CN104542265A - Mutation breeding method for cunninghamia lanceolata - Google Patents
Mutation breeding method for cunninghamia lanceolata Download PDFInfo
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Abstract
The invention discloses a mutation breeding method for cunninghamia lanceolata. The method comprises the following steps: especially adopting ripen seeds of a cunninghamia lanceolata seed garden; carrying out low-temperature overwintering treatment, carrying out constant-temperature seed soaking stimulation on a gibberellin solution with specific concentration, immediately putting the gibberellin solution subjected to constant-temperature seed soaking stimulation into clear water, carrying out variable-temperature soaking for 3 days, and carrying out sprout promoting treatment; respectively carrying out colchicine mutation treatment, ethylmethane sulfonate (EMS) mutation treatment and sodium azide (NaN3) mutation treatment; and finally seeding and germinating the cunninghamia lanceolata seeds which are subjected to mutation treatment, so as to obtain a large group of cunninghamia lanceolata mutation seedlings. The cunninghamia lanceolata mutation seedlings obtained by the mutation method have the characteristics of obvious phenotypic characteristic variation, large population growth characteristic variation range and the like; a great number of mutated cunninghamia lanceolata materials can be formed within a short period of time by the method disclosed by the invention; and a foundation is laid for breeding of a new variety of cunninghamia lanceolata.
Description
Technical field
The present invention relates to a kind of method for mutation breeding of plants, particularly improve mutation rate, the mutagenic breeding method of initiative new material, belongs to the mutation breeding technologies field of China fir.
Background technology
China fir (Cunninghamia Lanceolata Lamb.Hook) is the peculiar afforestation commerical tree species of south China, and cultivation history is long, and distributed areas are wide, are the main study subjects of China's woody energy work always.Start provenance test so far from the 1950's, China fir high powder-content artificial sand is got bumper crops.In this Breeding Process, the economic indicators such as plant height, the diameter of a cross-section of a tree trunk 1.3 meters above the ground, wood property are often retained as important goal proterties, and some other such as drought resisting, the resistance proterties such as pest-resistant, barren-resistant are easily out in the cold, cause its genetic background more and more narrow.The change of the serious and weather of water and soil loss in addition, soil alkaline, in the urgent need to the China fir new germ plasm of polymerization fast-growing, wood property, the excellent economic characters such as degeneration-resistant.
Mutation breeding refers to morphs by the hereditary capacity of physics, chemical factor inducing plant, then selects to meet people certain individual plant required or individuality from variation colony, and then cultivates into the breeding method of new germ plasm and even new varieties.It is the GENERALIZATION OF MODERN BREEDING TECHNIQUE grown up after selection and use and crossbreeding.Early stage forest genetics is main contents mainly with routine works such as provenance selection, select superior tree, crossbreeding, seed orchard and progeny tests greatly.Compare conventional breeding methods, mutation breeding can significantly improve the aberration rate being mutagenized material, create abundant variation for us and improve the chance of the excellent new germ plasm of seed selection, for effectively changing the indivedual bad proterties of high quality tree species, the wood property of raising forest and resistance etc. are laid a good foundation.This technology is a kind of approach of quick creation forest genetics resource, gives full play to himself advantage, can shorten the time cycle obtaining mutative material, make up the deficiency of traditional breeding method to a certain extent.In recent years, the radiation breeding research of China in crops, fruit tree and flowers etc. has greater advance and achieves more excellent achievement, but also few about the report of forest mutation breeding, be detected in the seeds such as locust tree, mao bamboon, yellow horn, acer monoes, plane tree, Huashan pine, China fir, elm and Chinese white poplar at present.
Due to the uncertainty of induced mutations, want to obtain satisfactory beneficial mutation proterties, need large group to select Breeding strategy by force, it is one of crucial for therefore obtaining large mutagenesis colony, can be screening like this and meets the variation that we require and provide higher probability.Therefore, in the selection of research material, the mutative material that can obtain compared with large group, and simply easy to operate, reproducible etc.Chinese fir seeds very meets above-mentioned requirements, Chinese fir seeds grain is less, general thousand kernel weight i.e. 5-7 gram, and Chinese fir seeds is very easy to obtain simultaneously, particularly China has built up the Chinese Fir Seed Orchards of more advanced lines, can obtain a large amount of improvement Chinese fir seeds improving genetic gain.Because Chinese fir seeds is little, easily obtain, so be mutant materials with Chinese fir seeds, larger mutagenesis colony can be obtained when being mutant materials compared with the larger material of China fir nursery stock equal-volume, for Variant selection provides more chance.Meanwhile, employing Chinese fir seeds is mutant materials, also has a major reason, and be process and simple to operate, easy nursery, simply, disposal cost is also very low for the required condition of test process.Further, be used in the seed of China fir high powder-content artificial sand, also ensure that the starting point of this mutagenic treatment work is higher, in conjunction with existing Breeding of Chinese fir resource, carry out seed selection targetedly, be conducive to solving and produce upper urgent problem.So this research take seed as test material.
Report only for one section (Co-60r radiation China fir offspring amount of growth variation research) with the pertinent literature of the technical method initiative screening China fir new germ plasm of mutation breeding up till now.This research carries out for 1976, employing be gamma Rays China fir grow directly from seeds natural forest elite stand gather seed.Compare the filial generation that high powder-content artificial sand brings out, its filial generation owing to often not exclusively not possessing the economic characters such as the fast-growing required by some afforestation commerical tree species through seed selection, and is compared decades ago, and present climatic environment there occurs larger change.
For the excellent genetic resources of China fir of initiative screening polymerization fast-growing, the proterties such as degeneration-resistant, enrich China fir sudden change, obtain larger variation colony, and be China fir new germ plasm and the new varieties of screening merit, the present invention, in conjunction with China fir conventional seedbed system technology, adopts colchicine, ethylmethane sulfonate (EMS), sodium azide (NaN
3) three kinds of mutagen method process Chinese fir seeds, summing up a set of being applicable to Chinese fir seeds is the induced-mutation technique method for examination material, and obtains 52340 strain mutagenesis seedling colonies, for seed selection China fir new germ plasm is laid a good foundation.
