CN101982065A - Method for simplifying saving of seedless grape embryo - Google Patents
Method for simplifying saving of seedless grape embryo Download PDFInfo
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- CN101982065A CN101982065A CN 201010278552 CN201010278552A CN101982065A CN 101982065 A CN101982065 A CN 101982065A CN 201010278552 CN201010278552 CN 201010278552 CN 201010278552 A CN201010278552 A CN 201010278552A CN 101982065 A CN101982065 A CN 101982065A
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Abstract
The invention relates to a method for simplifying saving of a seedless grape embryo, which is achieved by saving an in vitro embryo of the seedless grape embryo and comprises the following steps: preparing a synthetic medium, selecting and sterilizing young fruit, carrying out isolated culturing on the ovule, embryo stripping and embryo culture; selecting seed abortion seedless grape young fruit 72 days after flower blooming, washing the young fruit and sterilizing the young fruit at a clean bench respectively by corrosive sublimate and alcohol and stripping the ovule under aseptic conditions; then, inoculating the young fruit on the synthetic medium for carrying out dark culture, wherein the pH value of culture medium is 5.8-6.2 and culture temperature is 25-30 DEG C; carrying out embryo stripping treatment on the ovule after the ovule is cultured for 40-60 days; and continuing to inoculate the ovule to the synthetic medium and carrying out illumination culture on the ovule for 2000-4000LX. When the illumination culture is carried out on the ovule for 20-30 days, then embryo begins to germinate, a radicle gradually grows at the lower part of the embryo and two cotyledons gradually grown at the upper part of the embryo and finally the seedling is obtained. According to the method in the invention, a growth culture medium, a germination culture medium and a seedling formation culture medium are integrated into a whole and the saving of the seedless grape embryo is simplified and accelerated.
Description
Technical field
The present invention relates to a kind of method that the currant embryo is saved of simplifying, effectively simplify incubation step, quickened one-tenth seedling process.
Background technology
Embryo rescue techniques is promptly prepared a kind of medium---and constituent class is similar to the nutrient component of surrounding tissue supply embryo in the blastular environment, carries out cultured in vitro before the zygotic embryo abortion, makes the embryo of abortion soon continue to grow, and finally sprouts into whole plant.This technology realized the conventional hybridization breeding the seedless plant that can't obtain.Since Haberlandt in 1902 supposed according to cell theoretical proposition plant cell " totipotency ", plant embryos was cultivated and is able to fast development.Hanning in 1904 has successfully done systemic plant embryos with the embryo of radish and horseradish dish for the first time and has cultivated on synthetic medium.And nineteen twenty-nine, Laibach utilizes embryo culture to save the hybridization embryo of Iinum peerenne L and Austrian flax, and intergeneric cross is sterile lays a good foundation for the embryo culture solution of exsomatizing is planted.In the 30-40 age in 20th century, this technology begins to be widely used in the embryo redemption research of fruit tree.On multiple fruit tree species such as the apricot of citrus, grape, apple, pears and drupe class, plum, peach, cherry, succeed so far.The application of embryo rescue techniques has not only overcome embryo abortion problem, has also shortened breeding cycle greatly, has brought into play important function to promoting fruit breeding.
The existing a lot of reports of the research that relevant currant embryo is saved start from the beginning of the eighties the earliest.As nineteen eighty-two, reported first such as Ramming utilize improvement White medium to carry out the currant embryo ovule cultivate, and obtained 2 strain seedlings.Later Tian Lili etc., Hao Yan etc., Wang Fei etc., Pan Xuejun etc., Liu Xiaoning etc. have carried out the embryo redemption to currant, and obtained seedling, but all need design three kinds of medium successively in above all redemption incubation---grow medium, germination medium, one-tenth seedling medium, could finally save successfully.Up to the present, only carry out the currant embryo from start to finish and save the report that yet there are no that can successfully obtain seedling with a kind of medium.
