CN105145341B - Tidal land Spartina alterniflora and the positive and negative distant hybridization method of paddy rice - Google Patents

Abstract

The invention discloses a kind of tidal land Spartina alterniflora and the positive and negative distant hybridization method of paddy rice, including the step such as whole fringe, emasculation, pollination during orthogonal, reciprocal cross.The present invention is used（1）It is orthogonal：Paddy rice ♀ × Spartina alterniflora ♂：Paddy rice ♀ hot gas gametocide promotees flower and inhales the in good time pollination methods of powder Spartina alterniflora ♂, spike of rice is handled with hot gas and starch absorbing device absorbs flower pesticide, paddy rice female parent is set to remove lukewarm water gametocide from and cut clever emasculation process, in good time pollination make Spartina alterniflora's male parent remove from cut grain husk promote flower process, it is paddy to hybridize obtained seed, rather than the grain of rice, seed circularity is good；（2）Reciprocal cross：Spartina alterniflora ♀ × paddy rice ♂：The continuous producing Crossbred methods of Spartina alterniflora ♀.Take while emasculation, while pollinate, Spartina alterniflora ♀ is removed lukewarm water gametocide and paddy rice from and promote flower process, reduce the damage to grain husk flower.Two kinds of reciprocal cross methods substantially increase Cross fertile rate, emergence rate and operating efficiency.The present invention can cultivate Spartina alterniflora/Rice Salt Grain feed crop of suitable salt-soda soil growth.

Description

Tidal land Spartina alterniflora and the positive and negative distant hybridization method of paddy rice

Technical field

The present invention relates to a kind of tidal land Spartina alterniflora and the positive and negative distant hybridization method of paddy rice.

Background technology

At present, it is Spartina alterniflora (Spartina alterniflora that the careless distribution area of China coast rice is most wide Loisel.), it is grass family rice grass category Spartina Schreb. perennial herb C4 plants, 1979 by Nanjing University from U.S. State introduces China.Spartina alterniflora has very strong salt resistance ability, most suitable to be born in seabeach damp band.Plant stalk is up to 1-3m, directly Footpath is in more than 1cm, and grass yield is high, is adapted to the plant-eating animals such as free range husbandry cattle and sheep.Spartina alterniflora's panicle long 20-45cm is small Spike length about 1cm；The column cap of gynoecium two is very long, white featheriness, is exposed when blooming；Longitudinal cracking when stamen flower pesticide is ripe, Pollen yellow.The 6-11 months bloom successively, and the seed usual 8-12 months are ripe, and its seed has the characteristics of dehydration loses germination. The flower of Spartina alterniflora is hermaphrodite flower, and protogyny, its column cap stretches out before pollen bag splits, to receive the pollen of precocious flower, Therefore cross-pollination is conducive to.

Author Chen Qikang, in the case where Nanjing University professor Zhong Chongxin instructs, introduces a fine variety Spartina alterniflora in 1980, creates China the The careless pasture of one rice.Undertake State Scientific and Technological Commission《Test in beach pasture》Scientific research project, makees feed year using rice grass and raises cattle and sheep 3200 Bull, beach pasture experimental identification is domestic initiation.Find within 2005, Spartina alterniflora has the good characteristics such as salt tolerant, but seed Small, setting percentage is low, it is difficult to supply the nutritional need for raising cattle and sheep as feed.So making a decision to excavate the salt tolerant of Spartina alterniflora Deng fine genes.

Genus Oryza grass family Oryza cultigen, variety source enriches, and the full yield of seed is high, and straw can serve as cattle and sheep feeding again Material.Paddy contains 8% thick protein, and NFE content is occupied first of various cereal, effective energy value also highest, is a kind of good Good energy feed.Paddy rice provides excellent germ plasm resource for improvement rice grass, and paddy rice distant hybridization can import paternal plant Beneficial gene, there is close hybridization incomparable diversity and new type, expand the hereditary basis of paddy rice.

In order to excavate tidal land Spartina alterniflora salt tolerant and rice high yield germ plasm resource, 2006-2010 has carried out Spartina alterniflora Tested with paddy rice distant hybridization.Because Spartina alterniflora's grain husk spends small, female and first ripe, gynoecium is again exposed, and hybridization difficulty is big, so to mutual Flower rice grass ♀ × paddy rice ♂ (reciprocal cross) experiments fail to carry out always, have only carried out (orthogonal) experiments of paddy rice ♀ × Spartina alterniflora ♂, and And only pollinated once with Spartina alterniflora's paternal pollen, hybridization fringe setting percentage also lacks depth in 5.4-9.37% on hybridizing method Enter research.Paddy rice ♀ × Spartina alterniflora ♂, it is necessary to gametocide is carried out to paddy rice female parent, existing paddy rice gametocide method respectively has advantage and disadvantage： Cut that clever emasculation method workload is very big, the damage to grain husk flower is larger, reduces setting percentage；Lukewarm water gametocide method setting percentage is higher, and technology will Ask also higher, accomplish both all to kill stamen, and do not cause gynoecium damage；Vavuum pump gametocide method speed is fast, but needs to match somebody with somebody Complete equipment, cost is high；Cover black paper bag emasculation method convenient, but bagging time is difficult to grasp.Distant hybridization as a kind of breeding technique, The useful gene of category not of the same race can be introduced, an important channel is provided to improve existing kind.1. population constantly increases at this stage It is long；2. grain aggregate demand is continuously increased at this stage；3. cultivated area is reduced increasingly；4. the situation of freshwater resources wretched insufficiency Under, excavated and innovation research by the excellent economic characters of paddy rice and Spartina alterniflora's resistant gene of salt germ plasm resource, salt tolerant can be cultivated Transition silos New Crop Varieties.How setting percentage, the emergence rate of Spartina alterniflora and paddy rice distant hybridization, the wound of hybridizing method are improved It is newly crucial, accelerates initiative salt tolerant crop new germ plasm, solves the salt tolerant crop variety problem meaning for being badly in need of solving in Saline-alkali Field Control Justice is great, has broad application prospects.Studied therefore, having carried out tidal land Spartina alterniflora with the positive and negative distant hybridization method of paddy rice, Explore orthogonal：Paddy rice ♀ × Spartina alterniflora ♂；Reciprocal cross：Spartina alterniflora ♀ × paddy rice ♂ distant hybridization innovative approach.

