CN107494254B - A kind of hybridizing method of wild rice - Google Patents
A kind of hybridizing method of wild rice Download PDFInfo
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- CN107494254B CN107494254B CN201710845523.XA CN201710845523A CN107494254B CN 107494254 B CN107494254 B CN 107494254B CN 201710845523 A CN201710845523 A CN 201710845523A CN 107494254 B CN107494254 B CN 107494254B
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- 230000008774 maternal effect Effects 0.000 claims description 31
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- 229920001131 Pulp (paper) Polymers 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 12
- 235000017166 Bambusa arundinacea Nutrition 0.000 claims description 10
- 235000017491 Bambusa tulda Nutrition 0.000 claims description 10
- 241001330002 Bambuseae Species 0.000 claims description 10
- 235000015334 Phyllostachys viridis Nutrition 0.000 claims description 10
- 239000011425 bamboo Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 230000035784 germination Effects 0.000 claims description 5
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- 235000007164 Oryza sativa Nutrition 0.000 abstract description 45
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- 239000000463 material Substances 0.000 abstract description 19
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- 238000011160 research Methods 0.000 abstract description 6
- 230000002068 genetic effect Effects 0.000 abstract description 5
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 244000118056 Oryza rufipogon Species 0.000 description 41
- 238000009331 sowing Methods 0.000 description 10
- 238000012163 sequencing technique Methods 0.000 description 6
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/027—Apparatus for pollination
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of hybridizing methods of wild rice.The present invention between reasonable processing different materials florescence difference, carefully the wild rice hybridization technique of a set of maturation is explored in identification etc. after emasculation operation, different hybridization tools, suitable environmental condition and hybridization.Be experimentally confirmed: hybridization technique success rate of the invention is high, it is more to obtain hybrid seed quantity, it is widely used between different type wild rice, hybridizes demand between wild rice and cultivated rice, help to push the genetic resources of wild rice to excavate, be that widow's mite is contributed in the diversification scientific research of rice.
Description
Technical field
The invention belongs to crossbreeding fields, and in particular to a kind of hybridizing method of wild rice.
Background technique
Asian Cultivated Rice is one of crops most ancient in the world, the main source of the nearly half population food in the world.With
Explosive population growth and Global climate change, people's different need that there is an urgent need to constantly update main breed to meet with rapid changepl. never-ending changes and improvements
It asks.During cultivating new crop varieties, crossbreeding as current mainstream breeding method, have been developed that it is a set of compared with
For perfect technology path, it is desirable to the advantage economical characters that hybrid generation can not only inherit parents, but also by appropriate
Increase the hereditary difference between parents, further obtains better genetic breeding resource (Yuan Longping, 1997) using hybrid vigour；
And in terms of basic scientific research, although developing rapidly in recent years using the novel research method of modern biotechnology, hybridization, which remains, is ground
The basic steps studied carefully complex character, carry out the researchs such as the positioning of quantitative character related gene and functional verification, are that other methods exist
It is irreplaceable at this stage；So hybridization suffers from very important important meaning to theoretical research and actual production, it is through
Key technology means therein.
Rice (Oryza sativa) is before 7000-8000 by its Wild related germplasm O.nivara and O.rufipogon
Domestication, during domestication, cultivated rice optimizes many economical characters, only remains 60% allele (Sunet
al.,2001；Zhang et al.,2006)；Contain a large amount of genetic resources with potential value in wild rice, these are potential
Genetic resources not only the environmental suitability to research wild rice and species formation play an important role, but also be valuable breeding money
Source is the natural gene library that genetic improvement is carried out to cultivated rice.Wild rice is significantly better than cultivated rice in terms of resistance, if can
The environmental suitability advantage of the yield heterosis of cultivated rice and wild rice is combined, the resistance base lost during domestication is regained
Cause, it is believed that centainly can provide more more preferable more detailed technical data to novel super cultivated rice is cultivated.
