CN115669535A - Method for efficiently inducing persimmon polyploidy by treating seeds with colchicine - Google Patents
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Abstract
The invention provides a method for efficiently inducing persimmon polyploidy by treating seeds with colchicine, which comprises the following steps: soaking persimmon seeds, then disinfecting the soaked seeds, then carrying out hormone treatment on the disinfected seeds, and finally carrying out sand storage and germination acceleration to obtain germinated seeds; treating the germinated seeds with colchicine, then sowing, watering thoroughly after sowing, then building an arched shed for heat and moisture preservation, regularly observing the germination condition of the seeds, manually peeling off the seeds if the seed coats are difficult to fall off after the seeds break, expanding the cotyledons of the seeds, observing the temperature and humidity in the arched shed, replenishing water and cooling in time, and ensuring the normal growth of the seedlings. The method overcomes the defect that the direct treatment and induction effect of the traditional colchicine on ungerminated seeds is extremely poor, adopts the colchicine to treat the seeds in the optimal germination state, and obviously improves the induction efficiency of the persimmon polyploidy.
Description
Technical Field
The invention belongs to the technical field of breeding, and particularly relates to a method for efficiently inducing persimmon polyploidy by treating seeds with colchicine.
Background
Persimmon (diospyrokikunb.) is a plant of the family Ebenaceae (Ebenaceae), genus Ebenus (Diospyros), and is an important economic forest tree species, called "iron stem crops". The persimmon fruit is rich in nutrition and unique in flavor, can be eaten fresh or made into dried persimmon to be eaten, and the persimmon calyx, the persimmon root, the persimmon leaf and the like can be used as medicines for clearing heat and removing toxicity, protecting liver and reducing blood pressure, so that the persimmon fruit has important application value and wide development prospect in the fields of food, health care, medicine, chemical industry and the like.
The vast majority of persimmon cultivars are hexaploid (2n =6x = 90), very few cultivars are nonaploid (2n =9x = 135), nonaploid persimmons are intraspecific triploid, the comprehensive traits are excellent, and for example, the optimal Japanese sweet persimmon variety 'autumn king' and the main-cultivated astringent persimmon variety 'Pinkaryon-Wu' are nonaploid cultivars, and the application value and the development potential are extremely high. The persimmon varieties in China are various, but are hexaploid.
At present, ploidy breeding of persimmons is mostly focused on the aspect of creating the asploid by using 2n gametes, but the induction rate of the 2n gametes is not high, and embryo abortion is caused by unbalanced endosperm after pollination, so that the induction of the asploid is difficult. The induction of the dodecaploid is also relevant research, the isolated leaves are mainly treated by colchicine to obtain a dodecaploid regeneration plant, but the dodecaploid tissue culture seedling has the problems of difficult rooting, low transplanting survival rate and the like due to high tannin content. The colchicine is used for treating the seeds to induce the dodecaploid, the seeds can directly grow into seedlings after germination, the problems of re-rooting and survival rate of transplanting seedlings do not exist, and the method is one of the methods for efficiently obtaining the persimmon dodecaploid.
