CN113287526A - Chinese cabbage type rape regeneration system establishment and anther culture method - Google Patents
Chinese cabbage type rape regeneration system establishment and anther culture method Download PDFInfo
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- CN113287526A CN113287526A CN202110758296.3A CN202110758296A CN113287526A CN 113287526 A CN113287526 A CN 113287526A CN 202110758296 A CN202110758296 A CN 202110758296A CN 113287526 A CN113287526 A CN 113287526A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention belongs to the technical field of plant culture, and discloses a method for establishing a cabbage type rape regeneration system and culturing anthers, which analyzes the influence of dark culture on seed germination indexes and growth indexes in different soaking times, different disinfection modes and different time through a single-factor experiment to obtain the optimal soaking time, disinfection mode and dark treatment time; and screening out the optimal differentiation culture medium by comparing the influence of different hormone combinations on the induction and differentiation rates of different explant calli. According to the method, sterile water soaking for 6h is obtained as the optimal soaking time through the influence of different soaking times on the seed germination indexes, so that the seed dormancy can be broken, and the rooting and germination of the seeds can be accelerated; different disinfection modes have different influences on the germination index and the growth index of the explant, and the invention obtains that the seeds soaked for 6 hours are disinfected in 3 percent sodium hypochlorite solution for 10 minutes and then are subjected to dark culture for 3 days, so that the aim of consistent seed germination and plant growth can be achieved.
Description
Technical Field
The invention belongs to the technical field of plant culture, and particularly relates to a method for establishing a cabbage type rape regeneration system and culturing anthers.
Background
At present, rape belongs to a cultivation sub-population in Brassica species (b.campestis L.) of Brassica of Cruciferae (Cruciferae), is one of important oil crops in China and is also the second largest oil crop in the world. In oil crops planted in China, rape is the most potential and hopeful variety, and is the optimal choice for guaranteeing and improving the self-sufficiency rate of edible oil in China, China is taken as a world with large oil production and consumption, and the self-sufficiency rate is less than 35%, so that the current breeders study the improvement of the yield and quality of rape and the cultivation of new disease-resistant and stress-resistant rape varieties as important contents, and the rape can be subjected to tissue culture by using hypocotyls, cotyledons, cotyledon stalks, roots, stems, anthers, microspores and the like as explants in the existing research reports. A relatively perfect rape in-vitro culture regeneration system is established at present, and a large amount of high-quality germplasm resources are obtained. However, in the tissue culture of rape, the optimum regeneration conditions of the tissue culture of rape are greatly different due to the difference of genotype and explant, and particularly, the research on the regeneration system of the rape is rarely reported. The Chinese cabbage type winter rape is not only a multifunctional crop, but also has the functions of strong cold resistance, less pest and disease damage, fertilizer and soil conservation and the like. Therefore, establishing high-efficiency isolated culture of the brassica rapa pekinensis with excellent variety characteristics by taking Nebi as an example is very important for subsequent genetic transformation of brassica napus. The research on the optimal conditions of the regeneration system of the cabbage type rape has important significance for establishing high-efficiency in-vitro culture and subsequent genetic transformation of the cabbage type rape of the cold-resistant variety.
In recent years, anther tissue culture techniques have received increasing attention. For example, the research on rice, flax, medicago falcata, tomatoes and the like is successful. The anther culture of rape obtains the regeneration plant as early as 1975, but the induction rate of the plant is very low, and the plant cannot be applied to breeding work. There have been few reports on the research on the Chinese cabbage type rape anther culture and breeding practice since the 70 s. At present, anther culture generally improves callus induction and shoot regeneration by adjusting the hormone content and combination, sugar type and concentration of a culture medium. The anther culture pretreatment can keep the microspore in stronger viability, delay and prevent pollen abortion and increase the number of activated pollen. The common anther culture pretreatment method comprises the modes of low temperature, high temperature, centrifugation, mannitol, colchicine and the like, and improves the induction rate of embryos or calluses.
Through the above analysis, the problems and defects of the prior art are as follows:
in the culture of rape tissues, the optimal regeneration conditions of the rape tissue culture are greatly different due to the difference of genotypes and explants. The genotype and the culture medium have certain influence on the induction rate of the callus and the regeneration rate of the green seedling, but no relevant research report is found at present.
The difficulty in solving the above problems and defects is:
the callus induction is mainly genotype dependence, the anther culture of different crops has no reproducibility, and the anther callus induction rate is influenced by the possible existence of different chemical components.
The significance of solving the problems and the defects is as follows:
the formation of anther callus is the basis of haploid plant survival and double haploid generation, and provides a pure line breeding material for genetic research of brassica campestris.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for establishing a cabbage type rape regeneration system and culturing anther.
The invention is realized in such a way that the method for establishing a cabbage type rape regeneration system and culturing anther comprises the following steps:
the influence of dark culture on the germination indexes and growth indexes of the seeds in different soaking time, different disinfection modes and different time is researched through a single-factor experiment, and the optimal soaking time, disinfection mode and dark treatment time are obtained; by comparing the influence of different hormone combinations on the induction and differentiation rates of calli of different explants, the hypocotyl, the optimal pre-culture medium of the cotyledon with stalk, the optimal differentiation culture medium of the hypocotyl and the optimal differentiation culture medium of the cotyledon with stalk of the cabbage type rape are screened out.
Further, the sterile water is soaked for 6 hours, the disinfection mode is that 3% sodium hypochlorite solution is disinfected for 10 minutes, and the dark treatment time is dark culture for 3 days.
