CN115316280A - Method for improving leaf-shaped embryo formation rate and one-step seedling rate of black cabbage - Google Patents
Method for improving leaf-shaped embryo formation rate and one-step seedling rate of black cabbage Download PDFInfo
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- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 26
- 240000007124 Brassica oleracea Species 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 21
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 title claims abstract description 9
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 title claims abstract description 9
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 title claims abstract description 9
- 210000001161 mammalian embryo Anatomy 0.000 title claims description 7
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 235000013824 polyphenols Nutrition 0.000 claims abstract description 25
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 24
- 150000008442 polyphenolic compounds Chemical class 0.000 claims abstract description 24
- 235000011303 Brassica alboglabra Nutrition 0.000 claims abstract description 12
- 235000011302 Brassica oleracea Nutrition 0.000 claims abstract description 12
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims abstract description 6
- 239000007787 solid Substances 0.000 claims abstract description 5
- 238000002635 electroconvulsive therapy Methods 0.000 claims abstract description 4
- 239000002609 medium Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 229910000831 Steel Inorganic materials 0.000 claims description 3
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000010959 steel Substances 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 230000013020 embryo development Effects 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 241000208173 Apiaceae Species 0.000 claims 2
- 239000000706 filtrate Substances 0.000 claims 1
- 239000000725 suspension Substances 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 7
- 230000001488 breeding effect Effects 0.000 abstract description 7
- 230000006698 induction Effects 0.000 abstract description 3
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- 244000269722 Thea sinensis Species 0.000 abstract 2
- 241001122767 Theaceae Species 0.000 description 11
- 230000000694 effects Effects 0.000 description 5
- 235000013311 vegetables Nutrition 0.000 description 5
- 239000003963 antioxidant agent Substances 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000003078 antioxidant effect Effects 0.000 description 3
- 238000012136 culture method Methods 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 235000008671 Calycanthus floridus Nutrition 0.000 description 2
- 235000008670 Calycanthus occidentalis Nutrition 0.000 description 2
- 244000148992 Lindera benzoin Species 0.000 description 2
- 235000004520 Lindera benzoin Nutrition 0.000 description 2
- 235000008262 Lindera benzoin var. benzoin Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000000408 embryogenic effect Effects 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 244000221633 Brassica rapa subsp chinensis Species 0.000 description 1
- 241000219193 Brassicaceae Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 241000146029 Lindera aggregata Species 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 210000002242 embryoid body Anatomy 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- -1 vitamin polyphenol Chemical class 0.000 description 1
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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for improving the formation rate of leaf-shaped embryos of black cabbage seeds and the one-step seedling rate, which relates to the technical field of genetic breeding, and is characterized in that in the bolting and flowering period, a black cabbage flower bud sample at the mononuclear border period is obtained, and purified microspores of the sample are obtained; adding 0-2.0 mg/L of tea polyphenol into NLN culture medium, and re-suspending the obtained purified microspore; step three, placing the product obtained in the step two at 33 ℃ for heat shock treatment for 24 hours, and then transferring to dark culture at 25 ℃; after the embryoid appears visible to the naked eye, placing the embryoid on a shaking table at 25 ℃ for shaking culture for 5 to 10 days, then transferring the embryoid to a solid MS culture medium for culture, and counting the seedling formation condition after 25 days. In the NLN induction culture medium added with 0-2.0 mg/L tea polyphenol, the formation rate of cotyledon-shaped embryos is improved by 1.14-1.54 times, the seedling rate of the cotyledon-shaped embryos is improved by 1.10-1.44 times, and the one-step seedling rate is improved by 1.19-1.80 times. The method can effectively improve the formation rate and the one-step seedling rate of cotyledon-shaped embryos of the brassica oleracea, shorten the breeding period and further improve the efficiency of creating breeding resources by using the brassica oleracea microspore culture technology.
Description
Technical Field
The invention relates to the technical field of genetic breeding, in particular to a method for improving the formation rate and the one-step seedling rate of cotyledonary embryos of black-boned vegetables.
Background
Wucai (Brassica campestris L.ssp. Chinensis) is a variety of Brassica subspecies of Brassicaceae, and has obvious heterosis in heterogamy pollinated crops, but long period of heterogamy cross breeding. The method adopts free microspore culture, can shorten breeding period, improve breeding character, increase adventitious inheritance and enrich the quality resources of the black-boned vegetable.