Although the hybridization patent between existing cultivated rice and cultivated rice, between cultivated rice and wild rice at present, wild rice
There are larger difference between cultivated rice, this allow in many cultivated rices tend to mature study on classics method can not be in wild rice
Smoothly carry out, the difference for being related to hybridization is mainly reflected in the following: a. cultivated rice is based on being selfed, and Different groups is wild
Rice has different types of mating system；B. cultivated rice anther is small, and the anther of most of kind is less than 2mm, and wild rice anther
Greatly, for part anther up to 6mm or more, pollen amount is big, and general gametocide method is hard to work；C. it is not taken off after cultivation rice is mature
It falls, and falls off after wild rice maturation；D. the wild rice of different regions is bloomed loose powder time irreversibility, slightly poor with cultivated rice
It is different；E. the intermolecular hybrid fertility instability of the wild rice of different regions.Exactly these differences allow wild rice not to be suitable for cultivated rice
Hybridizing method, and it is lower to hybridize success rate.Therefore, it under the severe situation of crisis in food not yet complete incidence graph, probes into wild
Rice hybridization method is extremely urgent.
Summary of the invention
It is an object of the present invention to provide a kind of hybridizing methods of wild rice.
The hybridization includes the steps that emasculation；The emasculation includes the following steps: to remove at the top of the maternal inflorescence to emasculation
Then the hypogenetic little Hua of little Hua and bottom opened selects stamen position no more than lemma length 1/2 and is located at flower
The little Hua (15-30) of sequence middle and upper part cuts the gap of a point from base portion to bran in lemma side, takes out from the gap
Anther obtains female parent after emasculation.
In the above method, scissors abutting lemma is carefully cut off and (is careful not to cut anther) in lemma side, cuts one
The gap of item point from base portion to bran goes out 6 pieces of anther with the careful gently folder of sharp mouth tweezers, i.e., completely takes out whole anther.If
It is caught broken anther and then discards this little Hua.
In the above method, with white sulfuric acid paper bag bagging after the emasculation；The white sulfuric acid paper bag bottom is closed up, to prevent
Mature seed falls off.Experiments have shown that: white sulfuric acid paper bag is more advantageous to seed development than kraft paper bag.
In the above method, maternal inflorescence does not need the step of squirting after the emasculation.
In the above method, if be maternal with wild rice, maternal inflorescence is fixed on bamboo pole in advance before the emasculation, and
Away from the water surface, or whole strain maternal plant is tied up with nylon rope at the florescence and is supported with bamboo pole, its tiller is made to keep straight
It is vertical；
In pollination, the maternal inflorescence second level branch is gathered；
In bagging, the white sulfuric acid paper bag is folded into acute triangle, to gather little Hua after emasculation together.
In the above method, in the progress emasculation in second day that maternal inflorescence is bloomed；Also it may be selected have the 1 small mother that the flowers are in blossom puts
This inflorescence second level branch carries out emasculation (the no small second level branch that the flowers are in blossom puts, gynoecium often undeveloped mature；Open little Hua is more
Second level branch, can be few with little Hua number on entire inflorescence and be distributed scattered, is unfavorable for pollinating).
In the above method, the time of the emasculation carries out in daily 17:00 to next day 8:30.
In the above method, pollination is further included the steps that after the emasculation.Inflorescence maternal after same Zhi Suoshu emasculation can be connected
Continuous pollination, pollination is primary daily.Since the florescence of the single inflorescence of wild rice is about 1 week, the odd-numbered day, open little Hua quantity was few, can be daily
Take the male parent inflorescence little Hua Reusability that the same day is open.In a specific embodiment of the present invention, to same Zhi Quxiong female parent inflorescence
Continuous pollination three days, can be improved setting percentage.
In the above method, the pollination may be selected directly to be seeded in bucket or room after calm indoor progress, seed are sprouted
In interior facility；It is transplanted in indoor container, is made if complete stool or part tiller in paddy field, can be connected root before and after the florescence and dug by kind
Matrix is to be uniformly mixed so as to obtain clay loam and river sand according to the ratio that mass ratio is 1:1.
In the above method, the time pollinated every time preferably select male parent it is small the flowers are in blossom release powder when, wild rice is bloomed and loose powder
Time has focused largely on the 11:00-13:00 of fair weather.In practical applications, pollination time may be selected male parent small the flowers are in blossom puts
Same day 11:00-13:00 carries out.
Pollination can specifically carry out as follows every time: to male parent it is small the flowers are in blossom puts, when anther stretches out and starts loose powder, use
Sharp mouth tweezers cooperation scissors gently removes open anther, whole or several little Hua, and inflorescence is being just in maternal sack after emasculation
About 5s is shaken in top, completes pollination.
Tweezers and scissors are squirted with watering can after the pollination, kill all pollen, carry out next plant of pollination.It is more than in temperature
30 DEG C of hot dry weather can be sprayed water to male parent inflorescence with watering can, be evaporated to droplet, male parent maturation little Hua starts loose powder.
