Optimization method of pea tissue culture regeneration system

Abstract

The invention discloses an optimization method of a pea tissue culture regeneration system. The optimization method comprises the following steps: step 1, preparing pea aseptic seedlings; step 2, preparing an explant; step 3, inducing cluster buds; 4, carrying out bud elongation culture; step 5, rooting culture; and 6, domesticating and transplanting regenerated seedlings. By systematically optimizing the tissue culture conditions of the peas, an efficient regeneration system is established and is suitable for multiple pea varieties, and the problem of low regeneration efficiency in the prior art is solved. By means of the optimization method, researchers can obtain high-efficiency regenerated pea plants in a short time, the success rate and rooting efficiency of tissue culture regeneration are improved, meanwhile, the survival rate of regenerated seedlings is increased, and an important technical guarantee is provided for molecular breeding and gene modification of peas and other leguminous crops.

Description

Technical Field

[0001] The invention relates to the field of plant tissue culture and plant regeneration, and in particular to an optimization method for a pea tissue culture regeneration system. Background Art

[0002] Pea (Pisum sativum) is one of the important leguminous crops, and the establishment of its tissue culture and regeneration system is of great significance in gene function research, genetic improvement and molecular breeding. Traditional pea tissue culture systems usually face problems such as low regeneration efficiency, unstable bud induction rate and rooting rate, which greatly limits the application of rapid regeneration and genetic modification of peas. In past studies, although some pea varieties have been regenerated into complete plants through tissue culture, due to the large differences in the responses of different varieties to explants, culture media and culture conditions, the existing technology is difficult to be widely applied to different varieties of peas, and problems such as low rooting rate and low survival rate often occur during the regeneration process.

[0003] The regeneration system of pea generally includes key steps such as explant selection, bud induction, bud elongation and rooting culture. The formulation of bud induction medium and rooting medium, culture conditions (such as light and temperature) and explant type all have a significant impact on the regeneration efficiency. However, the regeneration system in existing research has failed to fully optimize each link, resulting in significant differences in the regeneration efficiency of different explants, especially the low bud induction rate and rooting rate.

[0004] Therefore, optimizing the tissue culture regeneration system of peas and improving the bud induction rate, rooting rate and survival rate of regenerated seedlings of different explants are urgent problems to be solved in the study of pea gene function and genetic improvement. The present invention systematically optimizes the tissue culture conditions of peas to establish an efficient regeneration system suitable for multiple pea varieties, solving the problem of low regeneration efficiency in the prior art. Summary of the invention

[0005] In view of this, the present invention provides a method for optimizing a pea tissue culture regeneration system.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for optimizing a pea tissue culture regeneration system comprises the following steps:

[0008] Step 1: Preparation of sterile pea seedlings

[0009] Select pea seeds "ZW 6" and "ZQ 1" respectively and spread them in sterile culture dishes, place the culture dishes with peas in a well-sealed desiccator, place a NaClO solution in a small beaker, and place the small beaker in the desiccator, add concentrated hydrochloric acid to the beaker with the NaClO solution, quickly cover the lid of the desiccator, seal and disinfect, and then inoculate the pea seeds into the germination medium for culture;

[0010] Step 2: Explant preparation

[0011] Take out the germinated sterile seedlings, cut the epicotyl and hypocotyl into long segments, cut the cotyledonary nodes in half, and keep part of the hypocotyl;

[0012] Step 3: Inducing budding

[0013] The prepared explants are inoculated on a bud induction medium and cultured under light, the explants are removed and the main buds with obvious growth are cut off, and then transferred to a fresh bud induction medium for the second round of screening culture;

[0014] Step 4: Bud elongation culture

[0015] The epicotyls, hypocotyls and cotyledon nodes from which clustered buds have sprouted are selected respectively, new wounds are cut on the epicotyls and hypocotyls, the cotyledon nodes are removed, and the wounds are placed in a bud elongation medium, and fresh medium is replaced regularly;

[0016] Step 5: Rooting and cultivation

[0017] When the clustered buds grow to 2-3 cm, cut off the buds, dip the bud base in IBA solution, absorb the excess liquid at the bud base with sterilized filter paper, and move them to the rooting medium for rooting culture, and replace the fresh medium regularly;

[0018] Step 6: Acclimation and transplanting of regenerated seedlings

[0019] When the root length meets the requirements, open the lid of the tissue culture bottle, add sterile water to the bottle, harden the seedlings in the tissue culture room, then rinse the roots under tap water, transplant the seedlings into a matrix of vermiculite and nutrient soil, transfer them to a greenhouse for cultivation, and obtain pea tissue culture regenerated plants.