The content of the invention

Raised it is an object of the invention to provide a kind of Spartina alterniflora/Rice Salt grain that can cultivate suitable salt-soda soil growth The tidal land Spartina alterniflora of dual-purpose type crop and the positive and negative distant hybridization method of paddy rice.

The present invention technical solution be：

A kind of tidal land Spartina alterniflora and the positive and negative distant hybridization method of paddy rice, it is characterized in that：Comprise the following steps successively：

(1) selection of tidal land Spartina alterniflora and management：Hybridize the tide band selection from the beach of seashore at the beginning of first trimester, i.e., 5 month The growth Spartina alterniflora plant that vigorous, stalk is more, stalk is thick, whole strain band soil loads in the ceramic cylinder being made ready beforehand for；Ceramic cylinder bore 26 ㎝, high 30 ㎝；It is transplanted into beside rice test crop field, is easy to irrigate, pours the salt solution of 1% concentration, poured 1 time every 15 days later The salt solution of 1% concentration, the Spartina alterniflora needed for training improved crossing；Plant Spartina alterniflora plant quantity according to cross combination how much Measuring and calculating is determined；There is 20-30 flower spike measuring and calculating by every Porcelain Jar Spartina alterniflora plant strain growth phase.

(2) it is orthogonal：Paddy rice ♀ × Spartina alterniflora ♂：Paddy rice ♀ hot gas gametocide promotees flower and inhales the in good time pollination methods of powder Spartina alterniflora ♂

1. the maternal emasculation of paddy rice：Hybridize noon before that day, the maternal fringe portion of selection paddy rice exposes leaf sheath 2/3-3/4 spike of rice, Plant root is put into plastic tub by the paddy rice maternal plant for having just enter into full-bloom stage with roots and soil, is fetched and is watered in interior, basin； The next morning 7：00-7：30 whole fringes, cut off the clever flower opened and the young tender small ear of fringe bottom, leave the rice for going to hybridization Fringe；With preprepared lift paddy rice hot gas gametocide brandreth, a row can hang upside down five wide-mouth empty bottles with mesh bag, you can simultaneously By the maternal gametocide of five basin paddy rice, each wide-mouth bottle can fill 2 ㎏ warm water, and bottle is put into the transparent plastic bag of thickness；In wide-mouth bottle first 60 DEG C of hot water is filled, water is poured out during emasculation, (thermometer is inserted at transparent plastic bag sack, after tested at this moment in wide-mouth bottle Hot air temperature is just 45 DEG C or so, can be kept 7-9 minutes), projecting wide-mouth empty bottle is set on the spike of rice of emasculation, transparent plastic Sack is blocked together with spike of rice stalk, thermometer, wide-mouth empty bottle mouthful tying；Through 5 minutes, wide-mouth empty bottle is removed, by 20 minutes left sides Right spike of rice just opens grain husk, end of blooming in about 30 minutes, immediately with the rapid flower pesticide opened by fringe exhaustion of starch absorbing device successively；Then cut off All grain husk flowers do not opened, bagging isolation waits pollination；

This method gametocide method is easy, can not only kill pollen, and have Accelerate bloom effect.Bloomed completely in 30 minutes, can Whole wipe out is spent to distinguish the grain husk that will do not bloom, it is to avoid later selfing and hybridization to obscure；Meanwhile, inhaled with starch absorbing device by fringe The flower pesticide opened to the greatest extent, prevents gametocide from not exclusively, selfing phenomenon occur.

Paddy rice female parent gametocide promotees the former field practice of worker employed in a plant nursery's work and is changed to field experiment operation, improves work efficiency.

2. pollination in good time：8：30-11：30, clip is being bloomed the tactile male parent Spartina alterniflora's tassel for having pollen to shed, and is leaned on Maternal spike of rice top after nearly emasculation bagging, shakes off pollen.One the spike of rice same day, the next morning respectively pollinate once；

3. hybrid plant is handled：After pollination is finished, paddy rice maternal plant is transplanted into crop field；After 20-25 days, during seed maturity Hybrid seed is harvested in time.

(3) reciprocal cross：Spartina alterniflora ♀ × paddy rice ♂：The continuous pollination methods when emasculation, paddy rice ♂ are continuous of Spartina alterniflora ♀

1. Spartina alterniflora makees maternal selection and processing：Taken measures according to the Spartina alterniflora's property spent feature as follows：

Select fringe：The one afternoon, and selection Spartina alterniflora's fringe top has a small amount of milky white color masterbatch chapiter just to appear, i.e., in selection Portion's small ear has been bloomed, and middle part small ear has the single fringe of the Spartina alterniflora that can be bloomed after 1-2 days again；

Whole fringe：The small ear that top has been bloomed all is removed with scissors, then the branch of bottom is obstructed, wrapped in the intravaginal institute of fringe There is tender grain seed to cut, it is to prepare hybridization pollination part to leave the middle part small ear do not bloomed；

Bagging：Bagging time is write on fringe portion after trimming bagging immediately, bag；

2. the maternal emasculation of Spartina alterniflora and pollination：Next day after bagging, morning 7：00-8：30, while emasculation, while pollinate, check Whether stamen appearance is had in bagging, and one finds stamen, and stamen is wiped out immediately；One finds that gynoecium style occurs, and pollinates, hangs immediately Board, writes paternal rice kind, first time hybridization time；After first day, first time hybridization pollination, first is repeated daily later Its step, is pollinated with first time cross combination rice varieties always, and until finding that gynoecium style has dried up in bagging, stamen is no longer Occur, i.e., no longer pollinate and check, the fringe combined hybrid terminates.Simultaneously hybridization is write listed finally in clip big envelope sack top Date；

Continuous every morning 7 after whole strain bagging：00-8：30, check that situation occur in stamen, gynoecium style in bagging；As a result It was found that：(1) start 1-5 days gynoecium styles, stamens and daily can all have new to go out fringe；(2) the 4-5 days after bagging, find in bagging Gynoecium style has dried up, and stamen no longer occurs, i.e., no longer pollinate and check；It is whole that Spartina alterniflora makees maternal continuous artificial emasculation pollination Individual process needs complete for 5-6 days.

