CN104782474A - Method for increasing seedless grape crossed embryo rescue seedling rate - Google Patents
Method for increasing seedless grape crossed embryo rescue seedling rate Download PDFInfo
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Abstract
The invention belongs to the technical field of plant breeding, and discloses a method for increasing the seedless grape crossed embryo rescue seedling rate. According to the method, before abortion of seedless grape crossed embryos, physical measures of pinching and girdling are taken to promote in-vivo development of ovules and embryos, the appropriate time for embryo abortion rescue sampling of two Southern seedless grape hybridized combinations is confirmed, and content such as culture medium additives for culturing ovules and germinating embryos, test tube seedling dual-bud stem inoculation subculturing and subculturing culture medium additives are confirmed. Due to the adoption of the method, the seedless grape crossed embryo developing rate is high, the embryo rescue seedlings are tidy and strong, the embryo developing rate is up to 41.7%, the seedling rate is up to 18.3%, the subculturing culture seedling rate is up to 92%, the rate of strong and healthy seedlings is 81.8%, the test tube seedling transplanting survival rate is up to 97%, and the cardinal number of the crossed filial generation embryo rescue seedlings is greatly increased, so that a relatively great number of seedless crossed generation plant groups can be obtained, the seedless grape crossed embryo seedling rate in southern areas can be increased, and the method is high in operability and easy to master.
Description
Technical field
The present invention relates to field of plant variety breeding technology, particularly relate to a kind of method improving currant bybrid embryo redemption seedling.
Background technology
Currant due to instant very popular, world's Table Grape produce on occupy critical role, the Table Grape of the U.S. more than 80% is seedless variety.But in grape breeding, because currant fruit does not have seed, filial generation plant cannot be obtained as female parent, core kind can only have been utilized for a long time to hybridize with seedless variety as maternal, but its offspring occurs that the ratio of seedless plant is very low, and breeding cycle is longer.The nineties in last century, the raisin grape breeding that develops into of embryo rescue techniques brings new way, currant is cross-breeding as female parent and becomes possibility, not only increase considerably the probability that offspring is seedless, also breeding cycle is substantially reduced, to some extent solve a difficult problem for raisin grape breeding, be subject to the great attention of breeding men as a kind of effective raisin grape breeding method.But in rescue culture process, variety and genetype, sampling inoculation time, kinds of culture medium and phase, plant growth regulator, carbon source, culture environment, transplanting methods etc. all can have an impact to the germination rate of rescue culture and planting percent, and each factor interaction, and southern and northern regional disparity makes south grape reach maturity early, aborted embryo optimal period is different, increase the difficulty that Embryo Rescue of Seedless Grape technical system is built, result in currant is that after hybridization of female parent, in fruit, embryonic development rate is low, rescue culture seedling deficiency has become the bottleneck restricting southern efficiency of raisin grape breeding, how to improve the planting percent of Embryo Rescue of Seedless Grape breeding, expand rescue culture plant population, become the striving direction of grape breeding man.
Summary of the invention
The present invention is directed to existing shortcoming, disclose a kind of method improving currant bybrid embryo redemption seedling, thus effectively promote that currant bybrid embryo is grown, seedling can be grown under excised cotyledon condition, and embryo culture seedling is healthy and strong neat, rapid expansion plant population, improves embryo crash breeding efficiency in the raisin grape breeding of southern area.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals.
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 7-10 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation; In the present invention, currant female parent newly slightly grew to for 6 leaf phases, inflorescence stayed 1 leaf carry out pinching process, second time pinching process was carried out to top sublateral shoot in 7-10 days before spending, all the other sublateral shoots stay 1 leaf pinching, play following technique effect: control to nourish and grow, promote inflorescence development.
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 4-6 DEG C low temperature treatment, 3 days, strip ovule inoculation, select the B5 liquid nutrient medium adding the first additives, 25 DEG C of light culture 80-90 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 30-40 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 20-40g/L of 0.1-0.5mg/L, the active carbon of 2-3g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: hardening is carried out to test-tube plantlet.
As preferably, in step B, after pollination, the best of hybrid seed abortion gets the fruit phase be flame currant and cold fragrant honey hybridization is 33-36 days after pollination, or the sub-currant of Audi and the seedless hybridization of green perfume (or spice) are 38-40 days after pollination.In step B, carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, and carries out low temperature treatment, take out running water 30min, then in superclean bench 75% ethanol rinse 30s, aseptic water washing 1 time, then fruit grain is put into mercuric chloride solution sterilization 6min, aseptic water washing 3-5 time, strip ovule inoculation, select B5 liquid nutrient medium, 25 DEG C of light culture 80-90 days.
