CN103340150A - Method for mutagenizing barbadosnut seeds by ethylmethane sulfonate - Google Patents
Method for mutagenizing barbadosnut seeds by ethylmethane sulfonate Download PDFInfo
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Abstract
The invention discloses a method for mutagenizing barbadosnut seeds by ethylmethane sulfonate. The method disclosed by the invention comprises the following steps of: first, performing pre-bud forcing treatment at 30 DEG C on the barbadosnut seeds, selecting the seeds which are broken and exposed with unexpanded radicles after 3-7 days and soaking the seeds in a 0.9% EMS (Ethylmethane Sulfonate) liquor and soaking for 7 hours; forcing buds of the treated seeds at 28 DEG C and culturing for 3-4 days; and sowing mutagenized survival seeds in a soft peat soil substrate with radicles facing downward, and earthing by 1cm to breed. According to the method, the defects in the prior art are overcome, the mutagenizing concentration and time of EMS are reduced and the mutagenizing efficiency of the barbadosnut seeds by ethylmethane sulfonate is improved so as to facilitate selection by mutation of barbadosnut and promote industrialized development of the barbadosnut.
Description
Technical field
The present invention relates to Phytochemistry mutation breeding technical field, particularly the method for a kind of ethylmethane sulfonate (EMS) mutagenic treatment jatropha curcas seed.
Background technology
Energy-source plant Jatropha curcas (JatrophacurcasL.) claims the little seeds of a tung oil tree, cream paulownia, little tung oil tree etc. again, is Euphorbiaceae leprosy Pterostyrax defoliation small arbor or shrub, and is drought-enduring anti-thin and weak, mainly is distributed in the torrid zone and subtropical zone.Jatropha curcas is one of higher plant of seed oil content, is the desirable feedstock of internationally recognized preparation biodiesel, and has higher comprehensive utilization value.Jatropha curcas is given priority to object because its advantage with " do not strive ground, do not strive grain with the people with grain " is listed in biodiesel.Although the curcas biological diesel oil industry has presented fast-developing situation, realize that the curcas biological diesel oil industrialized development also needs the long cycle.This mainly is that wherein topmost uncertainty shows as the per unit area yield potentiality of Jatropha curcas because as a new industry, the Jatropha curcas development still is faced with numerous uncertainties.Because the Jatropha curcas germ plasm resource of various places plantation all is the wild or seminatural germ plasm resource from unmodified mostly, produce the Jatropha curcas improved seeds that lack high yield in large tracts of land, cause its seed production low and unstable, can't satisfy the demand of the extensive development of biodiesel industry.
Owing to adopt the Jatropha curcas hereditary basis comparatively narrow, but be difficult to obtain the high yielding variety that large tracts of land is promoted by resource screening and conventional breeding.Therefore, utilize the mutagenesis method to be conducive to improve the mutation rate of Jatropha curcas, enrich the hereditary basis of Jatropha curcas, for the acquisition of high yield Jatropha curcas mutant and the seed selection of Jatropha curcas high yielding variety (strain) from now on lays the foundation.Chinese patent application CN201010500008.6 discloses " method of the Jatropha curcas M_2 seed that a kind of High-efficient Production EMS handles ", it is characterized in that pre-treatment step, collects jatropha curcas seed, and seed is carried out pre-treatment; The mutagenic treatment step, utilize the EMS solution of debita spissitudo that jatropha curcas seed is carried out the mutagenesis processing suitably for a long time, in the mutagenesis processing procedure, also stir simultaneously, to guarantee the EMS solution concentration evenly and all jatropha curcas seeds can fully contact with EMS solution; Grow seedlings, transplant step, will grow seedlings and transplant in good time through the Jatropha curcas M_1 seed of mutagenic treatment, to cultivate Jatropha curcas M_1 plant; The pollination self step after the Jatropha curcas M_1 plant of cultivating enters flowering stage, is carried out pollination self to it, namely obtains Jatropha curcas M_2 seed after the fruit maturation.
Jatropha curcas is oil plants, and seed oil content is higher, and the factor that influences seed germination rate is more, and seed soaks by the optimization process time that proposes after the EMS mutagenic treatment and concentration shortage accuracy.Carry out the EMS processing but remove kind of shell again with the kind skin, cause the workload of handling early stage excessive.Therefore correlation technique and immature also has further perfect space in actual production.
Summary of the invention
At the deficiency that prior art exists, the object of the present invention is to provide the method for a kind of ethylmethane sulfonate (EMS) mutagenic treatment jatropha curcas seed, this method is conducive to obtain improved seeds, improves output.