Further, the optimal pre-culture medium for the hypocotyl and the cotyledon with stalk of the cabbage type rape is as follows: MS +1 mg/L2,4-D +2 mg/L6-BA; the optimal differentiation medium of the hypocotyls is as follows: MS +0.1mg/L IBA +2 mg/L6-BA +5mg/L AgNO3(ii) a The optimum differentiation culture medium of cotyledon with stalk is as follows: MS +0.1mg/L IBA +3 mg/L6-BA +5mg/L AgNO3。
Further, the method for establishing the cabbage type rape regeneration system specifically comprises the following steps:
(1) seed soaking treatment: selecting seeds which are free of insect damage and full in grains, soaking the seeds in sterile water for 0 hour, 3 hours, 6 hours, 12 hours and 24 hours, carrying out planting culture in 5 treatment modes respectively, repeating each treatment mode for three times, repeating each treatment mode for 20 seeds, recording the germination number of the seeds in 7 days every day, and calculating the germination vigor, the germination rate, the germination index and the vitality index after 7 days;
(2) seed disinfection: selecting seeds which are free of insect damage and have full grains, numbering the seeds in advance, soaking the seeds in sterile water in the same way as the optimal soaking method in the step (1), and sucking out the soaked sterile water by using a liquid transfer gun; sterilizing the surface of the container with 75% alcohol for 30s, and washing the container with sterilized water for 3 times, wherein the container is continuously shaken; after the liquid transferring gun sucks out the alcohol, the sterile water is washed for three times; the numbered seeds will then be individually assigned 0.1% HgCl2Soaking the solution in 3% sodium hypochlorite solution for 5min, 10min, and 15 min; repeatedly washing with sterilized water for 5 times, and recording the number of seeds germinated every day for 7 daysCalculating the germination vigor, the germination rate, the germination index, the vitality index and the pollution rate after 7 days;
(3) dark treatment and culture of seeds: selecting seeds which are free of insect damage and full in grains, wherein the soaking mode is the same as the optimal soaking method in the step (1), the disinfection mode is the same as the optimal disinfection method in the step (2), the planting mode is the same as the step (1), the seeds are placed in an incubator (25-27 ℃) for dark culture, 4 treatments are set, the dark culture is performed for 1, 2, 3 and 4d respectively, then the seeds are placed in an illumination incubator, the treatment is repeated for 3 times, the germination number of the seeds in 7 days is recorded, and the germination vigor, the germination rate, the germination index, the vigor index and the seedling rate are calculated after 7 days;
(4) cutting the explants: selecting aseptic seedlings with the age of 5-6d, cutting cotyledons with stalks and hypocotyls, and spreading all explants in a callus culture medium;
(5) effect of different hormone combinations on different explant cultures: selecting aseptic seedlings with the growth seedling age of 5-6d, cutting the explants, inoculating the well-grown explants into a pre-culture medium by adopting the method in the step (4), placing the explants in the pre-culture medium at the temperature of 25-28 ℃ and the illumination intensity of 60 mu mol-2.s-1And light: culturing for 5 days in a dark period of 16h to 8h, then respectively inoculating the explants on a differentiation culture medium, then placing the explants in an artificial tissue culture room, continuously culturing, wherein each repetition is 20 explants, and treating the explants for 36 kinds;
(6) rooting culture: adventitious shoots growing to about 1-2cm in differentiation medium were excised from the explant base on a clean bench and inserted vertically into rooting medium: placing 0.2mg/L IBA +1/2MS in an illumination incubator for culture, and counting the rooting rate;
(7) testing and counting the test data;
(8) analyzing the influence of different soaking modes, different disinfection modes and different dark treatment time on seed germination and the influence of different hormone combinations on hypocotyl budding of the cabbage type rape.
Further, in the step (1), the planting method adopted in the planting culture comprises:
firstly, washing selected seeds for 2 times by using distilled water, soaking the seeds in 75% alcohol for 30s, and continuously shaking the container to ensure that the alcohol can fully contact each seed; after sucking out alcohol by a liquid transfer gun, washing for 3 times by using sterile water; taking out and putting on sterilized filter paper to fully absorb water; MS basic culture medium is accessed into the superclean bench; culturing in a light incubator.
Further, in the step (7), the measurement method of measuring the test data is:
measuring the diameter of the seed by a vernier caliper;
the germination potential (%) -3 d total germination number/inoculated seed number x 100;
the germination rate (%) is equal to the number of germinated seedlings/inoculated seeds within 7d multiplied by 100;
germination Index (GI) ═ Σ (GT/Dt), where Dt represents the number of days of germination and GT represents the number of seeds germinated per day corresponding to Dt.
Callus induction (%) × 100 (number of calli/total number of explants inoculated).
By combining all the technical schemes, the invention has the advantages and positive effects that:
according to the method, the influence of different soaking times on the seed germination indexes is researched, and the sterile water soaking time of 6 hours is obtained as the optimal soaking time, so that the seed dormancy can be broken, and the rooting and germination of the seeds can be accelerated; different disinfection modes have different influences on the germination index and the growth index of the explant, and the invention has the advantages that the pollution rate is lowest and the effect is best after the seeds soaked for 6 hours are disinfected in 3 percent sodium hypochlorite solution for 10 min; the influence of dark culture on growth indexes of aseptic seedlings at different time is different, and the invention obtains that the seeds soaked for 6 hours are disinfected by 3 percent sodium hypochlorite solution for 10min and then are subjected to dark culture for 3d, so that the growth rate of cotyledons can be inhibited to a certain extent, and the purposes of consistent seed germination and plant growth can be achieved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flow chart of a method for establishing a cabbage type rape regeneration system according to an embodiment of the present invention.
FIG. 2 is a flow chart of the anther culture method of Brassica campestris L.var.campestris according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for establishing a cabbage type rape regeneration system and culturing anther, and the invention is described in detail with reference to the attached drawings.
As shown in fig. 1, the method for establishing a brassica campestris regeneration system according to the embodiment of the present invention includes:
s101, soaking seeds;
s102, disinfecting seeds;
s103, dark treatment and culture of seeds;
s104, cutting an explant;
s105, influence of different hormone combinations on different explant cultures;
s106, rooting culture;
s107, testing and counting the test data;
and S108, analyzing the influence of different soaking modes, different disinfection modes and different dark treatment times on seed germination and the influence of different hormone combinations on hypocotyl budding of the cabbage type rape.
As shown in fig. 2, the anther culture method of brassica napus provided by the embodiment of the present invention includes:
s201, sterilizing and darkening anthers;
s202, low-temperature pretreatment;
s203, mannitol pretreatment;
s204, sucrose pretreatment;
s205, measuring an index;
s206, analyzing data;
s207, observing the shapes of flower buds and anther callus of different parts of the cabbage type rape, and analyzing the influence of different induction culture mediums on the anther induction rate of different bud taking parts, the influence of low-temperature pretreatment on the anther induction rate of the cabbage type rape, the influence of mannitol treatment on the anther callus and the influence of cane sugar on the anther callus.
The invention is further described below in connection with specific tests.
Firstly, a method for establishing a cabbage type rape regeneration system comprises the following steps:
1. materials and methods
1.1 Experimental materials and instruments
Experimental materials: cabbage type rape Neib seeds were used as the test material. MS minimal medium (30g/L sucrose, 6g/L agar, pH 5.7-5.9).