Summary of the invention
Present situation in view of above-mentioned technology, the invention provides a kind of method that the currant embryo is saved of simplifying, be intended to and grow the method for the simplification medium that medium, germination medium, one-tenth seedling medium be integrated, handled easily process greatly, both simplify incubation step, be accelerated into the seedling process again.This for germplasm innovation, distant hybridization, triploid breeding, hybridization is not affine, shorten breeding cycle, breed improvement can guarantee high efficiency acquisition currant filial generation, for germplasm innovation, distant hybridization, triploid breeding aspect provide a kind of new technology platform.
A kind of method that the currant embryo is saved of simplifying of the present invention, its method step comprises:
1, the preparation of synthetic medium, choose the raw material of following concentration proportioning:
Potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, ironic citrate 0.001%, GA
30.00001%-0.0003%, IAA 0.00001%-0.0003% are mixed into the mixing mother liquor that contains hormone, to mix and add sucrose 0,003%-0,006%, agar 0.0003%-0,0006% in the mother liquor, mother liquor dissolving finishing constant volume to be mixed forms synthetic medium, with synthetic medium pH value modulation 5.8～6.5, the synthetic medium that branch is installed is put into high-pressure sterilizing pot and is sterilized then, sterilising conditions: pressure 1.05MPa, 121 ℃ of temperature, sterilization time 15～20min is standby;
2, explant is selected and preliminary treatment, chooses and contains the seeds abortion type currant young fruit of spending back 72d, and mercuric chloride, alcohol disinfecting are used in the flowing water flushing respectively before superclean bench, strip out ovule under the aseptic condition;
3, ovule is cultivated, and aseptic ovule is seeded on the synthetic medium, dark cultivation, medium pH 5.8～6.2,25～30 ℃ of cultivation temperature;
4, embryo culture after ovule is cultivated 40～60d, is shelled embryo to it and is handled, and continues to be seeded on the synthetic medium, and adopts illumination cultivation 2000～4000LX, 20～30d embryo to begin to sprout, and the bottom grows radicle, and top grows two cotyledons gradually, last Cheng Miao.
According to of the present invention be exactly scheme, the raw material components of the synthetic medium of indication and concentration proportioning are:
Potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, VB
50.000025%, ironic citrate 0.001%, GA
30.00001%-0.0003%, IAA 0.00001%-0.0003%, sucrose 0,003%-0,006%, agar 0.0003%-0,0006%.
Further, potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, VB
50.000025%, ironic citrate 0.001%, GA
30.00001%-0.0003%, IBA 0.00001%-0.0003%, sucrose 0,003%-0,006%, agar 0.0003%-0,0006%.
Further, potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, VB
50.000025%, ironic citrate 0.001%, GA
30.00001%-0.0003%, 6-BA0.00001%-0.0003%, sucrose 0,003%-0,006%, agar 0.0003%-0,0006%.
Further, potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, VB
50.000025%, ironic citrate 0.001%, GA
30.00001%-0.0003%, L-cysteine 0.0001%-0.0003%+ caseinhydrolysate 0.01%-0.06%, sucrose 0,003%-0,006%, agar 0.0003%-0,0006%o
Among the present invention, described explant kind is to select golden field imperial family currant and the gloomy currant of crith for use.Imperial currant is the seedless new varieties of late grape that originate from the new seed selection of Hebei Science ﹠ Technology Normal College in late-maturing golden field, this kind has been inherited the no nuclear properties in male parent imperial family autumn, and in seedless variety, belong to big grain kind, under chemicals treatment, the heavy 6.3g of average particle, soluble solid content reaches 19.6%.This variety seeds is degenerated evening, and stays the vestiges of nuclear when fruit maturation, but does not all influence mouthfeel.The gloomy currant of crith is a late-maturing Table Grape kind, so because appearance to have beautiful color to be also referred to as the gentlewoman red.Its fruit ear is medium bigger than normal, and average fringe weighs 500 grams, and average particle weighs 4 grams.Because the soluble solid of this grape reaches 19%, sugar-acid ratio can reach 20: 1, so special delicate fragrance sweetness, mouthfeel is fine.