Summary of the invention
The object of the invention is, for the deficiency of existing China fir mutagenic and breeding technology, to provide a kind of Study on mutagenesis breeding method of China fir, the inventive method is screening polymerization fast-growing, the excellent genetic resources of China fir of the proterties such as degeneration-resistant provides abundant mutative material.
For realizing object of the present invention, one aspect of the present invention provides a kind of China fir mutagenic breeding method, comprises and adopts mutagen to soak Chinese fir seeds, carry out mutagenic treatment to Chinese fir seeds, obtain mutagenesis Chinese fir seeds; Then by sowing after the cleaning of mutagenesis Chinese fir seeds, seedling management.
Wherein, described mutagen select colchicine solution, ethylmethane sulfonate solution or sodium azide solution.
Particularly, the concentration of described colchicine solution is 0.9-1.2% (w/v), is preferably 0.9% (w/v), and namely every 0.9-1.2g colchicine is dissolved in the colchicine solution making described concentration in 100mL water; The concentration of described ethylmethane sulfonate solution is 0.3-0.9% (w/v), is preferably 0.3% (w/v); The concentration of described sodium azide solution is 6-10mmol/L, is preferably 8mmol/L.
Wherein, described Chinese fir seeds selects third generation China fir garden China fir mature seed.
Particularly, described Chinese fir seeds uses after storing under cryogenic and surviving the winter.
Especially, the time of surviving the winter is 2-3 month; Described low temperature is 0-4 DEG C, is preferably 4 DEG C.
Particularly, also comprise and gibberellin immersion treatment is carried out to Chinese fir seeds, be soaked in Gibberellins solution by Chinese fir seeds.
Wherein, the concentration of described Gibberellins solution is 15-35mg/L, is preferably 25mg/L, and namely every 25mg gibberellin is dissolved in the Gibberellins solution making described concentration in 1000mL water.
Particularly, also comprise the Chinese fir seeds after to gibberellin immersion treatment and carry out water soaking process, be immersed in water by the Chinese fir seeds after gibberellin immersion treatment, and then carry out mutagen immersion treatment.
The present invention provides a kind of China fir mutagenic breeding method on the other hand, comprises the steps: 1) adopt colchicine solution to soak Chinese fir seeds, colchicine mutagenic treatment is carried out to Chinese fir seeds, obtains mutagenesis Chinese fir seeds; 2) by sowing after the cleaning of mutagenesis Chinese fir seeds, seedling management.
Wherein, step 1) described in the concentration of colchicine solution be 0.9-1.2% (w/v), be preferably 0.9% (w/v), namely every 0.9-1.2g colchicine is dissolved in the colchicine solution making described concentration in 100mL water.
Particularly, step 1) in the colchicine mutagenic treatment time be 1-4d (my god); Mutagenic treatment temperature is 25 ± 5 DEG C; In mutagenic treatment process, complete lucifuge, namely carries out described mutagenic treatment under dark condition.
Particularly, step 1) described in mutagenic treatment be adopt concentration be 0.9% (w/v) colchicine solution soak Chinese fir seeds 4d.
Particularly, employing gibberellin (GA is also comprised
3) solution immersion Chinese fir seeds, carry out gibberellin immersion treatment, and then carry out the mutagenic treatment of described colchicine solution.
Wherein, the concentration of described Gibberellins solution is 15-35mg/L, is preferably 25mg/L, and namely every 25mg gibberellin is dissolved in the Gibberellins solution making described concentration in 1000mL water.
Particularly, the described gibberellin immersion treatment time is 20-28h, is preferably 24h; Treatment temperature is 25 ± 5 DEG C, is preferably 25 DEG C; In gibberellin immersion process, complete lucifuge, namely carries out described gibberellin immersion treatment under dark condition.
Particularly, also comprise and water cleaning treatment is carried out to the Chinese fir seeds through gibberellin immersion treatment, and then carry out described colchicine mutagenic treatment.
Especially, described water cleaning treatment number of times is 3-5 time, is preferably 5 times.
Particularly, also comprise cleaned for water Chinese fir seeds to be immersed in distilled water and soak 48-72h, be preferably 72h.
Wherein, distilled water immersion Chinese fir seeds is carried out under dark lucifuge condition.
Particularly, adopt high temperature immersion treatment and low temperature immersion treatment interval to carry out to Chinese fir seeds in distilled water immersion processing procedure, wherein high temperature immersion treatment temperature is 28 ± 1 DEG C, and high temperature soak time is 12h; Described low temperature immersion treatment temperature is 20 ± 1 DEG C, and high temperature soak time is 12h, namely carries out, in distilled water immersion processing procedure, first carrying out high temperature immersion to Chinese fir seeds in Chinese fir seeds, then carries out low temperature immersion, and then carry out high temperature immersion, repetitive cycling.
Wherein, step 2) adopt water cleaning mutagenesis Chinese fir seeds 3-5 time after sow again.
Particularly, described sowing be mutagenesis Chinese fir seeds is planted in sphagna be culture matrix cultivation cup in carry out germinateing, nursery cultivates.
Wherein, described germination, nursery cultivation temperature are 25 ± 5 DEG C; Relative moisture is 60-70%; Intensity of illumination is 1500-2000lux, and periodicity of illumination is that 10-16h illumination/8-14h is dark.
Particularly, described sowing is by the drilling of mutagenesis Chinese fir seeds in outdoor, and sowing stripe pitch is 10cm.
Particularly, described Chinese fir seeds selection thousand kernel weight is the seed of 5-7g is seed selection mutant materials, and being preferably thousand kernel weight is 5.8-6.0g, and more preferably the seed of 5.93g is seed selection mutant materials.
