CN105284622A - Method for rapid acquisition of excellent hybrid clone of iris - Google Patents
Method for rapid acquisition of excellent hybrid clone of iris Download PDFInfo
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- CN105284622A CN105284622A CN201510773709.XA CN201510773709A CN105284622A CN 105284622 A CN105284622 A CN 105284622A CN 201510773709 A CN201510773709 A CN 201510773709A CN 105284622 A CN105284622 A CN 105284622A
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Abstract
The invention discloses a method for rapid acquisition of an excellent hybrid clone of iris, belonging to the fields of plant tissue culture and plant biotechnology. According to the method, a mature hybrid seed of iris is used as an object and undergoes induced sprouting and subculturing propagation in virtue of embryo culture technology so as to obtain a great number of hybrid rootless sprouts; a part of the hybrid rootless sprouts are subjected to subculturing so as to retain germplasm; the other part of the hybrid rootless sprouts are subjected to strong sprout rooting, acclimatization and transplant and field planting, and after a growth period, the excellent hybrid clone achieving breeding objectives is screened; and the screened excellent hybrid clone is rapidly propagated to a certain scale through recovery growth in virtue of the retained germplasm. Compared with a conventional method which sows seeds at first, then screens out excellent individual plants and finally carries out tissue culture and propagation, the method provided by the invention enables a great number of seedlings of the excellent hybrid clone to be obtained 2 years earlier, so breeding process is substantially shortened.
Description
Technical field
The invention belongs to Plant Tissue Breeding and plant biotechnology field, particularly a kind of method of quick acquisition iris hybridization choiceness.
Background technology
Jris (
iris), Iridaceae (Iridaceae) Perennial Flowers, plant and various in style.Pattern enriches, and flower shape is different, blade bule, and sight is extremely strong.Part kind fragrance of a flower gas is simple and elegant, and its root-like stock can refine essence, also can make Chinese medicine.Adopt plant division, seeding method breeding more.
The breeding research of China iris is from the nineties in 20th century.Iris intervarietal cross ripening rate is low, and seed ubiquity dormancy, resting stage is long, and emergence rate is low, and division propagation coefficient is low, and seeding propagation required time is long; Iris tissue cultures explant many employings bud, but seedling needs 2-3 to become flower, adds that basic material is few, is difficult to obtain a large amount of explant, greatly limit the progress of iris tissue culture expanding propagation in the short time.Therefore, have impact on the process of iris breeding to a great extent.
Summary of the invention
The object of this invention is to provide a kind of method of quick acquisition iris hybridization choiceness.The method effectively shortens the clonal cultivation period of iris superior hybrid, accelerates sapling multiplication efficiency, and choiceness Applicative time of blooming is shifted to an earlier date greatly.
For achieving the above object, the present invention is achieved through the following technical solutions:
A kind of method of quick acquisition iris hybridization choiceness, it is characterized in that: with the ripe hybrid seed of iris for object, embryo culture technique is utilized to obtain a large amount of hybrid without offspring, a part continues squamous subculture and retains kind of a matter, a part is after strengthening seedling and rooting, acclimatization and transplants, field planting, through a growth cycle, filter out the choiceness meeting breeding objective, retain kind of matter and can carry out choose reasonable according to actual needs with the ratio of carrying out cultivating.Utilize the choiceness that filters out the kind matter that retains to be expanded rapidly by restoration ecosystem and numerously reach certain scale.
Preferably, described embryo culture refers to that emerge through induction, subculture expands numerous, strengthening seedling and rooting, hardening, transplants and obtains a large amount of hybridal clone seedling with the ripe hybrid seed embryo of iris for explant.
Preferably, described hybrid seed is florescence artificial pollination gained seed between iris kind or kind.
Preferably, described induction comprises the steps: ripe for described iris hybrid seed sterilization in emerging, with the seed after tweezers gripping sterilization, open along abdomen seaming and cutting, take out complete embryo, respectively at 75%(v/v) gently dip in alcohol and sterile water and stir 1-2s, In vitro Embryo is inoculated in MS medium.