The beneficial effect that the present invention has is, by Ovule Culture of Seedless Grapes is being simplified on the medium, can obtain filial generation without the conversion medium from start to finish, realize to grow medium, germination medium, three steps of one-tenth seedling medium first and changed into a kind of synthetic medium, realized that promptly the embryo of currant saves on a kind of medium, utilized the method to simplify test procedure, do not needed repeatedly to change kinds of culture medium, saved manpower and materials.Therefore, this method is the handled easily process greatly, both simplified incubation step, be accelerated into the seedling process again, for setting up a cover system, perfect, currant embryo rescue techniques establish a firm foundation efficiently, this for germplasm innovation, distant hybridization, triploid breeding, hybridization is not affine, research of shortening aspects such as breeding cycle, breed improvement all has crucial meaning.
Embodiment
Be described further below in conjunction with embodiments of the invention.
Embodiment 1
At first select potassium nitrate 950mg/L, ammonium nitrate 720mg/L, potassium dihydrogen phosphate 68mg/L, magnesium sulfate 185mg/L, calcium chloride 166mg/L, boric acid 3mg/L, manganese sulphate 22.3mg/L, zinc sulphate 10.0mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.08mg/L, nicotinic acid 0.5mg/L, thiamine hydrochloride 0.5mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, VB5+ ironic citrate+GA for use
33.0mg/L+IAA3.0mg/L mother liquor and hormone, add sucrose 30g/L
-1, agar 3g/L
-1Behind medium dissolving the finishing constant volume pH value is modulated 6, the medium that branch is installed is put into high-pressure sterilizing pot and is sterilized then, sterilising conditions: pressure 1.05MPa, 121 ℃ of temperature, sterilization time 15min (reaching requirement pressure picks up counting later on) makes synthetic medium, chooses and contains the seeds abortion type currant young fruit of spending back 72d, adopts the flowing water flushing, before superclean bench, use mercuric chloride respectively, alcohol disinfecting, strip out ovule under the aseptic condition, aseptic ovule is seeded on the synthetic medium secretly cultivates medium pH 5.8,25 ℃ of cultivation temperature, ovule is cultivated 40d it is shelled the embryo processing, continues to be seeded on the synthetic medium, and adopts illumination cultivation 2000LX, embryo begins to sprout during 20d, the bottom is sent radicle gradually, and top grows two cotyledons gradually to be seen, last Cheng Miao.
Embodiment 2
The raw material components of synthetic medium and concentration proportioning are: potassium nitrate 950mg/L, ammonium nitrate 720mg/L, potassium dihydrogen phosphate 68mg/L, magnesium sulfate 185mg/L, calcium chloride 166mg/L+ boric acid 3mg/L, manganese sulphate 22.3mg/L, zinc sulphate 10.0mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.08mg/L+ nicotinic acid 0.5mg/L, thiamine hydrochloride 0.5mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L+VB50.25mg/L+ ironic citrate 10mg/L+GA
30.1～3.0mg/L+IAA 0.1～3.0mg/L, sucrose 30g/L
-1-60g/L
-1, agar 3g/L
-1-60g/L
-1The embryo rescue method step of its currant is identical with above-mentioned embodiment 1, and the Therefore, omited is described.
Embodiment 3
The raw material components of synthetic medium and concentration proportioning are: potassium nitrate 950mg/L, ammonium nitrate 720mg/L, potassium dihydrogen phosphate 68mg/L, magnesium sulfate 185mg/L, calcium chloride 166mg/L+ boric acid 3mg/L, manganese sulphate 22.3mg/L, zinc sulphate 10.0mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.08mg/L+ nicotinic acid 0.5mg/L, thiamine hydrochloride 0.5mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L+VB50.25mg/L+ ironic citrate 10mg/L+GA
30.1～3.0mg/L+IBA 0.1～3.0mg/L, sucrose 30g/L
-1-60g/L
-1, agar 3g/L
-1-60g/L
-1The embryo rescue method step of its currant is identical with above-mentioned embodiment 1, and the Therefore, omited is described.