Another aspect of the invention provides a kind of China fir mutagenic breeding method, comprises the steps: 1) adopt ethylmethane sulfonate (EMS) solution to soak Chinese fir seeds, ethylmethane sulfonate mutation process is carried out to Chinese fir seeds, obtains mutagenesis Chinese fir seeds; 2) by sowing after the cleaning of mutagenesis Chinese fir seeds, seedling management.
Wherein, step 1) described in the preparation method of EMS solution EMS is dissolved in pH=7.0, the phosphate buffer of 0.1mol/L.
Particularly, the concentration of described ethylmethane sulfonate solution is 0.3-0.9% (w/v), be preferably 0.3% (w/v), namely every 0.3-0.9g EMS is dissolved in 100mLpH=7.0, makes the EMS solution of described concentration in the phosphate buffer of 0.1mol/L.
Especially, described phosphoric acid buffer liquid making method is: get potassium dihydrogen phosphate 0.68g, adds 0.1mol/L sodium hydroxide solution 29.1mL, is diluted with water to 100mL.
Particularly, step 1) in the ethylmethane sulfonate mutation processing time be 8-16h; Mutagenic treatment temperature is 25 ± 5 DEG C, is preferably 24 ± 4 DEG C; In mutagenic treatment process, complete lucifuge, namely carries out described mutagenic treatment under dark condition.
Particularly, step 1) described in mutagenic treatment be adopt concentration be 0.3% (w/v) ethylmethane sulfonate solution soak Chinese fir seeds 8h.
Particularly, employing gibberellin (GA is also comprised
3) solution immersion Chinese fir seeds, carry out gibberellin immersion treatment, and then carry out the mutagenic treatment of described ethylmethane sulfonate solution.
Wherein, the concentration of described Gibberellins solution is 15-35mg/L, is preferably 25mg/L, and namely every 15-35mg gibberellin is dissolved in the Gibberellins solution making described concentration in 1000mL water.
Particularly, the described gibberellin immersion treatment time is 20-28h, is preferably 24h; Treatment temperature is 25 ± 5 DEG C, is preferably 25 DEG C; In gibberellin immersion process, complete lucifuge, namely carries out described gibberellin immersion treatment under dark condition.
Particularly, also comprise and water cleaning treatment is carried out to the Chinese fir seeds through gibberellin immersion treatment, and then carry out described ethylmethane sulfonate mutation process.
Especially, described water cleaning treatment number of times is 3-5 time, is preferably 5 times.
Particularly, also comprise cleaned for water Chinese fir seeds to be immersed in distilled water and soak 48-72h, be preferably 72h.
Wherein, distilled water immersion Chinese fir seeds is carried out under dark lucifuge condition.
Particularly, adopt high temperature immersion treatment and low temperature immersion treatment interval to carry out to Chinese fir seeds in distilled water immersion processing procedure, wherein high temperature immersion treatment temperature is 28 ± 1 DEG C, and high temperature soak time is 12h; Described low temperature immersion treatment temperature is 20 ± 1 DEG C, and high temperature soak time is 12h, namely carries out, in distilled water immersion processing procedure, first carrying out high temperature immersion to Chinese fir seeds in Chinese fir seeds, then carries out low temperature immersion, and then carry out high temperature immersion, repetitive cycling.
Wherein, step 2) adopt water to rinse EMS mutagenesis Chinese fir seeds 4-5h after sow again.
Particularly, described sowing be mutagenesis Chinese fir seeds is planted in sphagna be culture matrix cultivation cup in carry out germinateing, nursery cultivates.
Wherein, described germination, nursery cultivation temperature are 25 ± 5 DEG C; Relative moisture is 60-70%; Intensity of illumination is 1500-2000lux, and periodicity of illumination is that 10-16h illumination/8-14h is dark.
Particularly, described sowing is by the drilling of mutagenesis Chinese fir seeds in outdoor, and sowing stripe pitch is 10cm.
Particularly, described Chinese fir seeds selection thousand kernel weight is the seed of 5-7g is seed selection mutant materials, and being preferably thousand kernel weight is 5.8-6.0g, and more preferably the seed of 5.93g is seed selection mutant materials.
Another aspect of the invention provides a kind of China fir mutagenic breeding method, comprises the steps: 1) adopt sodium azide solution to soak Chinese fir seeds, sodium azide mutagenic treatment is carried out to Chinese fir seeds, obtains mutagenesis Chinese fir seeds; 2) by sowing after the cleaning of mutagenesis Chinese fir seeds, seedling management.
Wherein, step 1) described in the concentration of sodium azide solution be 6-10mmol/L, be preferably 8mmol/L.
Particularly, step 1) in the sodium azide mutagenic treatment time be 4-12h; Mutagenic treatment temperature is 25 ± 5 DEG C, is preferably 24 ± 4 DEG C; In mutagenic treatment process, complete lucifuge, namely carries out described mutagenic treatment under dark condition.
Particularly, step 1) described in mutagenic treatment be adopt concentration be 8mmol/L sodium azide solution soak Chinese fir seeds 12h.
Especially, the compound method of described sodium azide solution is 0.1mol/L phosphate buffer sodium azide being dissolved in pH=3.0.
Particularly, described phosphoric acid buffer liquid making method is: get potassium dihydrogen phosphate 0.68g, adds 0.1mol/L sodium hydroxide solution 29.1mL, is diluted with water to 100mL, then with 0.1mol/L hydrochloric acid, pH value is adjusted to 3.0.
Wherein, described sodium azide mutagenic treatment is soaked in sodium azide solution by Chinese fir seeds, in 25 ± 5 DEG C, under dark condition, and oscillation treatment 4-12h.
Particularly, the speed of described vibration is 120rpm.
Particularly, employing gibberellin (GA is also comprised
3) solution immersion Chinese fir seeds, carry out gibberellin immersion treatment, and then carry out the mutagenic treatment of described sodium azide solution.
