CN103262795A - 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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- CN103262795A CN103262795A CN2013101662654A CN201310166265A CN103262795A CN 103262795 A CN103262795 A CN 103262795A CN 2013101662654 A CN2013101662654 A CN 2013101662654A CN 201310166265 A CN201310166265 A CN 201310166265A CN 103262795 A CN103262795 A CN 103262795A
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- explant
- male sterility
- broccoli
- sterility line
- cytoplasm male
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- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 title claims abstract description 30
- 235000017647 Brassica oleracea var italica Nutrition 0.000 title claims abstract description 30
- 240000003259 Brassica oleracea var. botrytis Species 0.000 title claims abstract description 28
- 206010021929 Infertility male Diseases 0.000 title claims abstract description 19
- 208000007466 Male Infertility Diseases 0.000 title claims abstract description 19
- 210000000805 cytoplasm Anatomy 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 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
- 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
- 239000002609 medium Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 2
- 230000035755 proliferation Effects 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 3
- 239000008223 sterile water Substances 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 abstract description 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
- 239000000463 material Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 230000036541 health Effects 0.000 description 3
- 244000308180 Brassica oleracea var. italica Species 0.000 description 2
- 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
- 238000004519 manufacturing process Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 239000005972 6-Benzyladenine Substances 0.000 description 1
- 241000563913 Brassia Species 0.000 description 1
- 241000219193 Brassicaceae 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
- 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
- 238000000338 in vitro Methods 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
- 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
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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, relate in particular to the fast numerous method of a kind of broccoli cytoplasm male sterility line.
Background technology
Broccoli (Brassia oleracea L. var. varitalica Planch) has another name called broccoli, broccoli, tender stem broccoli, for the Cruciferae rape belongs to cole vegetables, being rich in multivitamin, mineral matter and natural cancer-resisting material, is the very high vegetables of nutritive value.Broccoli originates from the Mediterranean bank, domestic germ plasm resource scarcity, and seed is expensive, and external elite germplasm is difficult to again obtain, and these factors are restricting carrying out of domestic broccoli polymorphic type good variety selection work.High-quality, cultivation degeneration-resistant, disease-resistant variety are one of hot issues of breeding man concern always.And being seed selection, the primary key of cultivating improved seeds possesses the broccoli germ plasm resource of target property.
Utilize the trend of the not affine production broccoli of male sterile replacement selfing crossbreed day by day obvious.Utilize cytoplasm male sterility line preparing hybrid kind, can overcome the artificial bud pollination reproductive-cost of self incompatible line height, the decline of long-term selfing vitality continuously, hybrid rate and be subjected to environmental constraints to be difficult to reach defective such as 100%, it is simple to breed the parent than dominant genic male sterility system again.Simultaneously this male sterile line has that transformation is simple, sterility is thorough, and with the crossbreed of its preparation, the land for growing field crops hybrid purity can guarantee to reach 100%.In recent years, my unit has obtained some cytoplasm male sterility line materials in the field seed selection, but because fuzzy to its genetic background, do not find as yet and keep system preferably, and by the broccoli breeding fast of exsomatizing, can realize to its preservation and expand numerous, with the needs that satisfy breeding and produce.
Tissue about broccoli is cultivated, numerous research sees external Anderson(1977 the earliest soon) and Johnson(1978).And at home, Li Shuxuan etc. (1983) utilize the group training of explants such as blade, middle rib, have obtained good effect under the condition of additional a certain amount of hormone combination in minimal medium MS; Zhang Lili etc. (2008) are explant with broccoli band handle cotyledon, study its high frequency Regeneration in Vitro system, have obtained higher regeneration frequency and all have been higher than 80%.But adopt broccoli curd not appear in the newspapers as the research that explant carries out tissue-culturing rapid propagation.
Summary of the invention
The technical problem that solves:The invention provides the fast numerous method of a kind of broccoli cytoplasm male sterility line, realize to the preservation of cytoplasm male sterility line material and expand numerous, with the needs that satisfy breeding and produce.
Technical scheme:The invention provides the fast numerous method of a kind of broccoli cytoplasm male sterility line, mainly may further comprise the steps: fetch explant from field broccoli cytoplasm male sterility line plant, explant is cut into the fritter of 3～5cm, to adopt concentration be 75% absolute ethyl alcohol its surface sterilizing 30～60 seconds, be that 8% liquor natrii hypochloritis sterilized 18～20 minutes with volume fraction again, with aseptic water washing 5～6 times, then aseptic explant is inoculated on the proliferated culture medium, places 25 ± 2 ℃ of culturing room to cultivate; When cultivating 20～25 days, propagation forms new young shoot, separating aseptic being transferred to of new young shoot continues in the medium to cultivate, after repeating switching and cultivating 5～10 times group training seedling is transferred in the root media and takes root, process is tamed, is transplanted to the land for growing field crops, thereby has realized the fast numerous and production application of broccoli cytoplasm male sterility line material.
The band handle children tender bouquet of described explant for newly growing, every explant comprises at least one bud point and complete bouquet after the stripping and slicing.
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 the pH value is 5.8～6.0, and wherein NAA is methyl, and 6-BA is 6-benzyladenine.
Described root media is 1/2 MS+0.1～0.15 mg/L IBA+0.1～0.15 mg/L NAA+20～30g/L sucrose+1.0～1.3g/L agar, and the pH value is 5.8～6.0, and wherein NAA is methyl, 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 fast numerous method of a kind of broccoli cytoplasm male sterility line provided by the invention, 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, the sterilization back is cultivated in proliferated culture medium and is easy to growth formation sprouting, through repeatedly obtaining a large amount of group training seedlings fast after the switching, satisfy and produce needs, have easy and simple to handle, reproductive efficiency advantages of higher.(2) the adventitious bud proliferation coefficient of the present invention's acquisition is 5～6, and rooting rate 85～90% is on average taken root several 7～9, and group training transplantation of seedlings survival rate is 94～96%.Group training seedling regeneration plant shows in the field with seedling from seed to be compared, and does 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 the 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 the pH value is 6.0.
