Method for establishing pea genetic transformation system

Abstract

The invention discloses a method for establishing a pea genetic transformation system. The method comprises the following steps: step 1, transforming agrobacterium competent cells by using a plant expression vector; step 2, preparing and storing agrobacterium liquid; step 3, preparation of aseptic pea seedlings; step 4, infection and co-culture of the explants; step 5, restoring culture; step 6, bud induction culture; step 7, carrying out bud elongation culture; and step 8, rooting culture. According to the method, the pea variety Zhongwan No.6 which is easily subjected to a genetic transformation test is screened, the test verifies that the optimal explant is the cotyledonary node, and the bud induction rate of the cotyledonary node is 80% or above. According to the method, the problem of rooting difficulty is solved, a complete regenerated plant is obtained, the pea plant with whitening phenotype is successfully obtained by transforming the CRISPR / Cas9 gene editing carrier of the targeted pea PDS gene, the genetic transformation efficiency is improved to 5% or above, and the genetic transformation efficiency of peas is greatly improved.

Description

Technical Field

[0001] The invention relates to the field of establishing a pea genetic transformation system, and in particular to a method for establishing a pea genetic transformation system. Background Art

[0002] Pea (Pisum sativum L.) is one of the four major edible legume crops in the world, with a long history of cultivation. Pea has strong adaptability, is cold-resistant and barren-resistant, and is rich in nutrients. It can be used as food, vegetable, and feed. Pea has become an important intercropping crop for adjusting the structure of the planting industry due to its nitrogen fixation function. It can improve soil conditions, increase soil fertility, reduce fertilizer pollution, and increase crop yields. Pea is also one of the main winter crops in southern my country and one of the main early spring crops in northern my country. It has an important impact on the sustainable agricultural development of my country and the dietary structure of the Chinese people. Although the planting area of ​​pea is large, the total yield is still very low. Some biological and abiotic stresses seriously affect the yield of peas; there is a lack of high-yield, disease-resistant and high-quality varieties in pea breeding, especially the lack of frost-resistant pea varieties in the north. In addition, peas are self-pollinating and have a narrow genetic basis. Traditional conventional breeding has the problems of low efficiency, long breeding cycle, few available resistance resources, and often accompanied by poor agronomic traits. The functional verification and rapid transformation of important agronomic trait genes also face huge challenges. Therefore, using genetic engineering technology to improve germplasm is an effective way, and it also lays a solid foundation for the analysis of important gene functions discovered and cloned in recent years.

[0003] However, the transgenic of leguminous plants, especially peas, has always been a research problem worldwide. As early as the 1970s, Malmberg et al. began to study pea tissue culture. There have been reports of pea adventitious bud regeneration and somatic embryogenesis through protoplasts and a series of explants. However, the regeneration of leguminous plants is relatively difficult, and the research on pea regeneration has progressed slowly due to its dependence on genotype, low regeneration frequency, and poor repeatability. Many scholars at home and abroad have conducted research on pea tissue culture through somatic embryogenesis and organogenesis. Although some results have been achieved in the study of key influencing factors of pea tissue culture such as genotype, explant selection, culture medium type and exogenous hormones, there are still problems in the application of pea genetic transformation, such as rooting difficulties, low transformation rate, poor regeneration ability, complex and long callus regeneration, decreased fertility, phenotypic abnormalities, ploidy changes, and loss of transgenic activity in offspring. Therefore, pea transgenic technology is not perfect enough, the transformation efficiency is generally low, and the current research on genetic transformation of gene function is far from enough. Therefore, the need to establish a reliable and effective genetic transformation system is an important technical issue related to whether exogenous genes in peas can be efficiently introduced. It is also the main way to conduct functional verification and genetic research on important trait genes discovered, thereby achieving pea trait improvement. Summary of the invention

[0004] In view of this, the present invention provides a method for establishing a pea genetic transformation system.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for establishing a pea genetic transformation system comprises the following steps:

[0007] Step 1: Transform plant expression vector into Agrobacterium competent cells

[0008] Step 1.1, take EHA105 Agrobacterium competent cells and wait for them to partially melt, and insert them into an ice bath when they are in an ice-water mixture state;

