CN110226516A - A kind of pair of conventional method stablized Tetraploid Rice and carry out character improvement - Google Patents
A kind of pair of conventional method stablized Tetraploid Rice and carry out character improvement Download PDFInfo
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- CN110226516A CN110226516A CN201910548258.8A CN201910548258A CN110226516A CN 110226516 A CN110226516 A CN 110226516A CN 201910548258 A CN201910548258 A CN 201910548258A CN 110226516 A CN110226516 A CN 110226516A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
A kind of pair of conventional method stablized Tetraploid Rice and carry out character improvement, which comprises the following steps: (1) revert back to diploid rice using Tetraploid Rice anther callus induction；(2) the diploid rice maturation embryonal induction Autotetraploidy Rice cultivated in recycle step (1)；The Induction Process of the step (1) is as follows: a. materials and pre-treatment: choosing pulvinus from experimental plot away from the tetraploid spike of rice for 4-8cm, is placed in polybag and seals inside 4 DEG C of refrigerator cold treatment 7 days after being wrapped with wet gauze.The present invention has the advantage that improveing the character of A-4X while former homozygous stable Tetraploid Rice A-4X genotype is fully retained, and character changes, and is substantially shorter the material settling out time limit.
Description
Technical field
The present invention relates to the New Crop Varieties breeding technique fields of modern agriculture, and in particular to a kind of to stablize four times to conventional
The method of body rice progress character improvement.
Background technique
It is exactly at present crossbreeding and improvement, example to the homozygous most common method of character improvement for stablizing Tetraploid Rice breeding material
Such as to stablize Tetraploid Rice A-4X to homozygosis and carry out character improvement it is necessary to choose a B-4X material, stablize four times with homozygosis
Body rice A-4X does maternal or male parent and is hybridized with B-4X material.
Disadvantage of this is that the genotype for having upset original homozygous stable Tetraploid Rice A-4X completely, and are additionally added
The gene of B-4X material, thus increase the difficulty for choosing target gene, or even the phenotype that choosing is wanted less than breeding people, and
The most important point was that the material settling out time limit after improvement is very long, up to 10 years or more.
Summary of the invention
Character improvement is carried out to conventional Tetraploid Rice of stablizing technical problem to be solved by the invention is to provide a kind of
Method, which comprises the following steps:
(1) diploid rice is reverted back to using Tetraploid Rice anther callus induction；
(2) the diploid rice maturation embryonal induction Autotetraploidy Rice cultivated in recycle step (1)；
The Induction Process of the step (1) is as follows:
A. pulvinus materials and pre-treatment: is chosen from experimental plot away from the tetraploid spike of rice for 4-8cm, after being wrapped with wet gauze
It is placed in polybag and seals inside 4 DEG C of refrigerator cold treatment 7 days；
B. anther evoked callus: by the young fringe Jing Guo cold treatment, with 70% ethanol postincubation 30s on superclean bench
10min is handled with 0.1% mercuric chloride solution again afterwards, then the top light green of picking, the milky grain husk flower in lower part, are removed with sharp tweezers
Grain husk flower with anther is seeded in induced medium and cultivates callus by glume；
C. it differentiates seedling and obtains liploid plant: being transferred to differential medium progress when callus grows to a certain size
Optical culture, temperature are 27 DEG C；
D. it takes root and transplants: being transferred to root media after differentiation and seedling emergence, after root system is grown well, open sealing, be added clear
Water practices seedling one week or so in greenhouse, then takes out and the culture medium of root is rinsed well with clear water, transplanting to field；
The Induction Process of the step (2) is as follows:
A. Seeds preprocess: by mature rice seed scissors remove 3/4ths or more endosperm fractions, stay
The part of embryo is had down；In super-clean bench, material is poured into triangular flask, finishes writing number, first with 75% alcohol surface sterilization
40s or so is added 0.1% mercuric chloride and impregnates 15min or so, between wanting during immersion then with sterilized pure water wash clean
Every rock, after washed 3-4 times with pure water；
B. seed embryo evoked callus: the material after disinfection is connected in induced medium, about 8 every bottle, seals mouth
It is put between 26 DEG C of tissue culture and carries out dark culture, grow to soybean grain size to callus and just carry out colchicine and double to handle；
C. colchicine doubles to handle: callus access being doubled to rock in shaking table in liquid 48 hours, shaking table setting
It is 27 DEG C, 110r/min, callus wash clean and accesses the dark training of continuation in recovery media with sterile water after doubling
It supports, the time of recovery is about 7-15 days；
D. differentiate seedling and obtain tetraploid plant: callus is transferred to differential medium and carries out optical culture, temperature after restoring normal
Degree is 27 DEG C, about has callus greening once week after transdifferentiation, with that can long emergence slowly；
E. it takes root and transplants: being transferred to root media after differentiation and seedling emergence, after root system is grown well, open sealing, be added clear
Water practices seedling one week or so in greenhouse, then takes out and the culture medium of root is rinsed well with clear water, transplanting to field.
