CN103262795B - Method for rapid propagation of broccoli cytoplasm male sterility line - Google Patents
Method for rapid propagation of broccoli cytoplasm male sterility line Download PDFInfo
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- CN103262795B CN103262795B CN201310166265.4A CN201310166265A CN103262795B CN 103262795 B CN103262795 B CN 103262795B CN 201310166265 A CN201310166265 A CN 201310166265A CN 103262795 B CN103262795 B CN 103262795B
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- explant
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- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 title claims abstract description 27
- 235000017647 Brassica oleracea var italica Nutrition 0.000 title claims abstract description 27
- 240000003259 Brassica oleracea var. botrytis Species 0.000 title claims abstract description 24
- 206010021929 Infertility male Diseases 0.000 title claims abstract description 16
- 208000007466 Male Infertility Diseases 0.000 title claims abstract description 16
- 210000000805 cytoplasm Anatomy 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims abstract description 7
- 230000001954 sterilising effect Effects 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000000338 in vitro Methods 0.000 claims description 14
- 229920001817 Agar Polymers 0.000 claims description 10
- 229930006000 Sucrose Natural products 0.000 claims description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 10
- 239000008272 agar Substances 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 5
- 244000037666 field crops Species 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 239000008223 sterile water Substances 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 abstract description 2
- 238000004659 sterilization and disinfection Methods 0.000 abstract 2
- 239000005708 Sodium hypochlorite Substances 0.000 abstract 1
- 229960000935 dehydrated alcohol Drugs 0.000 abstract 1
- 239000012883 rooting culture medium Substances 0.000 abstract 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 abstract 1
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 6
- 238000011069 regeneration method Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 244000308180 Brassica oleracea var. italica Species 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 239000005972 6-Benzyladenine Substances 0.000 description 1
- 241000563913 Brassia Species 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 208000021267 infertility disease Diseases 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229940064880 inositol 100 mg Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000006870 ms-medium Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000005849 recognition of pollen Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 229940033203 vitamin b6 0.5 mg Drugs 0.000 description 1
Abstract
The present invention discloses a method for rapid propagation of a broccoli cytoplasm male sterility line. The method mainly comprises the following steps: taking an explant from a field broccoli cytoplasm male sterility line plant, cutting the explant into 3-5 cm small blocks, adopting dehydrated alcohol with a concentration of 75% to carry out sterilization on the surface for 30-60 seconds, adopting a sodium hypochlorite solution with a volume fraction of 8% to carry out disinfection for 18-20 min, rinsing 5-6 times with sterile water, inoculating the sterile explant on a proliferation culture medium, placing in a 23-27 DEG C culture chamber to culture, forming new young buds through proliferation after culturing for 20-25 days, separating the new young buds, sterilely transferring the separated young buds into a culture medium to continuously culture, repeating transferring culture 5-10 times, transferring tissue culture seedling into a rooting culture medium to root, carrying out acclimation, and transplanting into a field. The method has advantages of simple operation, high breeding efficiency and the like.
Description
Technical field
The present invention relates to field of plant tissue culture technique, particularly relate to a kind of method that broccoli cytoplasm male sterility line is numerous soon.
Background technology
Broccoli (Brassia oleracea L. var. varitalica Planch) has another name called broccoli, broccoli, tender stem broccoli, for Cruciferae Brassica genus cole vegetables, being rich in multivitamin, mineral matter and natural cancer-resisting material, is the vegetables that nutritive value is very high.Broccoli originates from Mediterranean, and domestic germ plasm resource is deficient, and seed price is expensive, and external elite germplasm is difficult to again obtain, and these factors govern carrying out of domestic broccoli polymorphic type good variety selection work.High-quality, cultivation that is degeneration-resistant, disease-resistant variety are one of hot issues of breeding man concern always.And the primary key of cultivating improved seeds is seed selection possesses the Broccoli Resources resource of target property.
