CN105918116B - A kind of plant haploid method for doubling - Google Patents
A kind of plant haploid method for doubling Download PDFInfo
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- CN105918116B CN105918116B CN201610327715.7A CN201610327715A CN105918116B CN 105918116 B CN105918116 B CN 105918116B CN 201610327715 A CN201610327715 A CN 201610327715A CN 105918116 B CN105918116 B CN 105918116B
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- young shoot
- radicle
- haploid
- monoploid
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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
- 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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Abstract
The present invention provides a kind of plant haploid method for doubling, including by treated plant haploid young shoot with mass percent for 0.02% 0.05% colchicine be equipped with 2% dimethyl sulfoxide (DMSO) mixed liquid dipping monoploid young shoot radicle part, processing time is 18 36h, and temperature is 14 18 DEG C.Doubling monoploids method provided by the invention, solve and the low technical barrier of rate is doubled in haploid breeding, and it is simple to operate, it is higher to human and environment security, it will effectively promote the scale process of haploid breeding.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of plant haploid method for doubling.
Background technology
With the increase and expanding economy of population, the demand of corn rises steadily.Cultivated Land Area Decrease and environment
Deterioration makes people increasingly urgent to high yield, high-quality, strong stress resistance corn variety demand.And obtained at present with conventional breeding methods
Substantial amounts of manpower and time need to be expended by obtaining the homozygous inbred lines of high-combining ability.Conventional breeding technique can not increasingly meet
The breeding demand of people.In recent years, the biotechnology such as haploid breeding technology, genetic engineering breeding, molecular mark
The development of means improves breeding efficiency, opens the new way of corn breeding.
Monoploid technology is applied on corn breeding and had the following advantages that:Quickly it is sheerly；It can simplify in monoploid
Interaction of genes, remove expressed superdominance effect, retain favourable additivity and additivity epistatic effect；It can also eliminate harmful, lethal and half
Lethal recessive gene.But there is also shortcoming for haploid breeding technology, that is, it is low to double rate in Haploid Breeding of Maize,
And operation is often more complicated, can not realize the scale of Haploid Breeding of Maize.
The content of the invention
It is an object of the invention to solve the defects of above-mentioned prior art is present, there is provided one kind can improve corn monoploid
The method that rate is doubled in breeding.
A kind of plant haploid method for doubling, including be with mass percent by treated plant haploid young shoot
0.02%-0.05% colchicine is equipped with the radicle part of the mixed liquid dipping monoploid young shoot of 2% dimethyl sulfoxide (DMSO), place
The reason time is 18-36h, and temperature is 14-18 DEG C.
Further, method as described above, handling the method for plant haploid includes
1) 26-30 DEG C of moisturizing vernalization in fine sand will be placed in after single times of seed sterilization of corn；
2) young shoot is taken out when radicle length 1-1.5cm, is placed in moisturizing light culture 8-12h under certain temperature；
3) young shoot is taken out, first cuts off radicle tip 1-2mm, then along one 1-2mm osculum of radicle rip cutting.
Further, method as described above, certain temperature described in step 2) are 4 DEG C.
Further, method as described above, after the young shoot that colchicine is handled, including：Young shoot taking-up flowing water
40-60min is rinsed, is transplanted into culture dish, intelligent greenhouse culture is carried out, is transplanted when seedling is grown to 3 one core of leaf to crop field.
Further, method as described above, the intelligent greenhouse culture include：Cultivation temperature is 26 DEG C, optical culture 8h
Or light culture 16 hours.
Further, method as described above, the plant haploid are vegetables, the monoploid of flowers.
Further, method as described above, the plant haploid are the monoploid of corn.
Beneficial effect：
Doubling monoploids method provided by the invention suitable for corn, the method, which is solved in Haploid Breeding of Maize, to be added
The low technical barrier of multiplying power, and it is simple to operate, it is higher to human and environment security, it will effectively promote Haploid Breeding of Maize
Scale process.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, below technical scheme in the present invention carry out it is clear
Chu, it is fully described by, it is clear that described embodiment is part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
Embodiment 1：
A kind of corn doubling monoploids method is present embodiments provided, is comprised the following steps：
1) 26-30 DEG C of moisturizing vernalization in fine sand will be placed in after single times of seed sterilization of corn；
2) young shoot is taken out when radicle length 1-1.5cm, is placed in 4 DEG C of moisturizing light culture 8-12h；
Its mitosis in 1-1.5cm of Corn Root Tip Cells length is in most active period, less than 1cm or is more than
1.5cm root can also be used, but effect is taken second place.Therefore the present embodiment selection radicle length is 1-1.5cm.
The main function of the step is to reduce Root-tip Cells of Zea mays split speed, allows most cells to be in mitosis
Phase, it is easy to that more diploid cells can be obtained during colchicine processing below, raising doubles rate.