Preferably, described embryo culture sprouting, subculture, root media are respectively MS, MS+6-BA2.0-4.0mg/L+IBA0.5-1.0mg/L, MS+PP
3330.2-1.0mg/L; Described hardening is that in greenhouse blake bottle being placed on scattered light 5000Lx, temperature 23-25 DEG C, 3d is cultivated in sealing; Described transplanting medium is vermiculite.
Preferably, the described every L of MS medium is containing 30g sucrose and 6g agar, and pH value is adjusted to 5.8-6.0.
Preferably, described cultivation temperature is 23-25 DEG C, illumination every day 12-14h, intensity of illumination 1000-1500Lx.
Preferably, described reservation kind matter carries out Mid-term preservation for iris crossbreed matter is oozed preservation method by height.
Preferably, it is on MS or 1/2MS, add 2-5% mannitol that described height oozes preservation method.
Preferably, described preserving seed environmental condition is: temperature 10-15 DEG C, intensity of illumination 500-1000Lx, light application time 8-10h.
Preferably, described breeding objective is the breeding objective of breeder, and Variety Selection method is selected voluntarily by breeder.
Preferably, described kind matter reverts to by reservation kind of matter by 20-30d in renewed vaccination to subculture medium, obtain restoration ecosystem without offspring, gained is cultivated at above-mentioned subculture, root media without offspring, obtains a large amount of whole plant.
Embryo culture is the effective way broken seed dormancy, raising seed germination rate and then affect plant breeding process.There is not dormancy in iris mature seed embryos, can obtain a large amount of Hybrid clones without offspring by embryo culture in 1-2, within the 3rd year, can obtain a large amount of seedling.Traditional seed propagation, 2-3 is bloomed, and uses bud tissue-culturing rapid propagation, also obtains the earliest and within the 4th year, could obtain a small amount of choiceness seedling, ability large-scale production in the 5th year.Therefore use Seed embryo culture technology, can breeding cycle be shortened, accelerate breeding process.
The present invention with the ripe hybrid seed embryo of iris for explant, obtain hybrid without offspring through embryo culture, a part continues squamous subculture and retains kind of a matter, and a part is after strengthening seedling and rooting, acclimatization and transplants, field planting, through a growth cycle, filter out the choiceness meeting breeding objective.Utilize the choiceness that filters out the kind matter that retains to be expanded rapidly by restoration ecosystem and numerously reach certain scale.Compared with traditional seed propagation, tissue cultures shifts to an earlier date 3 years start-up time, and the plant blossom time is suitable, and the time filtering out choiceness is suitable, obtains a large amount of superior hybrid crosses clone seedling time advance 2 years.
The present invention is by investigating the condition in embryo culture each stage, find the complete in vitro embryo stripped under anatomical lens after seed disinfection, gently dip in 75% alcohol and sterile water respectively and be inoculated into Initial culture base after stirring 1-2s, 23-25 DEG C, sprout time is 3d under the condition of 1000-1500Lx illumination every day 12-14h, pollution rate lower than 3.1%, germination rate more than 95%.In conjunction with specific medium and transplanting medium, make the survival rate of seedling of the present invention more than 85.81%, thus ensure the reproductive speed of iris clone seedling.
Under given conditions height is carried out to kind of matter and ooze preservation, make kind of a matter Subculture Time extend to 12-18 month, and do not affect the survival rate after restoration ecosystem.
Embodiment
Below in conjunction with iris kind " your lattice maiden " × " Bai Yuhuang ", the ripe hybrid seed specific embodiment of " India head " × " Bai Yulan ", describe the specific embodiment of the present invention in detail.
Embodiment 1
1. hybrid seed screening: get the ripe hybrid seed that " your lattice maiden " × " Bai Yuhuang " is dry, choose full, the seed without damage by disease and insect is subjects, give up go mouldy, shrivelled cur is sub.
2. embryo culture: 1. explant sterilization: dry seed first cleans up with washing powder, then after using tap water 30min, superclean bench first used 75% alcohol disinfecting 10min, aseptic water washing 3 times, then use 0.1% mercuric chloride (HgCl after soaking 2d with sterile water
2) after sterilization 8min, test after aseptic water washing 4 times, whole disinfecting process constantly shakes.
