CN117243119A - Method for rapidly obtaining tetraploid cowpea - Google Patents
Method for rapidly obtaining tetraploid cowpea Download PDFInfo
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- SXAMGRAIZSSWIH-UHFFFAOYSA-N 2-[3-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,2,4-oxadiazol-5-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NOC(=N1)CC(=O)N1CC2=C(CC1)NN=N2 SXAMGRAIZSSWIH-UHFFFAOYSA-N 0.000 description 1
- DFGKGUXTPFWHIX-UHFFFAOYSA-N 6-[2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]acetyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)C1=CC2=C(NC(O2)=O)C=C1 DFGKGUXTPFWHIX-UHFFFAOYSA-N 0.000 description 1
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- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
-
- 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
- A01H4/002—Culture media for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of tetraploid cowpea seeds, in particular to a method for quickly obtaining tetraploid cowpea. The cowpea tetraploid leaf is used as the explant, the material quantity of the explant is large and is easy to obtain, and a recombined raw material is provided for subsequent dedifferentiation and redifferentiation; then through a proper dedifferentiation medium and a redifferentiation medium, a large number of tetraploid cowpea seedlings can be obtained rapidly; wherein the callus induction rate reaches 94.6%, and the bud induction rate reaches 32.5%. Furthermore, the cowpea growing point is treated by colchicine and dimethyl sulfoxide together, so that a large quantity of stable tetraploid cowpeas can be rapidly obtained, and the induction rate reaches 28.89%.
Description
Technical Field
The invention relates to the technical field of tetraploid cowpea seeds, in particular to a method for quickly obtaining tetraploid cowpea.
Background
Cowpea (vigna uiculata (lin.) walp.) is an annual winding vine or near-vertical herb of the genus fabaceae. The cowpea has high nutritive value, is rich in nutritional ingredients such as protein, vitamins and the like, has strong heat resistance and drought resistance, has good environmental adaptability, and is planted in areas with more China. Along with the development of agriculture and the adjustment of industrial structures, the production of cowpea faces more and more problems, such as aggravation of diseases and insect pests, continuous cropping obstacle and the like, become serious. Therefore, the breeding of cowpea varieties with strong stress resistance, continuous cropping resistance and excellent quality is urgently needed in the current production, the doubling of the chromosomes of plants is an effective means for breeding excellent varieties, and the current polyploid breeding is increasingly applied to the vegetable variety breeding. However, most of the current polyploid breeding adopts chemical reagents to induce stem tip growth points in cotyledon stage, and the stem tip growth points are identified as tetraploids and then are directly cultivated into plants, so that the seed utilization rate is low due to low induction efficiency, and a large number of tetraploid cowpea seedlings cannot be obtained in a short time.
Disclosure of Invention
In order to solve the problems, the invention provides a method for quickly obtaining tetraploid cowpea, which can quickly obtain a large number of tetraploid cowpea seedlings.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for rapidly obtaining tetraploid cowpea, which comprises the following steps:
inoculating cowpea tetraploid leaves into a dedifferentiation culture medium, and dedifferentiating and culturing to obtain callus; the dedifferentiation culture medium takes an MS culture medium as a basic culture medium and further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.5-3.5 mg/L of 6-BA and 2.5-3.5 mg/L of naphthylacetic acid;
inoculating the callus into a redifferentiation culture medium, redifferentiating and culturing to obtain adventitious buds; the redifferentiation culture medium is based on an MS culture medium and further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.3-2.8 mg/L of 6-BA and 0.3-0.7 mg/L of naphthylacetic acid.
Preferably, the cultivation method of the cowpea tetraploid blade comprises the following steps: dripping the induction liquid on a growth point among cotyledons of the diploid cowpea to obtain the tetraploid cowpea leaf; the induction liquid comprises 0.18 to 0.22wt.% colchicine and 1.8 to 2.2wt.% dimethyl sulfoxide; the dripping time is 9:30-10:30 and 21:30-22:30.
Preferably, the pH value of the dedifferentiation medium is 6-7; the pH value of the redifferentiation culture medium is 6-7.
Preferably, the dedifferentiation culture temperature is 23-27 ℃, and the culture time is 10-20 d.
Preferably, the temperature of the redifferentiation culture is 23-27 ℃, the illumination intensity is 2500-3000 Lux, and the illumination time is 15.5-16.5 h/d.