In recent decades, some researchers at home and abroad do a lot of work on the culture of cruciferous vegetable free microspores, and are dedicated to optimizing microspore embryogenic and embryoid seedling systems, but at present, many genotype microspores have low embryogenic rate and embryoid seedling rate, so that the application range of the technology in practical breeding work is limited.
Specific antioxidants inhibit apoptosis and reduce reactive oxygen species levels, and in recent years, antioxidants have been added to induction media to enhance microspore embryogenesis and embryoid body seedling formation. The antioxidant commonly used at present comprises ascorbic acid, glutathione, tea polyphenol and the like.
Tea polyphenol, also known as antioxidant, vitamin polyphenol and Fanghailing, is a polyhydroxy compound contained in tea leaves, is abbreviated as TP, and has strong antioxidant effect.
Disclosure of Invention
The invention provides a method for improving the formation rate and the one-step seedling rate of leaf-shaped embryos of black-cabbage seeds, which aims to solve the problems in the prior art.
The invention is realized by the following technical scheme:
a method for improving the forming rate and the one-step seedling rate of leaf-shaped embryos of black-cabbage seeds comprises the following steps:
step one, selecting a black-cabbage bud sample in a mononuclear border stage in a bolting and flowering stage, and obtaining purified microspores of the sample;
step two, resuspending the purified microspores obtained in the step one by using an NLN culture medium;
step three, performing heat shock treatment on the product obtained in the step two at 33 ℃ for 24 hours, and then transferring the product to dark culture at 25 ℃; counting the embryo condition when visible embryoid appears, adding 0-2.0 mg/L Tea Polyphenol (TP) into NLN culture medium, placing on a shaker at 25 ℃ for shake culture for 5-10 days, transferring to solid MS culture medium for culture, and counting the seedling condition after 25 days.
Further, in the third step, the shaking culture rotating speed is 50rnp/min.
Further, the step 1 specifically comprises: collecting a Brassica Oleracea bud sample at the bolting and flowering stage, placing the sample in an environment of 4 ℃ for precooling for 1d, then selecting the bud sample in the mononuclear border stage, placing the bud sample in a beaker, and sequentially sterilizing with 70% alcohol for 30s and 0.1% HgCl 2 Shaking for 6min, washing with sterile water for 5min for 3 times, adding B5 culture medium, and grinding bud sample in beaker with glass rod to release microspore; then, filtering the microspore solution in the beaker into a new sterile beaker by using a 300-mesh steel wire screen, secondarily filtering the microspore solution into a centrifuge tube by using a 40-micron cell screen, and centrifuging the filtered microspore solution at the rotating speed of 1000 r/min; and finally, resuspending the centrifugal precipitate by using a B5 culture medium, and centrifuging again at the centrifugal rotating speed of 1000r/min to finally obtain the purified microspore.
Further, after the microspores were basic-suspended and purified by NLN medium in the second step, the density of microspores was adjusted to 1X 10 by cell counter 5 ～5×10 5 one/mL.
Compared with the prior art, the invention has the following advantages:
(1) The invention provides a method capable of remarkably improving the formation rate and the one-step seedling rate of cotyledonary embryos of brassica oleracea, which is characterized in that in the process of culturing brassica oleracea microspore embryos, tea Polyphenol (TP) with a certain concentration is added to an NLN culture medium and is placed on a shaking table for shake culture for 5-10 days and then transferred to an MS solid culture medium, so that the formation rate and the one-step seedling rate of brassica oleracea microspore cotyledonary embryos can be remarkably improved, the breeding process is accelerated, and the method contributes to the breeding industry.
(2) The method provided by the invention has the advantages of simple operation, high production efficiency and the like, and the tea polyphenol is non-toxic and harmless to the environment and has high safety.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The method for improving the formation rate and the one-step seedling rate of the leaf-shaped embryos of the black-cabbage seeds comprises the following specific steps:
(1) And in the bolting and flowering period, picking corresponding flower bud samples for subsequent microspore culture.
(2) And (3) pre-cooling the picked bud sample for 1d at the temperature of 4 ℃.
(3) After precooling treatment, selecting a bud sample in the mononuclear border period, and sequentially disinfecting 30s and 0.1% of HgCl by using 70% of alcohol 2 Shaking for 6min, and washing with sterile water for 5min for 3 times; grinding buds in a B5 culture medium by using a glass rod to release microspores; then, filtering the microspore solution into a centrifuge tube by using a 300-mesh steel wire mesh screen and a 40-micron cell mesh screen in sequence, and centrifuging at the rotating speed of 1000 r/min; and finally, resuspending and centrifuging the precipitate by using a B5 culture medium, and centrifuging again at the centrifugal rotating speed of 1000r/min to finally obtain the precipitate, namely the purified microspore required by the target.