The test instrument: the test is carried out in a Sujing Antai SW-CJ-2F clean bench, an electronic balance, an autoclave and a Zhujiang brand constant-temperature illumination incubator (except for dark culture, the test culture temperature of all the rest in the illumination incubator is 26.0 ℃, the illumination intensity is 2000LX, the illumination is carried out for 16 h/8 h, and the relative air humidity is about 60%).
1.2 test methods
1.2.1 seed soaking treatment
Selecting seeds which are free of insect pests and full in grains, soaking the seeds in sterile water for 0, 3, 6, 12 and 24 hours for 5 treatments, wherein the planting method comprises the following steps: firstly, washing selected seeds for 2 times by using distilled water, soaking the seeds in 75% alcohol for 30s, and continuously shaking the container to ensure that the alcohol can fully contact each seed; after sucking out alcohol by a liquid transfer gun, washing for 3 times by using sterile water; taking out and putting on sterilized filter paper to fully absorb water; MS basic culture medium is accessed into the superclean bench; culturing in a light incubator. Each treatment was repeated three times, 20 seeds each, and the number of sprouts per day was recorded for 7 days, and the germination vigor, germination percentage and germination index, vigor index were calculated after 7 days.
1.2.2 seed Sterilization
Selecting seeds which are free of insect damage and full in grains, numbering in advance, soaking in sterile water in the same optimal soaking method as that in 1.2.1, and sucking out the soaked sterile water by using a liquid transfer gun; sterilizing the surface of the container with 75% alcohol for 30s, and washing the container with sterilized water for 3 times, wherein the container is continuously shaken; after the liquid transferring gun sucks out the alcohol, the sterile water is washed for three times; the numbered seeds will then be individually assigned 0.1% HgCl2Soaking the solution in 3% sodium hypochlorite solution for 5min, 10min, and 15 min; and repeatedly washing with sterilized water for 5 times. The planting method is the same as 1.2.1, the number of seeds sprouting every day in 7 days is recorded, and the germination vigor, the germination rate, the germination index, the vitality index and the pollution rate are calculated after 7 days.
1.2.3 seed dark treatment culture
Selecting seeds which are free of insect pests and full in grains, wherein the soaking mode is the same as the optimal 1.2.1 soaking method, the disinfection mode is the same as the optimal 1.2.2 disinfection method, the planting mode is the same as 1.2.1, placing the seeds in an incubator (25-27 ℃) for dark culture, setting 4 treatments, performing dark culture for 1, 2, 3 and 4 days respectively, then placing the seeds in an illumination incubator, repeating the treatment for 3 times, recording the germination number of the seeds every day for 7 days, and calculating the germination vigor, the germination rate, the germination index, the vitality index and the seedling rate after 7 days.
1.2.4 excision of explants
Selecting aseptic seedlings with the age of 5-6 days. Cutting cotyledon with stalk to obtain cotyledon with stalk 0.2-0.4cm2And (3) paving all explants in a callus culture medium on average, and inoculating to ensure that the cotyledon faces upwards. The hypocotyl cutting method comprises the following steps: firstly, cutting off terminal buds, and then cutting the terminal buds into stem sections with the length of 0.5-0.8 cm. All explants were plated in callus medium.
1.2.5 Effect of different hormone combinations on different explant cultures
Selecting aseptic seedling with 5-6D of growth seedling age, cutting explant by 1.2.4. the method comprises inoculating well-grown explant into pre-culture medium (composed of MS minimal medium and certain concentration of 2,4-D, 6-BA) shown in Table 1, placing at 25-28 deg.C, and illumination intensity of 60 μmol.m-2.s-1And culturing in dark for 16h:8h for 5 days, inoculating into differentiation culture medium (composed of minimal medium and IAA, 6-BA and AgNO at certain concentration) shown in Table 23Composition), placing in an artificial tissue culture room (culture condition is same as that of preculture), continuously culturing, wherein each repetition is 20 explants, and treating for 36 kinds. Namely: A1B1, A1B2, A1B3, A1B4, A1B5, A1B6, A1B7, A1B8, A1B9, A2B1, A2B2, A2B3, A2B4, A2B5, A2B6, A2B7, A2B8, A2B9, A3B1, A3B2, A3B3, A3B4, A3B5, A3B6, A3B7, A3B8, A3B9, A4B1, and A4B 1.
TABLE 1 Pre-culture Medium for explants at different sites
TABLE 2 differentiation medium for explants at different positions
1.2.7 rooting culture
Adventitious shoots growing to about 1-2cm in differentiation medium were excised from the explant base on a clean bench and inserted vertically into rooting medium: 0.2mg/L IBA +1/2MS was cultured in a light incubator. And (5) counting the rooting rate.
1.3, determination and statistics
1.3.1 measurement index
Measuring the diameter of the seed by a vernier caliper;
the germination potential (%) -3 d total germination number/inoculated seed number x 100;
the germination rate (%) is equal to the number of germinated seedlings/inoculated seeds within 7d multiplied by 100;
germination Index (GI) ═ Σ (GT/Dt), where Dt represents the number of days of germination and GT represents the number of seeds germinated per day corresponding to Dt.
Callus induction (%) (number of calli/total number of inoculated explants) × 100;
1.4 statistical analysis
The test data were calculated and collated using Microsoft Excel 2007 software, and the analysis of variance was performed using SPSS 19.0 software
2. Results and analysis
2.1 Effect of different soaking modes on seed Germination
As shown in table 3, as the soaking time is prolonged, the germination rates of the treatments tend to increase first and then decrease as a whole, and when the soaking time is 6 hours, the germination rate is the highest (99%), and then the germination rate is 94.33% for 3 hours, and the germination rate for 6 hours is slightly higher than 3 hours, but there is no significant difference, but the germination rate for 6 hours is significantly higher than the germination rate for 3 hours, and the germination rate and the germination index are high, which indicates that the dormancy of the seeds can be effectively broken through after the seeds are soaked in sterile water for 6 hours, and the self-vitality and vitality of the seeds can be excited; when the soaking time is 24 hours, the germination rate is obviously reduced, and is reduced by 17.48 percent compared with a blank control, which indicates that certain damage is caused to the seeds when the sterile water is soaked for too long time. Therefore, the optimal soaking time should be 6 hours.
TABLE 3 Effect of immersion on the Germination of Brassica campestris seeds
Different lower case letters in the same column represent significant differences (p ≦ 0.05) for the test.