Fetch the tender band handle bouquet of broccoli children of growing from the field, on the superclean bench of laboratory, be cut into the fritter of 3cm with scalpel, be 75% absolute ethyl alcohol with concentration its surface sterilizing 30 seconds, be that 8% liquor natrii hypochloritis sterilized 18 minutes with volume fraction again, with aseptic water washing 5 times, then aseptic explant is inoculated on the proliferated culture medium, place 23 ℃ of culturing room to cultivate, form sprouting when cultivating 20 days, the switching sprouting continues to cultivate in proliferated culture medium, after repeating switching and cultivating 5 times group training seedling is transferred in the root media and takes root, the health band root seedling that forms is through domestication at last, be transplanted to the field.
The adventitious bud proliferation coefficient of present embodiment acquisition is 5, and rooting rate 85% is on average taken root several 7, and group training transplantation of seedlings survival rate is 94%, and group training seedling regeneration plant shows in the field with seedling from seed to be compared, and does 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 the 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 the pH value is 5.9.
Fetch the tender band handle bouquet of broccoli children of growing from the field, on the superclean bench of laboratory, be cut into the fritter of 4cm with scalpel, be 75% absolute ethyl alcohol with concentration its surface sterilizing 45 seconds, be that 8% liquor natrii hypochloritis sterilized 19 minutes with volume fraction again, with aseptic water washing 5 times, then aseptic explant is inoculated on the proliferated culture medium, place 25 ℃ of culturing room to cultivate, form sprouting when cultivating 25 days, the switching sprouting continues to cultivate in proliferated culture medium, after repeating switching and cultivating 8 times group training seedling is transferred in the root media and takes root, the health band root seedling replanting of Xing Chenging arrives the field at last.
The adventitious bud proliferation coefficient of present embodiment acquisition is 5.5, and rooting rate 87% is on average taken root several 8, and group training transplantation of seedlings survival rate is 95%, and group training seedling regeneration plant shows in the field with seedling from seed to be compared, and does 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 the 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 the pH value is 5.8.
Fetch the tender band handle bouquet of broccoli children of growing from the field, on the superclean bench of laboratory, be cut into the fritter of 5cm with scalpel, be 75% absolute ethyl alcohol with concentration its surface sterilizing 60 seconds, be that 8% liquor natrii hypochloritis sterilized 20 minutes with volume fraction again, with aseptic water washing 6 times, then aseptic explant is inoculated on the proliferated culture medium, place 25 ℃ of culturing room to cultivate, form sprouting when cultivating 25 days, the switching sprouting continues to cultivate in proliferated culture medium, after repeating switching and cultivating 10 times group training seedling is transferred in the root media and takes root, the health band root seedling replanting of Xing Chenging arrives the field at last.
The adventitious bud proliferation coefficient of present embodiment acquisition is 6, and rooting rate 90% is on average taken root several 9, and group training transplantation of seedlings survival rate is 96%, and group training seedling regeneration plant shows in the field with seedling from seed to be compared, and does not have notable difference.
Claims (4)
1. the fast numerous method of a broccoli cytoplasm male sterility line, it is characterized in that mainly may further comprise the steps: fetch explant from field broccoli cytoplasm male sterility line plant, explant is cut into the fritter of 3～5cm, to adopt concentration be 75% absolute ethyl alcohol its surface sterilizing 30～60 seconds, be that 8% liquor natrii hypochloritis sterilized 18～20 minutes with volume fraction again, with aseptic water washing 5～6 times, then aseptic explant is inoculated on the proliferated culture medium, places 25 ± 2 ℃ of culturing room to cultivate; When cultivating 20～25 days, breed and form new young shoot, separate aseptic being transferred to of new young shoot and continue in the medium to cultivate, 5-10 the back of cultivation of repeating to transfer is transferred to group training seedling in the root media takes root, and process is tamed and is transplanted to the land for growing field crops.
2. the fast numerous method of a kind of broccoli cytoplasm male sterility line according to claim 1 is characterized in that the band handle children tender bouquet of described explant for newly growing, and every explant comprises at least one bud point and complete bouquet after the stripping and slicing.
3. the fast numerous method of a kind of broccoli cytoplasm male sterility line according to claim 1, it is characterized in that 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, the pH value is 5.8～6.0.
4. the fast numerous method of a kind of broccoli cytoplasm male sterility line according to claim 1, it is characterized in that described root media is 1/2 MS+0.1～0.15 mg/L IBA+0.1～0.15 mg/L NAA+20～30g/L sucrose+1.0～1.3g/L agar, the 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109169278A (en) * | 2018-09-18 | 2019-01-11 | 江苏省农业科学院 | A kind of method for tissue culture improving broccoli in-vitro regeneration efficiency |
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 |
-
2013
- 2013-05-08 CN CN201310166265.4A patent/CN103262795B/en active Active - Reinstated
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 |
|---|
| 唐征: "青花菜细胞质雄性不育系和保持系的选育研究", 《中国优秀硕士学位论文全文数据库》, 18 January 2011 (2011-01-18) * |
| 张振超等: "青花菜雄性不育系组培快繁技术研究", 《江苏农业学报》, no. 03, 30 June 2011 (2011-06-30) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109169278A (en) * | 2018-09-18 | 2019-01-11 | 江苏省农业科学院 | A kind of method for tissue culture improving broccoli in-vitro regeneration efficiency |
| 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 |
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