CN104521748B - A kind of method improving polyploid poplar gamete fertility - Google Patents

Abstract

The invention discloses a kind of method improving polyploid poplar gamete fertility, belong to plant genetics and breeding field.The inventive method includes: observe the flower bud development situation of polyploid poplar, when observing that its sporocyst was grown to the regular period, applies finite concentration chemical agent mutagenic treatment, wait to continue to be developed to pollen maturation, collect pollen, carry out pollen germination, observe pollen germination rate and judge fertility level.Each bar chromosome that the inventive method is polyploid plant provides homologue, it is helped to complete pairing, overcome the problem that chromosome imbalance separates during polyploid material meiosis, significantly improve gamete fertility, realize the sexual utilization of polyploid poplar kind, improve willow new germ plasm wound and educate efficiency.

Description

A kind of method improving polyploid poplar gamete fertility

Technical field

The present invention relates to a kind of method utilizing existing polyploid plant resource, improve polyploid particularly to one and plant The method of thing gamete fertility, belongs to plant genetics and breeding field.

Background technology

Utilize polyploid material as parent be Plant chromosomal engineering breeding, to educate polyploid new germ plasm the most direct for wound Means.But, due to polyploid plant meiosis process homologue joint conference and the erratic behavior of separation, connection Meeting multivalent oriented when mid-term I is irregular, and the unequal separation of first anaphase chromosome produces a large amount of chromosome The aneuploid gamete of number, when lacking certain tool Functional genomics in gamete, although or not lacking the dyeing of tool function Body, and when intergenic harmony is affected, gamete abortion will necessarily be caused, result in and normally can educate gamete Probability extremely low, be unfavorable for sexual hybridization produce offspring.

The existing important channel obtaining polyploid plant is to utilize physics and chemistry mode to process so that chromosome doubling, and induction is planted Thing non-subtrahend (2n) gamete carries out hybridization and forms.In willow ploidy breeding is studied, Zhang Zhiyi, Kang Xiangyang etc. are equal 2n pollen yield (Zhang Zhiyi, the Li Feng of up to more than 80% was obtained by the mode such as colchicin and high-temperature process Blue. white poplar chromosome doubling technical research and triploid Breeding (I) doubling of pollen chromosome technology. forestry in Beijing College journal, 1992,14 (increasing 3): 52-58；Zhang ZY,Li FL,Zhu ZT,Kang XY.Doubling technology of pollen chromosome of Populus tomentosa and its hybrid.J.Beijing For. Univ.(English Ed.),1997,6(2):9-20；Kang Xiangyang, Zhu Zhiti, Lin Huibin. Poplar Pollen chromosome doubling The research of effective treatment period. forest-science, 1999,35 (4): 21-24), and utilize and double rear pollen and carry out hybridization and obtain Populus tremula offspring must be crossed.

But, compared with the normal pollen of corresponding seeds, it is slow generally to there is sprouting process in the non-subtrahend pollen after doubling The problem such as slow, owing to there is the competition of normal haploidy pollen, cause the competitiveness participating in fertilization low (Kang Xiangyang, Zhu loves and respect one's elder brother. white poplar 2n pollen vitality assay method and the research of germination characteristics. and Yunnan plant is studied, and 1997,19 (4): 402-406), directly results in the triploid yield (less than 0.1%) after pollination hybridization well below for pollination 2n pollen ratio in pollen mixture.This low reason of pollen viability that doubles is deemed likely to and doubles pollen The factors such as the impact of the colchicum liquid that the imbalance in self heredity is remaining with in pollen are relevant.Similar non-subtrahend Pollen germination rate is low, sprout slow phenomenon has reported in the material such as camellia azalea, the bark of eucommia (Wang Youbing, Xu Bin, Zhang Fangqiu, etc. camellia azalea 2n pollen naturally-occurring and induction Primary Study thereof. Annual, 2011,27(6):9-15；Mao Yanke. high temperature induction bark of eucommia gametic chromosome doubling techniques research. Beijing Forestry University Master thesis, 2012), although showing to double to process to obtain higher 2n pollen yield, but set with corresponding The normal pollen planted is compared, and its viability but may be remarkably decreased.Just because of this, in recent years, seeds such as willows Study on Inducing Polyploid in, the most less employing physics and chemistry process induction doubling of pollen chromosome, and utilize double pollen The mode carrying out hybridizing is to obtain polyploid, and generally selects the oogamete chromosome doubling that triploid induction rate is higher Approach (Kang Xiangyang, Zhang Pingdong, Gao Peng, etc. the discovery of new way of poplar triploids induced with colchicine after pollination. Beijing woods Industry college journal, 2004,26 (1): 1-4；Wang J,Li DL,Kang XY.Induction of unreduced megaspores with high temperature during megasporogenesis in Populus.Annals of Forest Science,2012,69(1):59-67；Xi XJ,Guo LQ,Xu WT,et al.Megasporogenesis, megagametogenesis,and induction of 2n eggs with colchicine in poplar section Aigeiros. Scandinavian Journal of Forest Research,2014,29(6):527-536.)。