[0009] Step 1.2, add CRISPR / Cas9 gene editing vector plasmid DNA, mix well, and place on ice, in liquid nitrogen, in a water bath, and in an ice bath in sequence;

[0010] Step 1.3, add LB liquid medium without antibiotics and culture with shaking;

[0011] Step 1.4, collect the bacteria by centrifugation, keep the supernatant, blow and resuspend the bacteria, spread it on the LB plate containing kanamycin and rifampicin resistance, and invert it to culture in the incubator in the dark;

[0012] Step 2: Preparation and storage of Agrobacterium culture solution

[0013] Step 2.1, pick a single colony of Agrobacterium, streak it onto an LB plate containing kanamycin and rifampicin resistance, and place it upside down in an incubator for dark culture;

[0014] Step 2.2, use an inoculation loop to dip the Agrobacterium colony into LB liquid medium containing kanamycin and rifampicin resistance, and shake and culture;

[0015] Step 2.3, pipette the bacterial solution into a centrifuge tube, add glycerol, mix well, put it in liquid nitrogen, and put the frozen Agrobacterium bacterial solution in a refrigerator for later use;

[0016] Step 3: Preparation of sterile pea seedlings

[0017] Step 3.1, sterilize the peas in a closed environment;

[0018] Step 3.2, inoculating pea seeds into germination medium for cultivation;

[0019] Step 4. Infection and co-cultivation of explants

[0020] Step 4.1, activating the Agrobacterium culture solution twice;

[0021] Step 4.2, take out the germinated sterile seedlings, cut the cotyledons in half, and leave the hypocotyl;

[0022] Step 4.3, take the activated Agrobacterium bacterial solution, measure its absorbance at a wavelength of 600nm, centrifuge the remaining Agrobacterium bacterial solution, collect the bacteria, pour out the supernatant, resuspend the bacteria with the infection liquid culture medium, and activate the bacteria; put the explant in the resuspended and activated bacterial solution, let it stand and then shake it continuously, pour out the bacterial solution, put the explant on a culture dish lined with sterile filter paper, absorb and blow dry the excess bacterial solution, and then inoculate it on the co-cultivation medium for culture;

[0023] Step 5: Resume culture

[0024] The explants were washed with washing solution, placed on a culture dish lined with sterile filter paper, excess liquid was sucked off and blown dry, and then inoculated on recovery culture medium for culture;

[0025] Step 6: Bud induction culture

[0026] The explants are inoculated into a bud induction medium for bud induction and screening culture. After one round of screening, the cotyledon nodes are removed and the embryonic buds with obvious growth are cut off and transferred into a fresh bud induction medium for the second round of screening culture.

[0027] Step 7: Bud elongation culture

[0028] Select the cotyledon nodes that have sprouted clustered buds, remove the cotyledons, cut new wounds on the hypocotyls, and inoculate them in bud elongation medium for cultivation;

[0029] Step 8: Rooting culture

[0030] When the clustered buds grow to about 2-3 cm, cut off the buds and transfer them to the rooting medium for rooting culture.

[0031] Preferably, in step 3, the method for disinfecting peas is: selecting plump, clean and well-growing pea seeds and spreading them in a sterile culture dish, placing the culture dish containing the peas in a well-sealed dryer, placing 200 mL of sodium hypochlorite solution in a 500 mL small beaker, and placing the small beaker in the dryer, taking 10 mL of concentrated hydrochloric acid with a pipette and adding it to the beaker containing the sodium hypochlorite solution, and quickly covering the lid of the dryer, and sealing and disinfecting for more than 8-10 hours.

[0032] Preferably, in step 3, the germination medium formula is:

[0033]

[0034] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25 °C.

[0035] Preferably, in step 4, the method of performing two activations of Agrobacterium bacterial liquid is as follows: three days before infection, the frozen Agrobacterium bacterial liquid is streaked onto an LB plate containing kanamycin and rifampicin resistance, and placed upside down in a 28°C incubator for dark culture for 2-3 days for the first bacterial cell activation. The night before infection, the activated bacterial cells are dipped into an LB liquid culture medium containing kanamycin and rifampicin resistance using an inoculation loop, and cultured overnight at 200 rpm and 28°C for secondary activation.