Wherein, the concentration of described Gibberellins solution is 15-35mg/L, is preferably 25mg/L, and namely every 15-35mg gibberellin is dissolved in the Gibberellins solution making described concentration in 1000mL water.
Particularly, the described gibberellin immersion treatment time is 20-28h, is preferably 24h; Treatment temperature is 25 ± 5 DEG C, is preferably 25 DEG C; In gibberellin immersion process, complete lucifuge, namely carries out described gibberellin immersion treatment under dark condition.
Particularly, also comprise and water cleaning treatment is carried out to the Chinese fir seeds through gibberellin immersion treatment, and then carry out described sodium azide mutagenic treatment.
Especially, described water cleaning treatment number of times is 3-5 time, is preferably 5 times.
Particularly, also comprise cleaned for water Chinese fir seeds to be immersed in distilled water and soak 48-72h, be preferably 72h.
Wherein, distilled water immersion Chinese fir seeds is carried out under dark lucifuge condition.
Particularly, adopt high temperature immersion treatment and low temperature immersion treatment interval to carry out to Chinese fir seeds in distilled water immersion processing procedure, wherein high temperature immersion treatment temperature is 28 ± 1 DEG C, and high temperature soak time is 12h; Described low temperature immersion treatment temperature is 20 ± 1 DEG C, and high temperature soak time is 12h, namely carries out, in distilled water immersion processing procedure, first carrying out high temperature immersion to Chinese fir seeds in Chinese fir seeds, then carries out low temperature immersion, and then carry out high temperature immersion, repetitive cycling.
Wherein, step 2) adopt water cleaning mutagenesis Chinese fir seeds 3-5 time after sow again.
Particularly, described sowing be mutagenesis Chinese fir seeds is planted in sphagna be culture matrix cultivation cup in carry out germinateing, nursery cultivates.
Wherein, described germination, nursery cultivation temperature are 25 ± 5 DEG C; Relative moisture is 60-70%; Intensity of illumination is 1500-2000lux, and periodicity of illumination is that 10-16h illumination/8-14h is dark.
Particularly, described sowing is by the drilling of mutagenesis Chinese fir seeds in outdoor, and sowing stripe pitch is 10cm.
Particularly, described Chinese fir seeds selection thousand kernel weight is the seed of 5-7g is seed selection mutant materials, and being preferably thousand kernel weight is 5.8-6.0g, and more preferably the seed of 5.93g is seed selection mutant materials.
China fir mutagenic breeding method tool of the present invention has the following advantages:
1, the aberration rate of inventive method mutagenic and breeding China fir is high, and mutagenesis seedling emergence rate is high, and mutagenesis seedling stability is high, and spectrum of variation is wide, is beneficial to the improvement of China fir kind.
2, to solve the breeding methods existed in the work of China fir conventional breeding few for the inventive method, the technical problem that aberration rate is low, can obtain large variation colony, the China fir new germ plasm of merit and new varieties.
3, the inventive method is screening polymerization fast-growing, the excellent genetic resources of China fir of the proterties such as degeneration-resistant, pest-resistant provides abundant mutative material.
4, the inventive method is disposable can process mutant materials in enormous quantities, reduces mutagenic and breeding processing cost, and method is simple, easily manipulates.
Accompanying drawing explanation
Fig. 1 is that in embodiment 1, colchicine mutagenic treatment Chinese fir seeds is cultivated and to be contrasted (ck) and variating seedling after 3 weeks and (a, b, c) to compare;
Fig. 2 is the Germination of Chinese fir Seeds result of the test figure of EMS mutagenic treatment Chinese fir seeds in embodiment 2;
Fig. 3 is the China fir mutagenesis seedling change of height comparison diagram of EMS mutagenic treatment Chinese fir seeds in embodiment 2;
Fig. 4 is the Germination of Chinese fir Seeds result of the test figure of sodium azide mutagenic treatment Chinese fir seeds in embodiment 3;
Fig. 5 be in embodiment 3 sodium azide process to China fir mutagenesis seedling plant height observed result figure;
Fig. 6 be in embodiment 3 sodium azide process to China fir mutagenesis seedling leading thread observed result figure.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Technical scheme:
1, seed source
The present invention selects the thousand kernel weight of seed orchard collection to be that the ripe China fir breeding of 5-7g is as mutant materials.In November, 2011 gathers the ripe cone of China fir in Qiangyue County of Fujian Province state-owned forest farms Chinese Fir Seed Orchards, and the seed that collection airing sheds is for subsequent use.The dry seeds thousand kernel weight that airing sheds is 5.8-6.0g, and average thousand kernel weight is 5.93g.
2, seed gibberellin immersion treatment
First the dry seeds that 2-1) airing sheds carries out 4 DEG C of deepfreeze process 3 months, proceeds as follows after taking out in February, 2012.
2-2) under lucifuge dark, room temperature (20-28 DEG C) condition, first China fir dry seeds is immersed in the gibberellin (GA that concentration is 15-35mg/L
3) in solution, seed soaking stimulates to improve seed germination effects, in seed soaking process, seed will be kept all the time to be fully immersed in liquid; The Chinese fir seeds distilled water that will soak after 20-28h (being preferably 24h) cleans 3-5 time, removes the gibberellin Liquid Residue of the surface of the seed.
In the embodiment of the present invention with concentration be 25mg/L Gibberellins solution seed soaking be example, other concentration are that the Gibberellins solution of 15-35mg/L is all applicable to the present invention.
Post-stimulatory Chinese fir seeds of 2-3) being soaked seed by GA3 again puts into the seed that distilled water continuation immersion 48-72h (being preferably 72h) obtains sprouting.Distilled water immersion Chinese fir seeds, under dark lucifuge condition, first soaks 12h, i.e. high temperature immersion treatment 12h under temperature is 28 ± 1 DEG C of conditions; Then reduce temperature and soak 12h under remaining 20 ± 1 DEG C of conditions, i.e. low temperature immersion treatment 12h; Then repeat high temperature immersion treatment 12h, low temperature soaks 12h, until soak 48-72h.Namely in distilled water immersion processing procedure, high temperature immersion treatment and low temperature immersion treatment interval are carried out, and interval time is 12h.
