CN109924127A - A kind of polyploid breeding method based on negative pressure technique - Google Patents
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Abstract
The invention discloses a kind of polyploid breeding methods based on negative pressure technique, comprising the following steps: to the seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select germinating seedlings；Colchicin, broneomycin, sodium nitrate, potassium dihydrogen phosphate, ethylenediamine tetra-acetic acid and lithium chloride are dissolved in water, silica solution is then added and is stirred, mutagens are made；The above-mentioned germinating seedlings selected are placed in the culture dish for filling mutagens, and culture dish is placed in the negative-pressure cup of negative pressure system, are handled；The seedling handled well is placed in solid medium and is cultivated, is transplanted after growing young leaves.The plant leaf that method provided by the invention obtains is thick, and leaf color is deep, high survival rate, and easy to operate, at low cost.
Description
Technical field:
The present invention relates to agricultural technology fields, are specifically related to a kind of polyploid breeding method based on negative pressure technique.
Background technique:
Breeding is by creating hereditary variation, improvement hereditary capacity, to cultivate the technology of excellent animals and plants new varieties.With
Science of heredity is theoretical basis, and a variety of subject knowledges such as integrated application ecology, physiology, biochemistry, pathology and biometrics.To development
Animal husbandry and planting industry have a very important significance.The target of plant breeding is to obtain high yield, stable yields, high-quality, efficient work
Object.But specific breeding objective will comprehensively consider the status of native breed, breeding basis, natural environment, cropping system, cultivation water
The factors such as flat, economic condition, and constantly adjusted with the development of production.Will also be larger with this area cultivated area or there is representative
For several kinds of property as standard, the direction for clearly requiring holding or raising, improving or overcoming embodies breeding objective.
For crop, main breeding method is main at present are as follows: a mutation breeding: mutation breeding, which refers to, utilizes induced mutations
Method obtain the breeding methods of biological new varieties, main method of mutagenesis is radioinduction, laser, chemical substance mutagenesis, too
The methods of empty Induced variation.This method can effectively improve variation frequency, accelerate breeding process, significantly improve certain crop shapes
Shape, range of variation is wide, but its favorable variation is few, and the direction of mutagenesis and property not can control.Two, crossbreeding refers to benefit
Hybridized with (or not of the same race) bion of the same race that different genes form, phenotype type required for obtaining is educated
Kind method.The controlled shape of crop made from this method, but the time is longer, needs to find Optimality in time in breeding process
Shape.Three, haploid breeding: it is to obtain haplobiont using vitro anther culture technology, then induce its chromosome doubling, from
And the breeding method of pure lines plant required for obtaining.This method mainly utilizes chromosomal variation, and breeding time is short, mainly
It is to pass through method for tissue culture.But party's law technology is more complex, and rain crossbreeding is needed to combine.Four, polyploid breeding, the party
Method mainly utilizes chromosomal variation to obtain, and can cultivate new varieties, and the plant organ cultivated is big, and yield is high, and nutrition is rich
It is rich.But the plant setting percentage that this method is cultivated is low.Five, cell engineering breeding: refer to miscellaneous with the method acquisition of cell fusion
Kind of cell is crossbreeded the method for plant with the method for tissue cultures using the totipotency of cell.This method can overcome remote edge
The incompatibility of hybridization, purposefully cultivates improved seeds, but technology is also more complicated simultaneously, and difficulty is big.Six, genetic engineering
Breeding.