At present, no report is found about relevant research on induction of dodecaploid by treating persimmon seeds with colchicine. Therefore, it is important to provide a method for efficiently inducing persimmon polyploidy by treating seeds with colchicine.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for efficiently inducing persimmon polyploidy by treating seeds with colchicine, which overcomes the defect that the direct treatment and induction effect of the traditional colchicine on ungerminated seeds is extremely poor.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for efficiently inducing persimmon polyploidy by treating seeds with colchicine comprises the following steps:
s1, accelerating germination of seeds:
s101, seed soaking: selecting healthy and plump persimmon seeds, soaking the persimmon seeds in warm water for 3 days, changing water for 1 time every day, and airing water after soaking to obtain soaked seeds;
s102, disinfection: soaking the seeds soaked in the S101 solution for 4 hours by using a potassium permanganate solution, then washing the seeds by using clear water, and airing moisture to obtain disinfected seeds;
s103, hormone treatment: soaking the disinfected seeds obtained in the step S102 in a GA3 solution with the concentration of 1g/L for 15 hours, and then washing the seeds with clear water; soaking the mixture in a 6-BA solution with the concentration of 40mg/L for 3 hours, and washing the soaked mixture with clear water; finally, soaking the seeds in a 2,4-D solution with the concentration of 100mg/L for 30min, washing the seeds with clear water, and airing the water to obtain the seeds after hormone treatment;
s104, accelerating germination of sand-stored seeds: after impurities in the sand are screened out, spraying 800 times of diluted Junbiqing solution for wetting and disinfection to obtain disinfected sand; spreading a layer of disinfected coarse sand on the ground of a greenhouse, spreading and flatly spreading the hormone-treated seeds obtained in the step S103 on the disinfected coarse sand, covering a layer of disinfected middle sand, and storing the seeds in sand under the conditions of heat preservation and moisture preservation; in the sand storage process, observing the disinfected coarse sand and the disinfected medium sand at intervals of 1d, after the sand storage is carried out for 10d, checking the germination condition of the hormone-treated seeds for the first time, and then checking the hormone-treated seeds once a day until the hormone-treated seeds germinate, so as to finally obtain the germinated seeds;
s2, colchicine treatment of seeds: soaking the germinated seeds obtained in the step S104 in colchicine for 24 hours in a dark place, and then washing the seeds with clear water to obtain treated seeds;
s3, sowing and seedling raising: mixing soil, a matrix and vermiculite according to a mass ratio of 5; and (3) watering thoroughly after sowing, then building an arched shed for heat preservation and moisture preservation, regularly observing the germination condition of the treated seeds, manually peeling off the seeds if the seed coats are difficult to fall off after the seeds are broken, unfolding the cotyledons of the seeds, observing the temperature and humidity in the arched shed, replenishing water and reducing the temperature in time, and ensuring the normal growth of the seedlings.
Preferably, the temperature of the warm water in S101 is 37 ℃; the mass fraction of the potassium permanganate solution in the S102 is 0.2%.
Preferably, the thickness of the disinfected coarse sand in the S104 is 10cm, and the particle size is 0.5 mm-1 mm; the thickness of the disinfected medium sand is 5cm, and the particle size is 0.25 mm-0.5 mm.
Preferably, the temperature of the heat preservation and moisture preservation in S104 is more than or equal to 25 ℃, and the humidity is 50%.
Preferably, the concentration of colchicine in S2 is 0.1%.
Preferably, the heel length of the treated seed in S3 is 0.1cm to 0.5cm.
Preferably, the temperature in the arched shed in S3 is 25-30 ℃, and the humidity is 60%.
Compared with the prior art, the invention has the following advantages:
1. at present, persimmon polyploid breeding technology mainly focuses on the induction and utilization of 2n gametes and the induction of polyploidy by using colchicine to treat isolated tissues, and no report is found on the aspect of polyploidy creation by using seeds as materials. And when other species utilize colchicine to treat seeds to induce polyploidy, the ungerminated seeds are mostly treated, the seed coats and endosperm tissues of the persimmon seeds are thicker, and the direct treatment inducing effect is extremely poor when the seeds are ungerminated, so that the invention innovates and optimizes the optimum period of colchicine treatment (namely the optimum germination state of the persimmon seeds), the optimum colchicine concentration, the treatment time and other conditions, and the inducing efficiency of the persimmon polyploidy is obviously improved.
2. According to the method, persimmon seeds are subjected to combined treatment of hormone treatment, sand storage seed germination acceleration and colchicine treatment, the induction efficiency of polyploid persimmons can reach 13%, the seedling rate of the treated seeds after germination is high (> 95%), the problems of difficult rooting of tissue culture seedlings, low survival rate of transplanted seedlings and the like do not exist, and the ploidy breeding efficiency of persimmons is remarkably improved.
The technical solution of the present invention is further described in detail by the accompanying drawings and examples.
Drawings
FIG. 1 is a graph showing the ploidy determination by the flow cytometer in example 1.
