The temperature in the culture room was 26°C, the light was 16 hours a day, and the light intensity was 2500lx. After 3 weeks, the bud induction rate was counted, and the growth and differentiation of the buds were observed and recorded. The data processing method is shown in Table 2:

[0051] Induction rate = (number of explants forming single buds / number of inoculated explants) × 100%;

[0052] Table 2 Screening of primary culture medium for male fig trees

[0053]

[0054]

[0055] As can be seen from Table 2, different concentrations of 6-BA and TDZ have different effects on the induction of single buds in fig male trees. The results show that 6-BA has a better effect on bud induction than TDZ, among which 1.5 mg / L 6-BA has the most significant effect, with the highest induction rate of 100%, and the explant buds grow robustly and the leaves are dark green. In summary, the optimal 6-BA concentration for bud induction in fig male trees is 1.5 mg / L. The primary culture medium is: MS+6-BA1.5.mg / L+NAA0.2mg / L+sucrose30g / L+agar6g / L, pH is 5.8.

[0056] Example 3

[0057] This example provides an experiment on the effects of different hormone types and concentration ratios on the proliferation of fig male tissue culture seedlings. After the buds are successfully induced, they are transferred to a proliferation medium when they grow to 1.0 cm. Subculture is performed once every 30 days, and repeated multiple times to allow them to continue to proliferate.

[0058] The experiment was conducted on a basic culture medium, with the added plant hormone concentrations of 0.5 mg / L and 1.0 mg / L for cytokinin 6-BA, and 0.05 mg / L, 0.1 mg / L, and 0.15 mg / L for auxin NAA. The subculture experiment was set up with two factors for cytokinin concentration and auxin concentration. 30 bottles were inoculated for each treatment, and 3 explants were inoculated in each bottle, with 3 replicates. The experiment was placed under a light intensity of 3000 lx and a culture temperature of 26 ° C for 16 hours per day. The proliferation times of the buds were counted every 30 days, and the growth and differentiation of the bud tissue were observed and recorded. The specific experimental treatments are shown in Table 3.

[0059] (1) Bud number = number of explants that formed a single bud;

[0060] (2) Average number of regenerated buds from explants = total number of adventitious buds regenerated from explants / number of explants with regenerated adventitious buds;

[0061] (3) Induction rate = (number of explants forming single buds / number of inoculated explants) × 100%;

[0062] Table 3 Screening of fig male tree proliferation culture medium

[0063]

[0064] Table 3 shows the bud induction rate, average number of regenerated buds and bud height of male fig trees under different hormone concentrations after 30 days of culture. After 30 days of inoculation, the average number of regenerated buds in treatment 2 was the highest, 11.30, and the highest height was 3.18 cm. The proliferation medium was: MS + 6-BA 0.5 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8.

[0065] (4) Embodiment 4

[0066] This example provides an experiment on the effects of different hormone types and concentrations on the rooting of male fig tissue culture seedlings.

[0067] The experiment was based on the basic culture medium. When the cluster buds grew to 2.5 cm in height, the proliferated buds were cut and transferred to the rooting culture medium on the clean bench. The plant hormone added was the auxin IBA at concentrations of 0 mg / L, 0.5 mg / L, 1.0 mg / L, and 1.5 mg / L. Three buds were inoculated in each bottle, and 30 buds were inoculated in each treatment. The experiment was repeated 3 times. The light was illuminated for 16 hours a day, the light intensity was 3000 lx, and the culture temperature was 26 ° C until rooting. After 30 days, the effects of different hormones on the rooting of sterile seedlings of fig male trees were compared.

[0068] Table 4 Screening of rooting medium for male fig trees

[0069]

[0070]

[0071] The effects of different growth hormones on the rooting culture of male fig trees are shown in Table 4. Under IBA treatment, male fig trees have obvious rooting phenomenon. When the rooting induction culture was about 30 days, when the NAA concentration was 1.0 mg / L, the rooting rate reached the maximum value, which was 100%; the average number of roots reached the maximum value, which was 5.08. In summary, the rooting medium is: 1 / 2MS+IBA1.0 mg / L sucrose 30g / L+agar 6g / L, pH 5.8. Hardening and transplanting: Remove the bottle cap of the rooted tissue culture seedlings about 4.0-6.0 cm high and place them under natural light for 5-7 days. Then take the tissue culture seedlings out of the culture bottle, wash off the root culture medium, and transplant them into the substrate containing different ratios. After 30 days, the transplant survival rate was counted.