[0036] Preferably, in step 4, the formula of the infection liquid culture medium is:

[0037]

[0038]

[0039] Preferably, in step 4, the culture medium formula is:

[0040]

[0041] The culture conditions were: darkness, culture temperature 28°C.

[0042] Preferably, in step 5, the recovery medium formula is:

[0043]

[0044]

[0045] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

[0046] Preferably, in step 6, the bud induction medium formula is:

[0047]

[0048] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

[0049] Preferably, in step 7, the bud elongation medium formula is:

[0050]

[0051] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

[0052] Preferably, in step 8, the rooting medium formula is:

[0053]

[0054]

[0055] The culture conditions were: 12 h light / 12 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

[0056] Compared with the prior art, the present invention has achieved the following technical effects:

[0057] (1) The present invention screened out the pea variety "Zhongwan No. 6" which is easy to conduct genetic transformation experiments, and verified through experiments that the best explant is the cotyledon node, and the bud induction rate of the cotyledon node is above 80%;

[0058] (2) The present invention overcomes the problem of difficult rooting and obtains complete regenerated plants;

[0059] (3) The present invention successfully obtained pea plants with an albino phenotype by transforming the CRISPR / Cas9 gene editing vector targeting the pea PDS gene, and increased the genetic transformation efficiency to more than 5%, greatly improving the genetic transformation efficiency of peas. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The present invention is a flowchart of pea genetic transformation of a method for establishing a pea genetic transformation system. DETAILED DESCRIPTION

[0061] 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.

[0062] A method for establishing a pea genetic transformation system comprises the following steps:

[0063] Step 1: Transform plant expression vector into Agrobacterium competent cells

[0064] Step 1.1, take the EHA105 Agrobacterium competent cells stored at -80℃ and place them in room temperature or ice water bath for a while until they are partially thawed, and insert them into ice bath when they are in ice water mixing state;

[0065] Step 1.2, add 1ug of CRISPR / Cas9 gene editing vector plasmid DNA to every 100uL competent medium, stir the bottom of the tube by hand to mix, and then place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes;

[0066] Step 1.3, add 800uL of LB liquid medium without antibiotics and culture at 28℃ with shaking for 2-3 hours;

[0067] Step 1.4, centrifuge at 5000 rpm for 5 minutes to collect the bacteria, retain about 100uL of the supernatant, gently blow and resuspend the bacteria, spread it on the LB plate containing kanamycin and rifampicin resistance, and invert it in a 28℃ incubator for dark culture for 2-3 days;

[0068] Step 2: Preparation and storage of Agrobacterium culture solution

[0069] Step 2.1, pick 24 single colonies of Agrobacterium, streak them onto LB plates containing kanamycin and rifampicin resistance, and invert them in a 28°C incubator for dark culture for 2-3 days;

[0070] Step 2.2, use an inoculation loop to dip 24 Agrobacterium colonies into LB liquid medium containing kanamycin and rifampicin resistance, and culture at 200 rpm and 28°C for 2 days;

[0071] Step 2.3, pipette 500uL of bacterial solution into a centrifuge tube, add an equal volume of 60% glycerol, mix well, place in liquid nitrogen for 1 minute, and place the frozen Agrobacterium bacterial solution in a -80℃ refrigerator for later use;

[0072] Step 3: Preparation of sterile pea seedlings

[0073] Step 3.1, select plump, clean, and well-growing pea seeds and spread them flat in a sterile petri dish, place the petri dish containing the peas in a well-sealed desiccator, place 200 mL of sodium hypochlorite solution in a 500 mL small beaker, and place the small beaker in the desiccator, use a pipette to take 10 mL of concentrated hydrochloric acid and add it to the beaker containing the sodium hypochlorite solution, quickly cover the lid of the desiccator, and seal and disinfect for more than 8-10 hours;

[0074] Step 3.2, after disinfection, inoculate the pea seeds into the germination medium and culture for 5-7 days;

[0075] Among them, the germination medium formula is:

[0076]