(4) Adding 0-2.0 mg/L Tea Polyphenol (TP) into NLN culture medium, resuspending, diluting the purified microspore obtained in step (3), and adjusting the density to 1 × 105-5 × 105/mL by using a cell counter.
(5) Carrying out heat shock treatment on the culture obtained in the step (4) at the temperature of 33 ℃ for 24 hours, and then transferring to dark culture at the temperature of 25 ℃; when macroscopic embryoid appears, the embryo condition is counted and placed on a shaking table at 25 ℃ for shaking culture (the rotating speed is 50 rnp/min).
(6) And (5) placing the culture obtained in the step (5) in a shaking table for shake culture for 5-10 days, transferring the culture to a solid MS culture medium, and counting the seedling rate once after 25 days.
Example 2
This example is to demonstrate the effect of different concentrations of Tea Polyphenols (TP) on the rate of formation of cotyledonary embryos of different genotypes of brassica oleracea:
the method comprises the following steps: selecting the genotypes of the black-bone vegetables W16, W23, W27, HW, W15 and NH to culture the free microspores. In the process of dissociating microspores, tea Polyphenol (TP) with the concentration of 0, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0mg/L is prepared and respectively added into NLN culture media, the total embryo yield and the proportion of cotyledon-shaped embryos of each genotype are counted after 15 days, then cotyledon-shaped embryos in each genotype are selected to carry out a seedling test, and the influence of different concentrations of TP on the formation rate and the one-step seedling rate of the cotyledon-shaped embryos of the lindera aggregata is further researched. (the other steps are the same, refer to example 1).
The results are shown in table 1:
influence of different concentrations of TP on the germination and seedling conditions of the black-boned cabbage
TABLE 1
As can be seen from Table 1, in the NLN induction culture medium added with 0-2.0 mg/L of tea polyphenol, the microspore embryo-forming rate of different genotypes of the black-flower cabbage has no significant difference, but the formation rate of cotyledon-shaped embryos is improved by 1.14-1.54 times, and the seedling rate of cotyledon-shaped embryos is improved by 1.10-1.44 times. Thus, we can speculate that tea polyphenols may be
The formation of the cotyledon embryo of the free microspore of the black-boned vegetable is promoted, so that the one-time seedling rate is improved.
Example 3
In this embodiment, 210 microspore embryos of yunnan yellow cabbage (NH) are selected as the experimental material, 210 parts of the experimental material are averagely divided into 7 groups, 30 parts of the material are used in each group, each group is cultured according to the culture method provided in embodiment 1, and when the step of adding tea polyphenol into the culture medium in step (5) in embodiment 1 is performed in the culture process, the concentrations of the tea polyphenols used in each group are different, specifically as follows:
major group 1: no Tea Polyphenols (TP) was added to the medium as a control;
large group 2: adding 0.25mg/L Tea Polyphenols (TP) to the culture medium;
major group 3: adding 0.5mg/L Tea Polyphenols (TP) into the culture medium;
major group 4: adding 0.75mg/L Tea Polyphenols (TP) to the culture medium;
large group 5: adding 1.0mg/L Tea Polyphenols (TP) into the culture medium;
major group 6: adding 1.5mg/L Tea Polyphenols (TP) into the culture medium;
major group 7: 2.0mg/L Tea Polyphenols (TP) was added to the medium.
Next, each group was divided into three groups on an average, each group was divided into 10 parts of material, and each group was subjected to shake culture, which was different from time to time, when "the culture of step (5) was shake-cultured on a shaker for 5 to 10 days" in step (6) of example 1, specifically:
group 1: placing the culture obtained in the step (5) in a shaking table for shake culture for 5 days;
group 2: placing the culture obtained in the step (5) in a shaking table for shake culture for 8 days;
group 3: placing the culture obtained in the step (5) in a shaking table for shake culture for 10 days;
after each group completed step (6) in example 1, the groups were counted as shown in Table 2:
TABLE 2
As can be seen from Table 2, compared with the control group without TP, the addition of TP with different concentrations in the NLN medium for shake culture for 5-10 days can effectively improve the one-step seedling rate of the Brassica oleracea microspore embryos; especially under the combination of 0.5mg/L TP +8d, the seedling rate of the spicebush microspore reaches 62.53 percent. From the culture time, the effect of culturing for 8 days is better under the treatment of the same concentration; the effect is better when the culture is carried out for 8 days than 5 days and 10 days between different concentrations.