As preferably, in step B, the first additives is the sucrose of 6-BA, 50-65g/L of GA3,0.1-0.5mg/L of IBA, 0.5-1.5mg/L of 1-2.5mg/L, the active carbon of 0.5-1.5g/L.
As preferably, in step C, the second additives is the sucrose of 6-BA, 20-40g/L of 0.4-0.8mg/L, the active carbon of 1-2g/L.
As preferably, in step e, Trained Transition Methods of Chinese Dwarf is, test-tube plantlet is cultivated 2 days under moving on to Indoor Natural optical condition; Open bottle cap and cultivate 2 days; Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube installed with 70% thiophanate methyl and 1000 times of sterilizing peat, the moisturizing of seedling upper cover plastic cup, the time is 7 days; Grow field planting after 2 young leaves under moving on to facility condition, nursery soil dislikes mould spirit sterilization with 30%, namely obtains currant cross hybrid seedling plant.The present invention is in hardening process, and test-tube plantlet is cultivated 2 days under moving on to Indoor Natural optical condition, is to adapt to illumination.Opening that bottle cap cultivates 2 days is change to adapt to illumination, temperature and humidity.Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube installed with 70% thiophanate methyl and 1000 times of sterilizing peat, seedling upper cover plastic cup moisturizing cultivation is to adapt to light, temp. and humidity and peat growing medium.It is the adaptive training before transplanting under moving on to facility condition.
Compared with prior art, beneficial effect of the present invention is: the method applied in the present invention, effectively can promote that currant bybrid embryo continues to grow in vitro, improve ovule and embryonic development rate, promote embryo germination, add the radix of filial generation seedling, thus more filial generation can be obtained, and subculture to expand numerous rear embryo rescue seedlings healthy and strong neat, contribute to reducing the individual plant risk of missing and the qualification in later stage that cause because transplanting death.The currant bybrid embryo developmental rate that application the present invention obtains is high, embryo rescue seedlings is neatly healthy and strong, embryonic development rate reaches 41.7%, planting percent reaches 18.3%, and squamous subculture planting percent reaches 92%, and wherein healthy seedling reaches 81.8% with time healthy seedling, test-tube seedling transplanting survival rate reaches 97%, substantially increase the plant population of currant filial generation embryo crash breeding seedling, improve Embryo Rescue of Seedless Grape breeding efficiency, workable, easily grasp.
Embodiment
Embodiment 1
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 7 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation;
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 4 DEG C of low temperature treatment, 3 days, strip ovule inoculation, select to add the B5 liquid nutrient medium of the first additives, 25 DEG C of light culture 80 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 30 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 20g/L of 0.1mg/L, the active carbon of 2g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: hardening is carried out to test-tube plantlet.
In step B, the first additives is the sucrose of 6-BA, 50g/L of GA3,0.1mg/L of IBA, 0.5mg/L of 1mg/L, the active carbon of 0.5g/L.
In step C, the second additives is the sucrose of 6-BA, 20g/L of 0.4mg/L, the active carbon of 1g/L.
Embodiment 2
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 7-10 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation;
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 6 DEG C of low temperature treatment, 3 days, strip ovule inoculation, select to add the B5 liquid nutrient medium of the first additives, 25 DEG C of light culture 90 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 40 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 40g/L of 0.5mg/L, the active carbon of 3g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: Trained Transition Methods of Chinese Dwarf is, test-tube plantlet is cultivated 2 days under moving on to Indoor Natural optical condition; Open bottle cap and cultivate 2 days; Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube installed with 70% thiophanate methyl and 1000 times of sterilizing peat, the moisturizing of seedling upper cover plastic cup, the time is 7 days; Grow field planting after 2 young leaves under moving on to facility condition, nursery soil dislikes mould spirit sterilization with 30%, namely obtains currant cross hybrid seedling plant.
In step B, after pollination, the best of hybrid seed abortion really phase of getting is that flame currant and cold fragrant honey are hybridized for latter 33 days of pollination.
In step B, the first additives is the sucrose of 6-BA, 65g/L of GA3,0.5mg/L of IBA, 1.5mg/L of 2.5mg/L, the active carbon of 1.5g/L.
In step C, the second additives is the sucrose of 6-BA, 40g/L of 0.8mg/L, the active carbon of 2g/L.
