CN109744149B - Method for tissue culture and propagation of children vegetables by using bud blocks - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 17
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- 239000002609 medium Substances 0.000 claims abstract description 13
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- 230000006698 induction Effects 0.000 claims abstract description 8
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 8
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- 229940088597 hormone Drugs 0.000 claims description 9
- 239000005556 hormone Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 4
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 241000218093 Phedimus aizoon Species 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 3
- 239000012154 double-distilled water Substances 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 235000011332 Brassica juncea Nutrition 0.000 claims 1
- 244000178993 Brassica juncea Species 0.000 claims 1
- 235000014700 Brassica juncea var napiformis Nutrition 0.000 claims 1
- 235000021552 granulated sugar Nutrition 0.000 claims 1
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Abstract
The invention belongs to the technical field of baby vegetable planting, and discloses a method for tissue culture and propagation of baby vegetables by using bud blocks, which comprises the steps of cutting excellent plant axillary buds by using a scalpel, and filling the excellent plant axillary buds into a nylon mesh bag; cutting axillary buds into a plurality of small axillary bud blocks, and inoculating the small axillary bud blocks on a pre-culture medium 1/2 MS; inoculating in a solution containing 0.1 mg.L‑1NAA and 0.3 mg.L‑16-BA induction culture medium; adding 0.3 mg.L to MS proliferation medium with adventitious bud after 30 days of culture as explant‑16-BA and 0.1 mg. L‑1And (4) selecting strong seedlings to be inoculated into a rooting culture medium after NAA is subjected to subculture for 30 d. The invention selects single plants with excellent target characters as materials to carry out tissue culture and rapid propagation of the sedge, the test-tube plantlet is transplanted to a field after overwintering without vegetative growth and directly enters reproductive growth so as to obtain good-quality seeds, and the selective breeding of the good seeds of the sedge is carried out, thereby providing high-quality seeds for production.
Description
Technical Field
The invention belongs to the technical field of baby vegetable planting, and particularly relates to a method for tissue culture and propagation of baby vegetables by using buds.
Background
The baby cabbage is named Bazicai (Brassica juncea var. gemmifera Lin.) as a variety of Brassica mustard species in the family of cruciferae, commonly called "Kernel", sprout, Bazicai "and the like, and is named" Bazicai "in 1987 by Populus japonica in the names of" Bazicai "such as" Bazicai "in the name of" Bazicai ", Chencai forest, Suyunfu" and the like. When the seedling of the baby cabbage grows to a certain stage, the bud growing from the leaf axillary part continuously expands to form the bud (bud block) of the edible organ. The children vegetables have rich nutrition, tender quality, delicious taste and various eating methods, can be fried, stewed, fried, rinsed, cold mixed, made into soup and pickled, and are one of the special vegetables in southwest regions. The sedge belongs to a two-year plant, is sown in the current year for vegetative growth, and sprouts, flowers and fruits in summer and spring next year after overwintering. There are two main methods for reserving seeds of brassica napobrassica, namely, reserving seeds of large plants and reserving seeds of small plants. The large plant is easy to select for reserving seeds for target characters, but because the problems of poor stress resistance, serious stem rot and the like generally exist in the children vegetables, stems and bud blocks can rot in the hollow mode in the first year, so that seeds cannot be harvested in the second year or the quality of the seeds is poor; the small plant is difficult to select for the target character, which causes the seed purity of the brassica napobrassica not to reach the standard.
In summary, the problems of the prior art are as follows: the seedling reserving method has poor stress resistance and serious stalk rot; the selection of target characters is difficult, so that the seed purity of the brassica napobrassica does not reach the standard.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for tissue culture and propagation of children vegetables by using bud blocks.
The invention is realized in such a way, the method for tissue culture and propagation of the children vegetables by using the bud blocks uses a scalpel to cut excellent plant axillary buds and puts the excellent plant axillary buds into a nylon mesh bag; cutting axillary bud into multiple 1cm pieces3Inoculating the small axillary buds on a pre-culture medium 1/2MS for culture, and primarily screening the buds to obtain sterile buds with a good state; is inoculated inContains 0.1 mg.L-1NAA and 0.3 mg.L-16-BA induction culture medium; adding 0.3 mg.L to MS proliferation medium with adventitious bud after 30 days of culture as explant-16-BA and 0.1 mg. L-1NAA, obtaining a bud cluster, increasing the number of seedlings to achieve the purpose of propagation, and selecting strong seedlings to be inoculated into a rooting culture medium after subculture for 30 d.
Further, cutting good axillary buds with a scalpel, taking 15-20 axillary buds from each plant, placing into a nylon mesh bag, numbering, washing with running water overnight, adding into water solution containing detergent, stirring for 20min, dewatering, and washing with double distilled water for 3-5 times.