[0077] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, culture temperature 25 °C;

[0078] Step 4. Infection and co-cultivation of explants

[0079] Step 4.1: Three days before infection, streak the frozen Agrobacterium culture onto an LB plate containing kanamycin and rifampicin resistance, invert and place in a 28°C incubator for dark culture for 2-3 days for the first bacterial activation. The night before infection, use an inoculation loop to dip the activated bacteria into LB liquid culture medium containing kanamycin and rifampicin resistance, shake and culture at 200 rpm and 28°C overnight for secondary activation;

[0080] Step 4.2, take out the germinated sterile seedlings from the clean bench, cut the cotyledons in half, and leave 3-5 mm hypocotyls;

[0081] Step 4.3, take 3-5mL of activated Agrobacterium bacterial solution, adjust to zero with LB liquid medium containing kanamycin and rifampicin resistance, and measure its absorbance value (OD600) at a wavelength of 600nm; centrifuge the remaining Agrobacterium bacterial solution at 5000rpm for 10min, collect the bacteria, pour out the supernatant, resuspend the bacteria with infection liquid medium, make the bacterial solution OD600=0.1-0.2, and culture at 4℃ for more than half an hour to activate the bacteria; place the explant in the resuspended and activated bacterial solution, place it at room temperature for 30min, shake it continuously during the period, pour out the bacterial solution, place the explant on a culture dish padded with sterile filter paper, absorb and blow dry the excess bacterial solution, and then inoculate it on the co-cultivation medium (two layers of sterile filter paper are laid on the culture medium), and culture it in the dark at 28℃ for 3-4 days;

[0082] Among them, the formula of infection liquid culture medium is:

[0083]

[0084] The culture medium formula is:

[0085] Step 5: Resume culture

[0086] After the co-cultivation, the explants were washed 4-5 times with washing solution, and after washing, the explants were placed on a culture dish lined with sterile filter paper, excess liquid was sucked off and blown dry, and then inoculated on the recovery culture medium and cultured for 4-5 days;

[0087] Among them, the recovery medium formula is:

[0088]

[0089] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C;

[0090] Step 6: Bud induction culture

[0091] After the recovery culture is completed, the explants are inoculated into a bud induction medium for bud induction and screening culture. One round of screening lasts about 2 weeks. After one round of screening, the cotyledonary nodes are removed and the embryos with obvious growth are cut off and transferred into a fresh bud induction medium for the second round of screening culture for about 2 weeks.

[0092] Among them, the bud induction medium formula is:

[0093]

[0094]

[0095] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C;

[0096] Step 7: Bud elongation culture

[0097] After the second round of screening, the cotyledon nodes that sprouted clustered buds were selected, the cotyledons were removed, new wounds were cut on the hypocotyls, and the buds were inoculated into the bud elongation medium. The elongation culture lasted for about 8 weeks, and the fresh medium was replaced every 2 weeks.

[0098] Among them, the formula of the bud elongation medium is:

[0099]

[0100]

[0101] The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C;

[0102] Step 8: Rooting culture

[0103] When the clustered buds grow to about 2-3 cm on the elongation medium, the buds are cut off and transferred to the rooting medium for rooting culture;

[0104] Among them, the rooting medium formula is:

[0105]

[0106]

[0107] The culture conditions were: 12 h light / 12 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

[0108] Embodiment 1:

[0109] The present invention selects two pea varieties "Zhongwan No. 6" and "Zhongqin No. 1" as different genotype materials as receptors for genetic transformation of peas. The test results show that after infection with Agrobacterium and co-cultivation for several days, the cotyledon nodes of "Zhongqin No. 1" are seriously browned and seriously affected by Agrobacterium, and the cotyledons soften and turn yellow. In the subsequent differentiation and cultivation stage, they gradually die. The explants of "Zhongwan No. 6" are not affected by Agrobacterium infection, and the growth of Agrobacterium is well inhibited, and clustered buds can be differentiated, and the growth state is good.