In the above method, for guarantee parent's flower synchronization, can many times germination plant parent material, every batch of interval 10 days.
Since wild rice is different to photoperiod-sensitive degree, if wild rice O.rufipogon is light-sensitive material, need by
After nutrient growth at least about 80 days, can just it bloom under the stimulation of short-day, and wild rice O.nivara is unwise to the photoperiod
Sense, florescence are 60-80 days, do not need photoperiod stimulation.Therefore it for the wild rice to photoperiod-sensitive, needs to pass through short-day
Processing waits nature short-day mode that it is stimulated to bloom.Short-day sunshine treatment condition is as follows: light application time 10h/d, short-day
Processing 20 days.
If being female parent with wild rice Oryza rufipogon, wild rice Oryza nivara is that male parent is hybridized, with
Subject to the wild rice O.rufipogon florescence, in order to guarantee the two flower synchronization, it is carried out as follows plantation and processing:
Wild rice O.rufipogon is planted May to mid-June in Hainan, paddy field is transplanted after sprouting, grows to by the end of September
(may be otherwise and utilize spring short-day) is stimulated by natural short-day, about mid-October blooms.If being planted in other times,
Growth period day temperature is not less than 33 DEG C, and night temperature is not less than 29 DEG C, and short-day sunshine treatment induction wild rice can be carried out after growth 80 days and is mentioned
Early heading.
Wild rice O.nivara, every batch of interval 10 days are planted in three batches in early July to early August in Hainan.If other
Time plantation, growth period day temperature are not less than 33 DEG C, and night temperature is not less than 29 DEG C, blooms within about 70 days.
In the above method, selecting inflorescence, the mature wild rice that will be opened or cultivated rice select inflorescence as female parent
It just blooms after growing boot leaf leaf sheath completely and wild rice or cultivated rice that the awns at the top of all little Hua has removed is as male parent.This hair
Bright method is suitable for hybridizing between wild rice and wild rice, can equally be well applied to hybridizing between wild rice and cultivated rice,
At least one in parent is that wild rice is suitable for the invention hybridizing method.In a specific embodiment of the present invention, described
Male parent is specially wild rice Oryza nivara；The maternal specially wild rice Oryza rufipogon or cultivated rice Japan
It is fine.
It does not include other steps after the pollination in the above method, it can sowing after 20 days.Can be used after the sowing as
Lower method identifies hybrid generation: after hybrid generation seed is sprouted, taking the male parent and female parent and hybridization of hybrid generation respectively
The blade of filial generation extracts genomic DNA, using at least a pair of SSR primer carries out PCR amplification in table 1, obtains each pair of SSR primer pair
The PCR product of male parent, female parent and the hybrid generation answered.PCR product is sequenced respectively, respectively obtains each pair of SSR primer pair
Peak figure is sequenced in the PCR product of male parent, female parent and the hybrid generation answered.If the PCR that each pair of SSR primer amplification hybrid generation obtains is produced
Object sequencing peak figure exists simultaneously maternal peak and male parent peak, then the hybrid generation plant is cenospecies.In practical applications, may be used
With any pair in selection table 1 or wantonly two pairs or wantonly three pairs or wantonly four pairs or whole SSR primer are identified, used SSR
Primer logarithm is more, and qualification result is more accurate.
Compared with cultivated rice hybridizing method, difference is essentially consisted in:
A. wild rice pollen amount is big, and emasculation needs finely to carry out by rejecting complete unopened anther.It is not required to after rejecting
Squirt processing.
B. wild rice is bloomed, and loose powder is rapid, and pollination is needed in completely calm indoor progress.
C. with white sulfuric acid paper bag bagging after pollinating, setting percentage can be improved.
D. wild rice plant type is opened scattered, is easily lodged, is needed to be fixed with bamboo pole.
E. Part Wild rice inflorescence is opened scattered, and pollination or when bagging need to gather inflorescence.
F. it before and after wild rice blooms loose powder time concentration at noon, is had differences with cultivated rice.
