CN105284622B - A kind of method that quick acquisition Rhizoma Iridis Tectori hybridizes superior clone - Google Patents
A kind of method that quick acquisition Rhizoma Iridis Tectori hybridizes superior clone Download PDFInfo
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- CN105284622B CN105284622B CN201510773709.XA CN201510773709A CN105284622B CN 105284622 B CN105284622 B CN 105284622B CN 201510773709 A CN201510773709 A CN 201510773709A CN 105284622 B CN105284622 B CN 105284622B
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Abstract
The invention discloses a kind of method that quick acquisition Rhizoma Iridis Tectori hybridizes superior clone, is related to plant tissue culture and plant biotechnology field.The method is with Rhizoma Iridis Tectori maturation hybrid seed as object, emerged using embryo culture technique Jing inductions, subculture expanding propagation obtains a large amount of hybrids without root, a part continues successive transfer culture and retains germplasm, it is a part of Jing after strengthening seedling and rooting, acclimatization and transplantses, field planting, through a growth cycle, the superior clone for meeting breeding objective is filtered out.Superior clone to filtering out reaches certain scale by the rapid expanding propagation of restoration ecosystem using the germplasm for retaining.Sow with traditional seed, tissue culture expanding propagation is carried out after filtering out fine individual plant and is compared, the present invention obtains at least 2 years in advance time of a large amount of superior hybrid crosses clone seedlings, substantially reduces breeding process.
Description
Technical field
The invention belongs to plant tissue culture and plant biotechnology field, more particularly to a kind of quick acquisition Rhizoma Iridis Tectori hybridization
The method of superior clone.
Background technology
Jris（Iris）, Iridaceae（Iridaceae）Perennial Flowers, plant and various in style.Pattern enriches, and flower-shape is each
Different, blade bule, sight are extremely strong.Part kind fragrance of a flower gas is simple and elegant, and its root stock can refine essence, can also make Chinese medicine.
Bred using plant division, seeding method more.
The breeding research of China iris is from the beginning of the nineties in 20th century.Rhizoma Iridis Tectori interbreed setting percentage is low,
And seed generally existing dormancy, rest period is long, and emergence rate is low, and division propagation coefficient is low, long the time required to seeding propagation；
Adopt alabastrum more Rhizoma Iridis Tectori tissue culture explant, but seedling needs the 2-3 could be into flower, it is few to add basic material, the short time
It is interior to be difficult to obtain a large amount of explants, greatly limit the progress of Rhizoma Iridis Tectori tissue culture expanding propagation.Therefore, Rhizoma Iridis Tectori is have impact on to a great extent
The process of breeding.
The content of the invention
It is an object of the invention to provide a kind of method that quick acquisition Rhizoma Iridis Tectori hybridizes superior clone.The method effectively shortens
Rhizoma Iridis Tectori superior hybrid clonal cultivation period, accelerates sapling multiplication efficiency, makes superior clone application time of blooming big
It is big to shift to an earlier date.
For achieving the above object, the present invention is achieved through the following technical solutions：
A kind of method that quick acquisition Rhizoma Iridis Tectori hybridizes superior clone, it is characterised in that：With Rhizoma Iridis Tectori maturation hybrid seed it is
Object, obtains a large amount of hybrids without root using embryo culture technique, and a part continues successive transfer culture and retains germplasm, a part of Jing strong sprouts
Take root, acclimatization and transplantses, after field planting, through a growth cycle, filter out the superior clone for meeting breeding objective, retain germplasm
Reasonable selection is carried out according to actual needs can with the ratio cultivated.Superior clone to filtering out utilizes the germplasm for retaining
Certain scale is reached by the rapid expanding propagation of restoration ecosystem.
Preferably, described embryo culture refers to that Jing inductions are emerged, and subculture expands with Rhizoma Iridis Tectori maturation hybrid seed embryo as explant
Numerous, strengthening seedling and rooting, seedling exercising, transplanting obtain a large amount of hybridal clone seedlings.
Preferably, described hybrid seed is seed obtained by florescence artificial pollination between Rhizoma Iridis Tectori kind or kind.