Patent (CN201710317959.1) discloses a kind of breed cucumber method and polyploid based on induction polyploid
The identification method of plant, comprising: the cucumber seeds after sprouting are bred as seedling, the growth with colchicine solution to the seedling
Point carries out induction processing；Plant forms identification, Stomatal appraisal, flow cytometry then are carried out to diploid and variation plant
And cytological Identification.This method can effectively select the cucumber polyploidy novel strain of suitable production application, to make its economy
Benefit is improved.Patent (CN201010241253.X) discloses a kind of kelp breeding side based on induction polyploid gametocyte
Method, comprising the following steps: firstly, handle monoploid kelp gametophyte cell using mitosis Inhibitors, keep it intracellular
Chromosome doubling, so that haploid kelp gametophyte cell is induced the kelp gametophyte cell for polyploid；Recycle sea
Band gametophytic cell hybridization technique, cultivates the laminaria sporophytes of polyploid.The advantage of the invention is that by polyploid breeding,
Kelp gene content can be improved, increased Quantitative Trait Genes can enhance kelp physiological activity, to enable its economic characters
Improve；Chromosome doubling method of the invention not only can be applied to cultivate autopolyploid, but also can be applied to cultivate allopolyploid.
Patent (CN201410848092.9) discloses a kind of centella polyploid and its induced breeding method, comprising the following steps: will
Wild centella is transplanted to culture medium；Prepare derivant solution；The derivant solution is that concentration is 30~50mg/L amine sulphur
Spirit, and it is added with 1~3wt% dimethyl sulfoxide；Absorbent cotton is wrapped in centella to grow thickly leaf growing point, is soaked with derivant solution
Moisten absorbent cotton；It is handled 2~4 days under the conditions of natural lighting；The sample of tetraploid will be detected as through chromosome microscope, using nothing
Sexual reproduction expands numerous preservation.The present invention carries out living body induction to centella, and not only Induction Transformation rate is high, and in two polyploid centella asiatica
The content of medicinal active principle be greatly improved.Patent (CN93110536.6) discloses a kind of educating for polyploid cabbage
Kind method selects mutant strain using colchicine solution mutagenesis diploid Chinese cabbage, continuous to separate for 4 generations, obtains forth generation mutantion line
Two mutantion lines are combined seed collecting by prominent 1, prominent 2, are bred as tetraploid kind " excellent No. 2 of heat ".According to field trial and analysis measurement
As a result, the fast-growing of the excellent No. 2 existing polyploid cabbages of heat, the advantage that nutritional ingredient is high, marketing quality is good, and disease resistance is strong, and it is resistance to
High temperature is the ideal kind of summer plantation.Although the above-mentioned prior art can obtain good kind, the seedling cultivated is anti-
Inverse property is poor, and Induction Transformation rate is also required to further increase.
Summary of the invention:
The technical problem to be solved by the present invention is to the kind resistance cultivated in breeding process in the prior art is poor, lure
It is lower to lead conversion ratio, death rate height, poor quality.In order to solve the above technical problem, the present invention provides one kind to be based on negative pressure technique
Polyploid breeding method, this method first using induction plant seedlings, then under condition of negative pressure, at the same using medicament to children
Seedling carries out immersion treatment, and seedling carries out subsequent cultivation to treated.
In order to preferably solve above-mentioned technical problem, the invention adopts the following technical scheme:
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select germinating seedlings；
(2) colchicin, broneomycin, sodium nitrate, potassium dihydrogen phosphate, ethylenediamine tetra-acetic acid and lithium chloride are dissolved in water,
Then silica solution is added to be stirred, mutagens are made；
(3) the above-mentioned germinating seedlings selected are placed in the culture dish for filling mutagens, and culture dish is placed in negative pressure system
In the negative-pressure cup of system, handled；
(4) seedling handled well is placed in solid medium and is cultivated, transplanted after growing young leaves.
As a preferred embodiment of the above technical solution, in step (2), the dosage of each component is in the mutagens with weight percent
Meter specifically: 0.1-0.4 parts of colchicin, 0.2-0.6 parts of broneomycin, 1-5 parts of sodium nitrate, 1-3 parts of potassium dihydrogen phosphate, second two
0.1-0.3 parts of amine tetraacethyl, 0.02-0.03 parts of lithium chloride, 0.15-0.33 parts of silica solution, 40-60 parts of water.
As a preferred embodiment of the above technical solution, in step (2), the particle size analysis size of the silica solution is 10-20nm.
As a preferred embodiment of the above technical solution, in step (3), pressure when Negative pressure is for -40~-50KPa, processing
Time is 8-15min.