Example 4
In the embodiment, the brassica oleracea varieties with genotypes of W16 (the one-step seedling rate is 80%), W23 (the one-step seedling rate is 72%), W27 (the one-step seedling rate is 70%), HW (the one-step seedling rate is 62%), W15 (the one-step seedling rate is 38.8%), and NH (the one-step seedling rate is 35.7%) are cultured according to the culture method provided by the embodiment 1, 20 parts of each variety are taken and divided into two groups, namely a group A and a group B, and the two groups corresponding to each variety are cultured by the culture method provided by the embodiment 1; wherein:
group A: the concentration of TP added in the step (5) is 0mg/L, and the shaking culture time in the step (6) is 8d;
group B: the concentration of TP added in the step (5) is 0.5mg/L, and the shaking culture time in the step (6) is 8d;
after the culture is completed, the statistical results are shown in table 3;
TABLE 3
As can be seen from Table 3, for different genotypes of Brassica oleracea, "0.5mg/L TP +8d" can significantly improve the primary seedling rate.
In conclusion, the tea polyphenol can promote the one-step seedling formation of the leaf-shaped embryos of the free microspores of the black-boned cabbage by promoting the formation of the leaf-shaped embryos of the free microspores of the black-boned cabbage; in addition, shaking table shaking culture with TP of 0.5mg/L and 8d has obvious effect on the one-step seedling formation of the spicebush spores, and probably tea polyphenol enhances the antioxidation effect of the microspores, improves the respiration rate, reduces the generation of secondary metabolites and further improves the one-step seedling formation rate.
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 improving the leaf-shaped embryo formation rate and the one-step seedling rate of a black cabbage seed is characterized by comprising the following steps:
step one, selecting a black-cabbage bud sample in a mononuclear border stage in a bolting and flowering stage, and obtaining purified microspores of the sample;
step two, adding 0-2.0 mg/L Tea Polyphenol (TP) into the NLN culture medium and re-suspending the purified microspore obtained in the step one;
and step three, performing heat shock treatment on the product obtained in the step two at 33 ℃ for 24 hours, transferring the product to dark culture at 25 ℃, counting the embryo condition when visible embryoids appear, placing the embryoids on a shaking table at 25 ℃ for shake culture for 5-10 days, transferring the embryoids to a solid MS culture medium for culture, and counting the seedling formation condition after 25 days.
2. The method for improving the rate of formation of leaf shaped embryos from Brassica oleracea and the rate of seedling formation in one step according to claim 1, wherein the shaking speed of the shaking table in the third step is 50rnp/min.
3. A method for increasing the rate of leaf shaped embryos formation and the rate of one-step plantlet formation in Umbelliferae as claimed in claim 1 or 2,
the step 1 specifically comprises the following steps: collecting Brassica Oleracea bud sample at bolting and flowering stage, placing in 4 deg.C environment for pre-cooling for 1d, selecting bud at mononuclear border stage, placing in beaker, and sequentially sterilizing with 70% alcohol for 30s and 0.1% HgCl 2 Shaking for 6min, and washing with sterile water for 5min for 3 times; grinding bud with glass rod in B5 culture medium, and releasingPlacing microspores; sequentially filtering with 300 mesh steel wire mesh screen and 40 μm cell mesh screen, and centrifuging the collected filtrate at 1000 r/min; and finally, resuspending the centrifugal precipitate by using a B5 culture medium, and centrifuging again at the centrifugal rotating speed of 1000r/min to finally obtain the purified microspore.
4. The method for increasing leaf-shaped embryogenesis rate and one-step seedling rate of Umbelliferae as claimed in claim 3, wherein after basic suspension of microspores in NLN medium in step two, the density of microspores is adjusted to 1X 10 by cell counter 5 ～5×10 5 one/mL.
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| CN114916441A (en) * | 2022-05-24 | 2022-08-19 | 安徽农业大学 | Method for improving germ production rate of free microspores of lindera aggregate by using melatonin |
| CN114982632A (en) * | 2022-04-21 | 2022-09-02 | 安徽农业大学 | Method for reducing browning rate of regeneration plant of black-bone vegetable microspore |
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2022
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| CN113711920A (en) * | 2021-09-26 | 2021-11-30 | 安徽农业大学 | Method for improving one-time seedling rate of microspore embryoid of non-heading Chinese cabbage |
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