[0110] Embodiment 2:

[0111] The present invention selects pea cotyledon nodes, epicotyls and hypocotyls as explants for Agrobacterium-mediated genetic transformation. The test results show that the bud induction ability and the ability to resist Agrobacterium infection of the cotyledon nodes are greater than those of the epicotyls and hypocotyls respectively; in particular, the hypocotyls have almost no regeneration ability after Agrobacterium infection, and the bud induction ability of the epicotyls is also relatively weak. The bud induction rate of the cotyledon nodes is above 80%, and the cotyledon nodes have the ability to develop into complete regenerated plants.

[0112] Embodiment 3:

[0113] The invention sets different infection solution concentrations: OD600=0.1, OD600=0.2, OD600=0.6, OD600=0.8. Under the condition of controlling other variables to be the same, the test results show that as the infection solution concentration increases, the explant is more affected by Agrobacterium infection, and the growth of Agrobacterium is more difficult to be inhibited. Among them, when the concentration of Agrobacterium solution is OD600=0.1 and OD600=0.2, the explant is almost not affected by Agrobacterium infection, the growth of Agrobacterium can be basically inhibited in the bud induction and bud elongation stages, and there is no obvious effect on plant regeneration.

[0114] Embodiment 4:

[0115] The present invention sets different infection modes: ultrasonic infection and static infection at room temperature. Under the condition of controlling other variables to be the same, the test results show that after ultrasonic infection (ultrasonic time of 1 minute), the explant is greatly affected by Agrobacterium infection, it is difficult to inhibit the growth of Agrobacterium, and the induction and elongation of buds are affected; static infection at room temperature, accompanied by shaking during the period, after infection, the explant is slightly affected by Agrobacterium infection, the growth of Agrobacterium can be easily inhibited, and the induction and elongation of buds and plant regeneration are not affected.

[0116] Embodiment 5:

[0117] The present invention sets different room temperature static infection times: 15 minutes, 30 minutes, and 60 minutes. Under the condition of controlling other variables to be the same, test results show that when the infection time is 15 minutes and 30 minutes, the explant is slightly affected by the infection of Agrobacterium, the growth of Agrobacterium can be easily inhibited, and the induction and elongation of buds and the regeneration of plants are not affected; when the infection time is 60 minutes, the explant is greatly affected by the infection of Agrobacterium, the growth of Agrobacterium is difficult to inhibit, and a certain influence is produced on the induction and elongation of buds and the plants.

[0118] Embodiment 6:

[0119] The present invention sets different co-cultivation temperatures: 23°C, 25°C, and 28°C. When other variables are controlled to be the same, test results show that the degree of influence of Agrobacterium infection on explants, the difficulty of inhibiting Agrobacterium, bud induction, bud elongation, and plant regeneration are almost unrelated to the co-cultivation temperature. Even at 28°C, which is the most suitable temperature for the growth of Agrobacterium, the growth of Agrobacterium can be well inhibited after the infection of Agrobacterium liquid and the co-cultivation, and the regeneration of plants is not affected.

[0120] Embodiment 7:

[0121] The present invention sets different co-cultivation times: 2d, 3d, 4d and 5d. Under the condition of controlling other variables to be the same, the test results show that when the co-cultivation time is 2d, 3d and 4d, the degree of influence of Agrobacterium infection on the explant, the difficulty of inhibiting Agrobacterium, bud induction, bud elongation and plant regeneration are almost unrelated to the co-cultivation time; when the co-cultivation time is 5d, Agrobacterium grows more and is more difficult to control, so the preferred co-cultivation time is 3-4d.

[0122] Embodiment 8:

[0123] The present invention sets different concentrations of the screening agent hygromycin: 5 mg / L, 10 mg / L, 20 mg / L, and 50 mg / L. Under the condition of controlling other variables to be the same, the test results show that when the hygromycin concentration is 5 mg / L, although it has no effect on bud induction, the false positive rate of the screened regenerated buds is relatively high; when the hygromycin concentration is 10 mg / L, the transgenic positive seedlings can be better screened without affecting the regeneration of the plants; when the hygromycin concentration is 20 mg / L and 50 mg / L, although the positive rate of the screened transgenic seedlings is very high (almost all are transgenic positive seedlings), the screening pressure concentration at this time has seriously affected the regeneration of the plants.