Preferably, the method further comprises: inoculating the adventitious buds into a rooting culture medium, and performing rooting culture to obtain tissue culture seedlings; the rooting culture medium takes 1/2MS culture medium as basic culture medium, and further comprises 28-32 g/L of sucrose, 6-7 mg/L of agar and 1.3-1.7 mg/L of indoleacetic acid.
Preferably, the rooting culture temperature is 23-27 ℃, the illumination intensity is 2500-3000 Lux, and the illumination time is 15.5-16.5 h/d.
Preferably, the rooting medium has a pH of 6.
Preferably, the method further comprises: transplanting the tissue culture seedlings into a culture medium, and performing domestication culture to obtain cowpea tetraploid seedlings; the culture medium comprises the following components in parts by volume: 2.5 to 3.5 parts of turfy soil, 1.5 to 2.5 parts of vermiculite and 0.5 to 1.5 parts of perlite.
Preferably, the temperature of the domestication culture is 26-32 ℃.
The beneficial effects are that:
the invention provides a method for rapidly obtaining tetraploid cowpea, which comprises the following steps: inoculating cowpea tetraploid leaves into a dedifferentiation culture medium, and dedifferentiating and culturing to obtain callus; the dedifferentiation culture medium takes an MS culture medium as a basic culture medium and further comprises 8-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.5-3.5 mg/L of 6-BA and 2.5-3.5 mg/L of naphthylacetic acid; inoculating the callus into a redifferentiation culture medium, redifferentiating and culturing to obtain adventitious buds; the redifferentiation culture medium is based on an MS culture medium and further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.3-2.8 mg/L of 6-BA and 0.3-0.7 mg/L of naphthylacetic acid. According to the invention, cowpea tetraploid leaves are used as explants, each leaf can be cut into a plurality of fine leaves for induction, the material quantity of the explants is large and is easy to obtain, and a recombined raw material is provided for subsequent dedifferentiation and redifferentiation; the callus induction rate and bud induction rate can be improved through a proper dedifferentiation culture medium and a proper redifferentiation culture medium, wherein the callus induction rate reaches 94.6%, and the bud induction rate reaches 32.5%, so that a large number of tetraploid cowpea seedlings can be obtained rapidly.
Furthermore, the cowpea growing point is treated by colchicine and dimethyl sulfoxide together, so that a large quantity of stable tetraploid cowpeas can be rapidly obtained, and the induction rate reaches 28.89%.
Detailed Description
The invention provides a method for rapidly obtaining tetraploid cowpea, which comprises the following steps:
inoculating cowpea tetraploid leaves into a dedifferentiation culture medium, and dedifferentiating and culturing to obtain callus; the dedifferentiation culture medium takes an MS culture medium as a basic culture medium and further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.5-3.5 mg/L of 6-BA and 2.5-3.5 mg/L of naphthylacetic acid;
inoculating the callus into a redifferentiation culture medium, redifferentiating and culturing to obtain adventitious buds; the redifferentiation culture medium is based on an MS culture medium and further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.3-2.8 mg/L of 6-BA and 0.3-0.7 mg/L of naphthylacetic acid.
The cultivation method of the cowpea tetraploid blade preferably comprises the following steps: and (3) dripping the induction liquid on a growth point among the cotyledons of the diploid cowpea to obtain the cowpea tetraploid blade. In the present invention, the induction liquid preferably includes colchicine and dimethyl sulfoxide; the concentration of colchicine is preferably from 0.18wt.% to 0.22wt.%, more preferably 0.2wt.%; the concentration of dimethyl sulfoxide is preferably 1.8wt.% to 2.2wt.%, more preferably 2wt.%; the dripping time is preferably 9:30-10:30 and 21:30-22:30, more preferably 9:30-10:30 and 21:30-22:30 of the fifth day after sowing, and even more preferably 10:00 and 22:00 of the fifth day after sowing. The invention can rapidly obtain mass stable tetraploid cowpea by proper induction liquid and induction method, and the induction rate reaches 28.89%.
After obtaining the cowpea tetraploid leaves, the cowpea tetraploid leaves are inoculated into a dedifferentiation culture medium for dedifferentiation culture to obtain the callus. In the present invention, the length of the blade is preferably 1 to 1.5cm.