G. it falls off after wild rice is mature, hybridization bag needs to tie up bottom closing in.
H. subsequent processing is not needed after pollinating.
I. the florescence of different wild rices differs greatly, and needs to plant parent in batches.
The present invention successively plants a plurality of types of wild rices on Beijing, Hangzhou, Guangxi, Hainan and other places by studying for many years
And cultivated rice, a large amount of wild rice hybridization experiences are had accumulated, the wild rice hybridization technique of a set of maturation is explored.It is demonstrate,proved by experiment
Bright: hybridization technique success rate of the invention is high, and acquisition hybrid seed quantity is more, be widely used between different type wild rice,
Hybridize demand between wild rice and cultivated rice, help to push the genetic resources of wild rice to excavate, is the diversification section of rice
Grind contribution widow's mite.
Detailed description of the invention
Fig. 1 is the Molecular Identification of hybrid generation.Figure 1A be RM474 primer Molecular Identification result, wherein from top to bottom according to
The secondary sequencing peak figure for first familiar generation, female parent and male parent；Figure 1B is the Molecular Identification result of RM263 primer, wherein from top to bottom
It is followed successively by the sequencing peak figure of first familiar generation, female parent and male parent.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
Embodiment 1, a kind of hybridizing method of wild rice
With wild rice Oryza rufipogon be female parent, wild rice Oryza nivara be male parent as experimental material into
Row hybridization.Specific step is as follows:
One, many times germination material
Wild rice O.rufipogon is light-sensitive material, and wild rice O.rufipogon needs the nutrition by least about 80 days
After growth, can just it bloom under the stimulation of short-day；Wild rice O.nivara is insensitive to the photoperiod, and the florescence is 60-80 days, no
The photoperiod is needed to stimulate.It is subject to the wild rice O.rufipogon florescence, in order to guarantee the two flower synchronization, as follows
It is planted and is handled:
1, wild rice O.rufipogon
Wild rice O.rufipogon is planted May to mid-June in Hainan, the outdoor plantation of bucket, seedling stage are transplanted after sprouting
Pay attention to insect-and mouse-preventing and Routine Management, grow to is stimulated by natural short-day by the end of September, about blooms mid-October and (utilizes spring
Short-day also can), bucket is moved in greenhouse by the florescence.
2, wild rice O.nivara
Wild rice O.nivara, every batch of interval 10 days are planted in three batches in early July to early August in Hainan.
Two, the selection of parent material
1, prepare male parent material
It selects inflorescence to grow the wild rice O.nivara material just bloomed after boot leaf leaf sheath completely as male parent, and removes it
Awns at the top of all little Hua.
2, female parent material is selected
Selecting inflorescence, the mature wild rice O.rufipogon material that will be opened is as female parent, due to wild rice
The inflorescence that experiment needs is fixed on bamboo pole by O.rufipogon sprawl growth in advance, and away from the water surface, or at the florescence
When whole strain plant is tied up with nylon rope and is supported with bamboo pole, so that its tiller is kept upright.
Three, maternal emasculation
In second day or the existing 1 small second level branch that the flowers are in blossom the puts progress emasculation of selection that maternal inflorescence is bloomed, emasculation work
The strict control time is needed, is carried out in daily 17:00 to next day 8:30.Specific step is as follows for emasculation: removal is to emasculation
After the little Hua and the hypogenetic little Hua in bottom that have opened at the top of maternal inflorescence, stamen position is selected no more than lemma length
Scissors abutting lemma is carefully cut off in lemma side and (is careful not to cut by the 1/2 and little Hua 15-30 piece positioned at inflorescence middle and upper part
To anther), the gap of a point from base portion to bran is cut, goes out 6 pieces of anther with the careful gently folder of sharp mouth tweezers, i.e., completely takes
Whole anther out, it is firmly moderate, this little Hua is discarded if being caught broken anther.The inflorescence that emasculation is completed does not need to be squirted with watering can.
With white sulfuric acid paper bag bagging after emasculation, indicates maternal number, be fixed on spare on bamboo pole.It is fixed at the top of white sulfuric acid paper bag
On bamboo pole, bottom need to be fixed at least two pieces of paper clips or so, falls off to prevent mature seed, should avoid peduncle when fixed.
In addition, wild rice O.rufipogon inflorescence more open it is scattered, for guarantee whole little Hua can touch pollen, emasculation
When bagging, paper bag is folded into acute triangle, to gather emasculation little Hua together.
According to the method described above, white sulfuric acid paper bag is replaced with into kraft paper bag, and keeps other steps constant, as right
According to.