Seed is through GA
3solution and distilled water obtain gibberellin process seed, to promote Seed sprouting after soaking seed.
3, mutagenic treatment
Adopt colchicine, ethylmethane sulfonate (EMS), sodium azide (NaN
3) three kinds of mutagen carry out mutagenic treatment.With distilled water or phosphate buffer, three kinds of mutagen are mixed with the mutagenic treatment liquid of different concentration gradients, then will be immersed in completely in mutagenic treatment liquid through gibberellin process seed, and different time gradients be set and carry out mutagenic treatment.Mutagenic treatment process, at room temperature (20-28 DEG C), is carried out under dark condition.Chinese fir seeds distilled water after mutagenic treatment completes cleans or rinses in flowing water, sows after removing the surface of the seed raffinate, obtains mutagenesis Chinese fir seeds.
4, seeding and seedling raising
From the seed after often kind of mutagenic treatment, random selecting is seeded on a small quantity with in the careless tongue cultivation cup that is culture matrix, waters weekly once.Cultivation cup is placed in phytotron (normal temperature 25 DEG C, 70% relative moisture, 16h illumination) and carries out seed sprouting and situation of emerging observation.All the other seeds sow state-owned forest farms Seedlings nursery test block, Qiangyue County according to completely random district group experimental scheme, conventional seedbed system manages 9 months, carries out Chinese Fir Seedling that seedlings observation obtains for the early stage growing way observation of mutagenesis seedling colony and later stage variation screening after current growth stops.The nursery bedside 120cm of state-owned forest farms Seedlings nursery test block, Qiangyue County, high 20cm, sowing furrow width 2cm, dark 1cm, spacing 10cm.
Mutagenesis seedling colony observation in the present invention: the seed be seeded on a small quantity in careless tongue after 4 weeks, carries out the observation of seed sprouting situation through cultivation in the controlled environment chamber, and variating seedling is added up; All the other seeds be seeded in Seedlings nursery were cultivated through 9 months and carry out seedlings observation after current growth are stopped, for later stage variation screening is prepared.The suitableeest Mutagenesis Combinatorial of each mutagen is determined according to semilethal rate principle (compare photograph, after mutagenic treatment, percentage of seedgermination reduces half) or mutagenesis seedling growing state.
Embodiment 1 colchicine mutagenic treatment
1, seed source
In November, 2011 gathers the ripe cone of China fir in Qiangyue County of Fujian Province state-owned forest farms seed orchard, 4 DEG C of deepfreeze process 3 months are carried out, using the dry seeds (average thousand kernel weight is for 5.93g) after refrigerating as the mutagenesis seed material of this test after collecting the seed that sheds of airing.
2, gibberellin immersion treatment
In February, 2012 starts this research, and the seed that the low temperature treatment that starts February to learn from else's experience is crossed carries out gibberellin immersion treatment.
2-1 is constant room temperature (25 ± 5 DEG C, 25 DEG C are selected in the embodiment of the present invention) under condition, first China fir dry seeds being immersed in concentration is in the Gibberellins solution of 25mg/L, after soaking 24h, seed is cleaned 5 times with distilled water, remove the gibberellin Liquid Residue on Chinese fir seeds surface, obtain gibberellin seed soaking seed.
Seed soaking, to improve seed germination effects, in seed soaking process, will keep seed to be fully immersed in liquid all the time.
In gibberellin immersion treatment process, gibberellin concentration is 15-35mg/L, soak time 20-28h; Water wash number can also be 3-5 time.
3, distilled water immersion process
Under dark lucifuge condition, the Chinese fir seeds of gibberellin seed soaking is immersed in distilled water immediately and carries out distilled water immersion process, first under temperature is 28 ± 1 DEG C of conditions, soak 12h, i.e. high temperature immersion treatment 12h; Then reduce temperature and soak 12h under remaining 20 ± 1 DEG C of conditions, i.e. low temperature immersion treatment 12h; Then repeat high temperature immersion treatment 12h, low temperature soaks 12h, until soak 72h.Namely in distilled water immersion processing procedure, high temperature immersion treatment and low temperature immersion treatment interval are carried out, and interval time is 12h.
Gibberellin immersion treatment seed is immersed in outside time-triggered protocol high temperature in distilled water, low temperature interval immersion treatment 72h, and high temperature, low temperature interval immersion treatment time are 48-72h, are all applicable to the present invention.
4, mutagenic treatment
In room temperature (25 ± 5 DEG C) and completely under lucifuge condition (i.e. dark condition), colchicine mutagenic treatment is carried out to the Chinese fir seeds of distilled water immersion.
Test arrange 4 concentration gradients (0.3%, 0.6%, 0.9%, 1.2%w/v), 4 processing times (1d, 2d, 3d, 4d), with distilled water seed soaking in contrast; Be soaked in colchicine solution by gibberellin immersion treatment seed, carry out colchicine mutagenic treatment, then distilled water cleaning Chinese fir seeds 5 times, removes the colchicine Liquid Residue of the surface of the seed, obtains colchicine mutagenesis Chinese fir seeds.Every concentration per processing time processes 5000 seeds, repeats 3 times, and namely each combination amounts to 15000 seeds, and total Test comprises and amounts to 300,000 seeds to impinging upon 20 interior combinations.