[0020] Preferably, in step 1, the germination medium formula is as shown in Table 1;

[0021] Table 1 Germination medium formula

[0022]

[0023] The culture conditions are: 16h light / 8h dark, light intensity 3000Lux, culture temperature 25°C; culture time: 5-7 days.

[0024] Preferably, in step 1, the amount of NaClO solution added is 200-400 mL, the volume of the small beaker is 500 mL, the amount of concentrated hydrochloric acid taken is 10-30 mL, and the closed disinfection time is 10-12 h;

[0025] Preferably, the amount of NaClO solution added is 300 mL;

[0026] Preferably, the amount of concentrated hydrochloric acid is 20 ml.

[0027] Preferably, in step 2, the embryonic axes and hypocotyls are cut into 0.5 cm long segments, leaving 2-3 mm of the hypocotyls.

[0028] Preferably, in step 2, three groups of repetitions are performed on different explants of different pea varieties;

[0029] There were 30 explants per replicate for epicotyl and hypocotyl, and 60 explants per replicate for cotyledonary nodes.

[0030] Preferably, in step 3, the bud induction medium formula is as shown in Table 2;

[0031] Table 2 Bud induction medium formula

[0032]

[0033]

[0034] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25°C; the time for culture under light and transfer to fresh bud induction medium as the second round of screening culture was 2 weeks.

[0035] Preferably, in step 4, the bud elongation medium formula is as shown in Table 3;

[0036] Table 3 Bud elongation medium formula

[0037]

[0038] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25°C; fresh culture medium was replaced every 2 weeks.

[0039] Preferably, in step 5, the formula of the rooting medium is as shown in Table 4;

[0040] Table 4 Rooting medium formula

[0041]

[0042] The culture conditions were: 12 h light / 12 h dark, light intensity 2000 Lux, and culture temperature 25°C; fresh culture medium was replaced every 2 weeks.

[0043] Preferably, in step 5, the concentration of the IBA solution is 2 mg / mL, and the bud base is dipped in the IBA solution for 30 minutes.

[0044] Preferably, in step six, when the roots grow to 10 to 20 cm and have a certain number, the lid of the tissue culture bottle is opened, sterile water to a height of about 1 cm is added to the bottle, and the seedlings are hardened in the tissue culture room for 5 to 7 days.

[0045] Compared with the prior art, the present invention has achieved the following technical effects:

[0046] (1) Improved bud induction efficiency: By optimizing explant selection and culture medium formulation, especially differential treatment of epicotyls, hypocotyls, and cotyledon nodes, the bud induction efficiency of different pea varieties was significantly improved;

[0047] The experimental results show that the bud induction rate of the cotyledon node is the highest, reaching 86.67%, and the overall induction efficiency is better than the existing technology;

[0048] (2) Significantly improve the rooting rate: In order to solve the problem of low rooting rate in the pea regeneration system, the present invention successfully improves the rooting efficiency by adjusting the IBA concentration and the explant treatment method;

[0049] Research shows that the rooting rate can reach 22.99% to 29.21% under different treatment conditions, which is significantly higher than the traditional method;

[0050] (3) Improving the survival rate of regenerated seedlings: During the acclimatization and transplantation stage of regenerated seedlings, the present invention greatly improves the survival rate of regenerated seedlings by acclimatization treatment and reasonable selection of transplanting matrix;

[0051] The experimental data showed that the survival rate of regenerated seedlings was between 71.67% and 88.14%, which ensured the high success rate of pea tissue culture regeneration.

[0052] (4) Shortening the experimental period: The present invention optimizes the culture conditions at each stage, especially shortens the culture time during the bud induction and bud elongation process, so that the regeneration period of pea from explant treatment to complete plant is significantly shortened, thereby improving the experimental efficiency;

[0053] (5) Wide applicability: The method of the present invention is not only applicable to different pea varieties such as "ZW 6" and "ZQ 1", but also shows high regeneration efficiency in different explant types;

[0054] This indicates that the optimized system of the present invention has good applicability and is suitable for the tissue culture regeneration needs of various pea varieties;

[0055] (6) Stable regeneration system: The present invention establishes a stable pea tissue culture regeneration system, which not only solves the problems of low regeneration efficiency and complex explant processing in the traditional system, but also provides efficient and reliable technical support for the gene function research and genetic improvement of pea and other leguminous plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 A flow chart of a pea tissue culture regeneration system for an optimization method of a pea tissue culture regeneration system according to the present invention;

[0057] Figure 2 The present invention is a diagram showing the bud induction rate, rooting rate and survival rate of different pea varieties and different explants in an optimization method of a pea tissue culture regeneration system. DETAILED DESCRIPTION

[0058] 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.