Detailed Description
Example 1
The method for efficiently inducing persimmon polyploidy by treating seeds with colchicine in the embodiment comprises the following steps:
s1, accelerating germination of seeds:
s101, seed soaking: selecting healthy and plump persimmon seeds, soaking the persimmon seeds in warm water at 37 ℃ for 3 days, naturally cooling the warm water, changing water for 1 time every day, and airing the water after soaking to obtain the soaked seeds;
s102, disinfection: soaking the seed soaked in the S101 in a potassium permanganate solution with the mass fraction of 0.2% for 4 hours, then washing with clear water, and airing to obtain disinfected seed;
s103, hormone treatment: soaking the disinfected seeds obtained in the step S102 in a GA3 solution with the concentration of 1g/L for 15 hours, and then washing with clear water; soaking the mixture in a 6-BA solution with the concentration of 40mg/L for 3 hours, and washing the soaked mixture with clear water; finally, soaking the seeds in a 2,4-D solution with the concentration of 100mg/L for 30min, washing the seeds with clear water, and airing the seeds to obtain hormone-treated seeds;
s104, accelerating germination of sand-stored seeds: after impurities in the sand are screened out, spraying 800 times of diluted Junbiqing solution to the sand for wet disinfection to obtain disinfected sand; spreading a layer of disinfected coarse sand with the thickness of 10cm on the ground of a greenhouse (25 ℃), spreading and spreading the hormone-treated seeds obtained in S103 on the disinfected coarse sand, covering a layer of disinfected medium sand with the thickness of 5cm, then building an arched shed by using plastic cloth for heat preservation and moisture preservation, and preserving the sand under the conditions of heat preservation and moisture preservation, wherein the temperature of the sand preservation is kept at 25 ℃ and the humidity is kept at 50%; in the sand storage process, the sand humidity is observed every 1d, so that the sand can be held by hands to be gathered, and the sand can be scattered to be in an optimal humidity state after being held by hands, and water is timely supplemented; after 10d of sand storage, checking the germination condition of the hormone-treated seeds for the first time, and then checking the hormone-treated seeds once a day until the hormone-treated seeds germinate to finally obtain germinated seeds; the length of the seed heel of the germinated seed is 0.1 cm-0.5 cm; the grain size of the disinfected coarse sand is 0.5mm; the grain diameter of the disinfected medium sand is 0.25mm;
s2, colchicine treatment of seeds: soaking the germinated seeds obtained in the step S104 in 0.1% colchicine for 24h in a dark place, and then washing the seeds with clear water to obtain treated seeds;
s3, sowing and seedling raising: mixing soil, a matrix and vermiculite according to a mass ratio of 5; and (3) after sowing, watering thoroughly, then building an arched shed for heat preservation and moisture preservation, regularly observing the germination condition of the treated seeds, manually peeling off the seeds to expand cotyledons of the seeds if the seed coats are difficult to fall off after the seeds are broken, observing the temperature and humidity in the arched shed, replenishing water and cooling in time, keeping the temperature in the arched shed at 25 ℃ and the humidity at 60%, and ensuring the normal growth of the seedlings.
After the young seedling grows multiple main leaves, adopting new young leaf with 0.5cm 2 Placing in a culture dish, adding 400 μ l of lysis solution (CyStainUVPr 250tests kit, model 5002-02) onto the leaf, cutting the leaf with a blade, transversely cutting, longitudinally cutting, extracting for 3min, adding DAPI dye 160 into the cut leafMu.l, staining for 1min in the dark, filtering the liquid in the culture dish into a sample tube with a 30 μm filter screen, and performing ploidy identification with a CyFlowSpace type flow cytometer, wherein the results are shown in FIG. 1. The peak position corresponds to the genome size, the R2 peak is a hexaploid persimmon, and the R3 peak is a dodecaploid persimmon induced by colchicine treatment in this example.
Comparative example 1
The method for treating the seeds in this comparative example to induce persimmon polyploidy with high efficiency was the same as in example 1, except that the step S104 in example 1 was not performed, and only hormone-treated seeds, i.e., ungerminated seeds, were obtained.
The germinated seeds obtained in S104 of example 1 and the ungerminated seeds of comparative example 1 were subjected to colchicine treatment, setting a number of treatment combinations as shown in Table 1.