It is known that plant produces the enormous amount of pollen, once results pollen can meet substantial amounts of hybridization demand, If developing a kind of new technological means, it is thus achieved that while higher proportion doubles pollen, improve the life doubling pollen Power, will make tremendous contribution to the extensive initiative of plant polyploid.Kang Xiangyang etc. are it is proposed that utilize Different Ploidy Pollen, to gamma-ray radiation tolerance difference, improves method (Kang Xiangyang, Zhu of the sprouting competitiveness doubling pollen Love and respect one's elder brother, Lin Huibin. the radiosusceptibility of white poplar Different Ploidy pollen and application thereof. Acta Genetica Sinica, 2000,27 (1): 78-82.), and it is used in breeding, the triploid yield of this approach has been brought up to 12.9% (Kang Xiangyang, Zhu Love and respect one's elder brother, Zhang Zhiyi. silver gland poplar and Mao Xinyang reciprocal cross triploid seed selection. Beijing Forestry University's journal, 2000,22 (6): 8-11).But, this technical operation is loaded down with trivial details, and there is radiation hazard, and the dose of radiation of different tree species there are differences, Actual production is difficult to promote.Therefore, it is necessary to innovative technology means, physics and chemistry is promoted to process induction plant pollen Chromosome doubling seed selection polyploid technological progress, the while of improving pollen blodynamic after doubling, it is ensured that operation Convenience, security and practicality, thus eliminate the technology to doubling of pollen chromosome approach seed selection plant polyploid Prejudice.

Polyploid plant is used as breeding parent, and a feasible approach is induced synthesis height allotetraploid plant, Owing to allotetraploid plant is during meiosis, there's almost no the problem that homologue pairing is disorderly, Therefore can normally complete meiosis process, form the chromosome reduction gamete that can educate.But, allotetraploid The 2n female and male gametophyte that derived need utilizes source different merges, or adds distant hybrid enforcement somatic chromosome Times, induction difficulty is big, and, even if obtaining allotetraploid, in addition it is also necessary to wait its sexual utilization of ability of blooming, Cycle is long.Therefore, develop a kind of method instant, effective, raising polyploid poplar gamete fertility reliably, right In making full use of existing polyploid variety resource, improve polyploid new germ plasm wound by polyploid sexual hybridization approach and educate Efficiency, formulates lot of polyploid germ plasm resource, promotes poplar polyploid Research Advances on Breeding to have important value and reality Trample meaning, develop skill simultaneously for the polyploid plant gamete fertility of other similar plants and there is important reference meaning Justice.

Summary of the invention

It is an object of the invention to for existing polyploid plant gamete fertility low, gamete abortion rate is high, is unfavorable for sexual Hybridization produces the technical problem of offspring, it is provided that a kind of method significantly improving existing polyploid plant gamete fertility, real The sexual utilization of existing existing polyploid plant especially polyploid poplar kind, educates service for polyploid new germ plasm wound, And the inventive method has stronger instantaneity, validity, reliable in terms of polyploid poplar gamete fertility improving Property.

For realizing the purpose of the present invention, one aspect of the present invention provides a kind of method improving polyploid plant gamete fertility, Carrying out mutagenic treatment including to the sporocyst being in Meiosis, wherein said mutagenic treatment is to note bud Penetrate chemical agent.