Four, it pollinates
To male parent it is small the flowers are in blossom puts, when anther stretches out and starts loose powder, start to pollinate, it is continuous to same Zhi Quxiong female parent inflorescence
Pollination 3 days, pollination daily it is primary (pollination time about in same day 11:00-13:00), entire pollinating process it is calm it is indoor into
Row.The concrete operations pollinated every time are as follows: it is small gently to remove open anther, whole or several with sharp mouth tweezers cooperation scissors
Flower, about 5s is shaken in the surface of inflorescence in emasculation female parent sack, and pollination is completed.Tweezers are squirted with watering can after the completion of pollination and are cut
Knife kills all pollen, carries out next plant of pollination.In the hot dry weather that temperature is more than 30 DEG C, watering can be used to male parent flower
Sequence water spray, evaporates to droplet, and male parent maturation little Hua starts loose powder.
According to the method described above, it only pollinates once to same Zhi Quxiong female parent inflorescence, and keeps other steps constant, as right
According to.
Five, sowing and identification
It can be seen that ovary obviously expands elongation, column cap position is moved up within about 2 days after pollination；Seed can be obviously observed after 4 days
Grow lemma, after 20 days can sowing, Molecular Identification is carried out after sowing and counts hybrid seed quantity, setting percentage and success rate.
Setting percentage=mature seed number/emasculation little Hua number；Success rate=true F1 plant number/germination seed number.
The method of cenospecies Molecular Identification is as follows: after first familiar generation seed is sprouted, taking male parent, female parent and first familiar generation respectively
Blade extracts genomic DNA, and 5 pairs of SSR primers in table 1 are respectively adopted and carry out PCR amplification, PCR reaction condition is 94 DEG C of initial denaturations
3:00,35 circulations, 94 DEG C of denaturation 15s, 55 DEG C of annealing 25s and 72 DEG C of extension 45s, last 72 DEG C of extensions 1min.After PCR
PCR product is sequenced, the fluorescence peak figure after obtaining PCR product sequencing.What if each pair of SSR primer amplification hybrid generation obtained
PCR product sequencing peak figure exists simultaneously maternal peak and male parent peak, then the hybrid generation plant is cenospecies.Fig. 1 is part
The sequencing peak figure that primer amplification male parent, female parent and hybrid generation obtain.
Table 1, the primer information for identifying hybrid generation
| Title | Preceding primer | Primer afterwards |
| RM167 | gatccagcgtgaggaacacgt | agtccgaccacaaggtgcgttgtc |
| RM263 | cccaggctagctcatgaacc | gctacgtttgagctaccacg |
| RM474 | aagatgtacgggtggcattc | tatgagctggtgagcaatgg |
| RM267 | tgcagacatagagaaggaagtg | agcaacagcacaacttgatg |
| RM336 | cttacagagaaacggcatcg | gctggtttgtttcaggttcg |
The result shows that: hybridized according to above-mentioned hybridizing method, can get cenospecies, setting percentage average out to after pollination 20 days
27.5% (slightly has difference, reaches as high as 88%, minimum is about 10%) success rate 60.7% between different groups.With white
The cenospecies quantity that sulfuric acid paper bag obtains illustrates that white template bag is conducive to plant than kraft paper bag increase about 40% (2-4)
Son development, effect are better than kraft paper bag.Above (the 4- is doubled than 1 day obtained cenospecies quantity of only pollinating within continuous pollination 3 days
7), illustrate that increasing pollination number of days can be improved setting percentage.
Embodiment 2, a kind of hybridizing method of wild rice
It is female parent with cultivated rice OryzasativaLcv.Nipponbare, wild rice O.nivara is that male parent is hybridized as experimental material.Specific step
It is rapid as follows:
One, many times germination material
Cultivated rice OryzasativaLcv.Nipponbare and wild rice O.nivara are insensitive to the photoperiod, and the florescence is 60-80 days, do not need light
Period stimulating.Be subject to the wild rice O.nivara florescence, in order to both guarantee flower synchronization, be carried out as follows plantation and
Processing:
1, wild rice O.nivara
Wild rice O.nivara is planted in late July in Hainan, transplants paddy field after sprouting, seedling stage pays attention to insect-and mouse-preventing and normal
Reason is regulated, the tiller that the entire plant in field or single plant are more than half is connected root and dug out, is transplanted to standby in indoor bucket by the florescence
With.