As a preferred embodiment of the above technical solution, in step (3), negative pressure system include pressure pump, negative-pressure cup, vacuum table, into
Air valve, air outlet valve, air filter and culture dish.
As a preferred embodiment of the above technical solution, in step (4), the composition of the solid medium in parts by weight, specifically
Are as follows: MS minimal medium, 3-7mg/L amino acid, 6.5g/L agar, 0.35mg/L vitamin B1,80-100mg/L banana puree.
As a preferred embodiment of the above technical solution, in step (4), the actual conditions cultivated in solid medium are as follows: culture
Temperature is 22 DEG C, and the photoperiod is 15/9h period white/black, luminous intensity 1800-2200lx.
As a preferred embodiment of the above technical solution, it in step (4), also needs to carry out final-period management to the seedling after transplanting.
As a preferred embodiment of the above technical solution, the final-period management includes top dressing, watering, weeding, the prevention and control of plant diseases, pest control.
The invention has the following advantages:
In order to improve the survival rate of seedling, the present invention carries out induction processing using mutagens to seed germination seedling first,
And condition of negative pressure is used in processing, negative pressure can have slight damage to seed germination seedling, increase seed germination Seedlings Cell
For the rapid osmotic of mutagens, there is active influence hence for the growth and development of germinating seedlings, improve to a certain extent
Survival rate after seedling replanting.Size for the technology negative pressure value is key, if negative pressure value is too small, is sent out growth of seedling
Educate influence less, if but negative pressure value is excessive, can germinating seedlings be caused with irreversible damage, thus seriously affect transplanting children
The survival rate of seedling, the present invention limit negative pressure value as -40~-50KPa, and the processing time is 8-15min, substantially increases transplanting seedling
Survival rate.
The present invention also adds a certain amount of Nano silica sol in mutagens, is by unformed SiO2Particle is equal in water
The colloidal solution of even dispersion, element silicon can be efficiently entering the intracellular of germinating seedlings under condition of negative pressure, to improve transplanting
The resistance of seedling afterwards increases the survival rate of transplanting seedling, and the addition of silicon can also make the cell of germinating seedlings cell
Wall thickening, improves the activity of its internal enzyme, to increase disease-resistant, the impact resistance of transplanting seedling.And operation of the present invention is simple,
It is at low cost, it is economic and environment-friendly.
Specific embodiment:
In order to better understand the present invention, below by embodiment, the present invention is further described, and embodiment is served only for solving
The present invention is released, any restriction will not be constituted to the present invention.
By taking crape myrtle as an example, breeding method produced by the present invention is further described.
Embodiment 1
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the crape myrtle seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select the crape myrtle seedling of rudiment；
(2) in parts by weight, by 0.1 part of colchicin, 0.2 part of broneomycin, 1 part of sodium nitrate, 1 part of potassium dihydrogen phosphate,
0.1 part of ethylenediamine tetra-acetic acid and 0.02 part of lithium chloride are dissolved in 40 parts of water, and 0.15 part of silica solution is then added and is stirred, is made and lures
Become agent；
(3) the above-mentioned rudiment crape myrtle seedling selected is placed in the culture dish for filling mutagens, and culture dish is placed in negative
In the negative-pressure cup of pressure system, processing 8min is carried out under the pressure of -40~-50KPa；
(4) the crape myrtle seedling handled well is placed in MS minimal medium, 3mg/L amino acid, 6.5g/L agar, 0.35mg/L
Vitamin B1,80mg/L banana puree culture medium in cultivated, transplanted after growing young leaves, and to the seedling after transplanting
The final-period managements such as top dressing, watering, weeding, the prevention and control of plant diseases, pest control are carried out, after transplanting one month, using 500 plants of plants as research pair
As measuring its survival rate is 97.4%, and tetraploid induction rate is 76.5%.