2. first pickup kind: with the seed after tweezers gripping sterilization, open along abdomen seaming and cutting under anatomical lens, take out complete embryo, gently dip in 75% alcohol and sterile water respectively and stir 1-2s, being inoculated in by In vitro Embryo in MS medium, every bottle is labeled as the number of being, after having inoculated, be placed in culturing room and cultivate.Culturing room temperature 23-25 DEG C, illumination every day 12h, intensity of illumination 1000-1500Lx.Germination rate 95% after 3d, pollution rate 3.1%.
3. shoot proliferation: the seedling of getting about 2cm after sprouting, cuts off root system, be divided into budlet clump carefully in the mode peeled off between bud, be inoculated in MS+6-BA2.0mg/L+IBA0.5mg/L subculture medium.After having inoculated, be placed in culturing room and cultivate.Every 25d subculture once.
4. strengthening seedling and rooting: after subculture 4-5 time, the indefinite bud getting a part of about 5cm is inoculated into MS+PP
333culture of rootage is carried out in 0.5mg/L root media.After having inoculated, be placed in culturing room and cultivate.
5. tame hardening: when test tube shoot root grows to about 3cm, taken out by blake bottle from culturing room, be placed in the greenhouse of scattered light 5000Lx, temperature 23-25 DEG C, 3d is cultivated in sealing.
6. transplant: seedling of taking root takes out from blake bottle, with the agar of warm water washing its base portion clean, root is soaked 10s in 0.1% carbendazim solution, is transplanted in the vermiculite matrix of 0.5% potassium permanganate process (irrigating), covered rearing with plastic film moisturizing, add a cover sunshade net sunshade, regularly water bactericide sterilizing, note watering moisturizing, after 15d, take off film, strengthen the management after transplanting, plant shoot survival percent 85.81%.
3. kind of quality guarantee is stayed: remaining another part to be inoculated in the mode peeled off between bud without offspring and the medium of MS+5% mannitol carries out height to ooze preservation.Cultivation temperature 10-15 DEG C, intensity of illumination 500-1000Lx, light application time 8-10h.
4. elite plant strain screening: move into land for growing field crops plantlet in vitro after a growth cycle, filter out the choiceness meeting breeding objective.
5. kind of matter recover, numerous soon: the kind matter renewed vaccination that the choiceness filtered out is retained to 20-30d in MS+6-BA2.0mg/L+IBA0.5mg/L medium, obtain restoration ecosystem without offspring shoot proliferation repeatedly, obtain a large amount of whole plant.
Comparative example 1
Step in embryo culture 2. in the complete embryo that takes out from seed without 75% alcohol and sterile water process, be directly inoculated in MS medium, all the other are identical with embodiment 1.Germination rate 90.1% after 3d, pollution rate 9.2%.
From embodiment 1 and comparative example 1, with 75% alcohol and sterile water, In vitro Embryo is processed, effectively can reduce pollution rate and improve germination rate.
Comparative example 2
Step in embryo culture 2. in the complete embryo that takes out from seed gently dip in 75% alcohol and sterile water respectively and stir 10s, be then inoculated in MS medium, all the other are identical with embodiment 1.Germination rate 25.3% after 3d, pollution rate 1.5%.
From embodiment 1 and comparative example 2, by the overlong time of 75% alcohol and sterile water process, although the pollution rate of embryo reduces, germination rate reduces more obvious.
Embodiment 2
1. hybrid seed screening: get the ripe hybrid seed that " India head " × " Bai Yulan " is dry, choose full, the seed without damage by disease and insect is subjects, give up go mouldy, shrivelled cur is sub.
2. embryo culture: 1. explant sterilization: dry seed first cleans up with washing powder, then after using tap water 30min, superclean bench first used 75% alcohol disinfecting 10min, aseptic water washing 3 times, then use 0.1% mercuric chloride (HgCl after soaking 2d with sterile water
2) after sterilization 8min, test after aseptic water washing 4 times, whole disinfecting process constantly shakes.