2.2 Effect of different Disinfection modes on seed Germination
As can be seen from Table 4, 0.1% HgCl was used2And 3% NaClO had different effects on the germination of the seeds after the seeds were sterilized, respectively. 0.1% HgCl at the same sterilisation time as a whole2All of which are less than 3% NaClO, but the difference is not significant, wherein the germination vigor, germination rate and germination index of the seeds tend to decrease continuously with the increase of the disinfection time, at 0.1% HgCl2The germination vigor, the germination rate and the germination index are all the lowest when the mixture is sterilized by 3 percent NaClO for 15 min; 0.1% HgCl when the treatment time was the same2The pollution rates of the raw materials are all less than 3 percent of NaClOThe contamination rate of the seeds tended to decrease continuously with the increase of the disinfection time, with 0.1% HgCl2The pollution rate is lowest after 15min of treatment, but the difference with the pollution rate of 10min of 3 percent NaClO treatment is not obvious. In conclusion, the germination rate, the germination potential and the germination index of the 3 percent NaClO treated for 15min are all higher than 0.1 percent HgCl2The treatment time is 10min and 15min, but the pollution rate is 0.1 percent of HgCl2There was no significant difference in 10min treatment, so 3% NaClO treatment for 10min was the best mode of disinfection.
TABLE 4 Effect of Disinfection mode on seed Germination
Table3-4 Effects of disinfection methods on seed germination
Different lower case letters in the same column represent significant differences (p ≦ 0.05) for the test.
2.3 Effect of different time dark treatments on seed Germination
As shown in table 5, the dark treatment for a certain period of time on the seeds can improve the germination of the seeds in a certain range compared with 0d (light treatment), wherein the germination rate, the germination vigor and the germination index of the dark treatment for 3d are all the highest and are respectively 17.45%, 19.21% and 22.98% higher than those of the light control, and the seedling rate, the seedling length and the vitality index are respectively 41.67%, 34.44% and 36.73% higher than those of the light treatment. The germination indexes of 1d and 2d of dark culture have no obvious difference, and the influence on seed germination is small; although the germination rate, the germination index and the germination potential are not significantly different from those of 3d when the hypocotyls are cultured in dark for 4d, the seedling rate and the vitality index of 3d cultured in dark are significantly higher than those of 4d, the growth potential of 1d cultured in dark is not significantly different from that of a control, and the hypocotyls cultured in dark for 2d are obviously elongated compared with the hypocotyls cultured in dark for 1 d; the seedlings subjected to dark treatment for 4d have uneven overall growth conditions, narrow and thin leaves and weak overall; the seeds cultured in the dark for 3d have regular buds, and the seedling rate and the seedling height are higher than those of other treatments, so the optimal dark culture treatment time is 3 d.
TABLE 5 Effect of dark treatment on seed Germination
Different lower case letters in the same column represent significant differences (p ≦ 0.05) for the test.
2.4 Effect of different hormone combinations on hypocotyl sprouting of Brassica campestris
The hypocotyl is inoculated into a pre-culture medium for culturing for 5-7 days, then the callus with better growth vigor is inoculated into differentiation culture media with different treatments, and the hypocotyl can generate buds after 40 days, but the differentiation rate has obvious difference due to different hormone combination treatments. As can be seen from tables 6 and 7, hypocotyls treated with the hormone combinations A1B1, A1B2, A1B3 and A1B4 in the pretreatment medium without 6-BA were low in callus induction rate and germination rate, and they were experimentally observed to be contained in the formed callus at a relatively slow pace, and the treatment with 6-BA was relatively high in differentiation rate, short in callus formation time and high in final germination rate. When B is not changed, namely the differentiation medium is not changed, the pre-culture medium is changed from A1 to A4, namely the mass concentration of 6-BA is increased, the callus induction rate of hypocotyls of the combination of B1, B2, B3 and A1 to A4 is in a continuously increasing trend, and the callus induction rate of the hypocotyls of the combination of the rest differentiation medium (B) and the pre-culture medium (A) is increased and then reduced, and reaches a maximum value of 87.19 percent in A3B5 (2 mg/L of 6-BA). AgNO when A is unchanged, i.e.the pre-culture medium is unchanged, the differentiation medium is changed from B1 to B3 or from B4 to B6 or from B7 to B93When the mass concentration of 6-BA is increased for fixing the mass concentration, the callus induction rate and the germination rate of the hypocotyl are increased firstly and then reduced, and maximum values appear at B5 and B6 respectively. Wherein, in A3B5, A3B6 and A4B6, the germination rate of hypocotyls reaches 46.41 percent and 23.21 percent. 22.99 percent. When the 2,4D in the pre-culture medium is 1mg/L and the 6-BA is 2mg/L, the concentration of IBA in the differentiation culture medium is 0.1mg/L, the 6-BA is 2mg/L, and AgNO3When the mass concentration of (A) is 3mg/L,that is, the differentiation rate of treatment A3B5 reached as high as 46.41%.
TABLE 6 Effect of different hormone combinations on hypocotyl callus induction
Different lower case letters in the same column represent significant differences (p ≦ 0.05) for the test.
The same goes for
TABLE 7 Effect of different hormone combinations on hypocotyl callus differentiation
2.5 Effect of different hormone combinations on cotyledon budding of Brassica campestris
The cotyledon does not have buds on all the treated culture mediums, the callus induction rate and the germination rate have obvious difference due to different treatments, as shown in table 8, the callus induction rate of the cotyledon treated by the hormone combination of the pretreatment culture medium (A) and B1 and B2 reaches more than 52.56%, but the germination rate is 0, and the callus formed by the cotyledon is slow and seriously browned through experimental observation. When B is not changed, namely the differentiation medium is not changed, and the pre-culture medium is changed from A1 to A4, namely the mass concentration of 6-BA is increased, the callus induction rate of the pedunculate cotyledons is increased and then decreased, and reaches the maximum value of 91.93 percent at A3 (2 mg/L of 6-BA). As shown in Table 9, AgNO was obtained when A was unchanged, i.e.the pre-culture medium was unchanged, and the differentiation medium was changed from B1 to B3 or from B4 to B6 or from B7 to B93For a fixed mass concentration, 6-BA substanceWhen the quantitative concentration is increased, the callus induction rate and the germination rate of the cotyledon with stalk also increase first and then decrease, and the maximum values appear at B5, B6 and B9 respectively. Wherein, the germination rate of the cotyledon with stalk reaches 30% in A3B5, A3B6 and A3B 9. The cotyledon under each treatment can generate callus, the callus induction rate is greater than that of the hypocotyl, but the germination rate is less than that of the hypocotyl. Therefore, the hypocotyl of the cabbage type rape is more suitable to be used as the explant material of the regeneration system than the hypocotyl with the stem leaves. The invention shows that when the 2,4-D in the pre-culture medium is 1mg/L and the 6-BA is 2mg/L, the concentration of IBA in the differentiation culture medium is 0.1mg/L, the concentration of 6-BA in the differentiation culture medium is 2mg/L, and AgNO3When the mass concentration of (A3B 6) is 3mg/L, the differentiation rate of the treated A3B6 is up to 30.9%.