Wherein, described chemical agent is colchicine solution.

Particularly, the weight percent concentration of described colchicine solution is 0.1-0.5%, preferably 0.5%.

Wherein, during described mutagenic treatment, in bud, inject the colchicine solution of 4-6 time；Adjacent two The time interval of secondary injection is 3-5h；During mutagenic treatment, environment temperature is 5-10 DEG C.

Particularly, during described mutagenic treatment, in bud, inject the colchicine solution of 5 times；Adjacent two The time interval of secondary injection is 4h.

Wherein, described polyploid plant is polyploid poplar.

Particularly, described polyploid poplar selects triploid willow hybrid or autotetraploid willow.

Especially, described triploid willow hybrid selects triploid ' poplar in silver ' (Populus alba × P. Berolinensis ' Yinzhong '), triploid white poplar hybrid, triploid Cathay poplar hybrid, triploid black poplar hybrid；Institute State tetraploid willow and select tetraploid white poplar hybrid or tetraploid Cathay poplar hybrid.

Wherein, described sporocyst is in maiotic pachytene or diakinesis.

Particularly, determine in accordance with the following steps described in be in the sporocyst of Meiosis: first gather male flower Bud, is fixed 30 minutes with FarmerShi fixer in 4 DEG C；Then flower pesticide is taken off from bud and be placed in On slide；Then dye with 2% acetic acid magenta and be caught broken flower pesticide, in light field basis of microscopic observation, really Determine the developmental stage of sporocyst.

Especially, described FarmerShi fixer is made up of 95% ethanol that ratio is 3:1 and the acetic acid of volume.

Particularly, the male flower bud after also including mutagenic treatment is cultivated to male flower under conditions of temperature is 15-25 DEG C Sequence is reached maturity.

The present invention improves the method for polyploid plant gamete fertility and has the advantage that

The gamete fertility of the polyploid poplar after 1, the inventive method processes significantly improves, and pollen germination rate significantly carries Height, the germination rate through the pollen of the inventive method process is 9.9 times for normal polyploid poplar pollen germination rate Above, even up to 18.7 times.

2, the diameter difference using the pollen of the polyploid poplar of the inventive method process is notable, deposits higher in pollen The big pollen of ratio, and the polarization of pollen diameter is substantially, and diameter frequency distribution is bimodal distribution；Side of the present invention Method processes and ' poplar in silver ' partial pollen diameter is increased, and produces the unreduced big pollen of higher proportion chromosome number, Big pollen ratio reaches 44.7%.

3, each bar chromosome that the inventive method is polyploid poplar provides homologue artificially, helps it complete Become pairing, overcome polyploid material meiosis process chromosome pairing, separate disorderly problem, significantly improve and join Sub-fertility, thus realize the sexual utilization of polyploid poplar kind, improve willow new germ plasm wound and educate efficiency.

4, the inventive method suppression polyploid poplar subtrahend first division process spindle fiber is formed, and stops polyploid Material meiosis process chromosome imbalance separates the balance ensureing subtrahend second division process sister chromatids Separate, so that it is guaranteed that form ortholoidy and the gamete of chromosome quantity balance, significantly improve gamete fertility, it is achieved many The sexual utilization of times body Poplar Varieties, improves willow new germ plasm wound and educates efficiency.

5, the inventive method achieves the sexual utilization to existing polyploid plant especially polyploid poplar kind, And the inventive method has stronger instantaneity, validity, reliable in terms of polyploid poplar gamete fertility improving Property.

6, the inventive method have simple to operate, the cycle is short, use instrument be easy to get, cost is less, it is easy to behaviour Make, reliable results, contribute to polyploid plant sexual hybridization is bred.

7, the method that the present invention improves polyploid poplar gamete fertility, for making full use of existing polyploid variety money Source, improves polyploid new germ plasm wound by polyploid sexual hybridization approach and educates efficiency, initiative lot of polyploid kind matter money Source, promotes poplar polyploid Research Advances on Breeding to have important value and practice significance, is similar to simultaneously for other and plants The polyploid plant gamete fertility of thing develops skill and has important reference.