Distant hybridization method is one of biological evolution, the key factor for creating variation, is to form novel species and the weight of new type Want approach.Spartina alterniflora and the positive and negative distant hybridization of paddy rice, can select salt tolerant transition silos crop.1. population is continuous at this stage Increase；2. grain aggregate demand is continuously increased at this stage；3. cultivated area is reduced increasingly；4. feelings of freshwater resources wretched insufficiency Under condition, excavated and innovation research by the excellent economic characters of paddy rice and Spartina alterniflora's resistant gene of salt germ plasm resource, cultivate salt tolerant grain Raise dual-purpose crop new varieties.The setting percentage of Spartina alterniflora and paddy rice distant hybridization are how improved, accelerates crossbreeding process, hybridization The improvement of method is crucial.Spartina alterniflora and the innovation of the positive and negative distant hybridization method of paddy rice, greatly accelerate new germ plasm initiative speed Degree, accelerates cultivation salt tolerant transition silos New Crop Varieties process, can not only solve to be badly in need of in coastal development, Saline-alkali Field Control The salt tolerant crop variety problem of solution, and solve the problems, such as the smart roughage of plant-eating animal.Increase simultaneously for the utilization of resources, agricultural There is important scientific meaning and economic value in terms of effect, grain security, arable land strategy, have broad application prospects.

With reference to embodiment, the invention will be further described.

Embodiment

1 materials and methods

1.1 times and place

Time：In May, -2015 in April, 2014

Place：1st, Inst. of Agricultural science for Area along Yangtze River, Jiangsu Prov.'s field experiment：Crossing work original field practice is changed to experimental field And laboratory operation；2nd, Jiangsu Tongzhou gulf new district salt-soda soil field experiment, crossing work is former to take powder pollination to be changed to salt-soda soil in tidal land Field experiment implements crossing work.

1.2 material

1.2.1 tidal land Spartina alterniflora selects Spartina alterniflora's plant seedling on the emerging Huanghai Sea beach rice grassland of cultivating of Jiangsu Province's Qidong City, It is transplanted to plantation management in Inst. of Agricultural science for Area along Yangtze River, Jiangsu Prov.'s test site ceramic cylinder；At Jiangsu Tongzhou gulf new district test site Transplant Spartina alterniflora in salt-soda soil.The selection of tidal land Spartina alterniflora and management：Hybridize first trimester (at the beginning of 5 months) damp from the beach of seashore With the Spartina alterniflora plant that growth selection is vigorous, stalk is more, stalk is thick, the extra large soil of whole strain band is planted into the ceramic cylinder (interior diameter being made ready beforehand for 26 ㎝, high 30 ㎝) in, move into beside rice test crop field, be easy to irrigate, pour 1% salt solution, poured 1 time every 15 days later 1% salt solution.How much the quantity of plantation Spartina alterniflora plant calculates determination according to cross combination；By every Porcelain Jar Spartina alterniflora plant There is 20-30 flower spike measuring and calculating in growth period.

1.2.2 rice varieties 1, big China are fragrant glutinous；2nd, Tianjin rice 1007；3rd, Wu Xiang glutinous 8333；4th, salt round-grained rice 9；5th, silver perfume 18；6、 Salt round-grained rice 10；7th, military fortune round-grained rice 7；8th, military round-grained rice 15；9th, clear glutinous 218；10th, military round-grained rice 7；11st, peaceful round-grained rice 1；12nd, lead to and educate round-grained rice 14； 13rd, southern round-grained rice 45；14th, southern round-grained rice 46；15th, southern round-grained rice 47；16th, upper reality 400；17th, Nipponbare；18、7K339；19th, military round-grained rice 13；20th, salt rice No. 9；21st, Wuyunjing No.8；22nd, excellent spoke round-grained rice.

Rice varieties are planted in Inst. of Agricultural science for Area along Yangtze River, Jiangsu Prov. experimental plot and Jiangsu Tongzhou gulf new district test site. It is animated period of blooming because of Spartina alterniflora's 8-10 months, for flower synchronization, hybridization in good time, since 20 days April in 2014,22 water Rice varieties are a collection of every sowing in 15 days, point 4 batches of sowings.

1.2.3 instrument and equipment

(1) self-control lift paddy rice lukewarm water gametocide brandreth, the Plastic Drum (commercially available) that 12 ㎏ water can be filled, self-control lift paddy rice Hot gas gametocide brandreth, wide-mouth bottle (commercially available), thermometer, the timing clock that 2 ㎏ water can be filled；Experiment pencil, water-color paintbrush, survey chi, record Form, clip, waterproof sulfuric acid paper bag, scissors etc. must be cleaned, dried, and be put into standby in experiment dedicated box.

(2) starch absorbing device material：By power test, two kinds are compared (commercially available) 1. vacuum starch absorbing device, model Mc-DL200, weight Measure 1.3 ㎏, supply voltage 220V, rated power 400W；2. portable multi-function starch absorbing device, model GD-03, the ㎏ of weight 0.5, electricity Source voltage 220V, rated power 150W；

1.3 method

1.3.1 father and mother's parent's Observation on Flowering Habits

Flowering habit after Spartina alterniflora occurs with Rice Panicle is observed, to determine hybridizing method and producing Crossbred time.

1.3.2 orthogonal one group of paddy rice ♀ cuts clever pollination method using lukewarm water gametocide

Hybridize noon before that day, the maternal fringe portion of selection paddy rice exposes leaf sheath 2/3-3/4, that is, the paddy rice for having just enter into full-bloom stage is female Root is put into plastic tub and fetches interior this plant with roots and soil, is watered in basin；The next morning 7：00-7：30 whole strains, cut off The clever flower and the young tender small ear of fringe bottom opened, leave the spike of rice for suitably doing and hybridizing；Brandreth is lifted with self-control, gametocide will be prepared Rice plant root puts polybag, hangs upside down on brandreth, and a Plastic Drum for filling with warm water is put below, water temperature is adjusted to and controlled At 42 DEG C, spike of rice is immersed in warm water and handled 10 minutes；Take out plant to be put into plastic tub, by 20 minutes or so spikes of rice successively Grain husk is opened, after about 20 minutes, end of blooming；The grain husk flower do not opened must be now wiped out, having opened the flower pesticide of grain husk will also remove, and cut off grain husk Shell top about 1/3, to improve stigma exposing ratio and setting percentage；Gametocide cuts bagging after grain husk.Now just 8：30-11：30, male parent is mutual Dew has been done on the careless fringe of flower rice, just in flowering.

Pollination：Clip is being bloomed the tactile Spartina alterniflora's male parent tassel for having pollen to shed, the mother after emasculation bagging This spike of rice top, shakes off pollen.One the cross combination spike of rice same day, the next morning respectively pollinate once, and big envelope sack top is simultaneously It is listed to write cross combination and date.