The dedifferentiation culture medium disclosed by the invention takes an MS culture medium as a basic culture medium, further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.5-3.5 mg/L of 6-BA and 2.5-3.5 mg/L of naphthylacetic acid, more preferably comprises 30g/L of sucrose, 6.5mg/L of agar, 3mg/L of 6-BA and 3mg/L of naphthylacetic acid, and the pH value of the dedifferentiation culture medium is preferably 6-7, more preferably 6; the temperature of the dedifferentiation culture is preferably 23 to 27 ℃, more preferably 25 ℃; the dedifferentiation culture is preferably a dark culture, and the culture time is preferably 10 to 20d, more preferably 20d. The invention can improve the induction rate of the callus by a proper dedifferentiation culture medium, and the induction rate of the callus reaches 94.6 percent.
After obtaining the callus, the invention inoculates the callus into a redifferentiation culture medium, redifferentiation culture is carried out, and adventitious buds are obtained.
The redifferentiation culture medium is based on an MS culture medium and further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.3-2.8 mg/L of 6-BA and 0.3-0.7 mg/L of naphthylacetic acid, and more preferably comprises 30g/L of sucrose, 6.5mg/L of agar, 2.5mg/L of 6-BA and 0.5mg/L of naphthylacetic acid; the pH value of the redifferentiation medium is preferably 6 to 7, more preferably 6; the temperature of the redifferentiation culture is preferably 23-27 ℃, more preferably 25 ℃; the illumination intensity of the redifferentiation culture is preferably 2500-3000 Lux, more preferably 2500Lux, and the illumination time is preferably 15.5-16.5 h/d, more preferably 16h/d. The invention can improve the induction rate of the adventitious buds through a proper redifferentiation culture medium, and the induction rate of the adventitious buds reaches 32.5%.
After the adventitious buds are obtained, the adventitious buds are preferably inoculated into a rooting culture medium for rooting culture, so that tissue culture seedlings are obtained. In the invention, the rooting culture medium is preferably based on a 1/2MS culture medium, and preferably further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar and 1.3-1.7 mg/L of indoleacetic acid, more preferably comprises 30g/L of sucrose, 6.5mg/L of agar and 1.5mg/L of indoleacetic acid; the pH value of the rooting culture medium is preferably 6; the temperature of rooting culture is preferably 23-27 ℃, more preferably 25 ℃; the illumination intensity of the rooting culture is preferably 2500-3000 Lux, more preferably 2500Lux, and the illumination time is preferably 15.5-16.5 h/d, more preferably 16h/d.
After the tissue culture seedlings are obtained, the tissue culture seedlings are preferably transplanted into a culture medium, and domesticated and cultured to obtain cowpea tetraploid seedlings.
The cultivation substrate of the present invention preferably comprises 2.5 to 3.5 parts by volume of turfy soil, more preferably 3 parts by volume.
The cultivation substrate of the invention preferably comprises 1.5 to 2.5 parts, more preferably 2 parts, of vermiculite based on the volume parts of the turfy soil.
The cultivation substrate of the present invention preferably comprises perlite in an amount of 0.5 to 1.5 parts, more preferably 1 part, based on the volume parts of the peatmoss.
In the present invention, the temperature of the domestication culture is preferably 26 to 32 ℃, and more preferably 28 to 30 ℃, for further explanation of the present invention, a method for rapidly obtaining tetraploid cowpea according to the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A method for rapidly obtaining tetraploid cowpea comprises the following steps:
(1) Selecting diploid cowpea seeds with full and uniform particles, stirring in warm water at 55 ℃ for 20min, sterilizing, and soaking in clear water for 6h;
(2) Sowing the seeds after seed soaking in a culture medium, wherein the formula of the medium is turfy soil: vermiculite: perlite = 3:1:1 (volume ratio);
(3) Placing the sown seeds in a culture room, setting the temperature to be 27+/-2 ℃ in the daytime and 20+/-2 ℃ at night, and uniformly germinating the seeds after continuous 5 days for 12 hours;
(4) Colchicine treatment is carried out on the seedlings in the step (3) on the 5 th day, and the specific steps are as follows: preparing a premix of colchicine and dimethyl sulfoxide with distilled water in advance, wherein the concentration of colchicine is 0.2% and the concentration of dimethyl sulfoxide is 2%; watering the seedlings at night for full watering (slow watering around plants, taking water drops oozed out from the bottoms of the pots as the basis), and dripping (taking the condition that the seedlings can just stay on a growing point and cannot slide off as the basis) primary premix liquid on the growing point between two cotyledons of cowpea in the fifth day 10:00 and 22:00;