TABLE 8 Effect of different hormone combinations on cotyledon callus induction
TABLE 9 Effect of different hormone combinations on cotyledon callus differentiation
2.6, rooting
Adventitious shoots growing to about 1-2cm in differentiation medium were excised from the explant base and inserted vertically into rooting medium: 0.2mg/L IBA +1/2MS is placed in a light incubator for cultivation, and the rooting rate is 62.5%.
3、
Seed germination is a key step in plant growth and development. The method comprises the following steps of carrying out early-stage treatment on the cabbage type rape seeds in three aspects, namely, sterile water soaking time, different concentration disinfection modes and different time dark treatment. The experiment shows that the germination rate of the seeds is the highest after the seeds are soaked for 6 hours. When the soaking time is short, the dormancy of the seeds can not be completely broken, the seeds are in a semi-dormant state, and all indexes of the seeds are lower than those after soaking for 6 hours. When the soaking time is long, the seeds can not fully breathe in oxygen, so that the seeds can not breathe in oxygen, ethanol is greatly accumulated in the seeds, and the seeds are rotten and bud.
By comparing the influence of 6 different disinfection modes and different time treatments on seed germination, the germination rate, the germination potential and the germination index of the seeds treated by 3% NaClO for 10min are all higher than those of other groups, the pollution rate is lower than those of other groups, and therefore the treatment of the group is the best one. The most commonly used disinfection method before is HgCl2In recent years, it is found that the mercury bichloride treatment time is too long, which can cause certain damage to the explant, even inactivate the explant, and affect the effect of tissue culture. The present inventors have found that HgCl2The indexes of the treatment group are all worse than those of the NaClO treatment group, and one of the reasons for this may be that HgCl is used2During disinfection, embryo poisoning is caused, and the germination rate is further reduced.
The plant seeds are subjected to dark treatment for a proper time, so that the germination rate of the seeds can be improved, the cabbage type rape seeds are subjected to dark culture for 3 days in the experiment, the sprouts are neat, and the seedling rate and the seedling height are higher than those of other treatments. Because the light-sensitive pigment B (phyB) light receptor receives light signals and regulates the biosynthesis and signal transduction of endogenous hormones, the seed germination is further regulated and controlled. The phyB-RVE1-RGL2 module regulates seed dormancy and germination by integrating light signaling, GA synthesis, and GA signaling pathways. This provides a theoretical basis for why the plant seeds are dark treated for a certain period of time.
When the explant grows in an environment containing auxin or auxin analogs, the differentiation of the explant is promoted, and the differentiation rate is increased. The experiments show that 2,4-D has an indispensable effect on inducing callus formation, the concentration of 6-BA has different effects on callus generation, and in the experiments, we find that low-concentration 2,4-D and 6-BA have a promoting effect on the differentiation and growth of adventitious buds of rape, but have an inhibiting effect on the differentiation and growth of adventitious buds if the concentration is too high, but the differentiation and growth efficiency of adventitious buds is higher when the concentration proportion is combined properly, which may be possibly combined with internal and external aspectsThe attainment of a specific equilibrium state of the source hormone is associated with the presence of a state of saturation of the hormone receptors. AgNO addition to media of hypocotyls and peduncles of Brassica oleracea was indicated in previous reports3Can obviously improve the regeneration frequency of the buds, promote the early differentiation of the buds and also promote the regeneration frequency of hypocotyls. On the basis of the above, our research shows that: in both the hypocotyl budding experiment of Brassica campestris and cotyledon budding experiment, when 2,4-D and 6-BA in the pre-culture medium are 1mg/L and 2mg/L respectively, IBA concentration in the differentiation medium is 0.1mg/L, 6-BA is 2mg/L, and AgNO is3The highest differentiation rate was obtained at a mass concentration of 3 mg/L. Different explants are induced to grow on a culture medium with the highest differentiation rate, the formed callus has a compact structure and a tumor shape, and can form buds, but the regeneration capability is different, and in the experiment, the hypocotyl budding rate is more than the cotyledon budding rate, which is probably due to the different auxin content in the plant body. Therefore, the hypocotyl is more suitable for being used as a material for tissue culture and subsequent analysis of the cabbage type rape.
4, in the experiment, sterile water soaking for 6 hours is obtained as the optimal soaking time through the influence of different soaking times on the seed germination indexes, so that the dormancy of the seeds can be broken, and the rooting and germination of the seeds can be accelerated; different disinfection modes have different influences on the germination index and the growth index of the explant, and the invention has the advantages that the pollution rate is lowest and the effect is best after the seeds soaked for 6 hours are disinfected in 3 percent sodium hypochlorite solution for 10 min; the influence of dark culture on growth indexes of aseptic seedlings at different time is different, and the invention obtains that the seeds soaked for 6 hours are disinfected by 3 percent sodium hypochlorite solution for 10min and then are subjected to dark culture for 3d, so that the growth rate of cotyledons can be inhibited to a certain extent, and the purposes of consistent seed germination and plant growth can be achieved.
The hypocotyl and the cotyledon with the stalk of the cabbage type rape Neib are used as explants, and the optimal pre-culture medium suitable for different explants is screened by comparing the influence of different hormone combinations on the induction and differentiation rates of callus tissues of the explants: MS +1 mg/L2,4-D +2 mg/L6-BA. The optimal differentiation medium of the hypocotyls is as follows: MS +0.1mg/L IBA +2 mg/L6-BA +5mg/L AgNO3(ii) a Optimum differentiation medium of cotyledon with stalk: MS +0.1mg/L IBA +3 mg/L6-BA+5mg/L AgNO3。
Secondly, the influence of different pretreatments on the induction of anther callus of Brassica campestris L
1. Materials and methods
1.1, the experimental material takes Chinese cabbage type spring rape-Tianzhu small rape seeds in Tianzhu area of Gansu province as the material, the seeds are potted in Gansu agricultural university, and flower buds and anthers are taken as the experimental material when the flowering phase is up.