5, sowing, seedling management
After mutagenic treatment completes, from each treatment combination random sampling 150 planting seeds to sphagna be culture matrix cultivation cup in, water weekly once to keep sphagna moistening.Whole incubation be in the controlled environment chamber in complete, condition of culture is: 25 ± 5 DEG C, and relative moisture is 60-70%, and intensity of illumination is 1500-2000lux, and periodicity of illumination is that 16h illumination/8h is dark.Carry out the observation of seed sprouting situation after 3 weeks, the yield of statistical variation or dispersion seedling and variation features, determine the best mutagenic treatment concentration of colchicine and time.Variating seedling and normal development seedling are as shown in Figure 1.Colchicine mutagenic treatment Chinese fir seeds China fir aberration rate is as shown in table 1.
The process of table 1 colchicine is on the impact of China fir aberration rate
Note: different capitalization represents significant difference in 0.01 level
Table 1 result of the test shows: pretreated Chinese fir seeds, can obtain variating seedling through colchicine certain concentration range and the mutagenic treatment of time.Variating seedling yield can present along with the prolongation in the increase processing time of colchicine concentration the trend first raising and reduce afterwards generally.Emergence rate after process 0.9% and 1.2% concentration when 4d promote obviously emerging and and difference between other concentration reach pole significance level.The variating seedling quantity that in four processing times, 0.9% and 1.2% two concentration obtains is maximum, has been up to 85.7%.The variating seedling number that the same processing time obtains first raises rear reduction along with the increase of colchicine concentration mostly, except 2d, all reach maximum when 0.9% concentration.0.9% colchicine-induced variating seedling number and processing time correlation, namely the variating seedling of more looking more the processing time, from 150 seeds, obtain 12 strain variating seedling when processing time is 4d, aberration rate is 8.0%, and other concentration difference heteropoles in processing with other are remarkable.During process 4d, when colchicine concentration is 1.2%, not only suppress to emerge, and the variating seedling quantity of induction is compared 0.9% concentration and is sharply declined.Can draw, the colchicine concentration of 0.9% is the optimization process concentration of Chinese fir seeds induce variation.
Seedling (ck) is contrasted and three kinds of variating seedling compare after the mutagenic treatment of colchicine shown in Fig. 1.Plumular axis expands and does not extend, without radicle (a); Plumular axis expands and does not extend, and has radicle (b); Elongation is expanded, without radicle (c) bottom plumular axis
The present invention adopts the concentration of mutagen colchicine solution in colchicine mutagenic and breeding China fir method to select 0.9-1.2% (w/v), and the mutagenic treatment time is 1-4d, and mutagenesis seedling emergence rate is high, reach more than 36%, and variating seedling is many, aberration rate is high, and mutagenesis seedling stability is high.Colchicine is suitable for use in China fir mutation breeding.
Planting seed after mutagenic treatment is except doing except matrix with sphagna on a small quantity, also carry out the direct sowing (sowing of large sample amount) of seedling bed, be sowed at state-owned forest farms Seedlings nursery test block, Qiangyue County for the early stage growing way observation of mutagenesis seedling colony by remaining 4850 the direct bars of seed of each treatment combination.
The seed repeated test of large sample amount (4850/treatment combination), sowing condition changes direct bar into by sphagna matrix and is sowed at state-owned forest farms Seedlings nursery test block, Qiangyue County (nursery bedside 120cm, high 20cm, sowing furrow width 10cm, dark 1cm).The 31190 strain mutagenesis seedlings obtained at present complete afforestation, for later stage seed selection.
Embodiment 2 ethylmethane sulfonate (EMS) mutagenic treatment Chinese fir seeds
1, seed source
Identical with " seed source " in embodiment 1.
2, gibberellin immersion treatment
Identical with " gibberellin immersion treatment " in embodiment 1.
3, distilled water immersion process
Identical with " distilled water immersion process " in embodiment 1.
4, mutagenic treatment
In room temperature (25 ± 5 DEG C) and completely under lucifuge (i.e. dark) condition, EMS mutagenic treatment is carried out to the Chinese fir seeds of distilled water immersion process.
Test arrange 4 concentration gradients (0.3%, 0.6%, 0.9%, 1.2%w/v), 3 processing times (8h, 12h, 16h), wherein EMS is dissolved in pH=7.0 by the preparation method of EMS solution, the phosphate buffer of 0.1mol/L.By 0.3g, 0.6g, 0.9g, 1.2gEMS solution 100mLpH=7.0 respectively, the phosphate buffer of 0.1mol/L makes the EMS mutagen in the present embodiment, wherein, pH=7.0, the phosphate buffer method of 0.1mol/L is by potassium dihydrogen phosphate 0.68g, add 0.1mol/L sodium hydroxide solution 29.1mL, be diluted with water to 100mL and get final product.
Gibberellin immersion treatment seed is soaked in EMS solution, carries out EMS mutagenic treatment, then the seed after mutagenic treatment is rinsed 4h under flowing water, remove the EMS solution that the surface of the seed is residual, to prevent raffinate to the murder by poisoning of seed, obtain EMS mutagenesis Chinese fir seeds.
With distilled water seed soaking in contrast.
5, sowing cultivation, seedling management
EMS mutagenesis Chinese fir seeds is dried in the shade sowing in time after exosper to sphagna be culture matrix cultivation cup in, water in time to keep sphagna moistening.Whole incubation be in the controlled environment chamber in complete, condition of culture is: temperature 25 ± 5 DEG C, and relative moisture is 60-70%, and intensity of illumination is 1500-2000lux, and periodicity of illumination is that 16h illumination/8h is dark.Add up seed germination situation every day until off-test.Test arranges 3 repetitions, each process 100 seeds.The China fir germination test result of EMS mutagenic treatment Chinese fir seeds as shown in Figure 2.
The result of the test of Fig. 2 shows: pretreated Chinese fir seeds, and in certain density EMS solution, after mutagenic treatment a period of time, germination rate obviously reduces.Three processing times, its downward trend was obvious all along with the increase germination rate of EMS concentration reduces gradually.Show that mutagen EMS process obviously inhibits Germination of Chinese fir Seeds.Compare the germination rate of control treatment, when EMS concentration reaches 1.2%, the mutagenic treatment germination rate of 12h and 16h is reduced to less than 6%, is respectively 5.5% and 2.7%, close to lethal concentration.According to medial lethal dose principle, three optimum concentration of processing time EMS, all between 0.3%-0.9%, are respectively 8h process 0.9%, 12h process 0.6%, 16h process with 0.3%.