Embodiment 3
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 7-10 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation;
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 5 DEG C of low temperature treatment, 3 days, strip ovule inoculation, select to add the B5 liquid nutrient medium of the first additives, 25 DEG C of light culture 85 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 35 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 30g/L of 0.2mg/L, the active carbon of 2.5g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: test-tube plantlet is cultivated 2 days under moving on to Indoor Natural optical condition; Open bottle cap and cultivate 2 days; Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube installed with 70% thiophanate methyl and 1000 times of sterilizing peat, the moisturizing of seedling upper cover plastic cup, the time is 7 days; Grow field planting after 2 young leaves under moving on to facility condition, nursery soil dislikes mould spirit sterilization with 30%, namely obtains currant cross hybrid seedling plant.
In step B, after pollination, the best of hybrid seed abortion really phase of getting is that flame currant and cold fragrant honey are hybridized for latter 36 days of pollination.
In step B, the first additives is the sucrose of 6-BA, 52g/L of GA3,0.2mg/L of IBA, 1.2mg/L of 1.5mg/L, the active carbon of 0.8g/L.
In step C, the second additives is the sucrose of 6-BA, 25g/L of 0.5mg/L, the active carbon of 1.2g/L.
Embodiment 4
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 7-10 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation;
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 5 DEG C of low temperature treatment, 3 days, strip ovule inoculation, select to add the B5 liquid nutrient medium of the first additives, 25 DEG C of light culture 85 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 35 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 30g/L of 0.2mg/L, the active carbon of 2.5g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: test-tube plantlet is cultivated 2 days under moving on to Indoor Natural optical condition; Open bottle cap and cultivate 2 days; Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube installed with 70% thiophanate methyl and 1000 times of sterilizing peat, the moisturizing of seedling upper cover plastic cup, the time is 7 days; Grow field planting after 2 young leaves under moving on to facility condition, nursery soil dislikes mould spirit sterilization with 30%, namely obtains currant cross hybrid seedling plant.
In step B, after pollination, the best of hybrid seed abortion really phase of getting is that flame currant and cold fragrant honey are hybridized for latter 35 days of pollination.
In step B, the first additives is the sucrose of 6-BA, 65g/L of GA3,0.5mg/L of IBA, 1.5mg/L of 2.5mg/L, the active carbon of 1.5g/L.
In step C, the second additives is the sucrose of 6-BA, 40g/L of 0.8mg/L, the active carbon of 2g/L.
Embodiment 5
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 7-10 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation;
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 5 DEG C of low temperature treatment, 3 days, strip ovule inoculation, select to add the B5 liquid nutrient medium of the first additives, 25 DEG C of light culture 85 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 35 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 30g/L of 0.2mg/L, the active carbon of 2.5g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: test-tube plantlet is cultivated 2 days under moving on to Indoor Natural optical condition; Open bottle cap and cultivate 2 days; Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube installed with 70% thiophanate methyl and 1000 times of sterilizing peat, the moisturizing of seedling upper cover plastic cup, the time is 7 days; Grow field planting after 2 young leaves under moving on to facility condition, nursery soil dislikes mould spirit sterilization with 30%, namely obtains currant cross hybrid seedling plant.
In step B, after pollination, the best of hybrid seed abortion really phase of getting is the sub-currant of Audi and the seedless hybridization of green perfume (or spice) is latter 38 days of pollination.
In step B, the first additives is the sucrose of 6-BA, 65g/L of GA3,0.5mg/L of IBA, 1.5mg/L of 2.5mg/L, the active carbon of 1.5g/L.
In step C, the second additives is the sucrose of 6-BA, 40g/L of 0.8mg/L, the active carbon of 2g/L.
Embodiment 6
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 8 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation;
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 4.5 DEG C of low temperature treatment, 3 days, strip ovule inoculation, select to add the B5 liquid nutrient medium of the first additives, 25 DEG C of light culture 82 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 32 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 21g/L of 0.2mg/L, the active carbon of 2.5g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: hardening is carried out to test-tube plantlet.
In step B, after pollination, the best of hybrid seed abortion really phase of getting is the sub-currant of Audi and the seedless hybridization of green perfume (or spice) is latter 39 days of pollination.
In step B, the first additives is the sucrose of 6-BA, 51g/L of GA3,0.2mg/L of IBA, 0.8mg/L of 1.5mg/L, the active carbon of 0.6g/L.
In step C, the second additives is the sucrose of 6-BA, 21g/L of 0.5mg/L, the active carbon of 1.2g/L.
Embodiment 7
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 9 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation;
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 5.5 DEG C of low temperature treatment, 3 days, strip ovule inoculation, select to add the B5 liquid nutrient medium of the first additives, 25 DEG C of light culture 89 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 39 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 39g/L of 0.4mg/L, the active carbon of 2.8g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: hardening is carried out to test-tube plantlet.