Utilize the trend of male sterile replacement Self-Incompatibility production broccoli crossbreed day by day obvious.Utilize cytoplasm male sterility line preparing hybrid kind, the decline of self incompatible line artificial bud pollination reproductive-cost high, long-term selfing vitality continuously can be overcome, hybrid rate is difficult to reach the defect such as 100% by environmental constraints, breed parent than dominant genic male sterility system again simple.Simultaneously this male sterile line has that transformation is simple, sterility thorough, and with the crossbreed of its preparation, land for growing field crops hybrid purity can ensure to reach 100%.In recent years, my unit obtains some cytoplasm male sterility line materials in field seed selection, but due to fuzzy to its genetic background, not yet find good maintainer, and by broccoli Vitro Quick Reproduction, can realize preserving it and expanding numerous, to meet the needs on breed and production.
External Anderson(1977 is seen the earliest about the tissue cultures of broccoli, research numerous soon) and Johnson(1978).And at home, Li Shuxuan etc. (1983) utilize the group of the explant such as blade, middle rib to train, add the condition of a certain amount of hormone combination in minimal medium MS under, achieve good effect; Zhang Lili etc. (2008) for explant, study its High Frequency Regeneration system with broccoli Cotyledons with petiole, obtain higher regeneration frequency all higher than 80%.But the research adopting broccoli curd to carry out tissue-culturing rapid propagation as explant has no report.
Summary of the invention
the technical problem solved:the invention provides a kind of method that broccoli cytoplasm male sterility line is numerous soon, realize the preservation of cytoplasm male sterility line material and expand numerous, to meet the needs on breed and production.
technical scheme:the invention provides a kind of method that broccoli cytoplasm male sterility line is numerous soon, mainly comprise the following steps: fetch explant from field broccoli cytoplasm male sterility line plant, explant is cut into the fritter of 3 ~ 5cm, concentration is adopted to be that the absolute ethyl alcohol of 75% was its surface sterilizing 30 ~ 60 seconds, sterilize 18 ~ 20 minutes with the liquor natrii hypochloritis that volume fraction is 8% again, with aseptic water washing 5 ~ 6 times, then aseptic explant is inoculated on proliferated culture medium, is placed in 25 ± 2 DEG C of culturing room and cultivates; When cultivating 20 ~ 25 days, propagation forms new young shoot, be separated aseptic being transferred in medium of new young shoot to continue to cultivate, after repeating switching cultivation 5 ~ 10 times, plantlet in vitro is transferred in root media and takes root, through domestication, be transplanted to land for growing field crops, thus achieve the numerous soon of broccoli cytoplasm male sterility line material and production application.
Described explant is the tender bouquet of band handle children newly grown, and after stripping and slicing, every block explant comprises at least one bud point and complete bouquet.
Described proliferated culture medium is MS+2 ~ 3mg/L 6-BA+0.05 ~ 0.1mg/L NAA+20 ~ 30g/L sucrose+1.0 ~ 1.3g/L agar, and pH value is 5.8 ~ 6.0, and wherein NAA is methyl α-naphthyl acetate, and 6-BA is 6-benzyladenine.
Described root media is 1/2 mg/L IBA+0.1 ~ 0.15, MS+0.1 ~ 0.15 mg/L NAA+20 ~ 30g/L sucrose+1.0 ~ 1.3g/L agar, and pH value is 5.8 ~ 6.0, and wherein NAA is methyl α-naphthyl acetate, and IBA is indolebutyric acid.
Described MS medium, in 1L, consists of: NH
4hO
31650 mg, KNO
31900 mg, CaCl
22H
2o 440 mg, KH
2pO
4170 mg, MgSO
47H
2o 370 mg, FeSO
47H
2o 27.8 mg, Na
2eDTA 37.3 mg, H
3bO
36.2 mg, MnSO
4h
2o 16.9 mg, ZnSO
47H
2o 8.6 mg, Na
2moO
42H
2o 0.25 mg, CuSO
45H
2o 0.025 mg, CoCl
26H
2o 0.025 mg, KI 0.83 mg, inositol 100 mg, glycine 2 mg, nicotinic acid 0.5 mg, vitamin B1 0.1 mg, the sterile water of vitamin B6 0.5 mg and surplus.