Each test treatment combination seed used is except 100 of randomly drawing are for except indoor germination statistics, remaining for each process 2000 bars are sowed at state-owned forest farms Seedlings nursery test block, Qiangyue County (nursery bedside 120cm, high 20cm, sowing furrow width 10cm, dark 1cm), test arranges 3 repetitions.Obtain 4370 strain mutagenesis seedling colonies altogether through statistics after 9 months, and complete in January, 2013 afforestation of going up a hill, carry out random sampling after 7 months and measure plant height, measuring and calculating mutagenesis seedling populational variation amplitude (maximum-minimum of a value).After EMS mutagenic treatment Chinese fir seeds, mutagenesis seedling colony early stage growing way variation amplitude result as shown in Figure 3.
The result of the test of Fig. 3 shows: complete in January, 2013 in the mutagenesis seedling colony growing state investigation in 7 months after afforestation, find that the EMS low concentration short time processes height of seedling impact the most remarkable.Present the promotion of low concentration short time, high concentration suppresses even lethal rule for a long time.Especially short time low concentration treatment combination 0.3%8h and 0.6%8h, mean value, minimum of a value, maximum and variation amplitude (maximum-minimum of a value) are all greater than contrast, and significant difference.Therefore we have screened the characteristic new germ plasm (its plant height all at 90cm more than, namely plant height be more than 2 times of average control plant height 44.4cm) of the excellent variation individual plant of 18 strain as primary election of exhibits excellent on plant height from these two kinds for the treatment of combinations.
The present invention adopts the concentration of mutagen EMS solution in EMS mutagenic and breeding China fir method to select 0.3-0.9% (w/v), and the mutagenic treatment time is 8-16h, creates impact to the Germination of Chinese fir Seeds after mutagenic treatment.The growth Early manifestation after mutagenesis seedling colony afforestation obtained goes out that indivedual plant Seedling height is outstanding, populational variation amplitude increases, and enriches material for the excellent variation features new germ plasm of seed selection provides.EMS is suitable for use in China fir mutation breeding.
Because EMS is volatile, whole process operation will carry out the security protection of testing crew, should carry out in fume hood.EMS reagent is purchased from Beijing Baeyer enlightening Bioisystech Co., Ltd.
Embodiment 3 sodium azide (NaN
3) mutagenic treatment Chinese fir seeds
1, seed source:
Identical with " seed source " in embodiment 1.
2, gibberellin immersion treatment
Identical with " gibberellin immersion treatment " in embodiment 1.
3, distilled water immersion process
Identical with " distilled water immersion process " in embodiment 1.
4, mutagenic treatment
In room temperature (25 ± 5 DEG C) and completely under lucifuge (i.e. dark) condition, sodium azide mutagenic treatment is carried out to distilled water immersion process Chinese fir seeds.
Test arrange 5 concentration gradients (2,4,6,8,10mmol/L, with pH=3.0, the phosphate buffer preparation of 0.1mol/L), gibberellin immersion treatment seed is soaked in sodium azide solution respectively, vibrate (120 revs/min) process 4h, 8h, 12h respectively under 25 DEG C of conditions, then the seed after mutagenic treatment is rinsed 2h under flowing water, remove the sodium azide solution that the surface of the seed is residual, to prevent raffinate to the murder by poisoning of seed, obtain sodium azide mutagenesis Chinese fir seeds.
Wherein, the phosphate buffer method of pH=7.0,0.1mol/L is by potassium dihydrogen phosphate 0.68g, adds 0.1mol/L sodium hydroxide solution 29.1mL, is diluted with water to 100mL and get final product.
By 2,4,6,8, the sodium azide of 10mmol is dissolved in pH=7.0, in the phosphate buffer of 0.1mol/L, the sodium azide mutagen in obtained the present embodiment.
With distilled water seed soaking in contrast.
5, sowing cultivation, seedling management
Sodium azide mutagenesis Chinese fir seeds is dried in the shade sowing in time after exosper to sphagna be culture matrix cultivation cup in, water in time to keep sphagna moistening.Whole incubation be in the controlled environment chamber in complete, condition of culture is: temperature 25 ± 5 DEG C, and relative moisture is 60-70%, and intensity of illumination is 1500-2000lux, and periodicity of illumination is that 16h illumination/8h is dark.Add up seed germination situation every day until off-test.Test arranges 3 repetitions, each process 100 seeds.Sodium azide mutagenic treatment Germination of Chinese fir Seeds result of the test as shown in Figure 4.
The result of the test of Fig. 4 shows: three processing times, and Germination of Chinese fir Seeds rate is all along with the increase of sodium azide concentration presents the rear downward trend that first rises.Little in the change of 2-6mmol/L scope germination rate, sharply decline afterwards, when concentration reaches maximum 10mmol/L, germination rate is also minimum.As in processed group 8h, the germination rate of China fir reaches maximum 36.7% when 2mmol/L, sharply declines more than after 8mmol/L, is only 1.7% when 10mmol/L.In processed group 4h and 12h, roughly the same, when wherein 12h sodium azide concentration is 10mmol/L, germination rate is 2.3%, and under this treatment combination, germination rate is minimum, and seed is close to lethal for the rule that the increase germination rate with sodium azide concentration presents and 8h process.According to medial lethal dose principle, three optimum concentration of processing time sodium azide are all between 6-10mmol/L.