2, cultivated rice OryzasativaLcv.Nipponbare
In Hainan in the mid-July to mid-August of growing and cultivation rice OryzasativaLcv.Nipponbare in three batches, every batch of interval 10 days.
Two, the selection of parent material
1, prepare male parent material
It selects inflorescence to grow the wild rice O.nivara material just bloomed after boot leaf leaf sheath completely as male parent, and removes it
Awns at the top of all little Hua.
2, female parent material is selected
Select the inflorescence cultivated rice OryzasativaLcv.Nipponbare material that maturation will open as female parent.
Three, maternal emasculation
In second day or the existing 1 small second level branch that the flowers are in blossom the puts progress emasculation of selection that maternal inflorescence is bloomed, emasculation work
The strict control time is needed, is carried out in daily 17:00 to next day 8:30.Specific step is as follows for emasculation: removal is to emasculation
After the little Hua and the hypogenetic little Hua in bottom that have opened at the top of maternal inflorescence, stamen position is selected no more than lemma length
Scissors abutting lemma is carefully cut off in lemma side and (is careful not to cut by the 1/2 and little Hua 15-30 piece positioned at inflorescence middle and upper part
To anther), the gap of a point from base portion to bran is cut, goes out 6 pieces of anther with the careful gently folder of sharp mouth tweezers, i.e., completely takes
Whole anther out, it is firmly moderate, this little Hua is discarded if being caught broken anther.The inflorescence that emasculation is completed does not need to be squirted with watering can.
With white sulfuric acid paper bag bagging after emasculation, indicates maternal number, be fixed on spare on bamboo pole.Peduncle should be avoided when fixed.
Four, it pollinates
To male parent it is small the flowers are in blossom puts, when anther stretches out and starts loose powder, start to pollinate, it is continuous to same Zhi Quxiong female parent inflorescence
Pollination 3 days, pollination daily it is primary (pollination time about in same day 11:00-13:00), entire pollinating process it is calm it is indoor into
Row.The concrete operations pollinated every time are as follows: it is small gently to remove open anther, whole or several with sharp mouth tweezers cooperation scissors
Flower, about 5s is shaken in the surface of inflorescence in emasculation female parent sack, and pollination is completed.Tweezers are squirted with watering can after the completion of pollination and are cut
Knife kills all pollen, carries out next plant of pollination.In the hot dry weather that temperature is more than 30 DEG C, watering can be used to male parent flower
Sequence water spray, evaporates to droplet, and male parent maturation little Hua starts loose powder.
Five, sowing and identification
It can be seen that ovary obviously expands elongation, column cap position is moved up within about 2 days after pollination；Seed can be obviously observed after 4 days
Grow lemma, after 20 days can sowing, Molecular Identification is carried out after sowing and counts hybrid seed quantity.
The method of Molecular Identification is the same as the method in embodiment 1.
The result shows that: according to above-mentioned hybridizing method, it can get cenospecies after pollination 20 days.Setting percentage is greater than 60%；Success
Rate is 100%.
The hybridizing method of comparative example 1, conventional cultivated rice and wild rice
It is female parent with cultivated rice (japonica rice OryzasativaLcv.Nipponbare or Zhong Han 3), wild rice (O.rufipogon) is male parent, according to such as
Lower method is hybridized:
A. it is stimulated to spring short-day, wild rice (O.rufipogon) experiences florescence and cultivated rice after short-day and meets.
It b. is female parent, the bagging after hybridizing emasculation in proxima luce (prox. luc) afternoon, hybridization with cultivated rice (japonica rice OryzasativaLcv.Nipponbare or Zhong Han 3)
The clever flower shears that female parent is not bloomed removes 1/4-3/5, breaks anther, does not damage column cap, and water spray accelerates pollen dead immediately, reaches and kills
Male effect；
C. with wild rice (O.rufipogon) for male parent, the morning 5:00-8:30 on the Yu Kaihua same day takes blooming for wild rice
Fringe clamps the anther of single little Hua with tweezers or is placed in the single small ear of scissors clip wait pollinate on maternal spike of rice, and use is handshaking
Little Hua or small ear pollination, pollen is shaken off and completes to pollinate on the cultivated rice tassel of emasculation, then bagging indicates the father of hybridization
The maternal and date；Or choose the anther of cultivated rice female parent with tweezers, it is collected after the anther of the cultivated rice female parent wait choose is dead wild
Dead japonica rice anther and wild rice pollen are mixed to obtain pollen mixture by the mass ratio of 1:1 by raw rice pollen, then with the mixing
Pollen pollinates to the aforementioned cultivated rice female parent for having chosen anther.Sowing after pollination 20 days, and according to molecule in embodiment 1
Identification method identifies cenospecies.