Preferably, the induction comprises the steps in emerging：By Rhizoma Iridis Tectori maturation hybrid seed sterilization, pressed from both sides with tweezers
Cancel the seed after poison, open along abdomen seaming and cutting, take out complete embryo, respectively 75%（v/v）Gently dip in ethanol and sterilized water and stir 1-
2s, In vitro Embryo is inoculated in MS culture medium.
Preferably, described embryo culture sprouting, subculture, root media be respectively MS, MS+6-BA2.0-4.0 mg/L+
IBA0.5-1.0 mg/L、MS+PP3330.2-1.0 mg/L；Described seedling exercising is that culture bottle is placed on scattered light 5000Lx, temperature
Sealing culture 3d in the greenhouse of 23-25 DEG C of degree；Described transplanting medium is Vermiculitum.
Preferably, per L containing 30 g sucrose and 6g agar, pH value is adjusted to 5.8-6.0 to described MS culture medium.
Preferably, described cultivation temperature is 23-25 DEG C, daily illumination 12-14h, intensity of illumination 1000-1500Lx.
Preferably, described reservation germplasm is that Rhizoma Iridis Tectori hybridization germplasm is carried out Mid-term preservation by hypertonic preservation method.
Preferably, described hypertonic preservation method is the addition 2-5% Mannitol on MS or 1/2MS.
Preferably, described preserving seed environmental condition is：Temperature 10-15 DEG C, intensity of illumination 500-1000Lx, during illumination
Between 8-10h.
Preferably, breeding objective of the described breeding objective for breeder, Variety Selection method are voluntarily selected by breeder.
Preferably, described germplasm is reverted to and will retain germplasm by renewed vaccination 20-30d in subculture medium, is obtained
Restoration ecosystem without root, gained is cultivated in above-mentioned subculture, root media without root, obtains a large amount of whole plants.
Embryo culture is to break seed dormancy, improve seed germination rate and then affect one of plant breeding process effectively on the way
Footpath.There is no dormancy in Rhizoma Iridis Tectori mature seed embryos, can obtain a large amount of Hybrid clones without root by embryo culture in 1-2, and the 3rd
A large amount of seedlings are obtained by year.Traditional seed propagation, 2-3 are bloomed, and use alabastrum tissue-culturing rapid propagation, also obtain the 4th year earliest
A small amount of superior clone seedling can be obtained, the 5th year could large-scale production.Therefore Seed embryo culture technology is used, can be shortened and be educated
In the cycle of kind, accelerate breeding process.
With Rhizoma Iridis Tectori maturation hybrid seed embryo as explant, Jing embryo cultures obtain hybrid without root to the present invention, and a part continues
Successive transfer culture retains germplasm, a part of Jing after strengthening seedling and rooting, acclimatization and transplantses, field planting, through a growth cycle, filters out and meets
The superior clone of breeding objective.Superior clone to filtering out is reached by the rapid expanding propagation of restoration ecosystem using the germplasm for retaining
To certain scale.Compared with traditional seed propagation, tissue culture's starting time 3 years in advance, the plant blossom time quite, screens
Go out the time of superior clone quite, obtain a large amount of superior hybrid crosses clone seedling time advances 2 years.
The present invention is investigated by the condition to embryo culture each stage, and discovery is stripped Jing after seed disinfection under anatomical lens
Complete in vitro embryo, gently dip in 75% ethanol and sterilized water respectively and be inoculated into Initial culture base after stirring 1-2s, in 23-
25 DEG C, sprout time is 3d under conditions of daily 1000-1500Lx illumination 12-14h, pollution rate is less than 3.1%, germination rate 95% with
On.With reference to specific culture medium and transplanting medium, make the survival rate of seedling of the present invention more than 85.81%, so as to ensure Rhizoma Iridis Tectori without
The reproductive speed of property system seedling.
Hypertonic preservation is carried out to germplasm under given conditions, is made germplasm Subculture Time extend to 12-18 month, and is not affected
Survival rate after restoration ecosystem.
Specific embodiment
Below in conjunction with Rhizoma Iridis Tectori kind " your lattice maiden " × " Bai Yuhuang ", " India head " × " Bai Yulan " maturation cenospecies
Sub- specific embodiment, describes the specific embodiment of the present invention in detail.