Purpose of the present invention is achieved through the following technical solutions:
A kind of ethylmethane sulfonate mutation of the present invention is handled the method for jatropha curcas seed, may further comprise the steps:
(1) the pre-vernalization of seed: choose full jatropha curcas seed, clear water soaks;
(2) seeds mutagenesis is handled: select the seed that step (1) obtains, be soaked in the ethylmethane sulfonate solution;
(3) mutagenesis presprouting of seeds: the seed that step (2) is obtained is put into clear water and is soaked, and obtains germination seed;
(4) grow seedlings: the germination seed kind that step (3) is obtained is grown seedlings in the plantation dish;
Seed in the described step (2) is the seed that broken shell shows money or valuables one carries unintentionally and radicle does not stretch out through preliminary vernalization.
Preferably, in the step (1), described full jatropha curcas seed is the full jatropha curcas seed of this season, and described clear water soaks for seed is placed distilled water, soaks after 24 hours under the room temperature, picks up to drain to cultivate vernalization 3-7 days under back 30 ℃.
Preferably, between above-mentioned 3-7 days culture period, after seed being got express developed with distilled water in 2 days, drain, carry out 30 ℃ again and cultivate vernalization down.
Preferably, in the step (2), described ethylmethane sulfonate solution for be 0.1% with the volume mass percent concentration, the pH value is that 7.0 phosphate buffer is prepared, the volume mass percent concentration of ethylmethane sulfonate is 0.9%.
Preferably, in the described step (2), the time of described immersion is 7 hours.
Preferably, in the described step (3), it is to clean 3-4 time with distilled water that seed is put into the clear water immersion, is soaked in distilled water again 10 minutes, pulls out to drain, and cultivates in 28 ℃ and carries out vernalization in 3-4 days.
Preferably, in the described step (4), with the germination seed kind in the plantation dish for the planting seed through surviving after the mutagenic treatment in soft peat soil matrix, radicle down, earthing 1cm waters, earthing does not expose to there being seed again.
Compared with prior art, advantage of the present invention is: the present invention selects the seed that broken shell shows money or valuables one carries unintentionally and radicle does not stretch out through preliminary vernalization for use, avoid the bigger influence of oilseed self germination rate variation, the conditional parameters such as concentration of Best Times, temperature, solution in the ethylmethane sulfonate mutation process have been determined, time and the concentration of ethylmethane sulfonate mutation jatropha curcas seed have been reduced, improved the efficiency of inducing mutation of ethylmethane sulfonate, be conducive to improve Jatropha curcas mutation breeding, promote the industrialized development of Jatropha curcas.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited thereto.
The improvement method of EMS mutagenic treatment jatropha curcas seed of the present invention carries out in south China, Tianhe District, Guangzhou City, Guangdong Province innovation center.
Embodiment 1 a kind of ethylmethane sulfonate mutation of the present invention is handled first preferred embodiment of the method for jatropha curcas seed
Present embodiment is the preferred embodiment that a kind of ethylmethane sulfonate mutation of the present invention is handled the method for jatropha curcas seed.Present embodiment adopts the improvement method of ethylmethane sulfonate mutation jatropha curcas seed, comprises following concrete steps:
(1) the pre-vernalization of seed: choose the full jatropha curcas seed of this season, seed is placed distilled water, soak after 24 hours under the room temperature, pick up to drain and cultivate vernalization 7 days under back 30 ℃, in another embodiment, cultivate vernalization 5 days; During this time, after seed being got express developed with distilled water in 2 days, drain, carry out 30 ℃ again and cultivate vernalization down;
(2) seeds mutagenesis is handled: select the seed that broken shell shows money or valuables one carries unintentionally and radicle does not stretch out of the preliminary vernalization of warp that step (1) obtains, be soaked in the ethylmethane sulfonate solution; Described ethylmethane sulfonate solution is 0.9% ethylmethane sulfonate solution for the volume mass percent concentration with the phosphate buffer preparation; The volume mass percentage of phosphate buffer is that 0.1%, pH value is 7.0, and the time of immersion is 7 hours;
(3) mutagenesis presprouting of seeds: the seed that step (2) is obtained is put into clear water and is soaked for cleaning 3 times with distilled water, is soaked in distilled water again 10 minutes, pulls out to drain, and cultivates in 28 ℃ and carries out vernalization in 3 days, obtains germination seed;
(4) grow seedlings: with the planting seed of step (3) through surviving after the mutagenic treatment in soft peat soil matrix, radicle down, earthing 1cm waters, earthing does not expose to there being seed again.