2. first pickup kind: with the seed after tweezers gripping sterilization, open along abdomen seaming and cutting under anatomical lens, take out complete embryo, gently dip in 75% alcohol and sterile water respectively and stir 1-2s, being inoculated in by In vitro Embryo in MS medium, every bottle is labeled as a strain, after having inoculated, be placed in culturing room and cultivate.Culturing room temperature 23-25 DEG C, illumination every day 12h, intensity of illumination 1000-1500Lx.Germination rate 96.2% after 3d, pollution rate 3.0%.
3. shoot proliferation: the seedling of getting about 2cm after sprouting, cuts off root system, be divided into budlet clump carefully in the mode peeled off between bud, be inoculated in MS+6-BA4.0mg/L+IBA1.0mg/L subculture medium.After having inoculated, be placed in culturing room and cultivate.Every 25d subculture once.
4. strengthening seedling and rooting: after subculture 4-5 time, the indefinite bud getting a part of about 5cm is inoculated into MS+PP
333culture of rootage is carried out in 0.6mg/L root media.After having inoculated, be placed in culturing room and cultivate.
5. tame hardening: when test tube shoot root grows to about 3cm, taken out by blake bottle from culturing room, be placed in the greenhouse of scattered light 5000Lx, temperature 23-25 DEG C, 3d is cultivated in sealing.
6. transplant: seedling of taking root takes out from blake bottle, with the agar of warm water washing its base portion clean, root is soaked 10s in 0.1% carbendazim solution, is transplanted in the vermiculite matrix of 0.5% potassium permanganate process (irrigating), covered rearing with plastic film moisturizing, add a cover sunshade net sunshade, regularly water bactericide sterilizing, note watering moisturizing, after 15d, take off film, strengthen the management after transplanting, plant shoot survival percent 90.5%.
3. kind of quality guarantee is stayed: remaining another part to be inoculated in the mode peeled off between bud without offspring and the medium of 1/2MS+2% mannitol carries out height to ooze preservation.Cultivation temperature 10-15 DEG C, intensity of illumination 500-1000Lx, light application time 8-10h.
4. elite plant strain screening: move into land for growing field crops plantlet in vitro after a growth cycle, filter out the choiceness meeting breeding objective.
5. kind of matter recover, numerous soon: by the choiceness filtered out by the kind matter renewed vaccination that retains to 20-30d in MS+6-BA4.0mg/L+IBA1.0mg/L medium, obtain restoration ecosystem without offspring shoot proliferation repeatedly, obtain a large amount of whole plant.
The present invention is utilized to obtain a large amount of excellent iris Hybrid clones seedlings.In artificial pollination then, utilize ripe hybrid seed embryo to carry out embryo culture for explant, within the 2nd year, a large amount of Hybrid clones seedling can be obtained, Some Species matter carries out Plantlet in vitro, Some Species matter land for growing field crops is cultivated, and after a growth cycle, filters out the choiceness meeting breeding objective.A large amount of superior hybrid crosses clone seedling within 4th year, can be obtained.Traditional seed propagation, 2-3 is bloomed, and after filtering out fine individual plant, uses bud tissue-culturing rapid propagation, also obtains the earliest and within the 4th year, could obtain a small amount of choiceness seedling, ability large-scale production in the 5th year.By comparison, tissue cultures shifts to an earlier date 3 years start-up time, and the plant blossom time is suitable, filters out the choiceness time suitable, obtains a large amount of Hybrid clones seedling time advance 2 years.Combine kind of a matter Plantlet in vitro by embryo culture, shorten the iris crossbreeding cycle, accelerate breeding process.In addition, by the optimization to each stage conditions of embryo culture, make embryo after 3d, namely have higher germination rate and lower pollution rate, and after transplanting, survival rate is higher.
Claims (10)
1. one kind obtains the method for iris hybridization choiceness fast, it is characterized in that, with the ripe hybrid seed of iris for object, embryo culture technique is utilized to obtain hybrid without offspring, described hybrid continues squamous subculture without an offspring part and retains kind of a matter, a part is after strengthening seedling and rooting, hardening, transplanting, field planting, through a growth cycle, filter out the choiceness meeting breeding objective, utilize the kind matter retained to carry out by restoration ecosystem expansions to the choiceness that filters out numerous, through taking root, hardening, transplanting obtain and hybridize choiceness seedling.