Preferably, the induced medium of inoculation grain husk flower includes fluid nutrient medium and solid medium in the step (1).
Preferably, the fluid nutrient medium is placed on 30 DEG C, dark culture three days in the shaking table of 100r/min, after turn down culture temperature
Degree is to 26 DEG C, the constant continuation dark culture of revolving speed, and flaxen little particle callus occurs in visible culture basal part within about 30 days or so,
After become larger.
Preferably, first dark culture three days in 30 DEG C of incubator of the film solid media, after be placed in 26 DEG C of culturing room
It cultivates, has callus on about 30 days or so the anther of visible cracking.
The present invention has the advantage that while former homozygous stable Tetraploid Rice A-4X genotype is fully retained and
Improve the character of A-4X.The present invention only need enchashment have homozygous stable Tetraploid Rice A-4X mid-late uninucleate stage pollen or not
Ovary of being fertilized carries out the culture of monoploid, is trained homozygous stable diploid rice A-2X, then to this diploid rice into
Genome of row doubles, and doubles into homozygous stable Tetraploid Rice A1-4X, and former homozygosis is fully retained and stablizes four times
The genotype of body rice A-4X, and character changes, and is substantially shorter the material settling out time limit.
Specific embodiment
One, diploid rice is reverted back to using Tetraploid Rice anther callus induction
Four steps can be divided by reverting back to diploid rice technical process using Tetraploid Rice anther callus induction:
(a) materials and pre-treatment；(b) anther evoked callus；(c) it differentiates seedling and obtains liploid plant；(d) it takes root and moves
It plants.
A. pulvinus materials and pre-treatment: is chosen from experimental plot away from the tetraploid spike of rice for 4-8cm, after being wrapped with wet gauze
It is placed in polybag and seals inside 4 DEG C of refrigerator cold treatment 7 days；It is intended to reduce the browning journey of incubation middle and later periods anther wall
Degree, while improving healing rate；
B. anther evoked callus: by the young fringe Jing Guo cold treatment, with 70% ethanol postincubation 30s on superclean bench
10min is handled with 0.1% mercuric chloride solution again afterwards, then the top light green of picking, the milky grain husk flower (mid-late uninucleate stage) in lower part,
Remove glume with sharp tweezers and colored be seeded in induced medium of the grain husk with anther is cultivated into callus；
The fluid nutrient medium for being inoculated with grain husk flower is placed on 30 DEG C, dark culture three days in the shaking table of 100r/min, after turn down culture
To 26 DEG C, the constant continuation dark culture of revolving speed, there is flaxen little particle and are cured temperature in about 30 days or so visible culture basal parts
Wound, after become larger.
The solid medium for being inoculated with grain husk flower is also handled as same in liquid medium, first in 30 DEG C of incubator
Dark culture three days, after be placed in 26 DEG C of culturing room and cultivate, have callus on about 30 days or so the anther of visible cracking.
C. it differentiates seedling and obtains liploid plant: being transferred to differential medium progress when callus grows to a certain size
Optical culture, temperature are 27 DEG C；Antherderived callus differentiation and seedling emergence speed is slower, and the time of different material differentiation and seedling emergences is different.