Hybrid plant processing：After pollination is finished, by paddy rice maternal plant Transplanting Test field.After 20-25 days, during seed maturity Harvest hybrid seed, prevents from going mouldy in time.

1.3.3 orthogonal two groups of paddy rice ♀ hot gas gametocide promotees the flower suction in good time pollination methods of powder Spartina alterniflora ♂

1. the maternal emasculation of paddy rice：Hybridize noon before that day, the maternal fringe portion of selection paddy rice exposes leaf sheath 2/3-3/4 spike of rice, Having just enter into the paddy rice maternal plant of full-bloom stage, root is put into plastic tub with roots and soil, is fetched and is watered in interior, basin；Second day Morning 7：00-7：30 whole fringes, cut off the clever flower opened and the young tender small ear of fringe bottom, leave the spike of rice for going to hybridization；With pre- First ready lift paddy rice hot gas gametocide brandreth a, row can hang upside down five wide-mouth empty bottles with mesh bag, you can while by five basins Paddy rice female parent gametocide, each wide-mouth bottle can fill 2 ㎏ hot water, and bottle is put into the transparent plastic bag of thickness；Upper 60 are first filled in wide-mouth bottle DEG C warm water, water is poured out during emasculation, (thermometer is inserted at transparent plastic bag sack, after tested at this moment hot gas temperature in wide-mouth bottle Degree just 45 DEG C or so, can keep 7-9 minute), hang upside down wide-mouth empty bottle and be set on the spike of rice of emasculation, transparent plastic sack together with Spike of rice stalk, thermometer, wide-mouth empty bottle mouthful tying are blocked；Through 5 minutes, wide-mouth empty bottle is removed, by 20 minutes or so spikes of rice just Grain husk, end of blooming in about 30 minutes, immediately with the rapid flower pesticide opened by fringe exhaustion of starch absorbing device are opened successively；Then cut off and all do not open Grain husk flower, bagging isolation, wait pollination；

This method gametocide method is easy, can not only kill pollen, and have Accelerate bloom effect.Bloomed completely in 30 minutes, can Whole wipe out is spent to distinguish the grain husk that will do not bloom, it is to avoid later selfing and hybridization to obscure；Meanwhile, inhaled with starch absorbing device by fringe The flower pesticide opened to the greatest extent, prevents gametocide from not exclusively, selfing phenomenon occur.

Paddy rice female parent gametocide promotees the former field practice of worker employed in a plant nursery's work and is changed to field experiment operation, can improve work efficiency.

2. pollination in good time：8：30-11：30, clip is being bloomed the tactile male parent Spartina alterniflora's tassel for having pollen to shed, and is leaned on Maternal spike of rice top after nearly emasculation bagging, shakes off pollen.One the spike of rice same day, the next morning respectively pollinate once；

3. hybrid plant is handled：After pollination is finished, paddy rice maternal plant is transplanted into crop field；After 20-25 days, during seed maturity Hybrid seed is harvested in time.

1.3.4 Spartina alterniflora makees maternal selection and processing, it is adaptable to which reciprocal cross 1-3 groups are tested below.

Taken measures according to the Spartina alterniflora's property spent feature as follows：(1) fringe is selected one afternoon, growth selection is vigorous, stalk is more, The thick Spartina alterniflora of stalk, see Spartina alterniflora's fringe top have on a small quantity (fringe top is about 3-5 ㎝) milky white color masterbatch chapiter just appear, i.e., Selection top small ear has been bloomed, and middle part small ear has the single fringe of the Spartina alterniflora that can be bloomed after 1-2 days again；(2) whole fringe scissors The small ear that top has been bloomed all is removed, then branch stalk, the bag of bottom are cut in the intravaginal all tender grain seeds of fringe, is left not It is to prepare hybridization pollination part to have the middle part small ear bloomed；3. bagging：Bagging is write on fringe portion after trimming bagging immediately, bag Time.

1.3.5 one group of reciprocal cross：Cut clever producing Crossbred method：Directly artificial single flower is carried out after the whole fringes of Spartina alterniflora ♀ with tweezers It is male.Glume top is cut off 1/4, removing flower pesticide is stretched into from breach with tweezers, is then enclosed on and has been gone on male tassel with paper bag, The listed dated emasculation date.

Pollination：2nd, 3 days see that milky white color masterbatch chapiter is appeared, continuous two bestowed by heaven paddy rice paternal pollen.After hybridization is finished, envelope Cover sack top and be listed and write cross combination and date.

1.3.6 two groups of reciprocal cross：The continuous producing Crossbred methods of Spartina alterniflora ♀

Next day after bagging, morning 7：00-8：30, a discovery stamen in bagging is checked, stamen is wiped out immediately；One finds gynoecium Style occurs, and pollinates, is listed immediately, writes paternal rice kind, write first time hybridization time.From first day, for the first time it is miscellaneous Hand over after pollination, repeat first day step daily later, pollinated always with first time cross combination rice varieties.Lime light：Every time Check bagging, it is necessary to first emasculation, rear pollination.Until finding that gynoecium style has dried up in bagging, stamen no longer occurs, i.e., no longer award Powder and inspection, the fringe combined hybrid terminate.Simultaneously listed write the hybridization last time date in big envelope sack top.

The maternal former pollination emasculation of Spartina alterniflora is operated in tidal land operation and is changed to experimental field and laboratory operation；Original pollination is once Not emasculation is changed to continuous emasculation, continuous pollination.

1.3.7 three groups of reciprocal cross：The continuous producing Crossbred method of Spartina alterniflora's ♀ lukewarm water gametocides

After the whole fringes of Spartina alterniflora ♀ directly with 38 DEG C, 39 DEG C, 40 DEG C, 41 DEG C, 42 DEG C, 43 DEG C, 44 DEG C, 45 DEG C, 46 DEG C, 47 DEG C, 48 DEG C of lukewarm waters leaching 10 minutes gametocides of fringe, fringe portion after gametocide bagging folding envelope immediately writes gametocide time and temperature on bag.Observation Spartina alterniflora ♀ is from 38-48 DEG C of lukewarm water gametocidal effect.

Continuous producing Crossbred method is with two groups of reciprocal cross after lukewarm water gametocide.