Embodiment 2
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the crape myrtle seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select the crape myrtle seedling of rudiment；
(2) in parts by weight, by 0.4 part of colchicin, 0.6 part of broneomycin, 5 parts of sodium nitrate, 3 parts of potassium dihydrogen phosphates,
0.3 part of ethylenediamine tetra-acetic acid and 0.03 part of lithium chloride are dissolved in 60 parts of water, and 0.33 part of silica solution is then added and is stirred, is made and lures
Become agent；
(3) the above-mentioned rudiment crape myrtle seedling selected is placed in the culture dish for filling mutagens, and culture dish is placed in negative
In the negative-pressure cup of pressure system, processing 15min is carried out under the pressure of -40~-50KPa；
(4) the crape myrtle seedling handled well is placed in MS minimal medium, 7mg/L amino acid, 6.5g/L agar, 0.35mg/L
Vitamin B1,100mg/L banana puree culture medium in cultivated, transplanted after growing young leaves, and to the children after transplanting
Seedling carries out the final-period managements such as top dressing, watering, weeding, the prevention and control of plant diseases, pest control, after transplanting one month, using 500 plants of plants as research
Object, measuring its survival rate is 96.8%, and tetraploid induction rate is 77.2%.
Embodiment 3
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the crape myrtle seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select the crape myrtle seedling of rudiment；
(2) in parts by weight, by 0.12 part of colchicin, 0.3 part of broneomycin, 2 parts of sodium nitrate, 1.5 parts of biphosphates
Potassium, 0.15 part of ethylenediamine tetra-acetic acid and 0.022 part of lithium chloride are dissolved in 45 parts of water, and 0.17 part of silica solution is then added and is stirred,
Mutagens are made；
(3) the above-mentioned rudiment crape myrtle seedling selected is placed in the culture dish for filling mutagens, and culture dish is placed in negative
In the negative-pressure cup of pressure system, processing 10min is carried out under the pressure of -40~-50KPa；
(4) the crape myrtle seedling handled well is placed in MS minimal medium, 4mg/L amino acid, 6.5g/L agar, 0.35mg/L
Vitamin B1,85mg/L banana puree culture medium in cultivated, transplanted after growing young leaves, and to the seedling after transplanting
The final-period managements such as top dressing, watering, weeding, the prevention and control of plant diseases, pest control are carried out, after transplanting one month, using 500 plants of plants as research pair
As measuring its survival rate is 98.2%, and tetraploid induction rate is 76.8%.
Embodiment 4
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the crape myrtle seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select the crape myrtle seedling of rudiment；
(2) in parts by weight, by 0.3 part of colchicin, 0.4 part of broneomycin, 2.5 parts of sodium nitrate, 2 parts of biphosphates
Potassium, 0.2 part of ethylenediamine tetra-acetic acid and 0.024 part of lithium chloride are dissolved in 50 parts of water, and 0.2 part of silica solution is then added and is stirred, makes
Obtain mutagens；
(3) the above-mentioned rudiment crape myrtle seedling selected is placed in the culture dish for filling mutagens, and culture dish is placed in negative
In the negative-pressure cup of pressure system, processing 11min is carried out under the pressure of -40~-50KPa；
(4) the crape myrtle seedling handled well is placed in MS minimal medium, 5mg/L amino acid, 6.5g/L agar, 0.35mg/L
Vitamin B1,90mg/L banana puree culture medium in cultivated, transplanted after growing young leaves, and to the seedling after transplanting
The final-period managements such as top dressing, watering, weeding, the prevention and control of plant diseases, pest control are carried out, after transplanting one month, using 500 plants of plants as research pair
As measuring its survival rate is 98.6%, and tetraploid induction rate is 77.5%.