(5) Carrying out flow identification on newly grown true leaves on the 30 th day, wherein the identification method is shown in [ Wang Yali, zhou Lili, wang Na, cheng Gongmei ], comparing the method for rapidly identifying cotton ploidy by using a flow cytometry with [ J ]. Biotechnology report, 2022,38 (12): 144-148 ], and confirming that the cotton ploidy is a tetraploid plant;
(6) Taking leaf explants from the tetraploid plants confirmed in the step (5) for plant tissue culture, wherein the specific steps are as follows: selecting identified tetraploid cowpea leaves, and washing for 30min in running water; soaking in 75% alcohol for 30s, soaking in 0.1% mercuric chloride for 8min, and washing with sterile water for three times; cutting the leaves into rectangles with the side length of 1-1.5 cm, inoculating the rectangles on a dedifferentiation medium, and culturing in a dark place; inoculating the callus to a redifferentiation culture medium after 20 days to induce adventitious buds; then the induced adventitious buds are inoculated into a rooting culture medium for rooting induction, and finally domestication and transplanting are carried out;
wherein the dedifferentiation culture medium is based on an MS culture medium, and further comprises 30g/L of sucrose, 6.5mg/L of agar, 3mg/L of 6-BA and 3mg/L of naphthylacetic acid (NAA), and the pH value is 6; the dedifferentiation culture is dark culture at 25+/-2 ℃;
the redifferentiation culture medium is based on an MS culture medium and further comprises 30g/L of sucrose, 6.5mg/L of agar, 6-BA2.5mg/L of naphthalene acetic acid and 0.5mg/L of naphthalene acetic acid, wherein the pH value is 6;
the rooting culture medium takes a 1/2MS culture medium as a basic culture medium, and also contains 30g/L of sucrose, 6.5mg/L of agar and 1.5mg/L of indoleacetic acid (IBA), wherein the pH value is 6;
the conditions of adventitious bud and rooting culture are: the light irradiation time is 16h/d, the light intensity is 2500Lux, the temperature is 25+/-2 ℃, the domestication method is that seedlings are simply inoculated on a rooting culture medium for 30d, then the seedlings are taken out, the culture medium is cleaned, then the seedlings are transplanted into a cave dish containing a culture medium, a built small arch shed is placed, the temperature is 26-32 ℃, and natural light irradiation is carried out; the culture medium is turfy soil: vermiculite: perlite=3:2:1 (volume ratio).
Comparative example 1
Inducing diploid cowpea seedlings with different concentrations of colchicine (0.1%, 0.2%, 0.3%, 0.4% and 0.5%) and different dripping times (2 times, 4 times and 6 times), the method of the different treatment groups referring to steps (1) to (5) in example 1, differing in that only colchicine solution is used for induction, the colchicine concentration is different and/or the dripping times are different; when the dripping times are 4 times, the dripping times are respectively 10:00 and 22:00 on the fifth day of sowing and 10:00 and 22:00 on the sixth day of sowing; when the dripping times are 6 times, the dripping times are respectively 10:00 and 22:00 on the fifth day of sowing, 10:00 and 22:00 on the sixth day of sowing, and 10:00 and 22:00 on the seventh day of sowing; the treatment and results for the different groups are shown in Table 1.
Table 1 results of inductances from different groups
As is clear from Table 1, the optimum concentration of colchicine was 0.2% and the optimum number of induction was 2.
Comparative example 2
Inducing diploid cowpea seedlings by adopting different dripping times (2 times or 4 times) and a tweezers-removing method, wherein the methods of different treatment groups refer to the steps (1) to (5) in the embodiment 1, and the difference is that the dripping times are different; when the dripping times are 4 times, the dripping times are respectively 10:00 and 22:00 on the fifth day of sowing and 10:00 and 22:00 on the sixth day of sowing; the method of tweezers decoring is referred to watermelon tetraploid induction method [ Zhao Shengjie, liu Wenge, yan Zhigong, etc. ], colchicine and amisulin herbicide induced small watermelon tetraploid study [ J ]. The Yangtze river vegetables, 2010 (08): 12-13 ]; the treatment and results for the different groups are shown in Table 2.
TABLE 2 Induction Rate results for different groups
| Number of times | Treatment of plant number | Tetraploid number | Induction rate |
| 2 times | 90 | 26 | 28.89％ |
| 4 times | 81 | 11 | 13.58％ |
| 2 times (with tweezers to remove heart) | 47 | 0 | 0 |
| 4 times (with tweezers to remove heart) | 44 | 0 | 0 |
| 2 times (with scissors to remove the heart) | 44 | 0 | 0 |
As shown in table 2, the heart-removing method with remarkable effect has low applicability to cowpea in the induction process of the watermelon tetraploid.