Embodiment 2 a kind of ethylmethane sulfonate mutations of the present invention are handled second preferred embodiment of the method for jatropha curcas seed
A kind of ethylmethane sulfonate mutation of the present invention is handled second preferred embodiment of the method for jatropha curcas seed, may further comprise the steps:
(1) the pre-vernalization of seed: choose full jatropha curcas seed, clear water soaks;
(2) seeds mutagenesis is handled: select the seed that broken shell shows money or valuables one carries unintentionally and radicle does not stretch out of the preliminary vernalization of warp in the step (1), be soaked in the ethylmethane sulfonate solution;
(3) mutagenesis presprouting of seeds: the seed that step (2) is obtained is put into clear water and is soaked, and obtains germination seed;
(4) grow seedlings: the germination seed kind that step (3) is obtained is grown seedlings in the plantation dish.
Embodiment 3 a kind of ethylmethane sulfonate mutations of the present invention are handled the 3rd preferred embodiment of the method for jatropha curcas seed
A kind of ethylmethane sulfonate mutation of the present invention is handled a preferred embodiment of the method for jatropha curcas seed, may further comprise the steps:
(1) the pre-vernalization of seed: choose the full jatropha curcas seed of this season, seed is placed distilled water, soak after 24 hours under the room temperature, pick up to drain and cultivate vernalization 7 days under back 30 ℃; Between above-mentioned 7 days culture period, after seed being got express developed with distilled water in 2 days, drain, carry out 30 ℃ again and cultivate vernalization down.
(2) seeds mutagenesis is handled: select the seed that broken shell shows money or valuables one carries unintentionally and radicle does not stretch out of the preliminary vernalization of warp that step (1) obtains, be soaked in the ethylmethane sulfonate solution; Described ethylmethane sulfonate solution for be 0.1% with the volume mass percent concentration, the pH value is that 7.0 phosphate buffer is prepared, the volume mass percent concentration of described ethylmethane sulfonate is 0.9%, the time of immersion is 7 hours;
(3) mutagenesis presprouting of seeds: the seed that step (2) is obtained is put into clear water and is soaked for cleaning 4 times with distilled water, is soaked in distilled water again 10 minutes, pulls out to drain, and cultivates in 28 ℃ and carries out vernalization in 4 days, obtains germination seed;
(4) grow seedlings: with the planting seed of step (3) through surviving after the mutagenic treatment in soft peat soil matrix, radicle down, earthing 1cm waters, earthing does not expose to there being seed again.
A preferred embodiment of embodiment 4 conventional mutagenic treatment methods
Conventional mutagenic treatment method is carried out as follows:
(1) the pre-vernalization of seed: choose full jatropha curcas seed, clear water soaks;
(2) seeds mutagenesis is handled: with the seed in the step (1), be soaked in the ethylmethane sulfonate solution;
(3) mutagenesis presprouting of seeds: the seed that step (2) is obtained is put into clear water and is soaked, and obtains germination seed;
(4) grow seedlings: the germination seed kind that step (3) is obtained is grown seedlings in the plantation dish.
The comparative experiments of 5 ethylmethane sulfonate different disposal times of embodiment
Be set, relatively percentage of seedgermination the different processing time of ethylmethane sulfonate.The concrete operations step is as follows:
(1) the pre-vernalization of seed: choose full jatropha curcas seed, clear water soaks;
(2) seeds mutagenesis is handled: select in the step (1) through the seed that broken shell shows money or valuables one carries unintentionally and radicle does not stretch out of preliminary vernalization, be soaked in 1.0% the ethylmethane sulfonate solution, soak time was made as 2,4,6,8,10,12 hours;
(3) mutagenesis presprouting of seeds: the seed that step (2) is obtained is put into clear water and is soaked for cleaning 3-4 time with distilled water, is soaked in distilled water again 10 minutes, pulls out to drain, and cultivates in 28 ℃ and carries out vernalization in 4 days, obtains germination seed;
(4) half lethal time is determined: investigation seed number, the seed number that causes death and percentage of seedgermination, utilize statistical software to determine half lethal time.
Table 1 is the percentage of seedgermination of ethylmethane sulfonate different disposal time.Table 2 is the medial lethal dose analysis of ethylmethane sulfonate different disposal time.The result of the test of table 2 shows that the soak time estimated value is can reach the seed semilethal after 6.988 hours, determines that therefore the soak time of ethylmethane sulfonate is 7 hours.