1.2 Experimental design and methods
1.2.1 anther Sterilization and dark treatment
TABLE 10 anther callus induction medium hormone combinations
And (3) taking buds of the inner disc, the middle disc and the outer disc of the bud disc at the top end of the main inflorescence at 9.00 am every day, and selecting full rape buds without insect pests as explants for culturing callus to carry out anther culture. Sterilizing flower buds with 75% alcohol for 30s, washing with sterile water for three times, sterilizing with 0.1% mercuric chloride for 8min, washing with sterile water for 4-5 times, placing the sterilized flower buds on sterile filter paper, sucking off surface water, taking out the anthers with sterile forceps, placing in B5 culture medium, adding 1.5mg/L2,4-D and 6 different induction culture media (table 10) composed of KT and 6-BA at different concentrations and proportions, repeating the treatment for 3 times, placing the culture dish in dark at 25-28 deg.C for 30D, counting the number of callus, and screening to obtain flower bud parts, sizes and induction culture media with good callus.
1.2.2, low-temperature pretreatment, namely placing the screened rape midbasal disc buds in 4 ℃ low-temperature dark treatment for 2,4, 6, 8 and 10 days respectively, taking the untreated buds as a control, transferring the treated buds to callus induction culture media screened in the table 10 for dark culture, and counting the callus induction rate.
1.2.3 mannitol pretreatment
Placing the screened rape anther in B5 culture media with mannitol concentration of 0.1mol/L, 0.2mol/L, 0.3mol/L and 0.4mol/L for pre-culturing for 2 days, 4 days and 6 days respectively, then transferring to the callus induction culture media screened in the table 10 for dark culture, and counting the callus induction rate.
1.2.4, soaking the flower buds of the central disc in sucrose pretreatment screening in sucrose solutions with the concentration of 0.05mol/L, 0.1mol/L, 0.15mol/L and 0.2mol/L for 30min, 60min and 90min respectively, taking out anthers and inoculating the anthers in a callus induction culture medium.
1.3 measurement of index
Callus induction (%) (number of calli/total number of inoculated explants) × 100;
browning rate (%) (number of browned anthers/total number of inoculated explants) × 100;
1.4, data analysis
All data were checked for homogeneity of variance at SPSS 19.0, data comparisons were performed using one-way analysis of variance (ANOVA), and the significance level of differences between treatments was checked using the Duncan new complex range method (P < 0.05).
2. Results and analysis
2.1 observing the shape of flower buds and anther calluses at different parts of the cabbage type rape
The rape bud discs are divided into an inner disc, a middle disc and an outer disc, and the shapes of the buds of the Chinese cabbage type rape at different development stages are different. The flower buds of the inner disc are mostly spherical, the color is light green, and the length is more than 1-2 mm; one end of the flower bud of the central disk begins to become sharp, the color is mostly green, and the length is more between 2 and 3 mm; the shape of both ends of the outer disc bud is gradually sharp to be an olive shape, the color is mostly yellow green, the length of the bud is more between 4 and 6mm, and the transverse diameter of the bud is gradually increased along with the change of the length of the bud. Inoculating anther into induction culture medium for 1-2 days, observing formation of drop-shaped substance at break of anther filament, and swelling to form white loose callus; after one week, the formation of cracks at the suture of the anther abdomen was observed, followed by the formation of pale yellow dense callus at this site.
2.2 influence of different induction culture media on anther induction rates of different bud taking parts
TABLE 11 Effect of different treatments on anther callus induction rates at different bud sites
Different lower case letters in the same column represent significant differences (p ≦ 0.05) for the test.
As shown in table 11, in the same medium, the anther callus induction rates of the flower buds of the middle disc part are all higher than those of the flower buds of the other parts, the anther induction rates of the flower buds of the inner disc, the middle disc and the outer disc are respectively 13.87% -18.29%, 14.59% -27.26% and 13.95% -20.20%, in different induction media, the average induction rates of the flower buds of the inner disc, the middle disc and the outer disc are respectively 16.83%, 21.13% and 18.64%, and the anther callus induction rate of the flower buds of the middle disc is significantly higher than that of the flower buds of the inner disc and the outer disc (p is less than or equal to 0.05), respectively by 25.54% and 13.38%, wherein the anther induction rate of the flower in the disk in a5 is 27.26%, and is 51.36% and 24.42% higher than that of the flower buds of the inner disc and the outer disc. The browning rate is gradually increased from the inner disk to the outer disk, the average browning rate of the anther in the flower bud at the outer disk is 74.76 percent, the average browning rate is 11.64 percent higher than that of the anther at the inner disk, and the average browning rate is 3.21 percent higher than that of the middle disk.
When the concentration of 2,4-D is 1.5mg/L KT and the concentration of 6-BA are the same, the callus formation rate of the KT induced anther is higher than that of 6-BA, along with the increase of the concentration of KT, the anther callus induction rate is in a trend of increasing firstly and then decreasing, wherein the effect of the 1mg/L KT is the best compared with that of other concentrations, and the callus induction rates are respectively 18.01%, 27.26% and 21.91% in an inner and outer tray; and the anther callus induction rate gradually increases with the increase of the concentration of 6-BA, but the anther callus induction rate is lower than the 1mg/L KT induction anther callus formation rate, so that the anther of the Tianzhu small rape at different bud taking parts is inoculated into the callus induction culture medium with different hormone types and concentrations, the induction rate of the flower bud at the middle disc part is higher than that of other parts, the KT has better effect than that of 6-BA, and B5+1.5mg/L2 and 4-D +1mg/L KT are the best induction culture medium.
2.3 influence of Low-temperature pretreatment on anther induction rate of Brassica campestris
The low-temperature pretreatment shown in Table 12 can effectively promote the generation of anther callus of the small oilseed rape, the anther color is different along with the prolonging of the low-temperature treatment time, the anther color is mostly light green in 0-2 days, yellow green in 4-6 days and yellow brown in 8-10 days. With the increase of the low-temperature treatment time, the callus induction rate shows a trend of increasing first and then decreasing, and the browning rate shows a trend of decreasing first and then increasing with the increase of the low-temperature treatment time. The anther induction rate of the treated 2d is not obvious from the control, while the callus induction rate of the treated 4d is the largest and the browning rate is the lowest, the induction rate is 60.27%, and the browning rate is 46.77%; the induction rate was lowest (20.67%) and the browning rate was greatest (74.71%) after 10 days of treatment. Therefore, the optimal time for inducing the callus by the anther of the brassica napus is 4 days after the low-temperature pretreatment at 4 ℃.