Further, after being filled into the nylon mesh bag, the following steps are required:
placing the sedum aizoon in a 70% alcohol solution for disinfection for 30s in a super clean bench, pouring out alcohol, washing with sterile water for 3 times, then placing in a 2% sodium hypochlorite solution with a few drops of Tween for disinfection for 20min, continuously stirring during the disinfection, pouring out the solution, and washing with sterile water for 4-5 times to obtain a sterile explant;
taking 1/2MS and MS as basic culture medium, adding 30g/L white sugar and 8g/L agar into all the culture medium, adjusting hormone concentration ratio according to culture stage, adjusting pH to 5.8, and autoclaving at 121 deg.C for 20 min. The culture temperature is 25 +/-2 ℃, the illumination is 12-14 hours per day, and the illumination intensity is 1500-2000 Lx.
Further, taking out axillary buds, placing on sterilized filter paper, removing 1-2 layers of leaves on the surface, transversely cutting open the axillary buds from the middle part, and cutting the axillary buds into multiple pieces of 1cm3Inoculating the small axillary bud blocks on a pre-culture medium 1/2MS for culture; each dish was inoculated with 8 explants.
Further, after preculture, non-contaminated explants were selected and inoculated with a medium containing 0.1 mg.L-1NAA and 0.3 mg.L- 16-BA in the induction medium, each bottle was inoculated with 5 explants.
Further, the adventitious bud after 30 days of culture was used as an explant, and 0.3 mg. multidot.L was added to the MS growth medium-16-BA and 0.1 mg. L-1NAA, 4 explants per flask; selecting strong seedling after 30 days of subculture, inoculating to rooting culture medium, and culturing other plantletsThe proliferation culture is continued.
Furthermore, the rooting medium takes MS as a basic medium and is added with 0.1 mg.L-1NAA；
And (3) performing bottle opening and seedling hardening on the obtained rooted seedlings for 2-3 days, cleaning to remove culture medium attached to roots, and transplanting the seedlings into a greenhouse.
The invention also aims to provide the children vegetable cultivated by the method for carrying out the tissue culture and propagation of the children vegetable by using the bud blocks.
The invention has the advantages and positive effects that: transplanting the test-tube plantlets to a greenhouse, vernalizing at low temperature, enabling the test-tube plantlets to bloom and bear fruits in the spring of the next year, and harvesting seeds in the greenhouse for the subsequent seed selection of the improved variety of the children vegetables. The invention uses the low-toxicity sodium hypochlorite solution to disinfect for 20min when the children vegetables are disinfected, has the best effect, replaces the high-toxicity mercury bichloride to disinfect, and is more environment-friendly and safer; 2. the existing rapid propagation technology of the children vegetables directly adopts seeds as explants, but the invention utilizes bud blocks as explant materials, can directly select individuals with excellent properties, has more definite target individuals, and can avoid the problem that seeds cannot be reserved due to rotten stems or bud blocks of the children vegetables; 3. the conventional seedling breeding technology of the brassica napobrassica aims to obtain more brassica napobrassica seedlings by using less materials and reduce the cost of seeds, and the method is applied to breeding of the brassica napobrassica, screens excellent individuals and quickly obtains brassica napobrassica seeds, and shortens the breeding period by 2-3 years compared with the traditional breeding method.
Drawings
FIG. 1 is a flow chart of a method for tissue culture and propagation of children vegetables by using sprout pieces according to an embodiment of the 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.
The invention selects single plants with excellent target characters as materials to carry out tissue culture and rapid propagation of the sedge, transplants the test-tube plantlet into the field after overwintering, leads the test-tube plantlet not to carry out vegetative growth and directly enters reproductive growth so as to obtain good-quality seeds, carries out fine seed selection of the sedge and provides high-quality seeds for production.
EXAMPLE 1 acquisition of suitable Induction Medium formulations
Explants without contamination after preculture were selected and inoculated on MS induction medium containing different hormones. The screening of the optimal hormone proportion adopts a random block design, 9 treatments of hormone concentration combination 6-BA (0.1, 0.3,1.0 mg.L-1) and NAA (0, 0.1,0.2 mg.L-1) are designed to be added, 10 bottles are treated, and 5 explants are inoculated in each bottle. The callus formation rate was counted after 10 days of culture and the results are shown in Table 1. Obtaining a proper induction culture medium formula: MS +0.1 mg. L-1NAA+0.3mg·L-16-BA
EXAMPLE 2 preparation of suitable rooting Medium formulation
Selecting strong seedlings after subculture for 30 days, and inoculating the strong seedlings on a 1/2MS rooting medium containing different hormones. The screening of the optimal hormone proportion adopts a random block design, and 6 treatments of adding hormone concentration combination 6-BA (0, 0.5 mg. L-1) and NAA (0.1, 0.5, 1.0 mg. L-1) are designed. Each bottle was treated with 20 bottles, and each bottle was inoculated with 2 strong seedlings. And observing and counting the rooting condition after culturing for 10 days. The results are shown in Table 2. Obtaining a proper rooting medium formula: MS +0.1 mg. L-1NAA
As shown in fig. 1, a method for tissue culture and propagation of children vegetables using sprout pieces comprises the following steps:
s101: cutting good axillary buds with a scalpel, taking 15-20 axillary buds from each plant, placing into a nylon mesh bag, numbering, washing with running water overnight, adding into water solution containing detergent, stirring for about 20min, dewatering, and washing with double distilled water for 3-5 times;
s102: placing the sedum aizoon in a 70% alcohol solution for disinfection for 30s in a super clean bench, pouring out alcohol, washing with sterile water for 3 times, then placing in a 2% sodium hypochlorite solution with a few drops of Tween for disinfection for 20min, continuously stirring during the disinfection, pouring out the solution, washing with sterile water for 4-5 times to obtain a sterile explant for later use;
s103: taking 1/2MS and MS as basic culture medium, adding 30g/L white sugar and 8g/L agar into all the culture medium, adjusting hormone concentration ratio according to culture stage, adjusting pH to 5.8, and autoclaving at 121 deg.C for 20 min. The culture temperature is 25 +/-2 ℃, the illumination is 12-14 hours per day, and the illumination intensity is 1500-2000 Lx;
s104: taking the axillary buds, placing on sterilized filter paper, removing 1-2 layers of leaves on the surface, transversely cutting the axillary buds from the middle part, cutting the axillary buds into a plurality of small axillary bud blocks of 1cm3, and inoculating on a pre-culture medium 1/2MS for culture; 8 explants were inoculated on each dish;
s105: after a period of pre-culture, non-contaminating explants were selected and inoculated in a medium containing 0.1 mg.L-1NAA and 0.3 mg.L-1Inoculating 5 explants per bottle on 6-BA induction medium; all materials begin to form light green granular callus after about 7 days, and have differentiation capacity; after about 7 days, adventitious buds begin to differentiate;
s106: adding 0.3 mg.L to MS proliferation medium with adventitious bud after 30 days of culture as explant-16-BA) and 0.1 mg. L-1NAA, 4 explants per flask; after 30d of subculture, selecting strong seedlings to be inoculated into a rooting culture medium, and continuously carrying out enrichment culture on the rest seedlings;
s107: the rooting culture medium adopts MS as basic culture medium, and 0.1 mg.L is added-1Growing white thick and strong roots around NAA and 7 d;
s108: and (3) performing bottle opening and seedling hardening on the obtained rooted seedlings for 2-3 days, cleaning to remove the culture medium attached to the roots, transplanting the seedlings into a greenhouse, observing after one week, and enabling the seedlings to survive completely.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (4)
1. A method for using bud block to carry on tissue culture and propagation of the children, characterized by, cut and get the good plant axillary bud with the scalpel, each plant gets 15-20 axillary buds, pack into a nylon mesh bag and number, wash overnight through running water, put into water solution with detergent and stir 20 minutes, wash 3-5 times with double distilled water after dehydrating;
placing the sedum aizoon in a 70% alcohol solution for disinfection for 30s in a super clean bench, pouring out alcohol, washing with sterile water for 3 times, then placing in a 2% sodium hypochlorite solution with a few drops of Tween for disinfection for 20min, continuously stirring during the disinfection, pouring out the solution, and washing with sterile water for 4-5 times to obtain a sterile explant;
taking 1/2MS and MS as basic culture media, adding 30g/L white granulated sugar and 8g/L agar into all the culture media, adjusting the hormone concentration ratio according to the culture stage, adjusting the pH to 5.8, and autoclaving at 121 ℃ for 20 min; the culture temperature is 25 +/-2 ℃, the illumination is 12-14 hours per day, and the illumination intensity is 1500-2000 Lx;
cutting axillary bud into multiple 1cm pieces3Inoculating the small axillary bud blocks on a pre-culture medium 1/2MS for culture;
inoculating in a solution containing 0.1 mg.L-1NAA and 0.3 mg.L-16-BA in MS inducing culture medium; adding 0.3 mg.L to MS proliferation medium with adventitious bud after 30 days of culture as explant-16-BA and 0.1 mg. L-1NAA, selecting strong seedlings to be inoculated into a rooting culture medium after subculture for 30 d; the rooting culture medium takes MS as a basic culture medium, and 0.1 mg.L of the basic culture medium is added-1NAA；
And (3) performing bottle opening and seedling hardening on the obtained rooted seedlings for 2-3 days, cleaning to remove culture medium attached to roots, and transplanting the seedlings into a greenhouse.
2. The method for tissue culture and propagation of sprouts according to claim 1, wherein the axillary buds are taken out after sterilization and placed on sterilized filter paper, and 1-2 layers of leaves on the surface are peeled off,cutting axillary bud into multiple 1cm pieces3Inoculating the small axillary bud blocks on a pre-culture medium 1/2MS for culture; each dish was inoculated with 8 explants.
3. The method for tissue culture and propagation of sprouts using sprouts according to claim 1, wherein after preculture, non-contaminating explants are selected and inoculated with a medium containing 0.1 mg-L-1NAA and 0.3 mg.L-16-BA in MS induction medium, each bottle inoculated with 5 explants.
4. The method for tissue culture and propagation of brassica juncea using sprouts of claim 1, wherein the adventitious bud after 30 days of culture is used as an explant, and 0.3 mg-L is added to MS proliferation medium-16-BA and 0.1 mg. L-1NAA, 4 explants per flask; after 30 days of subculture, selecting strong seedlings to be inoculated into a rooting culture medium, and continuously carrying out enrichment culture on the rest seedlings.
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