[0124] Embodiment 9:

[0125] The present invention sets different antibiotic types and concentration combinations: (1) monoclonal antibody: carbenicillin, the concentration is 100mg / L, 200mg / L, 400mg / L; (2) monoclonal antibody: cephalosporin, the concentration is 100mg / L, 200mg / L, 400mg / L; (3) dual antibody: carbenicillin and cephalosporin, the concentration is 100mg / L, 150mg / L, 200mg / L, 250mg / L, 300mg / L, 400mg / L. Under the condition of controlling other variables to be the same, the test results show that adding a single antibiotic, the three different concentrations can not effectively inhibit the growth of Agrobacterium; when two antibiotics are added, and the antibiotic concentration is 250mg / L, respectively, the growth of Agrobacterium can be effectively inhibited, and the ability of plant regeneration is not affected.

[0126] Comparative Example 1:

[0127] Referring to the genetic transformation method of the leguminous crop alfalfa, an attempt was made to obtain regenerated plants of pea through somatic embryogenesis. After infecting pea explants and co-cultivating, the materials were transferred to callus maintenance medium containing antibiotics and cultured for 2 weeks. The culture conditions were: dark culture and culture temperature of 28°C.

[0128] The formula of callus maintenance medium is:

[0129]

[0130]

[0131] After 2 weeks, white callus tissue was generated. The well-growing callus tissue was clipped into small pieces and transferred to a new callus maintenance medium containing antibiotics for further 2 weeks. The culture conditions were: dark culture at 28°C.

[0132] The new callus maintenance medium formula is:

[0133]

[0134]

[0135] After 2 weeks, the well-growing callus tissue was clipped into small pieces and transferred to a new differentiation medium containing antibiotics for 2 weeks, with one subculture. The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28°C.

[0136] The differentiation medium formula is:

[0137]

[0138] Although pea callus was induced, the callus could not differentiate into buds, and during the differentiation culture process the callus gradually turned brown and hardened until it died.

[0139] This control experiment shows that pea, as a leguminous crop, has a regeneration pattern that is more similar to the organ regeneration pathway of soybeans, and it is difficult to regenerate plants through the somatic embryo pathway.

[0140] The present invention screened out the pea variety "Zhongwan No. 6" which is easy to conduct genetic transformation test through the above experiment, verified that the best explant is the cotyledon node, and the bud induction rate of the cotyledon node is above 80%. By controlling the experimental variables, the optimal condition combination for the pea genetic transformation experiment was determined, namely, when the concentration of the infection solution was OD600 = 0.1-0.2, the cotyledon node was infected for 30 minutes, and the cells were cultured in the dark at 28°C for 3-4 days. Regenerated seedlings were obtained in a shoot induction medium (added with 2mg / L 6-BA) supplemented with 10mg / L hygromycin, 250mg / L carbenicillin and 250mg / L cephalosporin, and a shoot elongation medium (added with 0.5mg / L GA3, 0.1mg / L IAA, 1mg / L ZT, 50mg / L asparagine, 50mg / L pyroglutamic acid). The regenerated seedlings successfully took root in a rooting medium (added with 1mg / LIBA, 50mg / L asparagine, 50mg / L pyroglutamic acid), thus overcoming the problem of difficult rooting and obtaining complete regenerated plants. By transforming the CRISPR / Cas9 gene editing vector targeting the pea PDS gene, pea plants with albino phenotype were successfully obtained, and the genetic transformation efficiency was increased to more than 5%, greatly improving the genetic transformation efficiency of peas.