TABLE 12 Effect of Low temperature on anther callus induction in Brassica campestris
Different lower case letters in the same column represent significant differences (p ≦ 0.05) for the test.
2.4 Effect of mannitol treatment on anther calli
Under the condition of the same concentration of mannitol treatment, the anther callus induction rate is in a trend of increasing firstly and then decreasing along with the extension of the treatment time under the condition of lower concentration of mannitol, and the anther callus induction rate is gradually decreased along with the extension of the treatment time under the condition of higher concentration of mannitol treatment. After the anther is pretreated by mannitol culture media with different concentrations, the induction rate of the callus is different in influence, the induction rate of the callus is in a trend of increasing firstly and then decreasing with the increase of the mannitol concentration below 4d of treatment, the induction rate of the anther callus is gradually decreased with the increase of the mannitol concentration when the anther is pretreated for 6d, and the browning rate is increased with the increase of the treatment time. The average induction rate of the callus is increased firstly and then decreased along with the increase of the mannitol concentration, the average browning rate is increased, and the induction rate difference of the anther callus is not significant when the mannitol concentration is 0.2mol/L and the callus is the highest (39.11%), the induction rate of the anther callus is 0.1mol/L for treating 4d and 0.2mol/L for treating 2d and 4d for treating mannitol, so that the mannitol treatment concentration range is 0.1-0.2mol/L, and the induction rate is 48.07% and the browning rate is the lowest (48.95%) when the mannitol treatment is 0.2mol/L for treating 4d, therefore, the effect of inducing the anther callus of small rape to form is the best when the mannitol treatment is 0.2mol/L for treating 4 d. As in table 13.
TABLE 13 Effect of mannitol on anther callus induction of Brassica napus
Different lower case letters in the same column represent significant differences (p ≦ 0.05) for the test.
2.5 Effect of sucrose on anther calli
As can be seen from Table 14, the anther callus induction rate decreased with the treatment time under the same concentration sucrose solution pretreatment; in addition to the 0.05mol/L sucrose concentration, the browning rate at other concentrations increases with the treatment time. Under the condition that the sucrose concentration is different and the treatment time is the same, the anther callus induction rate is increased along with the increase of the sucrose concentration, and the anther callus induction rate is reduced, wherein the anther callus induction rate is increased firstly after the treatment time is 30min and then is reduced along with the increase of the sucrose solution concentration, the anther callus induction rate is reduced after the treatment time is 60min and 90min, the induction rate is reduced to 14.18 percent when 0.2mol/L sucrose solution is treated for 90min, after the treatment time is 30min by 0.1mol/L sucrose solution, the anther callus induction rate reaches 54.8 percent at most, and has no significant difference with the treatment time of 0.05mol/L sucrose solution for 30min, but the browning rate is lower than 53.03 percent when the treatment time is 30min by 0.1mol/L sucrose solution, so that the anther callus is induced to form and is treated by 0.1mol/L sucrose solution for 30 min.
TABLE 14 Effect of sucrose on anther callus induction in Brassica napus
Different lower case letters in the same column represent significant differences (p ≦ 0.05) for the test.
3. Most crucifer microspores are subjected to anther culture when being in late mononuclear stage, and the callus induction rate is high. According to the previous researches, the length and petal-to-drug ratio of the flower bud are reliable indexes for judging the development stage of the microspore, so that the development stage of the microspore can be judged by integrating the appearance indexes such as the shape, size, color and the like of the flower bud and the anther, and the culture efficiency of the anther and the microspore is favorably improved. The invention shows that a relatively strong plant is selected in the initial flowering phase of rape, the flower buds of the middle disc of the bud disc at the top end of the main inflorescence are taken, and the anther of the rape with the length of 2-3mm is selected as the explant for culturing callus, which is favorable for the formation of the callus, such as Hovenia hernanensis, Begonia et al[118-120]The results were consistent.
Hormones are essential key substances for anther culture, and although the addition amount of hormones in a culture medium is small, the hormones play an essential role in inducing explant callus or embryoid. Currently, hormones commonly used in anther culture are generally classified into auxins and cytokinins. The auxin mainly promotes cell growth and cell elongation, and the cytokinin promotes cell growth and cell division, and researches show that the variety and the proportion of the exogenous hormone have great difference on the culture of the anther. The invention shows that the induction rate of the anther callus in the treatment of the 2,4-D + KT hormone combination is higher than that of the 2,4-D +6-BA combination, wherein the optimal induction culture medium screened out is B5+1.5 mol/L2, 4-D +1mol/L KT.
Temperature stress (low or high temperature) can promote the transition of certain plants from the gametophytic developmental pathway to the sporophytic developmental pathway. Research reports show that the low-temperature pretreatment in anther culture can not only prevent the formation of spindle silk, but also inhibit the decay of pollen microspores after anther in vitro to a certain extent, thereby prolonging the survival time of the microspores, promoting more microspores to start the development of androgens and increasing the induction rate of anther callus or embryoid.
The invention shows that: the optimum concentration of anthers of the Chinese cabbage type rape tianzhu small rape pretreated by the mannitol is 0.1-0.2mol/L, when the concentration of the mannitol reaches 0.3mol/L, the browning rate of the anthers is obviously increased, and when the concentration of the mannitol reaches 0.4mol/L, the induction rate of anther calluses is obviously reduced.
The sugar in the anther culture can be used as a carbon source, can also maintain the osmotic pressure of a culture medium, and provides energy required for the growth and development of explants. In the experiment, sucrose solutions with different concentrations are used for soaking the flower buds of the Chinese cabbage type rape skybrass small rape before inoculation, and then the flower buds are inoculated into an induction culture medium, and the experiment result shows that: the best effect of inducing anther callus by treating the anther callus for 30min with 0.1mol/L sucrose solution is 54.8%. The use of sucrose in the pretreatment is shown to be beneficial for improving the callus induction rate.
