CN110199877A - A method of the stable homozygous coix lacryma-jobi resource of phenotype is obtained by microspores culture - Google Patents
A method of the stable homozygous coix lacryma-jobi resource of phenotype is obtained by microspores culture Download PDFInfo
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- CN110199877A CN110199877A CN201910598934.2A CN201910598934A CN110199877A CN 110199877 A CN110199877 A CN 110199877A CN 201910598934 A CN201910598934 A CN 201910598934A CN 110199877 A CN110199877 A CN 110199877A
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- culture
- jobi
- resource
- coix lacryma
- phenotype
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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
- A01H4/001—Culture apparatus for tissue culture
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
Abstract
The invention discloses a kind of methods for obtaining the stable homozygous coix lacryma-jobi resource of phenotype by microspores culture, comprising the following steps: (1) selection of coix lacryma-jobi small ear and pretreatment；(3) microspore callus induces；(4) callus proliferation culture；(5) processing and embryo callus Multiplying culture are doubled；(6) seedling differentiation again；(7) acclimatization and transplants；(8) Ploidy Identification is preferentially carried out, applicable resource is continued to employ.Pass through the breeding method of science, accurate condition of culture, utilize microspores culture, the stable homozygous coix lacryma-jobi resource of systematic, high efficiency acquisition phenotype, the whole flow process used time is shorter, and obtained coix lacryma-jobi resource character is stable, consistent, with good application value, the expanding production in Job's tears region can be substantially pushed, increases household income, has a extensive future.
Description
Technical field
The invention belongs to technical field of plant cultivation more particularly to a kind of stable pure of phenotype is obtained by microspores culture
The method for closing coix lacryma-jobi resource.
Background technique
Homozygous genotype is that coix lacryma-jobi phenotype generation-inter- is stable, the consistent basis of group, since the application scale of coix lacryma-jobi is compared with rice and kernel
The gramineous crops grass family such as corn is small, and resource purification work is done less, much more current ground Job's tears kind domestication is insufficient,
Not system.Coix lacryma-jobi production is main in the remote regions, and common resource purifying is mainly based upon the sieve that form and statistical discrepancy carry out
Choosing takes this to obtain long period used in the application resource that phenotype economic characters are stable, Group Consistency is high, and efficiency is lower.This
Invention can get the coix lacryma-jobi resource of homozygous genotype by microspore culture.
Summary of the invention
The object of the invention is to remedy the disadvantages of known techniques, provides a kind of steady by microspores culture acquisition phenotype
The method of fixed homozygous coix lacryma-jobi resource.
In order to achieve the above purpose, the present invention the following technical schemes are provided:
A method of the stable homozygous coix lacryma-jobi resource of phenotype is obtained by microspores culture, comprising the following steps:
(1) selection of coix lacryma-jobi small ear and pretreatment: coix lacryma-jobi male flower fringe extracts 2cm out, and when the long 3-4mm of anther, acquisition grain husk spends male branch to obstruct, and uses
The sterilizing of 75% alcohol, 12 DEG C of gnotobasis are stood for 24 hours；
(2) pollen separates: sterile working strips anther, stands 10 minutes in 6% sucrose solution, is placed on magnetic agitation instrument, low speed
Rotation, releases the microspore in anther, is then sterile filtered and collects, is adjusted to suitable density, can be used to cultivate；
(3) microspore callus induces: appropriate microspore solution inoculum is drawn in culture medium culture, condition is ventilating cover tissue culture bottle,
PH5.8, it is 21-23 DEG C of temperature, illumination 13h, 1200lx, former culture medium subculture 2-3 times rear out；
(4) callus proliferation culture: after subculture 2-3 times, access culture medium culture, PH5.8,21-23 DEG C of temperature, illumination 13h,
1500lx, again squamous subculture 2-3 times；
(5) processing and embryo callus Multiplying culture are doubled: all callus of acquisition being divided into two, portion is labeled as
A homozygote, through N6+ 0.005% colchicine culture medium culture 10d, transfer N6Culture medium, PH5.8,21-23 DEG C of temperature, illumination
13h, 3000lx culture obtain the embryo callus that growth is fine and close, is easy to induce differentiation；It is standby that another is labeled as B monoploid
Part, shoot proliferation；Two parts of embryo callus carry out step 6, step 7 processing simultaneously, and processing A, B at different levels are horizontal consistent；
(6) seedling differentiation again: the embryo callus of the growth green point of compact zone is transferred to culture medium, PH5.8, and 21-23 DEG C of temperature, light
Bud induction differentiation culture is carried out according to 13h, 3000lx；2-3cm is grown to adventitious bud, is transferred in root media, PH5.8, temperature
21-23 DEG C, illumination 13h, 3000lx culture of rootage；
(7) acclimatization and transplants: seedling grows to 6-7cm, selects root system to induce preferable seedling corkage hardening 5-7d, transplants after carbendazol root dipping
In matrix, 1/8 N6Culture solution sprays, and by day dehumidification hardening, moves back into greenhouse within 2 weeks, then moves to crop field after surviving and normally plant
Training management, sowing；
(8) Ploidy Identification is preferentially carried out, continues to employ and is applicable in resource: according to the control of the form of two plant of A, B and field shape and warp
Ji trait phenotypes compare, and preferentially carry out Ploidy Identification, continue to employ the high-quality homozygous coix lacryma-jobi resource obtained from A group.