Accompanying drawing explanation

Fig. 1 is that the normal control pollen of triploid in the embodiment of the present invention 1 ' poplar in silver ' is straight with mutagenic treatment pollen Footpath and diameter frequency distribution comparison diagram；Wherein: Figure 1A observes figure for comparison pollen aceto-camine dyeing；Figure 1B is The diameter histogram of comparison pollen；Fig. 1 C is mutagenic treatment pollen aceto-camine dyeing observation figure；Fig. 1 D is The diameter histogram of mutagenic treatment pollen.

Fig. 2 is that the normal control pollen of triploid in the embodiment of the present invention 1 ' poplar in silver ' is sprouted with mutagenic treatment pollen Condition of oestrusing comparison figure；Wherein: Fig. 2 A is the sprouting situation map of comparison pollen；Fig. 2 B is sprouting of mutagenic treatment pollen Send out situation map.

Fig. 3 is the maiotic ideograph of normal polyploid plant.

Fig. 4 is that the present invention improves the maiotic ideograph of polyploid plant.

Detailed description of the invention

Further describe the present invention, advantages of the present invention and feature below in conjunction with specific embodiment to will be with describing And it is apparent.But these embodiments are only exemplary, the scope of the present invention is not constituted any restriction.This Skilled person it should be understood that can be to the technology of the present invention side under without departing from the spirit and scope of the present invention The details of case and form are modified or replace, but these amendments and replacement each fall within protection scope of the present invention.

The experiment material of the present invention selects triploid staminiferous plant Poplar Varieties that the Northeast of China extensively cultivates ' in silver Poplar ', gather male flower branch water planting in greenhouse.

Polyploid plant meiosis process, homologue joint conference is abnormal and separates imbalance, in causing gametophyte Chromosome aneuploidy or dosage are uneven, have the feature of high abortion rate.Triploid ' poplar in silver ' chromosome number is 2n=3x=57, i.e. every chromosome has 3 homologues, during subtrahend first division, 3 homologies Chromosome necessarily can not averagely be separated to the two poles of the earth, causes the chromosome number of its pollen to be in non-multiple and imbalance State, pollen viability is low.

Embodiment 1

1, spray water planting processes

By triploid ' poplar in silver ' male flower branch water planting in greenhouse, the temperature in greenhouse remains 15-25 DEG C；Water planting Process changes a clear water in every 3 days, and rinses out the metabolin in incubation under running water pipe.From spray water planting Start, gathered male flower bud every 2-3 hour, with FarmerShi fixer (95% ethanol: acetic acid=3:1) in 4 DEG C it is fixed, after fixing 30 minutes, with pincet, flower pesticide is taken off from bud, be placed on slide, use 2% acetic acid magenta dyes and is caught broken flower pesticide, makes pollen mother cell discharge, and removes bulk impurity, covers Cover glass, in light field basis of microscopic observation.

2, pollen mutagenic treatment

When observe ' poplar in silver ' pollen mother cells proceed to the pachytene (chromosome substantially shortens thicker, And it is deeper to dye) time, immediately water planting spray is transferred in 5-10 DEG C of environment, in order to avoid its growth is too fast, simultaneously Using micro syringe to apply 0.5% colchicine solution in bud, carry out mutagenic treatment, frequency injection is 5 times, The time interval of adjacent double injection is 4 hours.After mutagenic treatment, spray is retracted greenhouse, continue water planting to male Inflorescence development is ripe, collects mutagenic treatment pollen.

Not carry out the pollen (comparison pollen) of the normal development of colchicine solution mutagenic treatment as comparison.

At the mutagenic treatment temperature (5-10 DEG C) of the relative low temperature of the present invention, cell development speed reduces, and improves Mutagenic Effect, prevents from causing cell development fast owing to temperature is high, and mutagenesis is not thorough, reduces gamete fertility, reduces Efficiency of inducing mutation.

3, result

1) pollen diameter

The pollen of mutagenic treatment and comparison pollen are respectively placed on slide, drip 1 aceto-camine dye liquor dye Look, observes, the effect that estimation doubles, and utilizes the full spherical pollen of eyepiece micrometer random measurement at least 1000 Diameter, observed result is as shown in Figure 1.