Embodiment 4
The raw material components of synthetic medium and concentration proportioning are: potassium nitrate 950mg/L, ammonium nitrate 720mg/L, potassium dihydrogen phosphate 68mg/L, magnesium sulfate 185mg/L, calcium chloride 166mg/L+ boric acid 3mg/L, manganese sulphate 22.3mg/L, zinc sulphate 10.0mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.08mg/L nicotinic acid 0.5mg/L, thiamine hydrochloride 0.5mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L+VB50.25mg/L+ ironic citrate 10mg/L+GA
30.1～3.0mg/L+6-BA0.1～3.0mg/L, sucrose 30g/L
-1-60g/L
-1, agar 3g/L
-1-60g/L
-1The embryo rescue method step of its currant is identical with above-mentioned embodiment 1, and the Therefore, omited is described.
Embodiment 5
The raw material components of synthetic medium and concentration proportioning are: potassium nitrate 950mg/L, ammonium nitrate 720mg/L, potassium dihydrogen phosphate 68mg/L, magnesium sulfate 185mg/L, calcium chloride 166mg/L+ boric acid 3mg/L, manganese sulphate 22.3mg/L, zinc sulphate 10.0mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.08mg/L+ nicotinic acid 0.5mg/L, thiamine hydrochloride 0.5mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L+VB50.25mg/L+ ironic citrate 10mg/L+GA
30.1～3.0mg/L+L-cysteine 1.0～3.0mg/L+ caseinhydrolysate 100～600mg/L, sucrose 30g/L
-1-60g/L
-1, agar 3g/L
-1-60g/L
-1The embryo rescue method step of its currant is identical with above-mentioned embodiment 1, and the Therefore, omited is described.
Claims (5)
1. simplify the method that the currant embryo is saved for one kind, its method step is:
(1) raw material potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, ironic citrate 0.001%, GA are at first chosen in the preparation of synthetic medium
30.00001%-0.0003%, IAA 0.00001%-0.0003% are mixed into the mixing mother liquor that contains hormone, adding sucrose 0,003%-0,006%, agar 0.0003%-0,0006% are mixed into the mother liquor that contains hormone in the mother liquor with mixing, treat that mother liquor dissolving finishing constant volume forms synthetic medium, with synthetic medium pH value modulation 5.8～6.5, the synthetic medium that branch is installed is put into high-pressure sterilizing pot and is sterilized then, sterilising conditions: pressure 1.05MPa, temperature 121, sterilization time 15～20min is standby;
(2) choose the seeds abortion type currant young fruit that Sheng is spent back 72d, mercuric chloride, alcohol disinfecting are used in the flowing water flushing respectively before superclean bench, strip out ovule under the aseptic condition;
(3) aseptic ovule is seeded on the synthetic medium dark cultivation, medium pH 5.8～6.2,25～30 ℃ of cultivation temperature;
When (4) ovule is cultivated 40～60d its stripping embryo is handled, continued to be seeded on the synthetic medium, and adopt illumination cultivation 2000～4000LX, embryo begins to sprout during 20～30d, and the bottom is sent radicle gradually, and top grows two cotyledons gradually, last Cheng Miao.
2. the method that simplification currant embryo according to claim 1 is saved, it is characterized in that the concentration proportioning of described step (1) synthetic medium raw material is: potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, VB
50.000025%, ironic citrate 0.001%, GA
30.00001%-0.0003%, IAA 0.00001%-0.0003%, sucrose 0,003%-0,006%, agar 0.0003%-0,0006%.
3. the method that simplification currant embryo according to claim 1 is saved, it is characterized in that the concentration proportioning of synthetic medium raw material is: potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, VB
50.000025%, ironic citrate 0.001%, GA
30.00001%-0.0003%, IBA 0.00001%-0.0003%, sucrose 0,003%-0,006%, agar 0.0003%-0,0006%.