[0059] Embodiment 1:

[0060] The invention discloses a method for optimizing a pea tissue culture regeneration system, comprising the following steps:

[0061] Step 1: Preparation of sterile pea seedlings

[0062] Select plump, clean and well-growing pea seeds "ZW 6" and "ZQ 1" respectively and spread them in sterile culture dishes. Place the culture dishes containing peas in a well-sealed desiccator. Place 300 mL of NaClO solution in a 500 mL small beaker, and place the small beaker in the desiccator. Use a pipette to take 20 mL of concentrated hydrochloric acid and add it to the beaker containing the NaClO solution. Quickly cover the lid of the desiccator and seal it for disinfection for 10-12 hours. After disinfection, inoculate the pea seeds into the germination medium and culture it for 5-7 days. The formula of the germination medium is shown in Table 1. The culture conditions are: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25 ° C.

[0063] Step 2: Explant preparation

[0064] Take out the germinated sterile seedlings from the clean bench, cut the epicotyl and hypocotyl into segments about 0.5 cm long, cut the cotyledonary nodes in half, and leave 2-3 mm of hypocotyl;

[0065] Different explants of different pea varieties were replicated in 3 groups, including 30 explants per replicate of epicotyl and hypocotyl, and 60 explants per replicate of cotyledon node;

[0066] Step 3: Inducing budding

[0067] The prepared explants were inoculated into the bud induction medium and cultured under light for 2 weeks. The explants were removed and the main buds with obvious growth were cut off, and then transferred into fresh bud induction medium for the second round of screening and culture for 2 weeks (which can be fine-tuned according to the growth). The formula of the bud induction medium is shown in Table 2: the culture conditions are: 16h light / 8h dark, light intensity 3000Lux, and culture temperature 25°C;

[0068] The number of explants induced to sprout under different treatments was counted, and the sprout induction efficiency of different explants of different pea varieties was calculated;

[0069] Step 4: Bud elongation culture

[0070] The epicotyls, hypocotyls and cotyledon nodes from which clustered buds were germinated were selected respectively, new wounds were cut on the epicotyls and hypocotyls, the cotyledon nodes were removed, and the bud elongation medium was inoculated, and the fresh medium was replaced every 2 weeks; the formula of the bud elongation medium is shown in Table 3: the culture conditions are: 16h light / 8h dark, light intensity 3000Lux, and culture temperature 25°C;

[0071] Step 5: Rooting and cultivation

[0072] When the clustered buds grow to about 2-3 cm on the elongation medium, cut off the buds, dip the bud base in a 2 mg / mL IBA solution for 30 min, absorb the excess liquid at the bud base with sterilized filter paper, and move to the rooting medium for rooting culture. Replace the fresh medium every 2 weeks; the formula of the rooting medium is shown in Table 4: the culture conditions are: 12 h light / 12 h dark, light intensity 2000 Lux, and culture temperature 25 ° C;

[0073] Step 6: Acclimation and transplanting of regenerated seedlings

[0074] When the roots grow to 10-20 cm and have a certain number, open the lid of the tissue culture bottle, add about 1 cm high of sterile water into the bottle, harden the seedlings in the tissue culture room for 5-7 days, then rinse the roots under tap water, transplant the seedlings into a matrix of vermiculite and nutrient soil, transfer to a greenhouse for cultivation, and obtain pea tissue culture regenerated plants.

[0075] The number of surviving rooted regenerated seedlings under different treatments was counted, and the survival rate of regenerated seedlings from different explants of different pea varieties was calculated.

[0076] Example 2: Experimental results

[0077] like Figure 1As shown, each picture from left to right represents explantation, shoot induction, shoot elongation, rooting, and acclimatization transplantation.

[0078] In order to optimize the pea tissue culture regeneration system, the present invention selected two pea varieties "ZW 6" and "ZQ 1" as test materials, and epicotyls, hypocotyls and cotyledon nodes as explants, all of which successfully regenerated complete pea plants.

[0079] like Figure 2 As shown, ZW6 is the abbreviation of Zhongwan No. 6, and ZQ1 is the abbreviation of Zhongqin No. 1. The orange column represents the bud induction rate, the green column represents the rooting rate, and the purple column represents the survival rate.

[0080] like Figure 2 As shown in Table 5, after culturing on the bud induction medium for 4 weeks, it was found that the bud induction efficiencies of the epicotyl, hypocotyl and cotyledon node of "ZW 6" were 67.78%, 50.00% and 86.67%, respectively, and there were extremely significant differences in the bud induction rates of these three explants.

[0081] The bud induction efficiencies of epicotyl, hypocotyl and cotyledon node of "ZQ 1" were 64.43%, 53.33% and 82.78% respectively. There was a significant difference between the bud induction rates of cotyledon node and hypocotyl, but no significant difference between epicotyl and hypocotyl. At the same time, there was no significant difference in the bud induction rate of the same explants among different varieties. The bud induction rate of cotyledon node was the highest, followed by epicotyl, and the lowest was hypocotyl.