2. method according to claim 1, is characterized in that, described embryo culture to refer to the ripe hybrid seed embryo of iris as explant, emerges, subculture expands numerous, strengthening seedling and rooting, hardening and transplanting obtains hybrid seedling through induction.
3. method according to claim 2, is characterized in that, described hybrid seed is florescence artificial pollination gained seed between iris kind or kind.
4. method according to claim 2, it is characterized in that, described induction comprises the steps: ripe for described iris hybrid seed sterilization in emerging, with the seed after tweezers gripping sterilization, open along abdomen seaming and cutting, take out complete embryo, respectively at 75%(v/v) gently dip in alcohol and sterile water and stir 1-2s, In vitro Embryo is inoculated in MS medium.
5. method according to claim 2, is characterized in that, described embryo culture sprouting, subculture, root media are respectively MS, MS+6-BA2.0-4.0mg/L+IBA0.5-1.0mg/L, MS+PP
3330.2-1.0mg/L; Described hardening is that in greenhouse blake bottle being placed on scattered light 5000Lx, temperature 23-25 DEG C, 3d is cultivated in sealing; Described transplanting medium is vermiculite.
6. method according to claim 5, is characterized in that, the described every L of MS medium is containing 30g sucrose and 6g agar, and pH value is adjusted to 5.8-6.0.
7. the method according to claim 1-6, is characterized in that, described embryo culture temperature is 23-25 DEG C, illumination every day 12-14h, intensity of illumination 1000-1500Lx.
8. method according to claim 1, is characterized in that, described reservation kind matter carries out Mid-term preservation for iris crossbreed matter is oozed preservation method by height, and it is on MS or 1/2MS, add 2-5% mannitol that described height oozes preservation method; Preserving seed environmental condition is: temperature 10-15 DEG C, intensity of illumination 500-1000Lx, light application time 8-10h.
9. method according to claim 1, is characterized in that, described breeding objective is the breeding objective of breeder, and Variety Selection method is selected voluntarily by breeder.
10. method according to claim 1, it is characterized in that, described kind matter reverts to by reservation kind of matter by 20-30d in renewed vaccination to subculture medium, obtain restoration ecosystem without offspring, gained proceeds subculture, culture of rootage without offspring, obtains whole plant.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106561457A (en) * | 2016-11-07 | 2017-04-19 | 宁德师范学院 | Heterophylla hybrid seed artificial cultivating and breeding method |
| CN114027188A (en) * | 2021-11-02 | 2022-02-11 | 江苏省中国科学院植物研究所 | Culture medium for obtaining iris interspecific hybridization progeny by utilizing immature embryos and application of culture medium |
| CN115428732A (en) * | 2022-09-05 | 2022-12-06 | 金华市农业科学研究院(浙江省农业机械研究院) | Culture medium for reducing subculture of anoectochilus formosanus and anoectochilus formosanus culture method |
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| CN103609224A (en) * | 2013-11-04 | 2014-03-05 | 浙江大学 | Louisiana iris hybrid seed germination method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106561457A (en) * | 2016-11-07 | 2017-04-19 | 宁德师范学院 | Heterophylla hybrid seed artificial cultivating and breeding method |
| CN106561457B (en) * | 2016-11-07 | 2019-03-15 | 宁德师范学院 | A kind of radix pseudostellariae hybrid seed manually cultivates selection |
| CN114027188A (en) * | 2021-11-02 | 2022-02-11 | 江苏省中国科学院植物研究所 | Culture medium for obtaining iris interspecific hybridization progeny by utilizing immature embryos and application of culture medium |
| CN115428732A (en) * | 2022-09-05 | 2022-12-06 | 金华市农业科学研究院(浙江省农业机械研究院) | Culture medium for reducing subculture of anoectochilus formosanus and anoectochilus formosanus culture method |
| CN115428732B (en) * | 2022-09-05 | 2023-10-03 | 金华市农业科学研究院(浙江省农业机械研究院) | Culture medium for reducing anoectochilus formosanus subculture and anoectochilus formosanus culture method |
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