[0141] 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 establishing a pea genetic transformation system, characterized in that: The following steps are involved: Step 1: Transform plant expression vector into Agrobacterium competent cells Step 1.1, take EHA105 Agrobacterium competent cells and wait for them to partially melt, and insert them into an ice bath when they are in an ice-water mixture state; Step 1.2, add CRISPR / Cas9 gene editing vector plasmid DNA, mix well, and place on ice, in liquid nitrogen, in a water bath, and in an ice bath in sequence; Step 1.3, add LB liquid medium without antibiotics and culture with shaking; Step 1.4, collect the bacteria by centrifugation, keep the supernatant, blow and resuspend the bacteria, spread it on the LB plate containing kanamycin and rifampicin resistance, and invert it to culture in the incubator in the dark; Step 2: Preparation and storage of Agrobacterium culture solution Step 2.1, pick a single colony of Agrobacterium, streak it onto an LB plate containing kanamycin and rifampicin resistance, and place it upside down in an incubator for dark culture; Step 2.2, use an inoculation loop to dip the Agrobacterium colony into LB liquid medium containing kanamycin and rifampicin resistance, and shake and culture; Step 2.3, pipette the bacterial solution into a centrifuge tube, add glycerol, mix well, put it in liquid nitrogen, and put the frozen Agrobacterium bacterial solution in a refrigerator for later use; Step 3: Preparation of sterile pea seedlings Step 3.1, sterilize the peas in a closed environment; Step 3.2, inoculating pea seeds into germination medium for cultivation; Step 4. Infection and co-cultivation of explants Step 4.1, activating the Agrobacterium culture solution twice; Step 4.2, take out the germinated sterile seedlings, cut the cotyledons in half, and leave the hypocotyl; Step 4.3, take the activated Agrobacterium bacterial solution, measure its absorbance at a wavelength of 600nm, centrifuge the remaining Agrobacterium bacterial solution, collect the bacteria, pour out the supernatant, resuspend the bacteria with the infection liquid culture medium, and activate the bacteria; put the explant in the resuspended and activated bacterial solution, let it stand and then shake it continuously, pour out the bacterial solution, put the explant on a culture dish lined with sterile filter paper, absorb and blow dry the excess bacterial solution, and then inoculate it on the co-cultivation medium for culture; Step 5: Resume culture Wash the explants with washing solution, place them on a petri dish lined with sterile filter paper, remove and blow dry the excess liquid, and then inoculate them on recovery medium for culture; Step 6: Bud induction culture The explants are inoculated into a bud induction medium for bud induction and screening culture. After one round of screening, the cotyledonary nodes are removed and the embryonic buds with obvious growth are cut off and transferred into a fresh bud induction medium for the second round of screening culture. Step 7: Bud elongation culture Select the cotyledon nodes that have sprouted clustered buds, remove the cotyledons, cut new wounds on the hypocotyls, and inoculate them in bud elongation medium for cultivation; Step 8: Rooting culture When the clustered buds grow to about 2-3 cm, cut off the buds and transfer them to the rooting medium for rooting culture.

2. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In the step 3, the method for disinfecting peas is as follows: plump, clean and well-growing pea seeds are selected and spread flat in a sterile culture dish, the culture dish containing the peas is placed in a well-sealed dryer, 200 mL of sodium hypochlorite solution is placed in a 500 mL small beaker, and the small beaker is placed in the dryer, 10 mL of concentrated hydrochloric acid is taken with a pipette and added to the beaker containing the sodium hypochlorite solution, and the lid of the dryer is quickly closed, and the dryer is sealed and disinfected for more than 8-10 hours.

3. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In step 3, the germination medium formula is: The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 25 °C.

4. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In step 4, the method for performing two activations of the Agrobacterium bacterial liquid is as follows: three days before infection, the frozen Agrobacterium bacterial liquid is streaked onto an LB plate containing kanamycin and rifampicin resistance, and inverted in a 28° C. incubator for dark culture for 2-3 days to perform the first bacterial cell activation. The night before infection, the activated bacterial cells are dipped into an LB liquid culture medium containing kanamycin and rifampicin resistance using an inoculation loop, and cultured overnight at 200 rpm and 28° C. for secondary activation.

5. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In step 4, the formula of the infection liquid culture medium is:

6. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In step 4, the culture medium formula is:

7. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In step 5, the recovery medium formula is: The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

8. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In step 6, the bud induction medium formula is: The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

9. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In step 7, the bud elongation medium formula is: The culture conditions were: 16 h light / 8 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

10. The method for establishing a pea genetic transformation system according to claim 1, characterized in that: In step 8, the rooting medium formula is: The culture conditions were: 12 h light / 12 h dark, light intensity 3000 Lux, and culture temperature 28 °C.

Images