Table 1
| Time (h) | Germination rate (%) |
| 2 | 82.76 |
| 4 | 83.33 |
| 6 | 66.67 |
| 8 | 41.67 |
| 10 | 25.00 |
| 12 | 3.13 |
Table 2
The comparative experiments that the ethylmethane sulfonate of embodiment 6 variable concentrations is handled
The ethylmethane sulfonate of variable concentrations is handled with a collection of seed, relatively seed germination rate.The concrete operations step is as follows:
(1) the pre-vernalization of seed: choose full jatropha curcas seed, clear water soaks;
(2) seeds mutagenesis is handled: the seed that broken shell shows money or valuables one carries unintentionally and radicle does not stretch out of selecting the preliminary vernalization of warp in the step (1), be soaked in the ethylmethane sulfonate solution, described ethylmethane sulfonate solution is the ethylmethane sulfonate solution that contains the phosphate buffer of certain volume mass percent concentration, in the present embodiment, this concentration is made as 0,0.5,1.0,1.2,1.5,2.0%, and soak time is 8 hours;
(3) mutagenesis presprouting of seeds: the seed that step (2) is obtained is put into clear water and is soaked for cleaning 3-4 time with distilled water, is soaked in distilled water again 10 minutes, pulls out to drain, and cultivates in 28 ℃ and carries out vernalization in 4 days, obtains germination seed;
(4) half lethal concentration is determined: investigation seed number, the seed number that causes death and percentage of seedgermination, utilize statistical software to determine half lethal concentration.
Table 3 is the percentage of seedgermination that the ethylmethane sulfonate of variable concentrations is handled seed.Table 4 is the medial lethal dose analysis of variable concentrations ethylmethane sulfonate.Experimental result shows, the concentration for the treatment of estimated value is can reach the seed semilethal after 0.9%, determines that therefore the concentration for the treatment of of ethylmethane sulfonate is 0.9%.
Table 3
| Concentration (%) | Germination rate (%) |
| 0.0 | 100.00 |
| 0.5 | 66.67 |
| 1.0 | 46.67 |
| 1.2 | 41.67 |
| 1.5 | 6.67 |
| 2.0 | 0.00 |
Table 4
Survive the comparative experiments of plant aberration rate behind embodiment 7 embodiment 1,2,3,4 the seed seedling-raising
Survive the aberration rate of plant behind comparing embodiment 1,2,3,4 the seed seedling-raising, comprise that a plurality of proterties such as plant height, leaf, male and female flowering, fecundity investigate variation.
Table 5 is the aberration rate comparative result.Result of the test shows that the aberration rate that survives plant of the seed that method of the present invention obtains than conventional method is more stable, and than the aberration rate height of conventional method.
Table 5
| Embodiment | Aberration rate (%) |
| Embodiment 1 | 19.64％ |
| Embodiment 2 | 18.92％ |
| Embodiment 3 | 19.78％ |
| Embodiment 4 | 15％ |
Claims (7)
1. an ethylmethane sulfonate mutation is handled the method for jatropha curcas seed, may further comprise the steps:
(1) the pre-vernalization of seed: choose full jatropha curcas seed, clear water soaks;
(2) seeds mutagenesis is handled: select the seed that step (1) obtains, be soaked in the ethylmethane sulfonate solution;
(3) mutagenesis presprouting of seeds: the seed that step (2) is obtained is put into clear water and is soaked, and obtains germination seed;
(4) grow seedlings: the germination seed kind that step (3) is obtained is grown seedlings in the plantation dish;
It is characterized in that,
Seed in the described step (2) is the seed that broken shell shows money or valuables one carries unintentionally and radicle does not stretch out through preliminary vernalization.
2. ethylmethane sulfonate mutation according to claim 1 is handled the method for jatropha curcas seed, it is characterized in that, in the described step (1), described full jatropha curcas seed is the full jatropha curcas seed of this season, described clear water soaks for seed is placed distilled water, soak after 24 hours under the room temperature, pick up to drain and cultivate vernalization 3-7 days under back 30 ℃.
3. ethylmethane sulfonate mutation according to claim 2 is handled the method for jatropha curcas seed, it is characterized in that, described cultivations be during 3-7 days, drains after seed being got express developed with distilled water in 2 days, carries out 30 ℃ of cultivation vernalization down again.