[0082] When the cluster buds grow to about 2 to 3 cm, the regenerated seedlings are transferred to the rooting medium for rooting culture. Roots begin to form after about 3 weeks, and the number of rooted explants increases after 4 weeks. By counting the number of rooted regenerated seedlings under different treatments, we found that the rooting rate was between 13.15% and 29.21%, but there was no significant difference between different varieties and different explants.

[0083] When the roots grow long enough (10-20 cm), they are acclimated and transplanted to obtain pea tissue culture regeneration plants. Statistical results show that the survival rate of regenerated seedlings after acclimation and transplantation is 71.67%-88.14%, and there is no significant difference between different varieties and different explants.

[0084] Table 5 Bud induction rate, rooting rate and survival rate of different explants of different pea varieties

[0085]

[0086] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for optimizing a pea tissue culture regeneration system, characterized in that: The following steps are involved: Step 1: Preparation of sterile pea seedlings Select pea seeds "ZW 6" and "ZQ 1" respectively and spread them in sterile culture dishes, place the culture dishes with peas in a well-sealed desiccator, place NaClO solution in a small beaker, and place the small beaker in the desiccator, add concentrated hydrochloric acid to the beaker with NaClO solution, quickly cover the lid of the desiccator, seal and disinfect, and then inoculate the pea seeds into the germination medium for culture; Step 2: Explant preparation Take out the germinated sterile seedlings, cut the epicotyl and hypocotyl into long segments, cut the cotyledonary nodes in half, and keep part of the hypocotyl; Step 3: Inducing budding The prepared explants are inoculated on a bud induction medium and cultured under light, the explants are removed and the main buds with obvious growth are cut off, and then transferred to a fresh bud induction medium for the second round of screening culture; Step 4: Bud elongation culture The epicotyls, hypocotyls and cotyledon nodes from which clustered buds have sprouted are selected respectively, new wounds are cut on the epicotyls and hypocotyls, the cotyledon nodes are removed, and the wounds are placed in a bud elongation medium, and fresh medium is replaced regularly; Step 5: Rooting and cultivation When the clustered buds grow to 2-3 cm, cut off the buds, dip the bud base in IBA solution, absorb the excess liquid at the bud base with sterilized filter paper, and move them to the rooting medium for rooting culture, and replace the fresh medium regularly; Step 6: Acclimation and transplanting of regenerated seedlings When the root length meets the requirements, open the lid of the tissue culture bottle, add sterile water to the bottle, harden the seedlings in the tissue culture room, then rinse the roots under tap water, transplant the seedlings into a matrix of vermiculite and nutrient soil, transfer them to a greenhouse for cultivation, and obtain pea tissue culture regenerated plants.

2. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In the step 1, the germination medium formula is shown in Table 1; Table 1 Germination medium formula The culture conditions are: 16h light / 8h dark, light intensity 3000Lux, culture temperature 25°C; culture time: 5-7 days.

3. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In step 1, the amount of NaClO solution added is 200-400 mL, and the volume of the small beaker is 500 mL. The amount of concentrated hydrochloric acid is 10-30mL, and the airtight disinfection time is 10-12h; Preferably, the amount of NaClO solution added is 300 mL; Preferably, the amount of concentrated hydrochloric acid is 20 - ml.

4. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In the step 2, the embryonic axis and hypocotyl are cut into 0.5 cm long segments, leaving 2-3 mm hypocotyl.

5. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In the step 2, three groups of repetitions are performed on different explants of different pea varieties; There were 30 explants per replicate for epicotyl and hypocotyl, and 60 explants per replicate for cotyledonary nodes.

6. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In the step 3, the bud induction medium formula is shown in Table 2; Table 2 Bud induction medium formula The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25°C; the time for culture under light and transfer to fresh bud induction medium as the second round of screening culture was 2 weeks.

7. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In the step 4, the bud elongation medium formula is as shown in Table 3; Table 3 Bud elongation medium formula The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25°C; fresh culture medium was replaced every 2 weeks.

8. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In the step 5, the formula of the rooting medium is as shown in Table 4; Table 4 Rooting medium formula The culture conditions were: 12 h light / 12 h dark, light intensity 2000 Lux, and culture temperature 25°C; fresh culture medium was replaced every 2 weeks.

9. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In the step 5, the concentration of the IBA solution is 2 mg / mL, and the bud base is dipped in the IBA solution for 30 minutes.

10. The method for optimizing the pea tissue culture regeneration system according to claim 1, characterized in that: In step 6, when the roots grow to 10 to 20 cm and have a certain number, the lid of the tissue culture bottle is opened, sterile water about 1 cm high is added to the bottle, and the seedlings are hardened in the tissue culture room for 5 to 7 days.

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