Each test treatment combination seed used is except 100 of randomly drawing are for except indoor germination statistics, remaining for each process 2000 bars are sowed at state-owned forest farms Seedlings nursery test block, Qiangyue County (nursery bedside 120cm, high 20cm, sowing furrow width 10cm, dark 1cm), test arranges 3 repetitions.Each mutagenic treatment seedling colony growing way in seedling stage (measure the footpath of seedling is high, leading thread) is observed after 9 months.The 16780 strain mutagenesis seedlings obtained at present complete afforestation, for later stage seed selection.After sodium azide mutagenic treatment Chinese fir seeds, seedling situation result is as shown in table 2.
Table 2 sodium azide is on the impact of Chinese fir seeds seedling situation
The result of the test of table 2 shows: soak after Chinese fir seeds carries out mutagenic treatment with sodium azide solution and cultivate through routine, the mutagenesis seedling quantity that each treatment combination obtains presents regular change with sodium azide concentration and the difference in processing time.Such as, when sodium azide concentration is 4-10mmol/L, the mutagenesis seedling quantity that the mutagenic treatment time more looks fewer.Especially the sodium azide process 12h of 10mmol/L, obtains 3 young plants only from 2000 seeds, and emergence rate is low to moderate 0.15%, closely its lethal dose, this also with the coming to the same thing of 100 small sample germination tests.
By finding mutagenesis seedling colony growing way in the seedling stage observation in Seedlings nursery, sodium azide process inhibits mutagenesis seedling Seedling height in seedling stage, on the impact of China fir mutagenesis seedling plant height as shown in Figure 5, sodium azide process to the shadow of China fir mutagenesis seedling leading thread as shown in Figure 6 in sodium azide process.
In the treatment combination of each concentration of sodium azide, mutagenesis seedling plant height during 2mmol/L and 10mmol/L is apparently higher than 4-8mmol/L, and especially the short time processed group Seedling Height Seedling height of 4h and 8h is obvious.4-10mmol/L sodium azide through the process of 4-12h, mutagenesis seedling plant height, stem all present first rise after downward trend, namely the average plant height of mutagenesis seedling that obtains of 8h processed group is higher than other two groups.Although the Seedling Height of sodium azide mutagenic treatment is lower than contrast, by finding through measuring each processed group seedling, warp is higher than contrast fifty-fifty for the mutagenesis seedling that 2mmol/L process 4h and 10mmol/L process 8h obtains, and especially 10mmol/L process 8h combines.Show sodium azide mutagenic treatment plant can be made to downgrade thick through increasing, change the phenotypic characteristic of original colony, the mutagenesis seedling that Chinese fir seeds obtains there occurs hereditary capacity and changes, and improves mutation rate.
The present invention adopts the concentration of mutagen sodium azide solution in sodium azide mutagenic and breeding China fir method to select 6-10mmol/L, and the mutagenic treatment time is 4-12h, creates impact to the Germination of Chinese fir Seeds after mutagenic treatment.The mutagenesis seedling colony that 10mmol/L process 8h obtains goes out average plant height at wheat seeding and becomes short through increasing thick phenotypic variation feature, enriches material for the excellent variation features new germ plasm of seed selection provides.Sodium azide is suitable for use in China fir mutation breeding.
Whole process operation will carry out the security protection of testing crew, should carry out in fume hood.Sodium azide reagent is enough in Beijing Baeyer enlightening Bioisystech Co., Ltd.
Claims (10)
1. a China fir mutagenic breeding method, is characterized in that comprising employing mutagen soaks Chinese fir seeds, carries out mutagenic treatment to Chinese fir seeds, obtains mutagenesis Chinese fir seeds; Then by sowing after the cleaning of mutagenesis Chinese fir seeds, seedling management.
2. a China fir mutagenic breeding method, is characterized in that comprising the steps: 1) adopt colchicine solution to soak Chinese fir seeds, colchicine mutagenic treatment is carried out to Chinese fir seeds, obtains mutagenesis Chinese fir seeds; 2) by sowing after the cleaning of mutagenesis Chinese fir seeds, seedling management.
3. mutagenic breeding method as claimed in claim 2, is characterized in that also comprising employing Gibberellins solution soaks Chinese fir seeds, carries out gibberellin immersion treatment, and then carries out the mutagenic treatment of described colchicine solution.
4. mutagenic breeding method as claimed in claim 3, is characterized in that the Chinese fir seeds also comprised through gibberellin immersion treatment carries out water cleaning treatment, and then carries out described colchicine mutagenic treatment.
5. a China fir mutagenic breeding method, is characterized in that comprising the steps: 1) adopt ethylmethane sulfonate solution to soak Chinese fir seeds, ethylmethane sulfonate mutation process is carried out to Chinese fir seeds, obtains mutagenesis Chinese fir seeds; 2) by sowing after the cleaning of mutagenesis Chinese fir seeds, seedling management.
6. mutagenic breeding method as claimed in claim 5, is characterized in that also comprising employing Gibberellins solution soaks Chinese fir seeds, carries out gibberellin immersion treatment, and then carries out the mutagenic treatment of described ethylmethane sulfonate solution.
7. method as claimed in claim 6, is characterized in that the Chinese fir seeds also comprised through gibberellin immersion treatment carries out water cleaning treatment, and then carries out described ethylmethane sulfonate mutation process.
8. a China fir mutagenic breeding method, is characterized in that comprising the steps: 1) adopt sodium azide solution to soak Chinese fir seeds, sodium azide mutagenic treatment is carried out to Chinese fir seeds, obtains mutagenesis Chinese fir seeds; 2) by sowing after the cleaning of mutagenesis Chinese fir seeds, seedling management.
9. mutagenic breeding method as claimed in claim 8, is characterized in that also comprising employing Gibberellins solution soaks Chinese fir seeds, carries out gibberellin immersion treatment, and then carries out the mutagenic treatment of described sodium azide solution.
10. mutagenic breeding method as claimed in claim 9, is characterized in that the Chinese fir seeds also comprised through gibberellin immersion treatment carries out water cleaning treatment, and then carries out described sodium azide mutagenic treatment.
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