In step B, after pollination, the best of hybrid seed abortion really phase of getting is the sub-currant of Audi and the seedless hybridization of green perfume (or spice) is latter 40 days of pollination.
In step B, the first additives is the sucrose of 6-BA, 64g/L of GA3,0.4mg/L of IBA, 1.4mg/L of 2.4mg/L, the active carbon of 1.4g/L.
In step C, the second additives is the sucrose of 6-BA, 39g/L of 0.7mg/L, the active carbon of 1.9g/L.
Embodiment 8
Improve the method that currant bybrid embryo saves seedling, comprise the following steps,
A. pinching band girdle: flame currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, before spending, 7-10 zenith sublateral shoot carries out secondary pinching process, and all the other sublateral shoots stay 1 leaf pinching, carries out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation pollination.
B. sampling after hybridization, ovule is inoculated: the maternal initial bloom stage of flame currant carries out artificial emasculation after low temperature treatment, artificial pollination is carried out for male parent so that cold fragrant honey is seedless, after pollination, namely aborted embryo optimal period is pollinated and is won young fruit in latter 35 days, carry out 5 DEG C of low temperature treatment of 3 days, running water 30min after taking out, then in superclean bench 75% ethanol rinse 30s, aseptic water washing 1 time, again fruit grain is put into mercuric chloride solution sterilization 6min, aseptic water washing 4 times, strip ovule, (additives of interpolation is the IBA of 2.0mg/L to be seeded in B5 liquid nutrient medium, the GA3 of 1mg/L, the 6-BA of 0.2mg/L, the sucrose of 60g/L, the active carbon of 1g/L) on, 25 DEG C of light culture 85d,
C. get naked embryonic breeding kind: ovule rip cutting strips white naked embryo, be inoculated on medium WPM (additives of interpolation is the sucrose of 6-BA, 30g/L of 0.6mg/L, the active carbon of 1.5g/L), 2000lx illumination 12h at 25 DEG C, Germination And Seedling.
D. subculture expands numerous: expand numerous by the rescue culture Plantlet subculture of 35 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM (additives added is the sucrose of IBA, 30g/L of 0.2mg/L, the active carbon of 2.5g/L), 25 DEG C of 3000lx illumination every day 16h, cultivate seedling;
E. acclimatization and transplants: embryo rescue seedlings adopts the sequencing Trained Transition Methods of Chinese Dwarf of ' in blake bottle airtight 2 days-blake bottle inner opening 2 days-go out blake bottle 10 days-natural conditions 7 days ', and namely test-tube plantlet moves on to Indoor Natural optical condition lower 2 days, adapts to illumination; Open bottleneck and unclamp bottle cap 2 days, adapt to illumination, temperature and humidity and change; Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube (12 × 10cm) installed with 70% thiophanate methyl, 1000 times of sterilizing peat, and the moisturizing of lid plastic cup, the time is 7 days, adapts to light, temp. and humidity and culture matrix; Move on to facility condition lower refining seedling within 7 days, transplant before adaptive training, field planting after 2 young leaves occurs, and nursery soil dislikes mould spirit sterilization with 30%, namely obtains currant cross hybrid seedling plant.
Embodiment 9
Improve the method that currant bybrid embryo saves seedling, comprise the following steps
A. pinching band girdle: the sub-currant female parent of Audi newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, before spending, 8 zenith sublateral shoots carry out secondary pinching process, all the other sublateral shoots stay 1 leaf pinching, control is nourished and grown, promote inflorescence development, before initial bloom stage artificial emasculation pollination, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot.
B. sampling after hybridization, ovule is inoculated: the maternal initial bloom stage of the sub-currant of Audi carries out artificial emasculation after low temperature treatment, artificial pollination is carried out for male parent so that green perfume (or spice) is seedless, after pollination, namely aborted embryo optimal period is pollinated and is won young fruit in latter 39 days, carry out 5 DEG C of low temperature treatment of 3 days, after taking out, flowing water rushes 30min, then in superclean bench 75% ethanol rinse 30s, aseptic water washing 1 time, again fruit grain is put into mercuric chloride solution sterilization 6min, aseptic water washing 5 times, strip ovule, (additives of interpolation is the IBA of 2.0mg/L to be seeded in B5 liquid nutrient medium, the GA3 of 1mg/L, the 6-BA of 0.2mg/L, the sucrose of 60g/L, the active carbon of 1g/L) on, 25 DEG C of light culture 82 days,
C. naked embryonic breeding kind is got: on aseptic operating platform, take out the ovule after cultivating, rip cutting direction strips the naked embryo of the white being positioned at beak portion, be inoculated on medium WPM (additives of interpolation is the sucrose of 6-BA, 30g/L of 0.6mg/L, the active carbon of 1.5g/L), 25 DEG C of 2000lx illumination every day 12h, until Germination And Seedling.