TABLE 1 statistics of results of different treatment combinations for induction of dodecaploid
Therefore, the ungerminated seeds are treated, the colchicine use concentration and the treatment time are the same as those of the method, but because the germination of the persimmon seeds is not hundred percent, the persimmon seeds cannot germinate in a positive state in a certain proportion, the seedling formation is low, the seed coats and endosperm tissues of the persimmon seeds are thick, the colchicine cannot be directly applied to embryos in the centers of the seeds by direct treatment, and the polyploid induction rate is extremely low.
In the embodiment 1, the induction efficiency of the polyploid persimmon is 13%, the seedling rate of the seed after germination is 100%, the problems of difficult rooting of tissue culture seedlings, low survival rate of transplanted seedlings and the like do not exist, and the ploidy breeding efficiency of the persimmon is remarkably improved.
Comparative example 2
High-efficiency induction of kaki dolichos in seeds treated in this comparative exampleThe ploidy method was the same as in example 1, except that 3 factors (GA 3, 6-BA and 2,4-D) were set, and 4 levels were set for each factor, in accordance with L16 (4) 3 ) Performing an orthogonal test, respectively treating the disinfected seeds obtained in the step S102, performing colchicine treatment and sowing after sand storage and germination acceleration, and counting the germination rate of each treatment combination; as shown in Table 2, the germination rate of the treated combination 15 is the highest, namely the treated combination is treated for 15h by 1000mg/LGA3, treated for 3h by 40mg/L of 6-BA and treated for 30min by 100mg/L of 2,4-D, the germination effect of the seeds is the best, and the germination rate is 64%.
TABLE 2 statistics of germination rates for seeds treated with different hormone concentrations
Example 2
The method for efficiently inducing persimmon polyploidy by treating seeds with colchicine in the embodiment comprises the following steps:
s1, accelerating germination of seeds:
s101, seed soaking: selecting healthy and plump persimmon seeds, soaking the persimmon seeds in warm water at 37 ℃ for 3 days, naturally cooling the warm water, changing water for 1 time every day, and airing the water after soaking to obtain the seeds after soaking;
s102, disinfection: soaking the seed soaked in the S101 in a potassium permanganate solution with the mass fraction of 0.2% for 4 hours, then washing with clear water, and airing to obtain disinfected seed;
s103, hormone treatment: soaking the disinfected seeds obtained in the step S102 in a GA3 solution with the concentration of 1g/L for 15 hours, and then washing the seeds with clear water; soaking the mixture for 3 hours by using a 6-BA solution with the concentration of 40mg/L, and washing the soaked mixture by using clear water; finally, soaking the seeds in a 2,4-D solution with the concentration of 100mg/L for 30min, washing the seeds with clear water, and airing the water to obtain hormone-treated seeds;
s104, accelerating germination of sand-stored seeds: after impurities in the sand are screened out, spraying 800 times of diluted Junbiqing solution to the sand for wet disinfection to obtain disinfected sand; spreading a layer of disinfected coarse sand with the thickness of 10cm on the ground of a greenhouse (24 ℃), spreading and spreading the hormone-treated seeds obtained in S103 on the disinfected coarse sand, covering a layer of disinfected medium sand with the thickness of 5cm, then building an arched shed by using plastic cloth for heat preservation and moisture preservation, and preserving the sand under the conditions of heat preservation and moisture preservation, wherein the temperature of the sand preservation is kept at 27 ℃ and the humidity is kept at 50%; in the sand storage process, the sand humidity is observed every 1d, so that the sand can be held by hands to be gathered, and the sand can be scattered to be in an optimal humidity state after being held by hands, and water is timely supplemented; after the seeds are stored in the sand for 10 days, the germination condition of the seeds treated by the hormone is checked for the first time, and then the seeds treated by the hormone are checked once a day until the seeds treated by the hormone germinate, and finally the germinated seeds are obtained; the length of the seed heel of the germinated seed is 0.1 cm-0.5 cm; the grain size of the disinfected coarse sand is 1mm; the grain size of the disinfected medium sand is 0.5mm;
s2, treating seeds with colchicine: soaking the germinated seeds obtained in the step S104 in 0.1% colchicine for 24h in a dark place, and then washing the seeds with clear water to obtain treated seeds;
s3, sowing and seedling raising: mixing soil, a matrix and vermiculite according to a mass ratio of 5; and (3) after sowing, watering thoroughly, then building an arched shed for heat and moisture preservation treatment, regularly observing the germination condition of the treated seeds, manually peeling off the seeds if the seed coats are difficult to fall off after the seeds are broken, unfolding the seed leaves, observing the temperature and humidity in the arched shed, replenishing water and cooling in time, keeping the temperature in the arched shed at 30 ℃ and the humidity at 60%, and ensuring the normal growth of the seedlings.