4. the method that simplification currant embryo according to claim 1 is saved, it is characterized in that the concentration proportioning of synthetic medium raw material is: potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, VB
50.000025%, ironic citrate 0.001%, GA
30.00001%-0.0003%, 6-BA0.00001%-0.0003%, sucrose 0,003%-0,006%, agar 0.0003%-0,0006%.
5. the method that simplification currant embryo according to claim 1 is saved, it is characterized in that the concentration proportioning of synthetic medium raw material is: potassium nitrate 0.095%, ammonium nitrate 0.072%, potassium dihydrogen phosphate 0.0068%, magnesium sulfate 0.0185%, calcium chloride 0.0166%, boric acid 0.0003%, manganese sulphate 0.00223%, zinc sulphate 0.001%, sodium molybdate 0.000025%, copper sulphate 0.000008%, nicotinic acid 0.00005%, thiamine hydrochloride 0.00005%, puridoxine hydrochloride 0.00005%, glycine 0.0002%, VB
50.000025%, ironic citrate 0.001%, GA
30.00001%-0.0003%, L-cysteine 0.0001%-0.0003%+ caseinhydrolysate 0.01%-0.06%, sucrose 0,003%-0,006%, agar 0.0003%-0,0006%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102668991A (en) * | 2012-06-14 | 2012-09-19 | 宁夏农垦西夏王实业有限公司 | Application of penicillin to simple test-tube breeding of grapes and novel technology for test-tube breeding of grapes |
| CN104782474A (en) * | 2015-03-27 | 2015-07-22 | 浙江省农业科学院 | Method for increasing seedless grape crossed embryo rescue seedling rate |
| CN104988179A (en) * | 2015-06-30 | 2015-10-21 | 中国农业科学院郑州果树研究所 | Biotechnological breeding method for obtaining antiviral seedless grapes |
| CN110089426A (en) * | 2018-01-29 | 2019-08-06 | 南京农业大学 | A method of cultivating Chinese cabbage microspore plant |
-
2010
- 2010-09-07 CN CN 201010278552 patent/CN101982065B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库》 20060215 唐冬梅 无核葡萄胚挽救育种技术及种质创建 摘要，9-10 1-5 , 第2期 2 * |
| 《中国优秀硕士学位论文全文数据库》 20081115 石艳 胚挽救技术创制无核及多倍体葡萄新种质 摘要、4 1-5 , 第11期 2 * |
| 《中外葡萄与葡萄酒》 20041231 王劲松等 胚挽救技术在葡萄无核育种上的应用 22-25 1-5 , 第2期 2 * |
| 《北方果树》 20090131 金钊等 无核葡萄胚挽救技术初探 13-15 1-5 , 第1期 2 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102668991A (en) * | 2012-06-14 | 2012-09-19 | 宁夏农垦西夏王实业有限公司 | Application of penicillin to simple test-tube breeding of grapes and novel technology for test-tube breeding of grapes |
| CN102668991B (en) * | 2012-06-14 | 2013-10-23 | 宁夏农垦西夏王实业有限公司 | Application of penicillin to simple test-tube breeding of grapes and novel technology for test-tube breeding of grapes |
| CN104782474A (en) * | 2015-03-27 | 2015-07-22 | 浙江省农业科学院 | Method for increasing seedless grape crossed embryo rescue seedling rate |
| CN104782474B (en) * | 2015-03-27 | 2017-05-31 | 浙江省农业科学院 | It is a kind of to improve the method that currant bybrid embryo saves seedling |
| CN104988179A (en) * | 2015-06-30 | 2015-10-21 | 中国农业科学院郑州果树研究所 | Biotechnological breeding method for obtaining antiviral seedless grapes |
| WO2017000089A1 (en) * | 2015-06-30 | 2017-01-05 | 中国农业科学院郑州果树研究所 | Biotechnological breeding method for obtaining antiviral seedless grapes |
| CN110089426A (en) * | 2018-01-29 | 2019-08-06 | 南京农业大学 | A method of cultivating Chinese cabbage microspore plant |
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