D. subculture expands numerous: expand numerous by the rescue culture Plantlet subculture of 32 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM (additives added is the sucrose of IBA, 30g/L of 0.2mg/L, the active carbon of 2.5g/L), 25 DEG C of 3000lx illumination every day 16h, cultivate seedling;
E. acclimatization and transplants: embryo rescue seedlings adopts the sequencing Trained Transition Methods of Chinese Dwarf of ' in blake bottle airtight 2 days-blake bottle inner opening 2 days-go out blake bottle 10 days-natural conditions 7 days ', and namely test-tube plantlet moves on to Indoor Natural optical condition lower 2 days, adapts to illumination; Open bottleneck and unclamp bottle cap 2 days, adapt to illumination, temperature and humidity and change; Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube (12 × 10cm) installed with 70% thiophanate methyl, 1000 times of sterilizing peat, and the moisturizing of lid plastic cup, the time is 7 days, adapts to light, temp. and humidity and culture matrix; Move on to facility condition lower refining seedling within 7 days, transplant before adaptive training, field planting after 2 young leaves occurs, and nursery soil dislikes mould spirit sterilization with 30%, namely obtains currant cross hybrid seedling plant.
In a word, the foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to the covering scope of patent of the present invention.
Claims (5)
1. improve the method that currant bybrid embryo saves seedling, it is characterized in that: comprise the following steps,
A. pinching band girdle: currant female parent newly slightly grew to for 6 leaf phases, inflorescence stay 1 leaf carry out pinching process, within 7-10 days before spending, carry out second time pinching process to top sublateral shoot, all the other sublateral shoots stay 1 leaf pinching, carry out the band girdle pushing up sublateral shoot pinching and fruit bearing shoot before initial bloom stage artificial emasculation;
B. ovule is inoculated after sampling, low temperature treatment after hybridization: carry out artificial emasculation pollination at initial bloom stage, after pollination, the best of hybrid seed abortion is got the fruit phase and is won young fruit, young fruit fetches rear 4-6 DEG C low temperature treatment, 3 days, strip ovule inoculation, select the B5 liquid nutrient medium adding the first additives, 25 DEG C of light culture 80-90 days;
C. get naked embryonic breeding kind: get ovule, ovule rip cutting strips white naked embryo, is inoculated into and adds on the WPM medium of the second additives, and light is cultivated;
D. subculture expands numerous: by the rescue culture test-tube plantlet of 30-40 days seedling ages, two leaf stem section is seeded on subculture medium 1/2WPM, subculture medium 1/2WPM adds the 3rd additives, additives is the sucrose of IBA, 20-40g/L of 0.1-0.5mg/L, the active carbon of 2-3g/L, and light cultivates to obtain test-tube plantlet;
E. hardening: hardening is carried out to test-tube plantlet.
2. raising currant bybrid embryo according to claim 2 saves the method for seedling, it is characterized in that: in step B, after pollination, the best of hybrid seed abortion gets the fruit phase be flame currant and cold fragrant honey hybridization is 33-36 days after pollination, or the sub-currant of Audi and the seedless hybridization of green perfume (or spice) are 38-40 days after pollination.
3. raising currant bybrid embryo according to claim 1 saves the method for seedling, it is characterized in that: in step B, the first additives is the sucrose of 6-BA, 50-65g/L of GA3,0.1-0.5mg/L of IBA, 0.5-1.5mg/L of 1-2.5mg/L, the active carbon of 0.5-1.5g/L.
4. raising currant bybrid embryo according to claim 1 saves the method for seedling, and it is characterized in that: in step C, the second additives is the sucrose of 6-BA, 20-40g/L of 0.4-0.8mg/L, the active carbon of 1-2g/L.
5. raising currant bybrid embryo according to claim 1 saves the method for seedling, and it is characterized in that: in step e, Trained Transition Methods of Chinese Dwarf is, test-tube plantlet is cultivated 2 days under moving on to Indoor Natural optical condition; Open bottle cap and cultivate 2 days; Take out test-tube plantlet, clean medium on root system, plant in the nutritive cube installed with 70% thiophanate methyl and 1000 times of sterilizing peat, the moisturizing of seedling upper cover plastic cup, the time is 7 days; Grow field planting after 2 young leaves under moving on to facility condition, nursery soil dislikes mould spirit sterilization with 30%, namely obtains currant cross hybrid seedling plant.
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