Example 3
The method for efficiently inducing persimmon polyploidy by treating seeds with colchicine in the embodiment comprises the following steps:
s1, accelerating germination of seeds:
s101, seed soaking: selecting healthy and plump persimmon seeds, soaking the persimmon seeds in warm water at 37 ℃ for 3 days, naturally cooling the warm water, changing water for 1 time every day, and airing the water after soaking to obtain the seeds after soaking;
s102, disinfection: soaking the seeds obtained in the step S101 in a potassium permanganate solution with the mass fraction of 0.2% for 4 hours, then washing with clear water, and airing moisture to obtain disinfected seeds;
s103, hormone treatment: soaking the disinfected seeds obtained in the step S102 in a GA3 solution with the concentration of 1g/L for 15 hours, and then washing the seeds with clear water; soaking the mixture for 3 hours by using a 6-BA solution with the concentration of 40mg/L, and washing the soaked mixture by using clear water; finally, soaking the seeds in a 2,4-D solution with the concentration of 100mg/L for 30min, washing the seeds with clear water, and airing the water to obtain hormone-treated seeds;
s104, accelerating germination of sand-stored seeds: after impurities in the sand are screened out, spraying 800 times of diluted Junbiqing solution to the sand for wetting and disinfection to obtain disinfected sand; spreading a layer of disinfected coarse sand with the thickness of 10cm on the ground of a greenhouse (23 ℃), spreading and spreading the hormone-treated seeds obtained in S103 on the disinfected coarse sand, covering a layer of disinfected medium sand with the thickness of 5cm, then building an arched shed by using plastic cloth for heat preservation and moisture preservation, and preserving the sand under the conditions of heat preservation and moisture preservation, wherein the temperature of the sand preservation is kept at 28 ℃ and the humidity is kept at 50%; in the sand storage process, the sand humidity is observed every 1d, so that the sand can be held by hands to be gathered, and the sand can be scattered to be in an optimal humidity state after being held by hands, and water is timely supplemented; after the seeds are stored in sand for 10 days, the germination condition of the seeds treated by the hormone is checked for the first time, and then the seeds treated by the hormone are checked once a day until the seeds treated by the hormone germinate, and finally the germinated seeds are obtained; the length of the heels of the germinated seeds is 0.1 cm-0.5 cm; the grain size of the disinfected coarse sand is 0.7mm; the grain size of the disinfected medium sand is 0.35mm;
s2, treating seeds with colchicine: soaking the germinated seeds obtained in the step S104 in 0.1% colchicine for 24h in a dark place, and then washing the seeds with clear water to obtain treated seeds;
s3, sowing and seedling raising: mixing soil, a matrix and vermiculite according to a mass ratio of 5; and (3) after sowing, watering thoroughly, then building an arched shed for heat preservation and moisture preservation, regularly observing the germination condition of the treated seeds, manually peeling off the seeds to expand cotyledons of the seeds if the seed coats are difficult to fall off after the seeds are broken, observing the temperature and humidity in the arched shed, replenishing water and cooling in time, keeping the temperature in the arched shed at 27 ℃ and the humidity at 60%, and ensuring the normal growth of the seedlings.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
Claims (7)
1. A method for efficiently inducing persimmon polyploidy by treating seeds with colchicine is characterized by comprising the following steps:
s1, accelerating germination of seeds:
s101, seed soaking: selecting healthy and plump persimmon seeds, soaking the persimmon seeds in warm water for 3 days, changing water for 1 time every day, and airing water after soaking to obtain soaked seeds;
s102, disinfection: soaking the seed soaked in the S101 in a potassium permanganate solution for 4 hours, then washing with clear water, and airing to obtain the disinfected seed;