Wherein: Figure 1A observes figure for comparison pollen aceto-camine dyeing, shows that normal pollen diameter is the most uniform； Figure 1B is the diameter histogram of comparison pollen, in approximate Gaussian distribution；Fig. 1 C is mutagenic treatment pollen acetic acid Figure is observed in fuchsin dyeing, deposits the big pollen of higher proportion in display pollen；Fig. 1 D is the diameter of mutagenic treatment pollen Histogram, in bimodal distribution, shows that process makes pollen diameter there occurs differentiation.

Understand from the above: through the pollen of colchicine solution mutagenic treatment, the frequency distribution of pollen diameter is with right Compare according to normal pollen and there is larger difference.Wherein, comparison pollen diameter the most homogeneous (such as Figure 1A), diameter frequency Rate is distributed in approximate Gaussian distribution (such as Figure 1B), and the pollen diameter difference after mutagenic treatment is notable, polarization Substantially (such as Fig. 1 C), diameter frequency distribution is bimodal distribution (such as Fig. 1 D), shows through colchicine solution mutagenesis Process makes ' poplar in silver ' partial pollen diameter increased, and creates higher proportion chromosome number unreduced greatly Pollen (chromosome number is 2n=3x=57), big pollen ratio reaches 44.7%.

2) pollen germination

By the mutagenic treatment pollen of fresh collection and pollen uniform broadcasting respectively is compareed in solid culture with clean writing brush On base, carrying out sprouting and cultivate in constant temperature and humidity incubator, wherein solid medium is: 1.5% agar+120g/L Sucrose+60mg/L boric acid+80mg/L calcium chloride；Sprouting cultivation temperature is 25 ± 1 DEG C, and incubation time is 18h, profit Observe with light field microscope, add up its germination rate, to evaluate its viability level.Observed result is as shown in Figure 2.

Fig. 2 A is the sprouting situation of comparison pollen, due to triploid ' poplar in silver ' Meiosis, causes flower Powder height abortion, the normal pollen sprouted is few, and germination rate is extremely low；Fig. 2 B is the sprouting feelings of mutagenic treatment pollen Condition, it was observed that the pollen tube that more sprouting produces, and the mostly big pollen sprouted, show suitable reason Change processes and the viability of pollen is significantly improved.

Pollen germination test result is as shown in table 1, finds from the result of the test of table 1, and comparison pollen is triploid ' silver Middle poplar ' pollen, there is the problems such as Meiosis, the germination rate of pollen is the lowest (Fig. 2 A), and only 1.77%； Mutagenic treatment pollen germination rate then significantly improves (Fig. 2 B), has reached 33.06%, and the overwhelming majority sprouted is Big pollen.

Embodiment 2

1, spray water planting processes

Same as in Example 1.

2, pollen induction

Proceed to the diakinesis by ' poplar in silver ' pollen mother cells when observing (bivalent high enrichment is also point Being scattered to the edge of core, kernel diminishes or even disappears) time, immediately water planting spray is transferred in 5-10 DEG C of environment, with Exempt from its growth too fast, use micro syringe to apply 0.5% colchicine solution in bud simultaneously, carry out at mutagenesis Reason, frequency injection is 5 times, and the time interval of adjacent double injection is 4 hours.After mutagenic treatment, spray is moved Rise again room, continue water planting and reach maturity to male inflorescence, collect mutagenic treatment pollen.

Not carry out the pollen (comparison pollen) of the normal development of colchicine solution mutagenic treatment as comparison.

3, result

The pollen and the comparison pollen that induction are processed are respectively placed on slide, drip 1 aceto-camine dye liquor dye Look, observes, the effect that estimation doubles, and utilizes the full spherical pollen of eyepiece micrometer random measurement at least 1000 Diameter；Simultaneously with clean writing brush by mutagenic treatment pollen and comparison pollen respectively uniform broadcasting on solid medium, Carrying out sprouting in constant temperature and humidity incubator and cultivate, wherein solid medium is: 1.5% agar+120g/L sucrose + 60mg/L boric acid+80mg/L calcium chloride；Sprouting cultivation temperature is 25 ± 1 DEG C, and incubation time is 18h, adds up it Germination rate, to evaluate its viability level.