Preferably, microspore callus induces used medium in the step 3 are as follows: N6+ 2.0mg/L 2,4-D+
500mg/L proline+300mg/L caseinhydrolysate+1.0mg/L AgNO3+ 60g/L sucrose.
Preferably, callus proliferation culture used medium in the step 4 are as follows: N6+ 1.0mg/L 2,4-D+
500mg/L proline+300mg/L caseinhydrolysate+0.5mg/L AgNO3+ 30g/L sucrose.
Preferably, differentiation culture used medium in the step 6 are as follows: N6+ 0.5mg/L IAA+2.0mg/L 6-BA
+ 500mg/L lactoalbumin hydrolysate (LH)+30g/L sucrose.
Preferably, culture of rootage used medium in the step 6 are as follows: N6+ 1.0mg/L IAA+500mg/L hydrolysis cream
Albumen (LH)+30g/L sucrose.
Preferably, the matrix in the step 7 is made of the 1:1:2 mixing by volume of wood sawdust, vermiculite, river sand.
The invention has the advantages that
For the present invention by scientific breeding method, accurate condition of culture is systematic, high efficiency using microspores culture
The stable homozygous coix lacryma-jobi resource of phenotype is obtained, the whole flow process used time is shorter, and obtained coix lacryma-jobi resource character is stable, consistent, has
Good application value can substantially push the expanding production in Job's tears region, increase household income, have a extensive future.
Specific embodiment
Below in conjunction with specific example, technical scheme is described further:
A method of the stable homozygous coix lacryma-jobi resource of phenotype is obtained by microspores culture, comprising the following steps:
1, the selection of coix lacryma-jobi small ear and pretreatment
Coix lacryma-jobi male flower fringe extracts 2cm out, and when the long 3-4mm of anther, acquisition grain husk spends male branch to obstruct, and is sterilized with 75% alcohol, 12 DEG C of gnotobasis
It stands for 24 hours.
2, pollen separates
Sterile working strips anther, stands 10 minutes in 6% sucrose solution, is placed on magnetic agitation instrument, low speed rotation makes anther
In microspore release, then be sterile filtered collect, be adjusted to suitable density, can be used to cultivate.
3, microspore callus induces
Appropriate microspore solution inoculum is drawn in culture medium (N6+ 2.0mg/L 2,4-D+500mg/L proline+300mg/L water
Solve casein+1.0mg/L AgNO3+ 60g/L sucrose) culture, ventilating cover tissue culture bottle, PH5.8, temperature (22 ± 1) DEG C, illumination
13h, 1200lx, it is former culture medium subculture 2~3 times rear out.
4, callus proliferation culture
After subculture 2~3 times, culture medium (N is accessed6+ 1.0mg/L 2,4-D+500mg/L proline+300mg/L hydrolyze junket egg
White+0.5mg/L AgNO3+ 30g/L sucrose) culture, PH5.8, temperature (22 ± 1) DEG C, illumination 13h, 1500lx, subculture again
Culture 2~3 times.
5, processing and embryo callus Multiplying culture are doubled
All callus of acquisition are divided into two, portion is labeled as A homozygote, through N6+ 0.005% colchicine culture medium
10d is cultivated, transfer N6Culture medium, PH5.8, temperature (22 ± 1) DEG C, illumination 13h, 3000lx culture obtain growth densification, are easy to
Induce the embryo callus of differentiation；Another is labeled as the backup of B monoploid, shoot proliferation.Two parts of embryo callus are simultaneously
Carrying out following 6, seedling differentiation, 7 acclimatization and transplants steps, processing A, B at different levels are horizontal consistent again.
6, seedling differentiation again
The embryo callus of the growth green point of compact zone is transferred to culture medium (N6+ 0.5mg/L IAA+2.0mg/L 6-BA+
500mg/L lactoalbumin hydrolysate (LH)+30g/L sucrose), PH5.8, temperature (22 ± 1) DEG C, illumination 13h, 3000lx carry out bud and lure
Lead differentiation culture；
2~3cm is grown to adventitious bud, is transferred in root media (N6+ 1.0mg/L IAA+500mg/L lactoalbumin hydrolysate
(LH)+30g/L sucrose), PH5.8, temperature (22 ± 1) DEG C, illumination 13h, 3000lx culture of rootage.
7, acclimatization and transplants
Seedling grows to 6,7cm, selects root system to induce preferable seedling corkage 5~7d of hardening, transplanting is in matrix (sawmilling after carbendazol root dipping
Bits, vermiculite, river sand volume ratio 1:1:2), 1/8 N6Culture solution sprays, and by day dehumidification hardening, moves back into greenhouse within 2 weeks, after surviving
Then move to the normal cultivation management in crop field, sowing.