4 DEG C belong to critical-temperature to maize radicel growth, meristematic zone mitosis.Less than 4 degree, the tip of a root produces Serious Cold
, there is serious aging, jaundice, death in evil；Higher than 4 DEG C, Meristernatic zone cell division can be still normally carried out, and be extremely difficult to majority
Cell is in the state in same division period.Microscopic findings show, after 4 DEG C of processing 8-12 hours, in same split coil method
The cell proportion of (mitosis metaphase) is higher than 4 DEG C in 25%-38%, temperature, and the cell proportion in mitosis metaphase is most
More 11%, therefore, the condition of the present embodiment selection processing is 4 DEG C of moisturizing light culture 8-12h.
3) young shoot is taken out, first cuts off radicle tip 1-2mm, then along one 1-2mm osculum of radicle rip cutting；
4) the above-mentioned monoploid young shoot handled well is put into 1.5ml centrifuge tubes, is 0.02%- with mass percent
0.05% colchicine is equipped with the radicle part of the mixed liquid dipping monoploid young shoot of 2% dimethyl sulfoxide (DMSO), processing time
For 18-36h, temperature is 14-18 DEG C；
1.5ml centrifuge tubes size can just be put into a corn seed, and its bottom is cone, and germination can be allowed by removing bottom
Corn seed root stretch out centrifuge tube, be easy to medicament after centrifuge tube is fixed only to soak root without influenceing seed.
What the present invention considered first is how corn doubling monoploids rate to be improved under the processing of low concentration colchicine.
Less than under forefathers' concentration for the treatment of (0.06%), by extend the immersion treatment time, reduce soak when temperature, on the one hand reduce autumn waters -- limid eyes
Harm of the celestial element to human body, monoploid seedling and environment, on the other hand allows the rate of doubling to be significantly increased with Natural double ratio.This is one
Individual organic whole, reduction process concentration, long time treatment, relatively low treatment temperature are indispensable.
Colchicine concentration：Forefathers study most colchicine concentrations (mass percent) in 0.06%-
0.08%, to monoploid seed (or seedling, root, stem apex) injury, larger to the potential threat of human and environment.Colchicine concentration is low
When 0.02%, to maize radicel not damaged, but double rate and at most also there was only 12%, with Natural double effect without significant difference；
When colchicine is more than 0.05%, the influence to radicle is concentrated mainly on late growing stage, knee, browning, growth largely occurs
Situations such as slow, dead seedling, lopsided seedling, weak seedling ratio is caused to rise.
The immersion treatment time：Here time be autumn waters -- limid eyes first plain concentration under 0.02%-0.05% concentration.Less than 18
Hour, doubling etticiency is not notable with Natural double difference；Higher than 36 hours, Maize Roots are influenceed with notable, reduction transplanting
Survival rate.
Soaking temperature：Temperature is less than 14 degree, and root-tip cell division activity slowly, doubles rate with Natural double without significance difference
It is different；Temperature between 14-18 degree, Root-tip Cells of Zea mays division is stable, and transplanting survival rate is most within the time of processing 18-36 hours
Height, young plant abnormal rate are minimum；Temperature is higher than 18 degree, and root-tip cell division speed, it is not notable that processing time is short doubles effect,
If processing time is grown, lopsided seedling is easily produced after rear transplanting.
5) the young shoot taking-up flowing water handled through colchicine is rinsed into 40-60min, be transplanted into special nutritional cup (with
The material of the compositions such as high-quality biological organic matter and mineral matter is cultivation medium, is handled by early stages such as decomposed pesticide-germicides, addition
The auxiliary materials such as vermiculite), intelligent greenhouse culture (16h light cultures, 8h illumination cultivations) is placed in, pays attention to keeping nutrient discs humidity, treats
Seedling is transplanted to crop field when growing to 3 one core of leaf.
The present invention cannot be only used for the cultivation of monoploid seedling, be additionally operable to gardening, vegetables, flowers field.
Embodiment 2：
1) corn monoploid germination：It will be placed in distilled water and soak 10 hours after monoploid seed disinfection；Will immersion
Seed afterwards is equably inserted in the germination disk for filling sand of humidity 75% or so (more than sand table thickness 10cm), spreads wet hair
Towel, light culture 2 days in 26 DEG C of incubator are placed on, and kept the skin wet in time.
Radicle top is cut off：When corn haploid root long to 1.5cm or so, corn monoploid young shoot radicle top is cut off
1-2mm is held, then along radicle direction rip cutting 1-2mm.
2) colchicine solution is prepared：The colchicine solution of 3 kinds of various concentrations (mass percent) is prepared respectively：
The dimethyl sulfoxide (DMSO) of 0.02% colchicine+2%；The dimethyl sulfoxide (DMSO) of 0.04% colchicine+2%；0.049% colchicine+2%
Dimethyl sulfoxide (DMSO)；The dimethyl sulfoxide (DMSO) of 0.05% colchicine+2%；
3) young shoot soaks：The young shoot radicle of step 1) processing, which is lowered into, adds various concentrations autumn waters -- limid eyes described in 300ul steps 2
In the 1.5ml centrifuge tubes of celestial plain solution, the radicle part of young shoot is set to soak in the solution, and not contact colchicine molten for seed
Liquid.Whole operation process is carried out at 10-24 DEG C, and soak time is in 12-36h.