4. In conclusion, the three pretreatments of low temperature, mannitol and sucrose have different influences on the induction of the anther callus of the cabbage type rape, wherein the anther callus at the low temperature of 4 ℃ has the highest induction rate (60.27%) and the later browning rate is 46.77%, compared with the pretreatment of mannitol and sucrose, the pretreatment is simpler and more convenient, and the optimal culture medium for screening the anther callus of the cabbage type rape is B5+1.5mg/L2 and 4-D +1mg/L KT.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for establishing a cabbage type rape regeneration system is characterized by comprising the following steps:
analyzing the influence of dark culture on seed germination indexes and growth indexes in different soaking times, different disinfection modes and different time by a single-factor experiment to obtain the optimal soaking time, disinfection mode and dark treatment time; by comparing the influence of different hormone combinations on the induction and differentiation rates of calli of different explants, the hypocotyl, the optimal pre-culture medium of the cotyledon with stalk, the optimal differentiation culture medium of the hypocotyl and the optimal differentiation culture medium of the cotyledon with stalk of the cabbage type rape are screened out.
2. The method for establishing a cabbage type rape regeneration system according to claim 1, wherein the soaking time is 6 hours of sterile water soaking, the disinfection method is 3% sodium hypochlorite solution disinfection for 10min, and the dark treatment time is 3 days.
3. The method for establishing a cabbage type rape regeneration system according to claim 1, wherein the optimal pre-culture medium for Neib hypocotyls and cotyledons with stalks of the cabbage type rape is as follows: MS +1 mg/L2,4-D +2 mg/L6-BA.
4. The method for establishing a brassica rapa regeneration system as claimed in claim 1, wherein the hypocotyl optimal differentiation medium is: MS +0.1mg/L IBA +2 mg/L6-BA +5mg/L AgNO3。
5. The method for establishing a brassica rapa regeneration system as claimed in claim 1, wherein the optimal differentiation medium of the cotyledon with stalk is as follows: MS +0.1mg/L IBA +3 mg/L6-BA +5mg/L AgNO3。
6. The method for establishing a brassica rapa napus regeneration system according to claim 1, wherein the method for establishing the brassica rapa napus regeneration system specifically comprises:
(1) seed soaking treatment: selecting seeds which are free of insect damage and full in grains, soaking the seeds in sterile water for 0 hour, 3 hours, 6 hours, 12 hours and 24 hours, carrying out planting culture in 5 treatment modes respectively, repeating each treatment mode for three times, repeating each treatment mode for 20 seeds, recording the germination number of the seeds in 7d every day, and calculating the germination vigor, the germination rate, the germination index and the vitality index after 7 d;
(2) seed disinfection: selecting seeds which are free of insect damage and full in grains, numbering in advance, and soaking in sterile water in the same way as the optimal soaking method in the step (1);
(3) dark treatment and culture of seeds: selecting seeds which are free of insect damage and full in grains, wherein the soaking mode is the same as the optimal soaking method in the step (1), the disinfection mode is the same as the optimal disinfection method in the step (2), the planting mode is the same as the step (1), the seeds are placed in an incubator to be subjected to dark culture, 4 treatments are set, the dark culture is performed for 1, 2, 3 and 4 days respectively, the seeds are placed in an illumination incubator, the treatment is repeated for 3 times, the number of the seeds sprouting each day in 7 days is recorded, and the sprouting potential, the sprouting rate, the sprouting index, the vigor index and the seedling rate are calculated after 7 days;
(4) cutting the explants: selecting aseptic seedlings with the age of 5-6d, cutting cotyledons with stalks and hypocotyls, and spreading all explants in a callus culture medium;
(5) effect of different hormone combinations on different explant cultures: selecting aseptic seedlings with the growth seedling age of 5-6d, cutting the explants, inoculating the well-grown explants into a pre-culture medium by adopting the method in the step (4), and placing the explants at the temperature of 25-28 ℃ and the illumination intensity of 60 mu mol.m-2.s-1And light; culturing for 5 days in a dark period of 16h to 8h, respectively inoculating the cultured cells on a differentiation culture medium, placing the cells in an artificial tissue culture room, and continuously culturing, wherein each repetition is 20 explants, and the total number of the explants is 36;
(6) rooting culture: adventitious shoots growing to about 1-2cm in differentiation medium were excised from the explant base on a clean bench and inserted vertically into rooting medium: placing 0.2mg/L IBA +1/2MS in an illumination incubator for culture, and counting the rooting rate;
(7) and (5) measuring and counting the test data.
7. The method for establishing a Chinese cabbage type rape regeneration system according to claim 6, wherein in the step (1), the planting method adopted in the planting culture comprises the following steps:
washing the selected seeds for 2 times by using distilled water, soaking the seeds for 30s by using 75% alcohol, and continuously shaking the container to ensure that the alcohol can fully contact each seed; after sucking out alcohol by a liquid transfer gun, washing for 3 times by using sterile water; taking out and putting on sterilized filter paper to fully absorb water; MS basic culture medium is accessed into the superclean bench; culturing in a light incubator.
8. The method for establishing a cabbage type rape regeneration system according to claim 6, wherein the soaking manner in the step (2) comprises: sucking out the soaked sterile water by using a liquid transfer gun; sterilizing the surface of the container with 75% alcohol for 30s, and washing the container with sterilized water for 3 times, wherein the container is continuously shaken; after the liquid transferring gun sucks out the alcohol, the sterile water is washed for three times; the numbered seeds were each assigned 0.1% HgCl2Soaking the solution in 3% sodium hypochlorite solution for 5min, 10min, and 15 min; and repeatedly washing the seeds for 5 times by using post-sterilized water, recording the germination number of the seeds every day in 7 days, and calculating the germination vigor, the germination rate, the germination index, the vitality index and the pollution rate after 7 days.
9. The method for establishing a brassica rapa regeneration system as claimed in claim 6, wherein in the step (7), the method for measuring the test data comprises the following steps:
measuring the diameter of the seed by a vernier caliper;
the germination potential (%) -3 d total germination number/inoculated seed number x 100;
the germination rate (%) is equal to the number of germinated seedlings/inoculated seeds within 7d multiplied by 100;
germination Index (GI) ═ Σ (GT/Dt), where Dt represents the number of days of germination and GT represents the number of seeds germinated per day corresponding to Dt.
Callus induction (%) × 100 (number of calli/total number of explants inoculated).
10. The method for establishing a brassica rapa regeneration system according to claim 6, wherein after the step (7), the method further comprises the following steps:
analyzing the influence of different soaking modes, different disinfection modes and different dark treatment time on seed germination and the influence of different hormone combinations on hypocotyl budding of the cabbage type rape.
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