Comparative example 3
A similar method to example 1 was carried out, except that the concentration of 6-BA in the dedifferentiated medium was 2mg/L and the NAA concentration was 2mg/L.
Comparative example 4
A similar method to example 1 was carried out, except that the concentration of 6-BA in the dedifferentiated medium was 2mg/L, NAA was replaced with 2,4-D, and the concentration of 2,4-D in the dedifferentiated medium was 2mg/L.
Comparative example 5
A similar method to comparative example 4 was conducted, except that the concentration of 6-BA in the dedifferentiated medium was 3mg/L and the concentration of 2,4-D was 3mg/L.
Comparative example 6
A method similar to example 1, except that the dedifferentiation medium was based on MS medium, further comprising 30g/L of sucrose, 6.5mg/L of agar, 6-BA2mg/L, NAA mg/L and 2, 4-D1 mg/L.
Comparative example 7
A method similar to comparative example 6, except that the dedifferentiated medium was 1mg/L in the concentration of 6-BA in the MS medium.
The inductivity of the calli of example 1 and comparative examples 3 to 7 was measured, 500ml of each dedifferentiated medium was prepared, 10 bottles were packed, and 12 pieces of explant leaves were inoculated per bottle, and the results are shown in Table 3.
TABLE 3 callus induction results from different groups
| Group of | 6-BA(mg/L) | NAA(mg/L) | 2,4-D(mg/L) | Induction rate |
| Comparative example 3 | 2 | 2 | - | 84.4％ |
| Comparative example 4 | 2 | - | 2 | 72.3％ |
| Example 1 | 3 | 3 | - | 94.6％ |
| Comparative example 5 | 3 | - | 3 | 65.5％ |
| Comparative example 6 | 2 | 1 | 1 | 32.1％ |
| Comparative example 7 | 1 | 1 | 1 | 25.9％ |
As shown in Table 3, the dedifferentiated media of the present invention showed the best effect of callus induction.
Comparative example 8
A method similar to example 1, except that the redifferentiation medium was based on MS medium, and further contained 30g/L of sucrose, 6.5mg/L of agar, 0.5mg/L of 2,4-D and 2mg/L of KT (kinetin).
Comparative example 9
A method similar to example 1, except that the redifferentiation medium was based on MS medium, and further contained 30g/L of sucrose, 6.5mg/L of agar, 0.5mg/L of NAA and 2mg/L of KT (kinetin).
Comparative example 10
A similar method to example 1, except that the redifferentiation medium was based on MS medium, and further contained 30g/L of sucrose, 6.5mg/L of agar, 1.5mg/L of 6-BA and 0.5mg/L of NAA.
Comparative example 11
A method similar to example 1, except that the redifferentiation medium was based on MS medium, and further contained 30g/L of sucrose, 6.5mg/L of agar, 1.5mg/L of 6-BA and 0.5mg/L of 2, 4-D.
Comparative example 12
A method similar to example 1, except that the redifferentiation medium was based on MS medium, and further contained 30g/L of sucrose, 6.5mg/L of agar, 6-BA2mg/L and 0.5mg/L of NAA.
Comparative example 13
A method similar to example 1, except that the redifferentiation medium was based on MS medium, and further contained 30g/L of sucrose, 6.5mg/L of agar, 6-BA2mg/L and NAA 1mg/L.
Comparative example 14
A similar method to example 1, except that the redifferentiation medium was based on MS medium, and further contained 30g/L of sucrose, 6.5mg/L of agar, 6-BA2.5mg/L and 1mg/L of NAA.
The induction rate of adventitious buds of example 1 and comparative examples 8 to 14 was measured, 500ml of each redifferentiation medium was prepared, 10 flasks were packed, and 15 calli were inoculated per flask, and the results are shown in Table 4.