D. it takes root and transplants: being transferred to root media after differentiation and seedling emergence, after root system is grown well, open sealing, be added clear
Water practices seedling one week or so in greenhouse, then takes out and the culture medium of root is rinsed well with clear water, transplanting to field；If (
Strong seedling culture base culture reincarnation root again for a period of time can be first transferred to if seedling is weaker).
Two, mature embryonal induction Autotetraploidy Rice is utilized:
Five steps: (a) Seeds preprocess can be divided into using the test process of mature embryonal induction Autotetraploidy Rice；
(b) seed embryo evoked callus；(c) colchicine doubles to handle；(d) it differentiates seedling and obtains tetraploid plant；(e) it takes root
And transplanting.
A. Seeds preprocess: by mature rice seed scissors remove 3/4ths or more endosperm fractions, stay
The part of embryo is had down；In super-clean bench, material is poured into triangular flask, finishes writing number, first with 75% alcohol surface sterilization
40s or so is added 0.1% mercuric chloride and impregnates 15min or so, between wanting during immersion then with sterilized pure water wash clean
Every rock, after washed 3-4 times with pure water；
B. seed embryo evoked callus: the material after disinfection is connected in induced medium, about 8 every bottle, seals mouth
It is put between 26 DEG C of tissue culture and carries out dark culture, grow to soybean grain size to callus and just carry out colchicine and double to handle；
C. colchicine doubles to handle: callus access being doubled to rock in shaking table in liquid 48 hours, shaking table setting
It is 27 DEG C, 110r/min, callus wash clean and accesses the dark training of continuation in recovery media with sterile water after doubling
Depending on the case where feeding, the time of recovery is about 7-15 days, specifically sees callus；
D. differentiate seedling and obtain tetraploid plant: callus is transferred to differential medium and carries out optical culture, temperature after restoring normal
Degree is 27 DEG C, about has callus greening once week after transdifferentiation, with that can long emergence slowly, different materials differentiates
The time of seedling is different；
E. it takes root and transplants: being transferred to root media after differentiation and seedling emergence, after root system is grown well, open sealing, be added clear
Water practices seedling one week or so in greenhouse, then takes out and the culture medium of root is rinsed well with clear water, transplanting to field (if
Strong seedling culture base culture reincarnation root again for a period of time can be first transferred to if seedling is weaker).
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., is all included in the scope of protection of the present invention.
Claims (4)
1. a kind of to the conventional method stablized Tetraploid Rice and carry out character improvement, which comprises the following steps:
(1) diploid rice is reverted back to using Tetraploid Rice anther callus induction；
(2) the diploid rice maturation embryonal induction Autotetraploidy Rice cultivated in recycle step (1)；
The Induction Process of the step (1) is as follows:
A. materials and pre-treatment: pulvinus is chosen from experimental plot away from the tetraploid spike of rice for 4-8cm, is placed on after being wrapped with wet gauze
It is sealed inside 4 DEG C of refrigerator cold treatment 7 days in polybag；
B. anther evoked callus: by the young fringe Jing Guo cold treatment, on superclean bench with after 70% ethanol postincubation 30s again
10min is handled with 0.1% mercuric chloride solution, then the top light green of picking, the milky grain husk flower in lower part, remove glume with sharp tweezers
Grain husk flower with anther is seeded in induced medium and cultivates callus；
C. it differentiates seedling and obtains liploid plant: being transferred to differential medium when callus grows to a certain size and carry out light training
It supports, temperature is 27 DEG C；
D. it takes root and transplants: being transferred to root media after differentiation and seedling emergence, after root system is grown well, open sealing, clear water is added,
Practice seedling one week or so in greenhouse, then takes out and the culture medium of root is rinsed well with clear water, transplanting to field；
The Induction Process of the step (2) is as follows:
A. Seeds preprocess: by mature rice seed scissors remove 3/4ths or more endosperm fractions, leave band
There is the part of embryo；In super-clean bench, material is poured into triangular flask, finishes writing number, it is first left with 75% alcohol surface sterilization 40s
The right side is added 0.1% mercuric chloride and impregnates 15min or so, what is be spaced during immersion shakes then with sterilized pure water wash clean
Shake, after washed 3-4 times with pure water；
B. seed embryo evoked callus: the material after disinfection is connected in induced medium, about 8 every bottle, mouth is sealed and is put into
Dark culture is carried out between 26 DEG C of tissue culture, is grown to soybean grain size to callus and is just carried out colchicine and doubles to handle；
C. colchicine doubles to handle: callus access being doubled to rock in shaking table in liquid 48 hours, shaking table is set as 27
DEG C, 110r/min callus wash clean and is accessed with sterile water after doubling and is continued dark culture in recovery media, extensive
The multiple time is about 7-15 days；
D. differentiate seedling and obtain tetraploid plant: callus is transferred to differential medium and carries out optical culture after restoring normal, and temperature is
27 DEG C, about there is callus greening once week after transdifferentiation, slowly can grow emergence with that；
E. it takes root and transplants: being transferred to root media after differentiation and seedling emergence, after root system is grown well, open sealing, clear water is added,
Practice seedling one week or so in greenhouse, then takes out and the culture medium of root is rinsed well with clear water, transplanting to field.