8, Ploidy Identification is preferentially carried out, applicable resource is continued to employ
Compared according to the control of the form of two plant of A, B and field shape and economic characters phenotype, preferentially carries out Ploidy Identification (stream
Formula cell instrument), continue to employ the high-quality homozygous coix lacryma-jobi resource obtained from A group.
Claims (6)
1. a kind of method for obtaining the stable homozygous coix lacryma-jobi resource of phenotype by microspores culture, which is characterized in that including following
Step:
(1) selection of coix lacryma-jobi small ear and pretreatment: coix lacryma-jobi male flower fringe extracts 2cm out, and when the long 3-4mm of anther, acquisition grain husk spends male branch to obstruct, and uses
The sterilizing of 75% alcohol, 12 DEG C of gnotobasis are stood for 24 hours；
(2) pollen separates: sterile working strips anther, stands 10 minutes in 6% sucrose solution, is placed on magnetic agitation instrument, low speed
Rotation, releases the microspore in anther, is then sterile filtered and collects, is adjusted to suitable density, can be used to cultivate；
(3) microspore callus induces: appropriate microspore solution inoculum is drawn in culture medium culture, condition is ventilating cover tissue culture bottle,
PH5.8, it is 21-23 DEG C of temperature, illumination 13h, 1200lx, former culture medium subculture 2-3 times rear out；
(4) callus proliferation culture: after subculture 2-3 times, access culture medium culture, PH5.8,21-23 DEG C of temperature, illumination 13h,
1500lx, again squamous subculture 2-3 times；
(5) processing and embryo callus Multiplying culture are doubled: all callus of acquisition being divided into two, portion is labeled as
A homozygote, through N6+ 0.005% colchicine culture medium culture 10d, transfer N6Culture medium, PH5.8,21-23 DEG C of temperature, illumination
13h, 3000lx culture obtain the embryo callus that growth is fine and close, is easy to induce differentiation；It is standby that another is labeled as B monoploid
Part, shoot proliferation；Two parts of embryo callus carry out step 6, step 7 processing simultaneously, and processing A, B at different levels are horizontal consistent；
(6) seedling differentiation again: the embryo callus of the growth green point of compact zone is transferred to culture medium, PH5.8, and 21-23 DEG C of temperature, light
Bud induction differentiation culture is carried out according to 13h, 3000lx；2-3cm is grown to adventitious bud, is transferred in root media, PH5.8, temperature
21-23 DEG C, illumination 13h, 3000lx culture of rootage；
(7) acclimatization and transplants: seedling grows to 6-7cm, selects root system to induce preferable seedling corkage hardening 5-7d, transplants after carbendazol root dipping
In matrix, 1/8 N6Culture solution sprays, and by day dehumidification hardening, moves back into greenhouse within 2 weeks, then moves to crop field after surviving and normally plant
Training management, sowing；
(8) Ploidy Identification is preferentially carried out, continues to employ and is applicable in resource: according to the control of the form of two plant of A, B and field shape and warp
Ji trait phenotypes compare, and preferentially carry out Ploidy Identification, continue to employ the high-quality homozygous coix lacryma-jobi resource obtained from A group.
2. the method according to claim 1 for obtaining the stable homozygous coix lacryma-jobi resource of phenotype by microspores culture, special
Sign is that microspore callus induces used medium in the step 3 are as follows: N6+ 2.0mg/L 2,4-D+500mg/L proline
+ 300mg/L caseinhydrolysate+1.0mg/L AgNO3+ 60g/L sucrose.
3. the method according to claim 1 for obtaining the stable homozygous coix lacryma-jobi resource of phenotype by microspores culture, special
Sign is, callus proliferation culture used medium in the step 4 are as follows: N6+ 1.0mg/L 2,4-D+500mg/L dried meat ammonia
Acid+300mg/L caseinhydrolysate+0.5mg/L AgNO3+ 30g/L sucrose.
4. the method according to claim 1 for obtaining the stable homozygous coix lacryma-jobi resource of phenotype by microspores culture, special
Sign is that used medium is cultivated in differentiation in the step 6 are as follows: N6+ 0.5mg/L IAA+2.0mg/L 6-BA+500mg/
L lactoalbumin hydrolysate (LH)+30g/L sucrose.
5. the method according to claim 1 for obtaining the stable homozygous coix lacryma-jobi resource of phenotype by microspores culture, special
Sign is, culture of rootage used medium in the step 6 are as follows: N6+ 1.0mg/L IAA+500mg/L lactoalbumin hydrolysate (LH)
+ 30g/L sucrose.
6. the method according to claim 1 for obtaining the stable homozygous coix lacryma-jobi resource of phenotype by microspores culture, special
Sign is that the matrix in the step 7 is made of the 1:1:2 mixing by volume of wood sawdust, vermiculite, river sand.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109169280A (en) * | 2018-09-20 | 2019-01-11 | 四川农业大学 | The preparation method and used medium of coix lacryma-jobi regeneration plant |
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Application publication date: 20190906 |