beneficial effect: (1)the method that a kind of broccoli cytoplasm male sterility line provided by the invention is numerous soon, what adopt is that the little bouquet of young tender band handle carries out tissue-culturing rapid propagation as explant, because the little bouquet of band handle contains growing point, cultivate in proliferated culture medium after sterilizing and be easy to growth formation sprouting, a large amount of plantlet in vitro can be obtained fast after repeatedly transferring, meet need of production, there is easy and simple to handle, reproductive efficiency advantages of higher.(2) the adventitious bud proliferation coefficient that the present invention obtains is 5 ~ 6, and rooting rate 85 ~ 90%, on average take root several 7 ~ 9, plantlet in vitro transplanting survival rate is 94 ~ 96%.Plantlet in vitro regeneration plant and seedling from seed, compared with variable rate technology, do not have notable difference.
Embodiment
embodiment 1
Proliferated culture medium is: MS+2mg/L 6-BA+0.05mg/L NAA+20g/L sucrose+1.0g/L agar, and pH value is 6.0; Root media is 1/2 MS+0.1mg/L IBA+0.15 mg/L NAA+20g/L sucrose+1.0g/L agar, and pH value is 6.0.
The tender band handle bouquet of broccoli children grown is fetched from field, laboratory superclean bench is cut into scalpel the fritter of 3cm, be that the absolute ethyl alcohol of 75% was its surface sterilizing 30 seconds by concentration, sterilize 18 minutes with the liquor natrii hypochloritis that volume fraction is 8% again, with aseptic water washing 5 times, then aseptic explant is inoculated on proliferated culture medium, be placed in 23 DEG C of culturing room to cultivate, sprouting is formed when cultivating 20 days, continue to cultivate in switching sprouting to proliferated culture medium, after repeating switching cultivation 5 times, plantlet in vitro is transferred in root media and takes root, the health care belt root seedling finally formed is through domestication, be transplanted to field.
The adventitious bud proliferation coefficient that the present embodiment obtains is 5, rooting rate 85%, and on average take root several 7, plantlet in vitro transplanting survival rate is 94%, and plantlet in vitro regeneration plant and seedling from seed, compared with variable rate technology, do not have notable difference.
embodiment 2
Proliferated culture medium is: MS+3mg/L 6-BA+0.1mg/L NAA+25g/L sucrose+1.15g/L agar, and pH value is 5.9; Root media is 1/2 MS+0.12 mg/L IBA+0.12 mg/L NAA+25g/L sucrose+1.15g/L agar, and pH value is 5.9.
The tender band handle bouquet of broccoli children grown is fetched from field, laboratory superclean bench is cut into scalpel the fritter of 4cm, be that the absolute ethyl alcohol of 75% was its surface sterilizing 45 seconds by concentration, sterilize 19 minutes with the liquor natrii hypochloritis that volume fraction is 8% again, with aseptic water washing 5 times, then aseptic explant is inoculated on proliferated culture medium, be placed in 25 DEG C of culturing room to cultivate, sprouting is formed when cultivating 25 days, continue to cultivate in switching sprouting to proliferated culture medium, after repeating switching cultivation 8 times, plantlet in vitro is transferred in root media and takes root, the health care belt root seedling replanting finally formed is to field.
The adventitious bud proliferation coefficient that the present embodiment obtains is 5.5, rooting rate 87%, and on average take root several 8, plantlet in vitro transplanting survival rate is 95%, and plantlet in vitro regeneration plant and seedling from seed, compared with variable rate technology, do not have notable difference.
embodiment 3
Proliferated culture medium is: MS+3mg/L 6-BA+0.08mg/L NAA+30g/L sucrose+1.3g/L agar, and pH value is 5.8; Root media is 1/2 MS+0.15 mg/L IBA+0.15 mg/L NAA+30g/L sucrose+1.3g/L agar, and pH value is 5.8.