Observed result is as follows: in mutagenic treatment pollen, the big pollen ratio of diameter noticeably greater than comparison pollen reaches 27.3%, the frequency distribution of mutagenic treatment pollen diameter presents bimodal distribution equally；Mutagenic treatment pollen viability reaches To 17.5%, it is significantly increased, as shown in table 1 compared with comparison pollen.

Table 1 ' poplar in silver ' normal control pollen and the germination rate comparing result of mutagenic treatment pollen

Sample	Germination rate
		Comparison pollen	1.77%
Embodiment 1	33.06%
		Embodiment 2	17.5%

From the result of Fig. 1,2 and table 1 it can be seen that in the meiosis process of triploid willow ' poplar silver ' Bud is injected colchicine solution, applies physics and chemistry mutagenic treatment, subtrahend first division can be suppressed to occur, this Sample during subtrahend second division, sister chromatids generation normal separation, Meiotic Chromosomes can be prevented The generation of uneven segregation phenomenon, it is ensured that the order of meiosis process, thus significantly improve polyploid plant Gamete fertility, the gamete fertility ideograph of the present invention is as shown in Figure 4.The inventive method improves polyploid plant The gamete fertility of gamete fertility, especially triploid willow, for utilizing existing polyploid plant resource to carry out hybridization wound Educate polyploid new germ plasm and establish technical foundation.Prove simultaneously, utilize physics and chemistry to process induction plant gametophyte chromosome doubling Technology can improve the gamete viability of polyploid plant, this technology of redefinition is in plant polyploid is induced Practicality, overcome the prejudice in recent years to this technology.

Triploid ' poplar in silver ' plant normally reduces division as it is shown on figure 3, individual big chromosome in each cell Being one group of homologue, individual little chromosome is one group of homologue, divides at triploid plant normal reductive During splitting, 3 homologue joint conferences, produce the not tight phenomenon of joint conference, during subtrahend first division, Article 3, unequal the dividing to the two poles of the earth of homologue, the homologue number causing each pole to be contained is uneven, and During subtrahend second division, the sister chromatids of every chromosome is that equalization separates, therefore in generation Gamete is formed the unbalanced result of dosage between non-multiple chromosome and coloured differently body, causes height abortion.

The present invention improves the method for polyploid poplar gamete fertility, and the bud of Meiosis is injected colchicin Solution, carries out mutagenic treatment, the formation of suppression subtrahend first division spindle fiber so that should uneven decile Can only jointly stay in cell equatorial region to the homologue at the two poles of the earth and not separate, so in subtrahend second time In fission process, after sister chromatids separates, formed containing complete chromosome number and the non-subtrahend of dosage balance Gamete, these gametes have fertility (such as Fig. 4).

Claims (4)

1. the method improving polyploid plant gamete fertility, it is characterised in that: include being in maiotic thick line The sporocyst in phase or diakinesis carries out mutagenic treatment, it is characterised in that described mutagenic treatment is to inject male flower bud Chemical agent, wherein:

Described polyploid plant is triploid willow hybrid or autotetraploid willow；Described chemical agent is weight Percent concentration is the colchicine solution of 0.1-0.5%；And during described mutagenic treatment, be 5-10 in temperature In bud, the colchicine solution of 4-6 time is injected at DEG C；The time interval of adjacent double injection is 3-5h.

2. the method for claim 1, it is characterised in that described triploid willow hybrid selects triploid ' in silver Poplar ' (Populus alba × P.berolinensis ' Yinzhong '), triploid white poplar hybrid, triploid Cathay poplar hybrid Or triploid black poplar hybrid.

3. method as claimed in claim 1 or 2, it is characterised in that described in be in the maiotic pachytene or become eventually The sporocyst of phase determines in accordance with the following steps: first gather male flower bud, carries out in 4 DEG C with FarmerShi fixer Fix 30 minutes；Then flower pesticide is taken off from bud and be placed on slide；Then with 2% aceto-camine dyeing Liquid dyes and is caught broken flower pesticide, in light field basis of microscopic observation, determines the developmental stage of sporocyst.

4. method as claimed in claim 1 or 2, it is characterised in that the male flower bud after also including mutagenic treatment is in temperature Degree is reached maturity for cultivating under conditions of 15-25 DEG C to male inflorescence.