s103, hormone treatment: soaking the disinfected seeds obtained in the step S102 in a GA3 solution with the concentration of 1g/L for 15 hours, and then washing with clear water; soaking the mixture in a 6-BA solution with the concentration of 40mg/L for 3 hours, and washing the soaked mixture with clear water; finally, soaking the seeds in a 2,4-D solution with the concentration of 100mg/L for 30min, washing the seeds with clear water, and airing the water to obtain hormone-treated seeds;
s104, accelerating germination of sand-stored seeds: after impurities in the sand are screened out, spraying 800 times of diluted Junbiqing solution for wetting and disinfection to obtain disinfected sand; spreading a layer of disinfected coarse sand on the ground of the greenhouse, scattering and spreading the hormone-treated seeds obtained in the step S103 on the disinfected coarse sand, covering a layer of disinfected medium sand, and storing the seeds in sand under the conditions of heat preservation and moisture preservation; in the sand storage process, observing the disinfected coarse sand and the disinfected medium sand at intervals of 1d, after the sand storage is carried out for 10d, checking the germination condition of the hormone-treated seeds for the first time, and then checking the hormone-treated seeds once a day until the hormone-treated seeds germinate, so as to finally obtain the germinated seeds;
s2, treating seeds with colchicine: soaking the germinated seeds obtained in the step S104 in colchicine for 24 hours in a dark place, and then washing the seeds with clear water to obtain treated seeds;
s3, sowing and seedling raising: mixing soil, a matrix and vermiculite according to a mass ratio of 5; and (3) watering thoroughly after sowing, then building an arched shed for heat preservation and moisture preservation, regularly observing the germination condition of the treated seeds, manually peeling off the seeds if the seed coats are difficult to fall off after the seeds are broken, unfolding the cotyledons of the seeds, observing the temperature and humidity in the arched shed, replenishing water and reducing the temperature in time, and ensuring the normal growth of the seedlings.
2. The method for efficiently inducing persimmon polyploidy by colchicine-treated seeds as claimed in claim 1, wherein the temperature of the warm water in S101 is 37 ℃; the mass fraction of the potassium permanganate solution in the S102 is 0.2%.
3. The method for efficiently inducing persimmon polyploidy by colchicine-treated seeds as claimed in claim 1, wherein the thickness of the disinfected coarse sand in S104 is 10cm, and the particle size is 0.5mm to 1mm; the thickness of the disinfected medium sand is 5cm, and the particle size is 0.25 mm-0.5 mm.
4. The method for efficiently inducing the persimmon polyploid through colchicine treatment of seeds as claimed in claim 1, wherein the temperature of the heat preservation and moisture retention in S104 is not less than 25 ℃ and the humidity is 50%.
5. The method of claim 1, wherein the concentration of colchicine in S2 is 0.1%.
6. The method for inducing persimmon polyploidy with high efficiency by using colchicine-treated seeds as claimed in claim 1, wherein the heel length of the treated seeds in S3 is 0.1 cm-0.5 cm.
7. The method for efficiently inducing persimmon polyploidy by colchicine-treated seeds as claimed in claim 1, wherein the temperature in the shed in S3 is 25 ℃ to 30 ℃ and the humidity is 60%.
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| CN107980620A (en) * | 2017-12-05 | 2018-05-04 | 沈阳农业大学 | A kind of the compound of plant chromosome doubles agent and method for doubling |
| CN110959527A (en) * | 2018-09-28 | 2020-04-07 | 河北农业大学 | Method for inducing tetraploid by treating radicle of pyrus betulaefolia seed with colchicine |
| CN114586682A (en) * | 2022-03-10 | 2022-06-07 | 中国林业科学研究院亚热带林业研究所 | Method for inducing chromosome doubling of camellia chrysantha pollen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107980620A (en) * | 2017-12-05 | 2018-05-04 | 沈阳农业大学 | A kind of the compound of plant chromosome doubles agent and method for doubling |
| CN110959527A (en) * | 2018-09-28 | 2020-04-07 | 河北农业大学 | Method for inducing tetraploid by treating radicle of pyrus betulaefolia seed with colchicine |
| CN114586682A (en) * | 2022-03-10 | 2022-06-07 | 中国林业科学研究院亚热带林业研究所 | Method for inducing chromosome doubling of camellia chrysantha pollen |
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