[0072] Table 5 Effects of different substrates on the survival rate of transplanted male fig trees

[0073]

[0074] The results showed that the survival rates of male fig trees in four substrates with a volume ratio of peat: vermiculite (1:1; 2:1; 3:1; 4:1) were all above 90%, and the survival rate of male fig trees in the peat:vermiculite = 3:1 ratio reached 100%.

[0075] By adopting the method, the reproduction period of fig male tree germplasm materials can be shortened from the traditional 12 months to 3 months, and the reproduction coefficient of seedlings is higher than that of the traditional reproduction method (cutting propagation); in addition, compared with the existing conventional tissue culture method of fig, the method can increase the proliferation rate of fig male tree tissue culture seedlings by 11.30 times, the rooting rate is 100%, the survival rate of seedling transplanting is 100%, and the explant contamination is reduced to 11.1%.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications and equivalents made within the spirit and principles of the present invention are hereby deemed to be correct.

Claims

1. A method for rapid propagation of fig male tree germplasm material tissue culture, characterized in that: The following steps are involved: (1) Collection and treatment of explants: The new shoots of the greenhouse are used as explants. After the explant materials are collected, the leaves are removed, the explants are cleaned in sequence, and the explants are trimmed into stem segments with axillary buds. They are treated to prevent browning and disinfected for later use. (2) Primary culture: Cut the above stem segments into 0.5-1.0 cm pieces, inoculate them onto primary culture medium, place them under light for 16 h per day, light intensity of 2500 lx, and culture temperature of 26°C until adventitious buds are induced to form. (3) Proliferation culture: The adventitious buds grown to 1.0 cm in height were transferred to a proliferation medium for proliferation culture, placed under a light intensity of 3000 lx for 16 h per day and a culture temperature of 26°C, and transferred once every 30 days. (4) Rooting culture: The above-mentioned clustered buds with a height of about 2.5 cm were cut and inoculated onto rooting medium for rooting culture. The culture medium was placed under a light intensity of 3000 lx for 16 h per day and a culture temperature of 26°C until roots were formed. (5) Hardening and transplanting: Remove the bottle caps of the rooted tissue culture seedlings about 4.0 to 6.0 cm in height and place them under natural light for 5 to 7 days. Then, take the tissue culture seedlings out of the culture bottles, wash off the root culture medium, plant them in a matrix mixed with peat and vermiculite (3:1) and transplant them into the field.

2. A method for rapid propagation of male fig trees by tissue culture according to claim 1, characterized in that: When collecting the explants in (1), it is necessary to select sunny days for more than 3 consecutive days. The new buds are the stem segments with buds at the top of the branches that are healthy and free of diseases and insect pests.

3. A method for rapid propagation of male fig trees by tissue culture according to claim 1, characterized in that: The cleaning method in (1) is: soak in a dilute detergent solution for 10 minutes, rinse with tap water, then soak in a 5 mg / L pentamethylenediamine·carbendazim solution for 2 hours, rinse with tap water, and finally rinse with running water for 1 hour.

4. A method for rapid propagation of male fig trees by tissue culture according to claim 1, characterized in that: The method for pruning into stem segments with axillary buds in (1) is: cutting new shoots into single axillary bud stem segments of 1.0 to 1.5 cm.

5. The method for rapid propagation of male fig germplasm tissue culture according to claim 1, characterized in that: The (1) anti-browning treatment is: soaking the single axillary bud stem segment in a 1200 mg / L vitamin C solution at 4-8° C. for 30 minutes.

6. A method for rapid propagation of male fig trees through tissue culture according to claim 1, characterized in that: The (1) disinfection treatment is as follows: sterilize on a clean bench, sterilize the surface with 75% alcohol for 30 seconds, rinse with sterile water for 3 times, soak and shake in 2% NaClO solution for 7 minutes, and rinse with sterile water for 5 times.

7. A method for rapid propagation of male fig trees through tissue culture according to claim 1, characterized in that: The primary culture medium in (2) is: MS+6-BA1.5mg / L+NAA0.2mg / L+sucrose30g / L+agar6g / L+cefotaxime sodium200mg / L, pH 5.

8.

8. A method for rapid propagation of male fig trees through tissue culture according to claim 1, characterized in that: The proliferation medium in (3) is: MS+6-BA0.5mg / L+NAA0.1mg / L+sugar 30g / L+agar 6g / L, with a pH of 5.

8.

9. A method for rapid propagation of male fig trees through tissue culture according to claim 1, characterized in that: The rooting medium of (4) is: 1 / 2MS+IBA1.0mg / L+sucrose 30g / L+agar 6g / L, pH 6.0.

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