Embodiment 1
1. hybrid seed screening：The ripe hybrid seed that " your lattice maiden " × " Bai Yuhuang " is dried is taken, chooses full, it is disease-free
The seed of insect pest is subjects, give up go mouldy, shrivelled cur.
2. embryo culture：1. explant sterilization:After dry seed is with sterilized water immersion 2d, first cleaned up with detergent, then
Rinsed after 30min with tap water, first with 75% alcohol disinfecting 10min on superclean bench, aseptic water washing 3 times, then with 0.1%
Mercuric chloride（HgCl2）After sterilization 8min, tested after aseptic water washing 4 times, whole disinfecting process constantly shakes.
2. pickup kind at the beginning of：The seed after sterilization is gripped with tweezers, is opened in anatomical lens lower edge abdomen seaming and cutting, take out complete embryo, point
1-2s gently not being dipped in and being stirred in 75% ethanol and sterilized water, In vitro Embryo is inoculated in MS culture medium, per bottle is labeled as one and is
Number, after the completion of inoculation, it is placed in culturing room and cultivates.Culture room temperature 23-25 DEG C, daily illumination 12h, intensity of illumination 1000-
1500Lx.Germination rate 95% after 3d, pollution rate 3.1%.
3. subculture multiplication：The seedling of 2cm or so after sprouting is taken, root system is cut off, is carefully divided in the way of peeling off between bud
Budlet clump, is inoculated in MS+6-BA2.0 mg/L+IBA0.5 mg/L subculture mediums.After the completion of inoculation, it is placed in culturing room
Culture.Every 25d subcultures once.
4. strengthening seedling and rooting：After subculture 4-5 time, the adventitious bud for taking a part of 5cm or so is inoculated into MS+PP3330.5mg/L gives birth to
Root culture is carried out in root culture medium.After the completion of inoculation, it is placed in culturing room and cultivates.
5. tame seedling exercising：When test tube seedling root grows to 3cm or so, culture bottle is taken out from culturing room, scattered light is placed in
In 5000Lx, the greenhouse of temperature 23-25 DEG C, sealing culture 3d.
6. transplant：Seedling of taking root is taken out from culture bottle, with the agar of clean its base portion of warm water washing, by root 0.1%
10s is soaked in carbendazim solution, the process of 0.5% potassium permanganate of Jing is transplanted to（Irrigate）Vermiculite matrix in, covered rearing with plastic film protect
It is wet, sunshade net sunshade is added a cover, antibacterial sterilizing is periodically poured, is noted moisturizing of watering, after 15d, take off film, strengthened the management after transplanting, plant
Shoot survival percent 85.81%.
3. germplasm retains：Remaining another part is inoculated into the training of MS+5% Mannitol in the way of peeling off between bud without root
Hypertonic preservation is carried out on foster base.Cultivation temperature 10-15 DEG C, intensity of illumination 500-1000Lx, light application time 8-10h.
4. elite plant strain screening：Land for growing field crops tissue cultured seedling is moved into after a growth cycle, is filtered out and is met breeding objective
Superior clone.
5. germplasm recover, it is fast numerous：By the germplasm renewed vaccination of the superior clone for filtering out reservation to MS+6-BA2.0mg/
20-30d in L+IBA0.5mg/L culture medium, obtains the multiple without root subculture multiplication of restoration ecosystem, obtains complete in a large number plant
Strain.
Comparative example 1
Step in embryo culture 2. in the complete embryo that takes out from seed without 75% ethanol and aseptic water process, directly connect
Plant in MS culture medium, remaining is same as Example 1.Germination rate 90.1% after 3d, pollution rate 9.2%.
From embodiment 1 and comparative example 1, In vitro Embryo is processed with 75% ethanol and sterilized water, can effectively reduce dirt
Dye rate and raising germination rate.
Comparative example 2
Step in embryo culture 2. in the complete embryo that takes out from seed in 75% ethanol and sterilized water gently dip in and stir respectively
Dynamic 10s, is then inoculated in MS culture medium, and remaining is same as Example 1.Germination rate 25.3% after 3d, pollution rate 1.5%.