1.3.8 Spartina alterniflora × paddy rice cross breeding seed insemination and emergence experiment

It is orthogonal：Paddy rice ♀ × Spartina alterniflora's ♂ hybrid seeds normal temperature is preserved；Reciprocal cross：Spartina alterniflora ♀ × paddy rice ♂ hybrid seeds Divide two kinds of preserving types：(1) normal temperature is preserved；(2) the salt water-soaked of 1.5% concentration of cenospecies, puts 0-5 DEG C of refrigerator storage into Deposit；Three groups of reciprocal cross is more because of 38 DEG C of -42 DEG C of seeds, so two kinds of preserving types of design；Control group Spartina alterniflora's seed is 2014 The seed gathered by the end of November in Jiangsu Tongzhou gulf new district tidal land；Hybridize seed 10-11 days in May, 2015 sowing saline and alkaline in Tongzhou gulf Ground, soil salt content 7-10 ‰.

2 results and analysis

2.1 Parent Observation on Flowering Habits results

2.1.1 the flower observation of Spartina alterniflora：(1) fringe occurs, and column cap just exposes after 3-7 days, if not pollinating, column cap can Growth 5-6 days；(2) maternal instinct style first goes out fringe, and white featheriness is exposed when blooming；(3) after column cap comes out, 4-5 days it After there is stamen, withered column cap is pressed against side；(4) stamen blooms in the morning 8：30-11：It is most when 30；(5) flower pesticide is stretching out When split, longitudinal cracking when flower pesticide is ripe, pollen grain shed within two hours, pollen yellow；(6) filigree extends quickly, and two Withered after hour.

2.1.2 the flower observation of paddy rice：(1) spike of rice is extracted out after leaf sheath, and grain husk flower is on the day of or the 2nd day starts to bloom, filigree Extend flower pesticide simultaneously to eject and loose powder of splitting, pollen yellow；(2) flowering time is in normal fine day, the morning 8：30-11：When 30 At most.

Spartina alterniflora and paddy rice difference：(1) Spartina alterniflora's maternal instinct style first goes out fringe, is exposed when blooming；(2) column cap Go out after fringe, occur stamen after 4-5 days.

Spartina alterniflora and paddy rice identical point：(1) if Spartina alterniflora does not pollinate, column cap can grow 5-6 days；Paddy rice pistil stigma Can still be pollinated fertilization in 3-5 days；(2) stamen blooms in the morning 8：30-11：It is most when 30.

Thus it can determine that：(1) Spartina alterniflora award paddy rice stamen pollen as female parent can be 4-5 days；(2) paddy rice awards mutual flower as female parent The careless stamen pollen of rice can be 2-3 days；(3) emasculation is 8：Before 00, pollination in the morning 8：30-11：30.

2.2 it is orthogonal：Paddy rice ♀ × Spartina alterniflora ♂

2.2.1 orthogonal one group of paddy rice ♀ lukewarm water gametocides cut grain husk pollination hybrid method

Once experiment in 2006 cuts grain husk with the maternal lukewarm water gametocide of 6 paddy rice, with the pollination of Spartina alterniflora's male parent once, hybridization 39 Fringe, 32 fringes of setting seeds must hybridize seed 115, and fringe is set seeds 3.59；Grain husk is cut with the maternal fringe lukewarm water gametocide of paddy rice within 2009, use Spartina alterniflora pollinates once, hybridizes 35 fringes, obtains hybrid seed 328, fringe is set seeds 9.37.

This Study On Rice ♀ lukewarm water gametocides cut grain husk pollination hybrid method, are pollinated with Spartina alterniflora secondary, hybridize 50 fringes, fringe of setting seeds 34 fringes, obtain hybrid seed 431, and fringe is set seeds 12.68, than 2006, are respectively increased 253.20%, 35.33% within 2009.It is miscellaneous Knot truth condition is shown in Table 1.

Paddy rice ♀ lukewarm water gametocides cut the problem of grain husk pollination hybrid method is present：Lukewarm water gametocide technical requirements are higher, it is necessary to control Suitable temperature and leaching fringe time, accomplish both all to kill stamen, and without for fear of gynoecium.Slightly neglect, otherwise it is exactly to reach Less than gametocidal effect, otherwise it is exactly that whole tassel all scalds withered；Cut clever emasculation workload very big, it is necessary to devote a tremendous amount of time, Influence hybridization speed and number of combinations.

2.2.2 orthogonal two groups of paddy rice ♀ hot gas gametocide promotees the flower suction in good time pollination methods of powder Spartina alterniflora ♂

Orthogonal two groups numbers 2723 of setting seeds, initial survey goes out the cenospecies grain of rice substantially as paddy rice female parent, rice husk gilvous during sowing With the visibly different pseudostationary of cenospecies 114,4.19% is accounted for, has been removed when harvesting and storing, number of actually setting seeds is 2609.It is orthogonal Two groups same secondary with Spartina alterniflora's pollination, hybridizes the fringe of spike number 102,52 fringes more than orthogonal one group of hybridization spike number are doubled, ties Seed fringe improves 34.09%, and fringe is set seeds 28.05, is set seeds 12.68 than orthogonal one group of fringe and improves 121.21%, hybridizes seed Grain does not go mouldy, and orthogonal one group of hybridization seed rate of going mouldy accounts for 8.35%.Cross-fertile situation is shown in Table 1

Two kinds of starch absorbing devices are compared by experiment, inhale powder emasculation instrument with portable multi-functional starch absorbing device, model GD-03 is light Just, flexibly, be applicable.It is lightweight：0.5 ㎏, suction powder is gently uniform, and rice leaf and grain husk flower, power are not damaged：150W.

The paddy rice ♀ of the table 1. × solid situation of Spartina alterniflora's ♂ distant hybridization

2.3 reciprocal cross：Spartina alterniflora ♀ × paddy rice ♂

2.3.1 one group of reciprocal cross：Spartina alterniflora ♀ cuts clever producing Crossbred hybrid method

Spartina alterniflora ♀ cuts secondary with the pollination of paddy rice male parent after clever emasculation.Because Spartina alterniflora's floral organ is small, manually cuts grain husk and go Male difficulty is big and takes a lot of work；Although maternal instinct style has no out fringe when cutting clever emasculation, unless you give your whole attention to it, floral organ, reduction knot will be damaged Real rate；Fringe of setting seeds accounts for 35%, and fringe is solid only 4.86, the rate 11.76% while hybridization seed goes mouldy.So, this method is unsuitable Using.Cross-fertile situation is shown in Table 2

2.3.2 two groups of reciprocal cross：The continuous producing Crossbred hybrid methods of Spartina alterniflora ♀

Two groups of reciprocal cross Spartina alterniflora ♀ continuous producing Crossbreds 4-5 days, hybridize the fringe of spike number 40, than one group of 20 fringe increase by one of reciprocal cross Times, fringe of setting seeds increases by 3.14 times, 20.62 times of number increase of setting seeds, and fringe is set seeds 4.21 times of number increase, and hybridization seed is without going mouldy.