2. according to claim 1 a kind of to the conventional method stablized Tetraploid Rice and carry out character improvement, feature exists
In: the induced medium of inoculation grain husk flower includes fluid nutrient medium and solid medium in the step (1).
3. according to claim 2 a kind of to the conventional method stablized Tetraploid Rice and carry out character improvement, feature exists
Be placed on 30 DEG C in: the fluid nutrient medium, dark culture three days in the shaking table of 100r/min, after turn down cultivation temperature to 26 DEG C, turns
The constant continuation dark culture of speed, about 30 days or so the flaxen little particle callus of visible culture basal part appearance, after become larger.
4. according to claim 2 a kind of to the conventional method stablized Tetraploid Rice and carry out character improvement, feature exists
In first dark culture three days in 30 DEG C of incubator of: the film solid media, after be placed in 26 DEG C of culturing room and cultivate, about 30 days
There is callus on the anther of the visible cracking in left and right.
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| CN201910548258.8A CN110226516A (en) | 2019-06-24 | 2019-06-24 | A kind of pair of conventional method stablized Tetraploid Rice and carry out character improvement |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114190268A (en) * | 2021-10-21 | 2022-03-18 | 湖北大学 | Ploidy cyclic breeding method for polyploid rice |
| CN115349444A (en) * | 2022-08-29 | 2022-11-18 | 湖北大学 | Method for breeding diploid rice by using polyploid rice as variation carrier and application thereof |
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| CN1341350A (en) * | 2001-09-30 | 2002-03-27 | 湖北大学 | Method for high-efficiency breeding rice polyploid by combination of tissue culture and chemical induction |
| CN104542299A (en) * | 2015-01-26 | 2015-04-29 | 云南省农业科学院生物技术与种质资源研究所 | Method for improving culture efficiency of anther of filial generation of Oryza rufipogon |
| CN107333652A (en) * | 2017-07-07 | 2017-11-10 | 湖北大学 | A kind of method that diploid rice strain is created using polyploid Anther Culture |
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Patent Citations (3)
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| CN1341350A (en) * | 2001-09-30 | 2002-03-27 | 湖北大学 | Method for high-efficiency breeding rice polyploid by combination of tissue culture and chemical induction |
| CN104542299A (en) * | 2015-01-26 | 2015-04-29 | 云南省农业科学院生物技术与种质资源研究所 | Method for improving culture efficiency of anther of filial generation of Oryza rufipogon |
| CN107333652A (en) * | 2017-07-07 | 2017-11-10 | 湖北大学 | A kind of method that diploid rice strain is created using polyploid Anther Culture |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114190268A (en) * | 2021-10-21 | 2022-03-18 | 湖北大学 | Ploidy cyclic breeding method for polyploid rice |
| CN115349444A (en) * | 2022-08-29 | 2022-11-18 | 湖北大学 | Method for breeding diploid rice by using polyploid rice as variation carrier and application thereof |
| CN115349444B (en) * | 2022-08-29 | 2024-02-13 | 湖北大学 | Method for breeding diploid rice by using polyploid rice as mutation vector and application thereof |
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