CN103392592A - Tetraploid Brassica oleracea var. botrytis L. cultivation method - Google Patents
Tetraploid Brassica oleracea var. botrytis L. cultivation method Download PDFInfo
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- CN103392592A CN103392592A CN2013101658593A CN201310165859A CN103392592A CN 103392592 A CN103392592 A CN 103392592A CN 2013101658593 A CN2013101658593 A CN 2013101658593A CN 201310165859 A CN201310165859 A CN 201310165859A CN 103392592 A CN103392592 A CN 103392592A
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- preserved egg
- tetraploid
- aseptic
- egg dish
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Abstract
The invention discloses a tetraploid Brassica oleracea var. botrytis L. cultivation method. The method comprises the following steps: choosing plump and healthy Brassica oleracea var. botrytis L. seeds, disinfecting, and inoculating the obtained aseptic seeds to an MS germination medium for germination; cutting to obtain aseptic seedlings having a proper size, putting the aseptic seedlings into a colchicine-containing MS liquid introduction medium for induction; cleaning with sterile water, cutting into a proper size, inoculating to a colchicine-free solid differentiation medium for culture, and differentiating to form buds; cutting to obtain regenerated buds, inoculating to a rooting medium for rooting culture, hardening seedlings, and transplanting to obtain regenerated plants; and taking the tender leaves of the regenerated plants, detecting the ploidies of all the regenerated plants, and determining the ploidy level of the plants from a regenerated plant colony.
Description
Technical field
The present invention relates to a kind of breeding method of tetraploid preserved egg dish, belong to field of plant variety breeding technology.
Background technology
The preserved egg dish (
Brassica oleraceaVar.
BotrytisL.) claiming again loose cauliflower, is that the Cruciferae wild cabbage belongs to a mutation of spending cabbage in vain, and the Yin Qilei branch is longer, and the flower layer is thinner, and when bouquet fully expands, form consolidation not, be loose shape with respect to common cauliflower, so gain the name.Compare with general compact real form cauliflower kind, the preserved egg dish has two distinguishing features: the one, and boiling fastness is good, and food flavor is delicious, and the vitamin C of preserved egg dish, soluble sugar content are obviously high than tight bouquet cauliflower, get consumer reception very much.
Polyploid makes polyploid breeding become gradually horticultural crop because of the characteristics of its " huge property ", especially one of main path take nutrition organs or succulence fleshiness fruit as the vegetable crop breeding of edible organs.Utilize at present the research of Colchicine-induced Tetraploid to have been reported on the various vegetables crop, yet about the initiative of preserved egg dish tetraploid new germ plasm, have no report.Therefore, utilize Chemical treatment dliploid preserved egg dish test material, mutagenesis obtains quality and yield and all is better than the red skin preserved egg of autodiploid tetraploid dish new germ plasm and has very important significance.
Summary of the invention
The technical problem that solves:For existing preserved egg vegetable matter and the lower problem of output, the invention provides a kind of breeding method of tetraploid preserved egg dish, select a collection of tetraploid preserved egg dish new germ plasm.
Technical scheme:The invention provides a kind of breeding method of tetraploid preserved egg dish, mainly comprise the following steps: (1) selects healthy full preserved egg colza, be that 70%～75% ethanol is to the surface of the seed sterilization 30 ~ 60s with concentration on superclean bench, then using concentration (volume fraction) is that 1% mercuric chloride solution is processed 12～15min, with aseptic water washing 3～5 times; (2) aseptic seed is inoculated on MS germination medium, being placed in 25 ℃, light application time is 14 hd
-1Lower cultivation 3～7 days; (3) cut the aseptic seedling that contains the cotyledon growing point and be inoculated in the MS liquid inducing culture that contains 50～200 mg colchicins, 14hd
-1Under illumination, vibration is induced and was cultivated 2 ~ 3 days; (4) aseptic seedling that will cross with colchicine treatment, with sterile water wash 2～3 times, in access solid differential medium, is 14 hd in 25 ℃ of room temperatures, light application time after cutting
-1, intensity of illumination is to cultivate under 2000 lux conditions, until differentiation, the regeneration bud; (5) cut regeneration bud and be seeded on root media and cultivate, the seedling of robust growth, well developed root system is transplanted after hardening, obtain regeneration plant; (6) according to botany proterties, cytology proterties and flow cytometer, the regeneration plant of field normal growth is carried out Ploidy Identification fast, obtain generation tetraploid preserved egg dish mutant strain.