From embodiment 1 and comparative example 2, with 75% ethanol and the overlong time of aseptic water process, although the pollution of embryo
Rate is reduced, but germination rate reduces becoming apparent from.
Embodiment 2
1. hybrid seed screening：The ripe hybrid seed that " India head " × " Bai Yulan " is dried is taken, chooses full, it is disease-free
The seed of insect pest is subjects, give up go mouldy, shrivelled cur.
2. embryo culture：1. explant sterilization:After dry seed is with sterilized water immersion 2d, first cleaned up with detergent, then
Rinsed after 30min with tap water, first with 75% alcohol disinfecting 10min on superclean bench, aseptic water washing 3 times, then with 0.1%
Mercuric chloride（HgCl2）After sterilization 8min, tested after aseptic water washing 4 times, whole disinfecting process constantly shakes.
2. pickup kind at the beginning of：The seed after sterilization is gripped with tweezers, is opened in anatomical lens lower edge abdomen seaming and cutting, take out complete embryo, point
1-2s gently not being dipped in and being stirred in 75% ethanol and sterilized water, In vitro Embryo is inoculated in MS culture medium, per bottle is labeled as a strain
System, after the completion of inoculation, is placed in culturing room and cultivates.Culture room temperature 23-25 DEG C, daily illumination 12h, intensity of illumination 1000-
1500Lx.Germination rate 96.2% after 3d, pollution rate 3.0%.
3. subculture multiplication：The seedling of 2cm or so after sprouting is taken, root system is cut off, is carefully divided in the way of peeling off between bud
Budlet clump, is inoculated in MS+6-BA4.0 mg/L+IBA1.0 mg/L subculture mediums.After the completion of inoculation, it is placed in culturing room
Culture.Every 25d subcultures once.
4. strengthening seedling and rooting：After subculture 4-5 time, the adventitious bud for taking a part of 5cm or so is inoculated into MS+PP3330.6mg/L gives birth to
Root culture is carried out in root culture medium.After the completion of inoculation, it is placed in culturing room and cultivates.
5. tame seedling exercising：When test tube seedling root grows to 3cm or so, culture bottle is taken out from culturing room, scattered light is placed in
In 5000Lx, the greenhouse of temperature 23-25 DEG C, sealing culture 3d.
6. transplant：Seedling of taking root is taken out from culture bottle, with the agar of clean its base portion of warm water washing, by root 0.1%
10s is soaked in carbendazim solution, the process of 0.5% potassium permanganate of Jing is transplanted to（Irrigate）Vermiculite matrix in, covered rearing with plastic film protect
It is wet, sunshade net sunshade is added a cover, antibacterial sterilizing is periodically poured, is noted moisturizing of watering, after 15d, take off film, strengthened the management after transplanting, plant
Shoot survival percent 90.5%.
3. germplasm retains：Remaining another part is inoculated into 1/2MS+2% Mannitol in the way of peeling off between bud without root
Hypertonic preservation is carried out in culture medium.Cultivation temperature 10-15 DEG C, intensity of illumination 500-1000Lx, light application time 8-10h.
4. elite plant strain screening：Land for growing field crops tissue cultured seedling is moved into after a growth cycle, is filtered out and is met breeding objective
Superior clone.
5. germplasm recover, it is fast numerous：By the superior clone for filtering out by the germplasm renewed vaccination of reservation to MS+6-
20-30d in BA4.0mg/L+IBA1.0mg/L culture medium, obtains the multiple without root subculture multiplication of restoration ecosystem, obtains a large amount of
Whole plant.