Continuous every morning 7 after whole strain bagging：00-8：00, check that situation occur in bagging stamen, gynoecium style.As a result send out It is existing：(1) start 1-5 days gynoecium styles, stamens and daily can all have new to go out fringe；(2) the 4-5 days after bagging, find female in bagging Stamen style has dried up, and stamen no longer occurs, i.e., no longer pollinate and check.It is whole that Spartina alterniflora makees maternal continuous artificial emasculation pollination Process needs complete for 5-6 days.Cross-fertile situation is shown in Table 2

The Spartina alterniflora ♀ of the table 2. × solid situation of paddy rice ♂ distant hybridization

2.3.3 three groups of reciprocal cross：The continuous producing Crossbred hybrid method of Spartina alterniflora's ♀ different temperatures lukewarm water gametocides

Experiment adds treatment of different temperature process on the basis of two groups of continuous producing Crossbred hybrid methods of reciprocal cross.(1) temperature At 38-42 DEG C, fringe of setting seeds accounts for 44.44-60.00%, the solid 17.13-19.78% of fringe, and optimum efficiency is reached with 42 DEG C； (2) when temperature is at 43-48 DEG C, the solid number of fringe of setting seeds, fringe is gradually reduced, to lukewarm water gametocide at 46-48 DEG C after most of fringe Son is withered at 1-2 days.Cross-fertile situation is shown in Table 3

Result of the test shows：(1) Spartina alterniflora ♀ treatment of different temperature reaches best effective with paddy rice ♂ distant hybridization with 42 DEG C Really, it is solid 19.78 that fringe of setting seeds accounts for 60.00%, fringe.But still it is solid to account for 72.5%, fringe than two groups of fringes of setting seeds of reciprocal cross 25.34 grain is low；(2) after different temperatures lukewarm water gametocide first day find temperature at 38-42 DEG C with two groups of kindred circumstances of reciprocal cross, open 1-5 days beginning gynoecium style, stamen can all have the new fringe that goes out daily, equally want continuous artificial emasculation pollination, illustrate the temperature of first day Soup gametocide does not work simultaneously, on the contrary minor injury's floral organ, influences fringe setting percentage；Temperature is at 43-48 DEG C, and fringe of setting seeds, fringe are tied Real number is gradually reduced situation analysis, major injury floral organ even whole tassel when temperature is too high；(3) different temperatures lukewarm water gametocide Lukewarm water gametocide process is added, chemical industry amount is big, and effect is still not so good as two groups of reciprocal cross.So, Spartina alterniflora's ♀ different temperatures lukewarm waters are killed Male continuous artificial emasculation pollination's method should not be used.

The Spartina alterniflora ♀ different temperatures lukewarm water gametocides of table 3. and the solid situation of paddy rice ♂ distant hybridization

2.4 Spartina alternifloras × paddy rice cross breeding seed insemination and emergence result

2.4.1 orthogonal systems seed air drying stores interpretation of result insemination and emergence of emerging and the results are shown in Table 4

Orthogonal two groups double than orthogonal one group of cross combination number (spike number), and sowing number increases by 5.6 times, number of emerging increase 9.57 times, emergence rate increase by 60.07%.2.4.2 reciprocal cross group seed air drying stores interpretation of result of emerging

Reciprocal cross group seed air drying storage sowing 7 groups, sow seed 375, control group sow 100, from May 1- Germination is showed no in 30 days, 3 months July and is emerged, checks that seed has rotted.Analysis：Cenospecies still heredity " mutually spend rice Its careless seed has the characteristics of dehydration loses germination ".2.4.3 reciprocal cross group seed salt solution refrigerates interpretation of result of emerging

Two groups of reciprocal cross is doubled than one group of cross combination number (spike number) of reciprocal cross, and sowing number increases by 23.5 times；Air drying Two groups are stored not emerge；Emergence rate 56.15% after the refrigeration of seed tape salt solution is planted, than one group of increase by 68.47% of reciprocal cross, compares control group 39.00% increase by 43.97%.

38 DEG C of -42 DEG C of seed salt solution of reciprocal cross three group refrigerate emergence rate in 39.33-45.76%；42 DEG C of two groups of emergence rates 45.76% is highest, than the increase of control group 42.00% 8.95%, but still than the low emergence rate of two groups of emergence rates of reciprocal cross 56.15% 22.71%.

The different preserving type insemination and emergence situations of 4. Spartina alternifloras of table × paddy rice cross breeding seed

Note：(1) hold mode 1：Seed harvest each cross combination seed waterproof sulfuric acid paper bag packaging seal after drying, often Temperature dries storage in May, 2015 sowing；(2) hold mode 2:Not dried after seed harvest each will use anti-by cross combination seed Water sulfuric acid paper bag packaging seal, with the salt water-soaked of 1.5% concentration, puts 0-5 DEG C of refrigerator into and stores in May, 2015 sowing； (3) control group Spartina alterniflora seed be Jiangsu Tongzhou gulf new district tidal land gather seed, in by the end of November, 2014 with test group simultaneously Sowing.

3 conclusions are with discussing

3.1 Observation on Flowering Habits determine Spartina alterniflora and paddy rice distant hybridization emasculation, Pollination Times and time.

(1) the flower protogyny of Spartina alterniflora, its column cap stretches out before pollen bag splits, to receive the flower of precocious flower Powder, therefore be conducive to receiving cross-pollination；(2) if Spartina alterniflora does not pollinate, column cap can grow 5-6 days；Paddy rice pistil stigma exists Can still be pollinated fertilization in 3-5 days.Determine that both sides parent can continuously pollinate, continuously can at least pollinate 2-3 times.