Described preserved egg colza is zygoid preserved egg dish, and loose, green stalk, product are of fine quality; Described germination medium is comprised of 1L MS medium, 20～30g sucrose and 8～9g agar, and pH is 5.8～6.0; Described liquid inducing culture is comprised of 1L MS medium, 0.1～0.2 mg NAA, 5.0～6.0mg 6-BA, 50～200 mg colchicins, and pH is 5.8～6.0; Described solid differential medium is comprised of 1L MS medium, 0.1～0.2 mg NAA, 5.0～6.0 mg6-BA, 20～30g sucrose, 8～9g agar, and pH is 5.8～6.0; Described root media is comprised of 1L MS medium, 0.2～0.3 mg NAA, 20～30g sucrose, 8～9 g agar, and pH is 5.8～6.0.
Described MS medium,, in 1L, consist of: NH
4HO
31650mg, KNO
31900mg, CaCl
22H
2O 440mg, KH
2PO
4170mg, MgSO
47H
2O 370mg, FeSO
47H
2O 27.8mg, Na
2EDTA 37.3mg, H
3BO
36.2mg, MnSO
4H
2O 16.9mg, ZnSO
47H
2O 8.6mg, Na
2MoO
42H
2O 0.25mg, CuSO
45H
2O 0.025mg, CoCl
26H
2O 0.025mg, KI 0.83mg, inositol 100mg, glycine 2mg, nicotinic acid 0.5mg, vitamin B1 0.1mg, the sterile water of vitamin B6 0.5mg and surplus.
Beneficial effect:Variation has all appearred in the stages that the tetraploid preserved egg dish that the present invention obtains is compared at plant strain growth with its autodiploid.The cell observation aspects such as the reproductive organs such as the nutrition organs such as tetraploid stem, leaf and floral organ, fleshy root and blade lower epidermis pore, guard cell's chloroplast, pollen grain all show the huge property of polyploid, vane thickness increases by 30%～45%, pollen and pore opening increase more than 30%, and difference is extremely remarkable.
Embodiment
Embodiment 1
(1) medium preparation (medium that comprises different cultivation stages, its component and each component in every liter of medium contained weight)
1) the germination medium is: MS medium 1L+ sucrose 20g+ agar 8g, pH5.8;
2) the liquid inducing culture is: MS medium 1L+0.1mg NAA+5.0mg 6-BA+50mg colchicin, pH5.8;
3) the solid differential medium is: MS medium 1L+0.15mg NAA+5.0mg6-BA+20g sucrose+8g agar, pH5.8;
4) root media is: MS medium 1L+0.2 mg NAA+20 g sucrose+8 g agar, pH5.8;
(2) tetraploid preserved egg dish is induced processing
1) selecting the preserved egg colza son of full health, is 70% ethanol to the surface of the seed 30s that sterilizes with concentration on superclean bench; Be 1% mercuric chloride (mercury chloride) solution-treated 12min with concentration, aseptic water washing 3 times;
2) aseptic seed is inoculated on MS germination medium, is placed in 25 ℃, 14 hd
-1Cultivated 5 days;
3) cut the growth aseptic seedling of 5 days and put in the MS liquid inducing culture that contains colchicin, 14 hd
-1Shaken cultivation 72 h under illumination;
4) aseptic seedling of colchicine treatment being crossed sterile water wash 2 times, be linked into after cutting in the solid differential medium that does not contain colchicin, in 25 ℃ of room temperatures, light application time, is 14 hd
-1, intensity of illumination is to cultivate under 2 000 lux conditions, until differentiation, the regeneration bud;
5) regeneration bud that cuts healthy growth is seeded on root media, carries out culture of rootage under 14h illumination every day, 25 ℃; After 3 weeks, the seedling of root growth stalwartness was carried out hardening 3 days; Clean group training shoot root section medium with clear water during transplanting, 800 times of 75% tpn soaked base portion 2 minutes; Rear plant is planted in grow seedlings of vegetable dedicated substrate cave dish, plastic foil covers moisturizing, cultivates in greenhouse and transplants afterwards in 10 days, obtains regeneration plant;
6) get the tender leaf of regeneration plant, with flow cytometer, detect each plant ploidy, the ploidy level of the plant of determining to morph;
(3) in step (2) 3) not add in the liquid inducing culture colchicin induces, other operation is with " (2) tetraploid preserved egg dish is induced processing ", preserved egg dish in contrast.