TABLE 4 results of adventitious bud induction in different groups
| Group of | 6-BA(mg/L) | NAA(mg/L) | 2,4-D(mg/L) | KT(mg/L) | Average induction per bottle bud |
| Comparative example 8 | - | - | 0.5 | 2 | 9.8 |
| Comparative example 9 | - | 0.5 | - | 2 | 11.6 |
| Comparative example 10 | 1.5 | 0.5 | - | - | 18.2 |
| Comparative example 11 | 1.5 | - | 0.5 | - | 12.5 |
| Comparative example 12 | 2 | 0.5 | - | - | 24.9 |
| Example 1 | 2.5 | 0.5 | - | - | 32.5 |
| Comparative example 13 | 2 | 1 | - | - | 25.0 |
| Comparative example 14 | 2.5 | 1 | - | - | 19.5 |
As shown in Table 4, the redifferentiation medium provided by the invention has the best induction effect on adventitious buds.
Comparative example 15
A method similar to example 1, except that the IBA concentration in the rooting medium was 0.5mg/L.
Comparative example 16
A method similar to example 1, except that IBA was present in the rooting medium at a concentration of 1mg/L.
Comparative example 17
A method similar to example 1, except that the IBA concentration in the rooting medium was 2mg/L.
The rooting numbers of example 1 and comparative examples 15 to 17 were measured, 500ml of each rooting medium was prepared, 10 bottles were packed, and 15 adventitious buds were inoculated per bottle, and the results are shown in Table 5.
TABLE 5 rooting results for different groups
| Group of | IBA(mg/L) | Rooting rate |
| Comparative example 15 | 0.5 | 15.6 |
| Comparative example 16 | 1 | 50.8 |
| Example 1 | 1.5 | 80.0 |
| Comparative example 17 | 2 | 65.0 |
As can be seen from Table 5, the rooting medium provided by the invention has the best rooting effect.
And transferring the rooted tetraploid seedlings into a matrix for normal management, wherein the survival rate is 100%.
In conclusion, the method provided by the invention can rapidly obtain a large number of tetraploid cowpea seedlings.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The method for rapidly obtaining the tetraploid cowpea is characterized by comprising the following steps of:
inoculating cowpea tetraploid leaves into a dedifferentiation culture medium, and dedifferentiating and culturing to obtain callus; the dedifferentiation culture medium takes an MS culture medium as a basic culture medium and further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.5-3.5 mg/L of 6-BA and 2.5-3.5 mg/L of naphthylacetic acid;
inoculating the callus into a redifferentiation culture medium, redifferentiating and culturing to obtain adventitious buds; the redifferentiation culture medium is based on an MS culture medium and further comprises 28-32 g/L of sucrose, 6.5-7.5 g/L of agar, 2.3-2.8 mg/L of 6-BA and 0.3-0.7 mg/L of naphthylacetic acid.
2. The method according to claim 1, wherein the cultivation method of cowpea tetraploid leaves comprises: dripping the induction liquid on a growth point among cotyledons of the diploid cowpea to obtain the tetraploid cowpea leaf; the induction liquid comprises 0.18 to 0.22wt.% colchicine and 1.8 to 2.2wt.% dimethyl sulfoxide; the dripping time is 9:30-10:30 and 21:30-22:30.
3. The method of claim 1, wherein the dedifferentiating medium has a pH of 6 to 7; the pH value of the redifferentiation culture medium is 6-7.
4. The method according to claim 1, wherein the dedifferentiated cultivation is carried out at a temperature of 23 to 27℃for a dark cultivation time of 10 to 20d.
5. The method according to claim 1, wherein the temperature of the redifferentiation culture is 23-27 ℃, the illumination intensity is 2500-3000 Lux, and the illumination time is 15.5-16.5 h/d.
6. The method according to claim 1, wherein the method further comprises: inoculating the adventitious buds into a rooting culture medium, and performing rooting culture to obtain tissue culture seedlings; the rooting culture medium takes 1/2MS culture medium as basic culture medium, and further comprises 28-32 g/L of sucrose, 6-7 mg/L of agar and 1.3-1.7 mg/L of indoleacetic acid.
7. The method according to claim 6, wherein the rooting culture temperature is 23-27 ℃, the illumination intensity is 2500-3000 Lux, and the illumination time is 15.5-16.5 h/d.
8. The method of claim 6, wherein the rooting medium has a pH of 6.
9. The method of claim 6, wherein the method further comprises: transplanting the tissue culture seedlings into a culture medium, and performing domestication culture to obtain cowpea tetraploid seedlings; the culture medium comprises the following components in parts by volume: 2.5 to 3.5 parts of turfy soil, 1.5 to 2.5 parts of vermiculite and 0.5 to 1.5 parts of perlite.
10. The method of claim 9, wherein the temperature of the acclimation culture is 26-32 ℃.
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