Innovative point：(1) find that the flowering habit of Spartina alterniflora is conducive to receiving the cross-pollination of the male powder of paddy rice, illustrate mutually flower Rice grass can carry out distant hybridization with paddy rice；(2) Spartina alterniflora is bloomed 8 with paddy rice stamen：30-11：When 30 at most, Determine emasculation 8：Before 00, pollinate 8：30-11：30.Awarded in good time 3.2 paddy rice ♀ hot gas gametocide promotees flower suction powder Spartina alterniflora ♂ Powder hybrid method is applied to paddy rice ♀ × Spartina alterniflora ♂ (orthogonal)

This law (orthogonal two groups) is compared with the maternal lukewarm water gametocide of paddy rice cuts grain husk pollination hybrid method (orthogonal one group), cross combination Number 102,52 more than orthogonal one group of cross combination number, adds one times；Number of combinations of setting seeds improves 34.09%；Fringe is set seeds 28.05,121.21% is improved than orthogonal one group 12.68；Hybridization seed does not go mouldy, and orthogonal one group of hybridization seed goes mouldy rate Account for 8.35%；Sowing number increases by 5.6 times, and number of emerging increases by 9.57 times, and emergence rate improves 60.07%；Improve hybridization speed 6-7 Times, the not enough problem of labour during crossbreeding is alleviated significantly.

Innovative point：(1) the former field practice of paddy rice female parent pollination work is changed to experimental field and laboratory operation；Original pollination is once It is changed to continuously to pollinate secondary；(2) promote flower with hot gas processing spike of rice and portable multi-function starch absorbing device is absorbed flower pesticide and combined closely Get up, paddy rice female parent is removed lukewarm water gametocide from and is cut clever emasculation process；(3) technology content of the maternal emasculation of paddy rice is improved, no Corollary equipment is needed, operation difficulty is reduced, working efficiency is improved；(4) the obtained seed of hybridization is paddy, rather than the grain of rice, Seed circularity is good, not mouldy, thus substantially increases setting percentage and emergence rate.

The continuous producing Crossbred hybrid methods of 3.3 Spartina alterniflora ♀ are applied to Spartina alterniflora ♀ × paddy rice ♂ (reciprocal cross)

This law (two groups of reciprocal cross) is compared with Spartina alterniflora ♀ cuts clever producing Crossbred hybrid method (one group of reciprocal cross), cross combination number 40, than reciprocal cross one group, 20 are doubled, and number of combinations of setting seeds increases by 3.14 times, 20.62 times of number increase of setting seeds, and fringe is set seeds number 4.21 times of increase, hybridization seed is without going mouldy；Sowing number increases by 23.5 times；Emergence rate 56.15% after the refrigeration of seed tape salt solution is planted, than anti- One group of increase by 68.47% is handed over, than the increase of control group 39.00% 43.97%.Improve 7-8 times of speed of hybridization.

Innovative point：(1) the maternal former pollination emasculation of Spartina alterniflora is operated in tidal land operation and is changed to experimental field and laboratory operation； Once not emasculation is changed to continuous emasculation, continuous pollination for original pollination.(2) according to Spartina alterniflora's flowering habit take continuous side emasculation, Side is pollinated, and Spartina alterniflora's female parent is removed lukewarm water gametocide from and is cut clever emasculation process；(3) improve the maternal emasculation of Spartina alterniflora, award The technology content of powder, it is not necessary to corollary equipment, reduces operation difficulty；(4) seed circularity is good, not mouldy, thus carries significantly High setting percentage, emergence rate.

3.4 next step projects

3.4.1 carry out Field Screening and Molecular Detection the hybridization new germ plasm of initiative is carried out salt-soda soil plantation field observation, The screening of cenospecies, differentiates documenting analysis, the Integrated Selection of the economical characters such as true and false hybrid, salt-tolerance character；It is miscellaneous to what is filtered out Hand over kind of a progress laboratory Molecular Detection checking.

3.4.2 in terms of the application scale and scope selection Crossing system, the parent that expand distant hybridization innovative approach：In remote edge During hybridization, reciprocal cross is then more easy to the different situation of Percentage bound occur, and the result of reciprocal cross is different, therefore advocates reciprocal cross all Do；, should be using cultigen as female parent when cultigen and wild species hybridize；In the different distant hybridization of chromosome number, to contaminate Female parent being done colour solid number more.Further expand from now on orthogonal：Paddy rice ♀ × Spartina alterniflora ♂；Reciprocal cross：Spartina alterniflora ♀ × paddy rice The application scale and scope of ♂ distant hybridization innovative approach, accelerate the more new germ plasms of initiative and salt tolerant transition silos New Crop Varieties.

Distant hybridization method is one of biological evolution, the key factor for creating variation, is to form novel species and the weight of new type Want approach.1. population constantly increases at this stage；2. grain aggregate demand is continuously increased at this stage；3. cultivated area subtracts increasingly It is few；4. in the case of freshwater resources wretched insufficiency, the excellent economic characters of paddy rice and Spartina alterniflora's resistant gene of salt germ plasm resource are passed through Excavate and innovation research, salt tolerant transition silos New Crop Varieties can be cultivated.This research for excavate tidal land Spartina alterniflora salt tolerant and Rice high yield germ plasm resource provides the innovative usability methods of positive and negative distant hybridization, accelerates initiative salt tolerant transition silos crop New varieties process, not only solves the salt tolerant crop variety problem for being badly in need of solving in alkaline land improvement, and solve plant-eating animal Smart roughage problem, simultaneously for the utilization of resources, growth of agricultural efficiency, grain security, arable land strategy in terms of have important science Meaning and economic value, have broad application prospects.

Claims (2)

1. a kind of tidal land Spartina alterniflora and the positive and negative distant hybridization method of paddy rice, it is characterized in that：Comprise the following steps successively：

(1) selection of tidal land Spartina alterniflora and management：Hybridize at the beginning of first trimester, i.e., 5 month the tide band growth selection from the beach of seashore The Spartina alterniflora plant that vigorous, stalk is more, stalk is thick, whole strain band soil loads in the ceramic cylinder being made ready beforehand for；The ceramic ㎝ of cylinder bore 26 is high 30㎝；Be transplanted into beside rice test crop field, be easy to irrigate, pour the salt solution of 1% concentration, poured later every 15 days 1 time it is 1% dense The salt solution of degree, the Spartina alterniflora needed for training improved crossing；How much the quantity of plantation Spartina alterniflora plant is calculated according to cross combination It is determined that；There is 20-30 flower spike measuring and calculating by every Porcelain Jar Spartina alterniflora plant strain growth phase；