4) young shoot is taken out from centrifuge tube, puts germination box into, and rinsed immediately with clear water, then by discarded colchicum
Plain solution is poured into fixed waste liquid barrel and handled.Clear water washing time 40-60min.
5) young shoot is transplanted in the nutrition cup equipped with Nutrition Soil is entered, is placed in intelligent greenhouse and cultivates.Cultivation temperature is
26 DEG C, optical culture 8h, light culture 16 hours.Pay attention to keeping Nutrition Soil humidity.When treating that seedling is grown to 2-3 leaves, crop field is transplanted into.
Field management is horizontal to be slightly above common crop field.
Embodiment 3：
This method is current most common method --- Germinating Seeds facture
Colchicine concentration：0.06%th, 0.08%, 0.1%
Monoploid germination：By monoploid seed disinfection after soaking 10h in distilled water；Seed is equably placed in
It is covered with the germination disk of wet towel, is placed on half-light culture 24h or so in 26 DEG C of control environment, is taken out after radicle cut.
Processing：The seed embryo of (radicle cut) of showing money or valuables one carries unintentionally is placed in germination disk downwards, and it is dense to add appropriate above-mentioned difference
The colchicine solution of degree, it is defined by just flooding seed embryo face.Processing time be respectively 4h, 8 hours, 12 hours.Processing temperature
Spend for normal temperature (22 degree or so).
Seed after processing rinses 40min or so with flowing water, is sowed in nutritive cube, treats that 3 leaf periods moved into crop field.In management
It is slightly above common horizontal.
Control group experiment 2：(Natural double method)
It will be directly sowed at after monoploid presprouting of seeds in nutritive cube, treat that 3 leaf periods moved into crop field.It is slightly above common in management
It is horizontal.
As a result such as following table：
Monoploid seedling abnormal rate (often handle and counted with 50 seedlings, without dead strain) after the different colchicine concentration processing of table 1
(with 50 plants of statistics, male flower cracking is calculated as dissipating haplobiont loose powder rate after the different colchicine concentration processing of table 2
Powder)
3 first groups of control abnormal rates of table and loose powder rate
| Abnormal rate | Loose powder rate | |||||
| Colchicine concentration | 4 hours | 8 hours | 12 hours | 4 hours | 8 hours | 12 hours |
| 0.06% | 24% | 38% | 66% | 16% | 10% | 6% |
| 0.08% | 58% | 72% | 92% | 8% | 6% | 4% |
| 0.10% | 88% | 100% | 100% | 0% | 0% | 0% |
Found according to table 1- tables 3, corn doubling monoploids technology provided by the present invention can significantly reduce colchicine pair
The murder by poisoning of Western corn, terateger rate are average in 15% or so (control average 50%)；Significantly increase that corn is haploid to be doubled
Rate, tassel loose powder rate reach as high as 34%, far above control (Natural double, average in 5-10%), and reduce colchicine
Concentration half.The method, which solves, doubles the low technical barrier of rate in Haploid Breeding of Maize, and simple to operate, to people and
Environmental safety is higher, will effectively promote the scale process of Haploid Breeding of Maize.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
The present invention is described in detail with reference to the foregoing embodiments, it will be understood by those within the art that：It still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic；
And these modification or replace, do not make appropriate technical solution essence depart from various embodiments of the present invention technical scheme spirit and
Scope.
Claims (7)
1. a kind of plant haploid method for doubling, it is characterised in that including the plant haploid young shoot quality hundred that will be treated
Divide the radicle of the mixed liquid dipping monoploid young shoot of the dimethyl sulfoxide (DMSO) than being equipped with 2% for 0.02%-0.05% colchicine
Part, processing time 18-36h, temperature are 14-18 DEG C.
2. according to the method for claim 1, it is characterised in that handling the method for plant haploid includes
1) 26-30 DEG C of moisturizing vernalization in fine sand will be placed in after single times of seed sterilization of corn；
2) young shoot is taken out when radicle length 1-1.5cm, is placed in moisturizing light culture 8-12h under certain temperature；
3) young shoot is taken out, first cuts off radicle tip 1-2mm, then along one 1-2mm osculum of radicle rip cutting.
3. according to the method for claim 2, it is characterised in that certain temperature described in step 2) is 4 DEG C.
4. according to the method for claim 1, it is characterised in that after the young shoot that colchicine is handled, including：Young shoot takes
Go out and rinse 40-60min with flowing water, be transplanted into culture dish, carry out intelligent greenhouse culture, transplanted when seedling is grown to 3 one core of leaf
To crop field.
5. according to the method for claim 4, it is characterised in that the intelligent greenhouse culture includes：Cultivation temperature is 26 DEG C,
Optical culture 8h, light culture 16 hours are alternately.
6. according to the method for claim 1, it is characterised in that the plant haploid is vegetables, the monoploid of flowers.
7. according to the method for claim 1, it is characterised in that the plant haploid is the monoploid of corn.
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| CN201610327715.7A CN105918116B (en) | 2016-05-17 | 2016-05-17 | A kind of plant haploid method for doubling |
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| CN201610327715.7A CN105918116B (en) | 2016-05-17 | 2016-05-17 | A kind of plant haploid method for doubling |
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