4. ethylmethane sulfonate mutation according to claim 1 is handled the method for jatropha curcas seed, it is characterized in that, in the described step (2), described ethylmethane sulfonate solution for be 0.1% with the volume mass percent concentration, the pH value is that 7.0 phosphate buffer is prepared, the volume mass percent concentration of described ethylmethane sulfonate is 0.9%.
5. ethylmethane sulfonate mutation according to claim 1 is handled the method for jatropha curcas seed, it is characterized in that in the described step (2), the time of described immersion is 7 hours.
6. ethylmethane sulfonate mutation according to claim 1 is handled the method for jatropha curcas seed, it is characterized in that, in the described step (3), seed is put into clear water to be soaked for cleaning 3-4 time with distilled water, be soaked in distilled water again 10 minutes, pull out and drain, cultivate in 28 ℃ and carried out vernalization in 3-4 days.
7. ethylmethane sulfonate mutation according to claim 1 is handled the method for jatropha curcas seed, it is characterized in that, in the described step (4), with the germination seed kind in the plantation dish for the planting seed through surviving after the mutagenic treatment in soft peat soil matrix, radicle down, earthing 1cm waters, and earthing does not expose to there being seed again.
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Cited By (7)
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| CN104542265A (en) * | 2013-10-25 | 2015-04-29 | 北京林业大学 | Mutation breeding method for cunninghamia lanceolata |
| CN105010131A (en) * | 2015-07-27 | 2015-11-04 | 杭州市农业科学研究院 | Operation method for salvia splendens EMS(Ethyl methane sulfonate) mutation breeding |
| CN105210862A (en) * | 2015-09-18 | 2016-01-06 | 云南省烟草农业科学研究院 | A kind of plant seed method of mutagenesis and device |
| CN105532406A (en) * | 2016-01-13 | 2016-05-04 | 四川农业大学 | White-pulp loquat new strain preparing method based on red-pulp loquat variety |
| CN105875404A (en) * | 2016-04-13 | 2016-08-24 | 广东省农业科学院作物研究所 | Method for mutagenizing sugarcane through ethylmethylsulfone |
| CN106068757A (en) * | 2016-06-14 | 2016-11-09 | 泰安市泰山林业科学研究院 | A kind of method of mutagenesis of wingceltis coloured silk leaf kind |
| CN111685036A (en) * | 2020-06-07 | 2020-09-22 | 广东省农业科学院作物研究所 | Method for mutagenizing andrographis paniculata by ethyl methanesulfonate |
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| CN104542265A (en) * | 2013-10-25 | 2015-04-29 | 北京林业大学 | Mutation breeding method for cunninghamia lanceolata |
| CN105010131A (en) * | 2015-07-27 | 2015-11-04 | 杭州市农业科学研究院 | Operation method for salvia splendens EMS(Ethyl methane sulfonate) mutation breeding |
| CN105210862A (en) * | 2015-09-18 | 2016-01-06 | 云南省烟草农业科学研究院 | A kind of plant seed method of mutagenesis and device |
| CN105210862B (en) * | 2015-09-18 | 2017-09-15 | 云南省烟草农业科学研究院 | A kind of vegetable seeds method of mutagenesis and device |
| CN105532406A (en) * | 2016-01-13 | 2016-05-04 | 四川农业大学 | White-pulp loquat new strain preparing method based on red-pulp loquat variety |
| CN105875404A (en) * | 2016-04-13 | 2016-08-24 | 广东省农业科学院作物研究所 | Method for mutagenizing sugarcane through ethylmethylsulfone |
| CN105875404B (en) * | 2016-04-13 | 2018-05-29 | 广东省农业科学院作物研究所 | A kind of method of ethylmethane sulfonate mutation processing sugarcane |
| CN106068757A (en) * | 2016-06-14 | 2016-11-09 | 泰安市泰山林业科学研究院 | A kind of method of mutagenesis of wingceltis coloured silk leaf kind |
| CN106068757B (en) * | 2016-06-14 | 2018-10-09 | 泰安市泰山林业科学研究院 | A kind of method of mutagenesis of wingceltis coloured silk leaf kind |
| CN111685036A (en) * | 2020-06-07 | 2020-09-22 | 广东省农业科学院作物研究所 | Method for mutagenizing andrographis paniculata by ethyl methanesulfonate |
| CN111685036B (en) * | 2020-06-07 | 2021-07-23 | 广东省农业科学院作物研究所 | Method for mutagenizing andrographis paniculata by ethyl methanesulfonate |
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