(2) it is orthogonal：Paddy rice ♀ × Spartina alterniflora ♂：Paddy rice ♀ hot gas gametocide promotees flower and inhales the in good time pollination methods of powder Spartina alterniflora ♂

1. the maternal emasculation of paddy rice：Hybridize noon before that day, the maternal fringe portion of selection paddy rice exposes leaf sheath 2/3-3/4 spike of rice, i.e., just Paddy rice maternal plant into full-bloom stage is put into plastic tub fetches interior with roots and soil, is watered in basin；The next morning 7：00- 7：30 whole fringes, cut off the clever flower opened and the young tender small ear of fringe bottom, leave the spike of rice for going to hybridization；With the liter being made ready beforehand for Drop formula brandreth, row's lift brandreth hangs upside down five wide-mouth empty bottles with mesh bag, you can while by the maternal gametocide of five basin paddy rice, each Wide-mouth bottle can fill 2 ㎏ warm water, and bottle is put into transparent plastic bag；60 DEG C of hot water is first filled in wide-mouth bottle, water is fallen during emasculation Go out, thermometer is inserted at transparent plastic bag sack, hot air temperature is 45 DEG C in wide-mouth bottle during emasculation, projecting wide-mouth empty bottle is set to On male spike of rice, transparent plastic sack is blocked together with Rice Neck stalk, thermometer, wide-mouth empty bottle mouthful tying；Through 5 minutes, remove Wide-mouth empty bottle, grain husk is just opened by 20 minutes spikes of rice successively, end of blooming for 30 minutes, has been opened immediately with rapid exhausted by fringe of starch absorbing device Flower pesticide；Then all grain husk flowers do not opened are cut off, bagging isolation waits pollination；

2. pollination in good time：8 on the day of emasculation：30-11：30, clip is being bloomed the tactile male parent Spartina alterniflora for having pollen to shed Tassel, the maternal spike of rice top after emasculation bagging, shakes off pollen；One the spike of rice same day, the next morning respectively pollinate once；

3. hybrid plant is handled：After pollination is finished, paddy rice maternal plant is transplanted into crop field；It is timely during seed maturity after 20-25 days Harvest hybrid seed；

(3) reciprocal cross：Spartina alterniflora ♀ × paddy rice ♂：The continuous pollination methods when emasculation, paddy rice ♂ are continuous of Spartina alterniflora ♀

1. Spartina alterniflora makees maternal selection and processing：Taken measures according to the Spartina alterniflora's property spent feature as follows：

Select fringe：The one afternoon, and selection Spartina alterniflora's fringe top has a small amount of milky white color masterbatch chapiter just to appear, that is, selects top small Fringe has been bloomed, and middle part small ear has the single fringe of the Spartina alterniflora that can be bloomed after 1-2 days again；

Whole fringe：The small ear that top has been bloomed all is removed with scissors, then branch stalk, the bag of bottom is intravaginal all tender in fringe Grain seed is cut, and it is to prepare hybridization pollination part to leave the middle part small ear do not bloomed；

Bagging：Bagging time is write on fringe portion after trimming bagging immediately, bag；

2. the maternal emasculation of Spartina alterniflora and pollination：Next day after bagging, morning 7：00-8：30, while emasculation, while pollinate, check bagging Inside whether there is stamen appearance, one finds stamen, and stamen is wiped out immediately；One finds that gynoecium style occurs, and pollinates, is listed immediately, writes Upper paternal rice kind, first time hybridization time；After first time hybridization pollination, first time hybridization pollination step is repeated daily later Suddenly, pollinated always with first time cross combination rice varieties, until finding that gynoecium style has dried up in bagging, stamen no longer goes out It is existing, i.e., no longer pollinate and check；The fringe combined hybrid terminates, and hybridization is simultaneously write finally in clip big envelope sack top listed Date；

Continuous every morning 7 after whole strain bagging：00-8：30, check that situation occur in bagging stamen, gynoecium style；As a result find： (1) start 1-5 days gynoecium styles, stamens and daily can all have new to go out fringe；(2) the 4-5 days after bagging, pistillate flower in bagging is found Post has dried up, and stamen no longer occurs, i.e., no longer pollinate and check；Spartina alterniflora makees maternal continuous artificial emasculation pollination's whole process Need complete within 5-6 days.

2. tidal land Spartina alterniflora according to claim 1 and the positive and negative distant hybridization method of paddy rice, it is characterized in that：Spartina alterniflora ♀ × paddy rice ♂：The continuous pollination method steps when emasculation, paddy rice ♂ are continuous of Spartina alterniflora ♀ 1. in, Spartina alterniflora makees maternal selection With processing：Select fringe one afternoon, the Spartina alterniflora that growth selection is vigorous, stalk is more, stalk is thick sees that Spartina alterniflora's fringe top has on a small quantity Milky white color masterbatch chapiter is just appeared, that is, selects top small ear to bloom, and middle part small ear has can be bloomed after 1-2 days mutual again The mono- fringes of Hua meter Cao；

Whole fringe：The small ear that top has been bloomed all is removed with scissors, then branch stalk, the bag of bottom is intravaginal all tender in fringe Grain seed is cut, and it is to prepare hybridization pollination part to leave the middle part small ear do not bloomed；

Bagging：Bagging time is write on fringe portion after trimming bagging immediately, bag；

Step 2. in, Spartina alterniflora female parent emasculation with pollination：First day after bagging, morning 7：00-8：30, while emasculation, while pollinate, Check whether there is stamen appearance in bagging, one finds stamen, and stamen is wiped out immediately；One finds that gynoecium style occurs, pollination immediately, It is listed, write paternal rice kind, first time hybridization time；After first time hybridization pollination, repeat daily later miscellaneous for the first time Pollination step is handed over, is pollinated always with first time cross combination rice varieties, until finding that gynoecium style has dried up in bagging, stamen No longer occur, i.e., no longer pollinate and check, the fringe combined hybrid terminates；Simultaneously hybridization is write listed in clip big envelope sack top The last time date；

Continuous every morning 7 after whole strain bagging：00-8：30, check that situation occur in bagging stamen, gynoecium style；As a result find： (1) start 1-5 days gynoecium styles, stamens and daily can all have new to go out fringe；(2) the 4-5 days after bagging, pistillate flower in bagging is found Post has dried up, and stamen no longer occurs, i.e., no longer pollinate and check；Spartina alterniflora makees maternal continuous artificial emasculation pollination's whole process Need complete within 5-6 days.