The tender band handle bouquet of broccoli children grown is fetched from field, laboratory superclean bench is cut into scalpel the fritter of 5cm, be that the absolute ethyl alcohol of 75% was its surface sterilizing 60 seconds by concentration, sterilize 20 minutes with the liquor natrii hypochloritis that volume fraction is 8% again, with aseptic water washing 6 times, then aseptic explant is inoculated on proliferated culture medium, be placed in 25 DEG C of culturing room to cultivate, sprouting is formed when cultivating 25 days, continue to cultivate in switching sprouting to proliferated culture medium, after repeating switching cultivation 10 times, plantlet in vitro is transferred in root media and takes root, the health care belt root seedling replanting finally formed is to field.
The adventitious bud proliferation coefficient that the present embodiment obtains is 6, rooting rate 90%, and on average take root several 9, plantlet in vitro transplanting survival rate is 96%, and plantlet in vitro regeneration plant and seedling from seed, compared with variable rate technology, do not have notable difference.
Claims (1)
1. the method that a broccoli cytoplasm male sterility line is numerous soon, it is characterized in that comprising the following steps: fetch explant from field broccoli cytoplasm male sterility line plant, explant is cut into the fritter of 3 ~ 5cm, concentration is adopted to be that the absolute ethyl alcohol of 75% was its surface sterilizing 30 ~ 60 seconds, sterilize 18 ~ 20 minutes with the liquor natrii hypochloritis that volume fraction is 8% again, with aseptic water washing 5 ~ 6 times, then aseptic explant is inoculated on proliferated culture medium, is placed in 25 ± 2 DEG C of culturing room and cultivates; When cultivating 20 ~ 25 days, propagation forms new young shoot, is separated aseptic being transferred in proliferated culture medium of new young shoot and continues to cultivate, and repeats after switching cultivates 5-10 time plantlet in vitro to be transferred in root media to take root, through rooting culture to land for growing field crops; Described explant is the children's tender band handle bouquet newly grown, and after stripping and slicing, every block explant comprises at least one bud point and complete bouquet; Described proliferated culture medium is MS+2 ~ 3mg/L 6-BA+0.05 ~ 0.1mg/L NAA+20 ~ 30g/L sucrose+1.0 ~ 1.3g/L agar, and pH value is 5.8 ~ 6.0; Described root media is 1/2 mg/L IBA+0.1 ~ 0.15, MS+0.1 ~ 0.15 mg/L NAA+20 ~ 30g/L sucrose+1.0 ~ 1.3g/L agar, and pH value is 5.8 ~ 6.0.
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| CN201310166265.4A CN103262795B (en) | 2013-05-08 | 2013-05-08 | Method for rapid propagation of broccoli cytoplasm male sterility line |
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| CN106305418A (en) * | 2016-08-22 | 2017-01-11 | 江苏丘陵地区镇江农业科学研究所 | Method for inhibiting endophytic bacterial contamination of broccoli tissue culture seedlings |
| CN109169278B (en) * | 2018-09-18 | 2021-12-10 | 江苏省农业科学院 | Tissue culture method for improving in-vitro regeneration efficiency of broccoli |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1180744A (en) * | 1996-10-25 | 1998-05-06 | 辽宁师范大学生物工程研究所 | Method of tissue culture quick breeding for white cauliflower and green cauliflower cross breed new strain test-tube seeding |
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2013
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1180744A (en) * | 1996-10-25 | 1998-05-06 | 辽宁师范大学生物工程研究所 | Method of tissue culture quick breeding for white cauliflower and green cauliflower cross breed new strain test-tube seeding |
Non-Patent Citations (2)
| Title |
|---|
| 青花菜细胞质雄性不育系和保持系的选育研究;唐征;《中国优秀硕士学位论文全文数据库》;20110118;第24-27页，第28页第3-4行，第32页第3段第3-4行 * |
| 青花菜雄性不育系组培快繁技术研究;张振超等;《江苏农业学报》;20110630(第03期);第680页左栏第2段第2-3行，第680-681页第1.1-1.2.5节 * |
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