Embodiment 5
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the crape myrtle seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select the crape myrtle seedling of rudiment；
(2) in parts by weight, by 0.3 part of colchicin, 0.5 part of broneomycin, 4 parts of sodium nitrate, 2.5 parts of biphosphates
Potassium, 0.25 part of ethylenediamine tetra-acetic acid and 0.026 part of lithium chloride are dissolved in 55 parts of water, and 0.25 part of silica solution is then added and is stirred,
Mutagens are made；
(3) the above-mentioned rudiment crape myrtle seedling selected is placed in the culture dish for filling mutagens, and culture dish is placed in negative
In the negative-pressure cup of pressure system, processing 12min is carried out under the pressure of -40~-50KPa；
(4) the crape myrtle seedling handled well is placed in MS minimal medium, 6mg/L amino acid, 6.5g/L agar, 0.35mg/L
Vitamin B1,90mg/L banana puree culture medium in cultivated, transplanted after growing young leaves, and to the seedling after transplanting
The final-period managements such as top dressing, watering, weeding, the prevention and control of plant diseases, pest control are carried out, after transplanting one month, using 500 plants of plants as research pair
As measuring its survival rate is 97.6%, and tetraploid induction rate is 77.0%.
Embodiment 6
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the crape myrtle seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select the crape myrtle seedling of rudiment；
(2) in parts by weight, by 0.35 part of colchicin, 0.55 part of broneomycin, 4.5 parts of sodium nitrate, 3 parts of biphosphates
Potassium, 0.3 part of ethylenediamine tetra-acetic acid and 0.028 part of lithium chloride are dissolved in 55 parts of water, and 0.3 part of silica solution is then added and is stirred, makes
Obtain mutagens；
(3) the above-mentioned rudiment crape myrtle seedling selected is placed in the culture dish for filling mutagens, and culture dish is placed in negative
In the negative-pressure cup of pressure system, processing 13min is carried out under the pressure of -40~-50KPa；
(4) the crape myrtle seedling handled well is placed in MS minimal medium, 6mg/L amino acid, 6.5g/L agar, 0.35mg/L
Vitamin B1,95mg/L banana puree culture medium in cultivated, transplanted after growing young leaves, and to the seedling after transplanting
The final-period managements such as top dressing, watering, weeding, the prevention and control of plant diseases, pest control are carried out, after transplanting one month, using 500 plants of plants as research pair
As measuring its survival rate is 98.2%, and tetraploid induction rate is 76.9%.
Comparative example 1
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the crape myrtle seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select the crape myrtle seedling of rudiment；
(2) in parts by weight, by 0.35 part of colchicin, 0.55 part of broneomycin, 4.5 parts of sodium nitrate, 3 parts of biphosphates
Potassium, 0.3 part of ethylenediamine tetra-acetic acid and 0.028 part of lithium chloride are dissolved in 55 parts of water, and 0.3 part of silica solution is then added and is stirred, makes
Obtain mutagens；
(3) the above-mentioned rudiment crape myrtle seedling selected is placed in the culture dish for filling mutagens, under normal temperature and pressure
Manage 13min；
(4) the crape myrtle seedling handled well is placed in MS minimal medium, 6mg/L amino acid, 6.5g/L agar, 0.35mg/L
Vitamin B1,95mg/L banana puree culture medium in cultivated, transplanted after growing young leaves, and to the seedling after transplanting
The final-period managements such as top dressing, watering, weeding, the prevention and control of plant diseases, pest control are carried out, after transplanting one month, using 500 plants of plants as research pair
As measuring its survival rate is 83.2%, and tetraploid induction rate is 59.2%.
Comparative example 2
A kind of polyploid breeding method based on negative pressure technique, comprising the following steps:
(1) it to the crape myrtle seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select the crape myrtle seedling of rudiment；
(2) in parts by weight, by 0.35 part of colchicin, 0.55 part of broneomycin, 4.5 parts of sodium nitrate, 3 parts of biphosphates
Potassium, 0.3 part of ethylenediamine tetra-acetic acid and 0.028 part of lithium chloride are dissolved in 55 parts of water, and mutagens are made；
(3) the above-mentioned rudiment crape myrtle seedling selected is placed in the culture dish for filling mutagens, and culture dish is placed in negative
In the negative-pressure cup of pressure system, processing 13min is carried out under the pressure of -40~-50KPa；
(4) the crape myrtle seedling handled well is placed in MS minimal medium, 6mg/L amino acid, 6.5g/L agar, 0.35mg/L
Vitamin B1,95mg/L banana puree culture medium in cultivated, transplanted after growing young leaves, and to the seedling after transplanting
The final-period managements such as top dressing, watering, weeding, the prevention and control of plant diseases, pest control are carried out, after transplanting one month, using 500 plants of plants as research pair
As measuring its survival rate is 88.3%, and tetraploid induction rate is 61.2%.