Result: detect and find through flow cytometer, preserved egg dish tetraploid induction rate reaches 26.3%.The cell observation aspects such as the reproductive organs such as the nutrition organs such as tetraploid stem, leaf and floral organ, fleshy root and blade lower epidermis pore, guard cell's chloroplast, pollen grain all show the huge property of polyploid, pollen and pore opening increase more than 30%, and difference is extremely remarkable.
Embodiment 2
(1) medium preparation: 1) the germination medium is: MS medium 1L+ sucrose 20g+ agar 9g, pH5.9; 2) the liquid inducing culture is: MS medium 1L+0.15mg NAA+5.5mg 6-BA+100mg colchicin, pH5.9; 3) the solid differential medium is; MS medium 1L+0.1mg NAA+5.5mg6-BA+20 g sucrose+9g agar, pH5.9; 4) root media is: MS medium 1L+0.25 mg NAA+20 g sucrose+8g agar, pH5.9;
(2) tetraploid preserved egg dish is induced processing
1) selecting the preserved egg colza son of full health, is 75% ethanol to the surface of the seed 60s that sterilizes with concentration on superclean bench; Be 1% mercuric chloride (mercury chloride) solution-treated 15min with concentration, aseptic water washing 5 times;
2) aseptic seed is inoculated on MS germination medium, is placed in 25 ℃, 14 hd
-1Cultivated 7 days;
3) cut the growth aseptic seedling of 7 days and put in the MS liquid inducing culture that contains colchicin, 14 hd
-1Shaken cultivation 48 h under illumination;
4) aseptic seedling of colchicine treatment being crossed sterile water wash 3 times, be linked into after cutting in the solid differential medium that does not contain colchicin, in 25 ℃ of room temperatures, light application time, is 14 hd
-1, intensity of illumination is to cultivate under 2 000 lux conditions, until differentiation, the regeneration bud;
All the other operations are with embodiment 1.
Result:Detect and find through flow cytometer, preserved egg dish tetraploid induction rate reaches 27.6%.The cell observation aspects such as the reproductive organs such as the nutrition organs such as tetraploid stem, leaf and floral organ, fleshy root and blade lower epidermis pore, guard cell's chloroplast, pollen grain all show the huge property of polyploid, pollen and pore opening increase more than 31%, and difference is extremely remarkable.
Embodiment 3
(1) medium preparation
1) the germination medium is: MS medium 1L+ sucrose 20g+ agar 8.5g, pH6.0; 2) the liquid inducing culture is: MS medium 1L+0.2mg NAA+6mg 6-BA+200mg colchicin, pH6.0; 3) the solid differential medium is: MS medium 1L+0.15mg NAA+6.0mg6-BA+20 g sucrose+9 g agar, pH6.0; 4) root media is; MS medium 1L+0.3 mg NAA+20 g sucrose+9g agar, pH6.0;
(2) tetraploid preserved egg dish is induced processing
1) selecting the preserved egg colza son of full health, is 73% ethanol to the surface of the seed 40s that sterilizes with concentration on superclean bench; Be 1% mercuric chloride (mercury chloride) solution-treated 13min with concentration, aseptic water washing 4 times;
2) aseptic seed is inoculated on MS germination medium, is placed in 25 ℃, 14 hd
-1Cultivated 6 days;
3) cut the growth aseptic seedling of 6 days and put in the MS liquid inducing culture that contains colchicin, 14 hd
-1Shaken cultivation 48 h under illumination;
4) aseptic seedling of colchicine treatment being crossed sterile water wash 3 times, be linked into after cutting in the solid differential medium that does not contain colchicin, in 25 ℃ of room temperatures, light application time, is 14 hd
-1, intensity of illumination is to cultivate under 2 000 lux conditions, until differentiation, the regeneration bud;
All the other operations are with embodiment 1.
Result:Detect and find through flow cytometer, preserved egg dish tetraploid induction rate reaches 26.6%.The cell observation aspects such as the reproductive organs such as the nutrition organs such as tetraploid stem, leaf and floral organ, fleshy root and blade lower epidermis pore, guard cell's chloroplast, pollen grain all show the huge property of polyploid, pollen and pore opening increase more than 32%, and difference is extremely remarkable.