Substantial amounts of excellent Rhizoma Iridis Tectori Hybrid clones seedling is obtained using the present invention.In artificial pollination then, using maturation
Hybrid seed embryo carries out embryo culture for explant, can obtain a large amount of Hybrid clones seedlings within the 2nd year, and part germplasm is carried out in vitro
Preserve, part germplasm land for growing field crops is cultivated, and after a growth cycle, filters out the superior clone for meeting breeding objective.4th year
A large amount of superior hybrid crosses clone seedlings can be obtained.Traditional seed propagation, 2-3 are bloomed, and after filtering out fine individual plant, use
Alabastrum tissue-culturing rapid propagation, also obtains earliest and could obtain within the 4th year a small amount of superior clone seedling, the 5th year could large-scale production.Therewith
Compare, tissue culture's starting time 3 years in advance, plant blossom time quite, filter out the superior clone time quite, obtain big
Amount Hybrid clones seedling time advance 2 years.Germplasm in vitro conservation is combined by embryo culture, Rhizoma Iridis Tectori cross-breeding week is shortened
Phase, accelerate breeding process.Additionally, by the optimization to each stage conditions of embryo culture, making embryo have after 3d higher
Germination rate and relatively low pollution rate, and after transplanting, survival rate is higher.
Claims (4)
1. a kind of method that quick acquisition Rhizoma Iridis Tectori hybridizes superior clone, it is characterised in that be right with Rhizoma Iridis Tectori maturation hybrid seed
As, using embryo culture technique obtain hybrid without root, the hybrid without root a part continue successive transfer culture retain germplasm, one
Lease making strengthening seedling and rooting, seedling exercising, transplanting, after field planting, through a growth cycle, filter out and meet the excellent asexual of breeding objective
System, carries out expanding propagation by restoration ecosystem using the germplasm for retaining to the superior clone that filters out, and Jing is taken root, seedling exercising, transplanting are obtained
Superior clone seedling must be hybridized；
Described embryo culture refers to that Jing inductions are emerged with Rhizoma Iridis Tectori maturation hybrid seed embryo as explant, the life of subculture expanding propagation, strong sprout
Root, seedling exercising and transplanting obtain hybrid seedling；
The induction comprises the steps in emerging：By Rhizoma Iridis Tectori maturation hybrid seed sterilization, after tweezers gripping sterilization
Seed, opens along abdomen seaming and cutting, takes out complete embryo, respectively 75%（v/v）1-2s is gently dipped in and is stirred in ethanol and sterilized water, will be in vitro
Embryo is inoculated in MS culture medium；
In described embryo culture induction emerge, subculture, root media be respectively MS, MS+6-BA2.0-4.0 mg/L+
IBA0.5-1.0 mg/L、MS+PP3330.2-1.0 mg/L ；Described seedling exercising be by culture bottle be placed on scattered light 5000Lx,
Sealing culture 3d in the greenhouse of temperature 23-25 DEG C；Transplanting medium is Vermiculitum；
Described embryo culture temperature is 23-25 DEG C, daily illumination 12-14h, intensity of illumination 1000-1500Lx；
Described reservation germplasm is that Rhizoma Iridis Tectori hybrid is carried out Mid-term preservation, described hypertonic preservation by hypertonic preservation method without root
Method is the addition 2-5% Mannitol in MS or 1/2MS；Preserving seed environmental condition is：Temperature 10-15 DEG C, intensity of illumination
500-1000Lx, light application time 8-10h.
2. the method according to claim 1, it is characterised in that described hybrid seed is the florescence between Rhizoma Iridis Tectori kind or kind
Seed obtained by artificial pollination.
3. the method according to claim 2, it is characterised in that described MS culture medium is per L sucrose containing 30g and 6g
Agar, pH values are adjusted to 5.8-6.0.
4. the method according to claim 1, it is characterised in that described restoration ecosystem is will to retain germplasm by again
Be inoculated into 20-30d in subculture medium, obtain restoration ecosystem without root, gained proceeds subculture without root, training of taking root
Support, obtain whole plant.
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| CN106561457B (en) * | 2016-11-07 | 2019-03-15 | 宁德师范学院 | A kind of radix pseudostellariae hybrid seed manually cultivates selection |
| CN114027188B (en) * | 2021-11-02 | 2023-04-07 | 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 |
| 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 |
| CN116784230A (en) * | 2023-08-14 | 2023-09-22 | 江苏省中国科学院植物研究所 | Method for rapidly obtaining iris Netherlands offspring seedlings and excellent seed balls |
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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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| CN103609224A (en) * | 2013-11-04 | 2014-03-05 | 浙江大学 | Louisiana iris hybrid seed germination method |
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