The result shows that: pollination does not harvest to obtain cenospecies after 20 days.
The hybridizing method of comparative example 2, conventional wild rice and wild rice
It is female parent with wild rice (O.rufipogon), wild rice (O.nivara) is male parent, is carried out as follows miscellaneous
It hands over:
A. it is stimulated to spring short-day, wild rice (O.rufipogon) experiences florescence and wild rice after short-day
(O.nivara) it meets；Annual wild rice (O.nivara) does not carry out short-day sunshine treatment.
It b. is female parent with wild rice (O.rufipogon), the bagging after hybridizing emasculation in proxima luce (prox. luc) afternoon, hybridization is maternal not to open
Colored clever flower shears removes 1/4-3/5, breaks anther, does not damage column cap, and water spray accelerates pollen dead immediately, reaches gametocidal effect.
C. with wild rice (O.nivara) for male parent, the morning 5:00-8:30 on the Yu Kaihua same day takes the fringe of blooming of wild rice,
The anther of single little Hua is clamped with tweezers or is placed in the single small ear of scissors clip wait pollinate on maternal spike of rice, with handshaking little Hua
Or small ear pollination, pollen is shaken off and completes to pollinate on the wild rice tassel of emasculation, then bagging indicates the Parent of hybridization
And the date；Or choose the anther of wild rice female parent with tweezers, wild rice is collected after the anther of the wild rice female parent wait choose is dead
Dead wild rice female parent anther and wild rice paternal pollen are mixed to get pollen mixture by the mass ratio of 1:1 by paternal pollen,
It is pollinated again with the pollen mixture to the aforementioned wild rice female parent for having chosen anther.Sowing after pollination 20, and according to reality
It applies method for identifying molecules in example 1 and identifies cenospecies.
The result shows that: pollination does not harvest to obtain cenospecies after 20 days.
Claims (8)
1. a kind of hybridizing method of wild rice, it is characterised in that:
With wild riceOryza rufipogonFor female parent, wild riceOryza nivaraHybridized for male parent, to guarantee parent
Flower synchronization, many times germination plant the wild riceOryza nivara, every batch of interval 10 days；And it is wild by short-day sunshine treatment
RiceOryza rufipogon,Condition is as follows: light application time be 10 h/d, short-day sunshine treatment 20 days；
The hybridization includes the steps that emasculation, the emasculation include the following steps:It is gone within second day what maternal inflorescence was bloomed It is male, the emasculation time is daily 17:00 to next day 8:30；Remove the little Hua opened at the top of the maternal inflorescence to emasculation and bottom
Then the hypogenetic little Hua in portion selects stamen position no more than lemma length 1/2 and is located at the little Hua of inflorescence middle and upper part, outside
Bran side cuts the gap of a point from base portion to bran, and whole anther are taken out from the gap, obtains female parent after emasculation；
With white sulfuric acid paper bag bagging after the emasculation.
2. according to the method described in claim 1, it is characterized by: further including the steps that pollination after the emasculation: to same branch
Maternal inflorescence is continuously pollinated three days after the emasculation, and pollination is primary daily.
3. according to the method described in claim 1, it is characterized by: the white sulfuric acid paper bag bottom is closed up, to prevent mature kind
Son falls off.
4. method according to any one of claim 1-3, it is characterised in that: maternal inflorescence does not need to spray after the emasculation
Wet step.
5. according to the method described in claim 4, it is characterized by: the pollination is in calm indoor progress.
6. according to the method described in claim 5, it is characterized by: maternal inflorescence is fixed on bamboo pole in advance before the emasculation
On, and away from the water surface, or whole strain maternal plant is tied up with nylon rope at the florescence and is supported with bamboo pole, protect its tiller
It holds upright；In pollination, the maternal inflorescence second level branch is gathered；In bagging, the white sulfuric acid paper bag is folded into
Acute triangle, to gather little Hua after emasculation together.
7. according to the method described in claim 6, it is characterized by: male parent it is small the flowers are in blossom release powder when pollinate.
8. according to the method described in claim 7, it is characterized by: the time of the pollination daily 11:00 to 13:00 into
Row.
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