Claims (5)
1. the breeding method of a tetraploid preserved egg dish is characterized in that comprising the following steps:
(1) select preserved egg colza son, with concentration be 70%～75% ethanol to its surface sterilization 30～60s, with concentration, be then 1% mercuric chloride solution processing 12～15min, use aseptic water washing 3～5 times;
(2) aseptic seed is inoculated on the germination medium, being placed in 25 ℃, light application time is 14 hd
-1Lower cultivation 3～7 days;
(3) aseptic seedling after cultivating is inoculated in the liquid inducing culture, 14hd
-1Under illumination, vibration is induced and was cultivated 2 ~ 3 days;
(4) sterile water wash 2～3 times of the aseptic seedling that will process, be linked into after cutting in the solid differential medium, in 25 ℃ of room temperatures, light application time, is 14 hd
-1, intensity of illumination is to cultivate under 2 000 lux conditions, until differentiation, the regeneration bud;
(5) cut regeneration bud and be seeded on root media and cultivate, the seedling of robust growth, well developed root system is transplanted after hardening, obtain regeneration plant;
(6) according to botany proterties, cytology proterties and flow cytometer, the regeneration plant of field normal growth is carried out Ploidy Identification fast, obtain generation tetraploid preserved egg dish mutant strain.
2. the breeding method of a kind of tetraploid preserved egg dish according to claim 1, it is characterized in that described preserved egg colza is zygoid preserved egg dish, described germination medium is comprised of 1L MS medium, 20～30g sucrose and 8～9g agar, and pH is 5.8～6.0.
3. the breeding method of a kind of tetraploid preserved egg dish according to claim 1, it is characterized in that described liquid inducing culture is comprised of 1L MS medium, 0.1～0.2mg NAA, 5.0～6.0mg 6-BA, 50～200 mg colchicins, pH is 5.8～6.0.
4. the breeding method of a kind of tetraploid preserved egg dish described according to right 1, it is characterized in that described solid differential medium is comprised of 1L MS medium, 0.1～0.2 mg NAA, 5.0～6.0mg 6-BA, 20～30g sucrose, 8～9g agar, pH is 5.8～6.0.
5. the breeding method of a kind of tetraploid preserved egg dish according to claim 1, is characterized in that described root media is comprised of 1L MS medium, 0.2～0.3 mg NAA, 20～30g sucrose, 8～9g agar, and pH is 5.8～6.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105766277A (en) * | 2016-02-24 | 2016-07-20 | 黄翠菊 | Method for planting brassica oleracea L.var .botrytis L. |
| CN107372103A (en) * | 2017-07-05 | 2017-11-24 | 张家港市松田创新农业科技有限公司 | A kind of preserved egg dish plant method for doubling |
| CN111567402A (en) * | 2020-06-17 | 2020-08-25 | 南通科技职业学院 | Method for cultivating efficient tetraploid pakchoi |
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| CN101473789A (en) * | 2009-01-13 | 2009-07-08 | 浙江大学 | Method for breeding haploidy Potherb mustard |
| CN101617631A (en) * | 2009-08-13 | 2010-01-06 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN102613074A (en) * | 2012-02-28 | 2012-08-01 | 江苏丘陵地区镇江农业科学研究所 | Breeding method of tetraploid radish |
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2013
- 2013-05-08 CN CN2013101658593A patent/CN103392592A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101473789A (en) * | 2009-01-13 | 2009-07-08 | 浙江大学 | Method for breeding haploidy Potherb mustard |
| CN101617631A (en) * | 2009-08-13 | 2010-01-06 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN102613074A (en) * | 2012-02-28 | 2012-08-01 | 江苏丘陵地区镇江农业科学研究所 | Breeding method of tetraploid radish |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105766277A (en) * | 2016-02-24 | 2016-07-20 | 黄翠菊 | Method for planting brassica oleracea L.var .botrytis L. |
| CN105766277B (en) * | 2016-02-24 | 2018-12-11 | 黄翠菊 | A kind of implantation methods of songhua |
| CN107372103A (en) * | 2017-07-05 | 2017-11-24 | 张家港市松田创新农业科技有限公司 | A kind of preserved egg dish plant method for doubling |
| CN111567402A (en) * | 2020-06-17 | 2020-08-25 | 南通科技职业学院 | Method for cultivating efficient tetraploid pakchoi |
| CN111567402B (en) * | 2020-06-17 | 2022-09-09 | 南通科技职业学院 | Tetraploid pakchoi cultivation method |
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Application publication date: 20131120 |