Induction processing is carried out to germinating seedlings under condition of negative pressure it can be seen from above-described embodiment and comparative example, can be had
Effect improves the survival rate and multiploid induction rate of seedling after transplanting, mainly since the negative pressure of certain condition has explantation tissue
Certain damage improves inductivity to increase the absorption to mutagens, but negative pressure value selection is larger, can be to explant
Tissue causes biggish damage, will affect the survival rate of seedling.The addition of Nano silica sol also improves children to a certain extent
The survival rate of seedling.
Although specific embodiments of the present invention are described, many other forms of the invention and change
Change will be apparent to those skilled in the art.It should be understood that appended claims and the present invention usually cover the present invention very
All these apparent forms and change in real spirit and scope.
Claims (9)
1. a kind of polyploid breeding method based on negative pressure technique, which comprises the following steps:
(1) it to the seed collected at the beginning of 11 months, will sprout by the end of October through sand storage, and select germinating seedlings；
(2) colchicin, broneomycin, sodium nitrate, potassium dihydrogen phosphate, ethylenediamine tetra-acetic acid and lithium chloride are dissolved in water, then
Silica solution is added to be stirred, mutagens are made；
(3) the above-mentioned germinating seedlings selected are placed in the culture dish for filling mutagens, and culture dish is placed in negative pressure system
In negative-pressure cup, handled；
(4) seedling handled well is placed in solid medium and is cultivated, transplanted after growing young leaves.
2. a kind of polyploid breeding method based on negative pressure technique according to claim 1, which is characterized in that step (2)
In, the dosage of each component is by weight percentage in the mutagens specifically: 0.1-0.4 parts of colchicin, broneomycin
0.2-0.6 parts, 1-5 parts of sodium nitrate, 1-3 parts of potassium dihydrogen phosphate, 0.1-0.3 parts of ethylenediamine tetra-acetic acid, lithium chloride 0.02-0.03
Part, 0.15-0.33 parts of silica solution, 40-60 parts of water.
3. a kind of polyploid breeding method based on negative pressure technique according to claim 1, which is characterized in that step (2)
In, as a preferred embodiment of the above technical solution, the particle size analysis size of the silica solution is 10-20nm.
4. as a preferred embodiment of the above technical solution, in step (3), pressure when Negative pressure is for -40~-50KPa, when processing
Between be 8-15min.
5. a kind of polyploid breeding method based on negative pressure technique according to claim 1, which is characterized in that step (3)
In, negative pressure system includes pressure pump, negative-pressure cup, vacuum table, intake valve, air outlet valve, air filter and culture dish.
6. a kind of polyploid breeding method based on negative pressure technique according to claim 1, which is characterized in that step (4)
In, the composition of the solid medium in parts by weight, specifically: MS minimal medium, 3-7mg/L amino acid, 6.5g/L fine jade
Rouge, 0.35mg/L vitamin B1,80-100mg/L banana puree.
7. a kind of polyploid breeding method based on negative pressure technique according to claim 1, which is characterized in that step (4)
In, the actual conditions cultivated in solid medium are as follows: cultivation temperature is 22 DEG C, and the photoperiod is 15/9h period white/black, light intensity
Degree is 1800-2200lx.
8. a kind of polyploid breeding method based on negative pressure technique according to claim 1, which is characterized in that step (4)
In, it also needs to carry out final-period management to the seedling after transplanting.
9. a kind of polyploid breeding method based on negative pressure technique according to claim 8, which is characterized in that the later period
Management includes top dressing, watering, weeding, the prevention and control of plant diseases, pest control.
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| CN109924127B (en) | 2020-12-22 |
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