CN106386480B - It is a kind of to use cytohistology technology determination oil palm immature inflorescence method for distinguishing - Google Patents

Abstract

The invention belongs to phytobiology fields, it is related to a kind of cytohistology technology determination oil palm immature inflorescence method for distinguishing, it is to choose the inflorescence in male and female not dominant III phase of phase, slice is fabricated to after fixation, dehydration and transparent, waxdip and embedding, slice and roasting piece, dewaxing and dyeing, slice is observed and taken pictures with microscope, petal piece former base number, petal the piece number and new petal piece former base number, flower primordium separate living tissue quantity are calculated, the gender of inflorescence is then judged according to the decision rule of oil palm immature inflorescence gender.The present invention is according to the female of oil palm, male inflorescence in morphology and the difference of cytohistology level, using cytohistology technology ahead of time in the not dominant phase judgement inflorescence gender of male and female, effectively shorten search time, accelerate flow of research, relationship further to study oil palm inflorescence Sex Differentiation mechanism and external factor provides basis, this is of great significance for realizing florescence and sex ratio regulation and control in being produced in oil palm, improving yield etc..

Description

It is a kind of to use cytohistology technology determination oil palm immature inflorescence method for distinguishing

Technical field

The invention belongs to phytobiology fields, are related to a kind of determination method of inflorescence gender, specifically a kind of to use groups of cells It knits and learns technology determination oil palm immature inflorescence method for distinguishing.

Background technology

The Sex Differentiation of plant inflorescence is the stage particularly important in flowering plant growth course, is that plant life continues Key link is that phenomenon occurs for a complicated morphogenesis process and a kind of special organ.Understand the inflorescence of plant Other atomization, the variation including cytohistology feature and related Endogenous Hormone Contents in Vitro, for understand its Developmental Biology and Reproductive biology realizes that florescence and sex ratio regulation and control, hybridization, heterosis utilization etc. have important meaning in production Justice.

Oil palm is important woody oleiferous plants crop, and the oil production of unit area leads and bounds ahead of other oil plants in the world Crop is referred to as " world oil king ".Can oil palm inflorescence development and the decision of Sex Differentiation process form high-quality female, male inflorescence and fruit Real, this is the precondition for obtaining high yield and high quality palm oil.Oil palm inflorescence development and Sex Differentiation process may be with planting sites The environmental factors such as temperature, humidity and abiotic factor (such as water stress), metabolic factor (such as organic C storage), fertilizer element kind The abiotic factors such as the factors such as class and level, hormonal readiness are related.By regulating and controlling various factors it is possible that realizing oil in production Brown florescence and female, male inflorescence sex ratio regulation and control, heterosis utilization, tissue cultures and the high-quality inflorescence of formation and fruit, with Phase improves oil palm yield, obtains high yield and high quality palm oil.Therefore carry out between oil palm inflorescence Sex Differentiation process and external factor Relationship research it is extremely important.

Since oil palm inflorescence is the different sequence of monoecism, time of 2~3 years will be undergone by germinating from inflorescence to opening (Corley, 1976), process is very long, after this is substantially distinguished from other most plants, and inflorescence opens, the inflorescence of female inflorescence Axis is made of about 150 rachillas, and the rachis of male inflorescence be made of 100~300 rachillas (Jacquemard, 1995).Conventional oil palm inflorescence sex identification could about carry out in inflorescence opening period, and the time is long, is unfavorable for studying.In consideration of it, Female, male inflorescence gender is judged ahead of time, is understood the relationship between inflorescence Sex Differentiation process and external factor in time, is effectively shortened Search time accelerates flow of research, is carried for the relationship further between research oil palm inflorescence Sex Differentiation process and external factor For basis.

The temperature in south China winter is more cold, and the oil palm locally cultivated is adaptable to environment, with equator The oil palm of cultivation has marked difference, but the country is so far without the relevant report of its inflorescence Sex Differentiation cytohistology research.

Invention content

Cytohistology technology determination oil palm immature inflorescence method for distinguishing is used the object of the present invention is to provide a kind of, according to Female, male inflorescence judges female, male inflorescence gender in morphology and the difference of cytohistology level in the male and female not dominant phase ahead of time, Relationship further to study oil palm inflorescence Sex Differentiation mechanism and external factor provides basis, this is for real in being produced in oil palm Existing florescence and sex ratio regulation and control, raising yield etc. are of great significance.

The technical principle of the present invention：

Oil palm into age tree each inflorescence all in the different puberties, the present inventor is remembered with shoot apical meristem It is 0, the inflorescence near it is denoted as N1, followed by N2, N3 ..., in order successively to inflorescence sequencing numbers, different inflorescences Number represents the inflorescence different stage of development.Length changing rule after the entire length of each inflorescence and removal bract is shown in Table 1, attached drawing 1, attached drawing 2.Inflorescence quality change rule after the total quality of each inflorescence and removal bract be shown in Table 2, Fig. 3, Fig. 4.

Length situation of change during 1 oil palm inflorescence development of table

Quality change situation during 2 oil palm inflorescence development of table

From table 1, table 2 and Fig. 1~4 as can be seen that N1~N44 stages, the rate of development of inflorescence are very slow.From N45 Start, inflorescence is developed rapidly, inflorescence length and the exponential notable growth of quality, this period is inflorescence development Exponential growth stage (the exponential growth stage of the inflorescence,EGSI).At the beginning of oil palm Flower sex dlfferentlation, Inflorescence starts to develop rapidly, into EGSI.Female, male inflorescence growth curve is roughly the same, and the length of generally male inflorescence is more than The length of female inflorescence.As the protection sexual organ of inflorescence, length and the quality growth of bract occupy important portion in inflorescence growth Point.The quality growth for removing the inflorescence of bract has hysteresis phenomenon relative to the quality growth of bract.As inflorescence is constantly developed, bud Piece continued propagation and lignifying.

It researchs and analyses and shows that the growth course of oil palm inflorescence can be divided into the not dominant phase (N1~N46) of male and female and male and female are dominant Two Main Stages of phase (N47~N56), wherein male and female not dominant phase development time phase more dominant than male and female are much longer, account for about total The 80% of period.

The male and female not dominant phase：Including I phase, II phase, III phase：I phase was the gender undifferentiated phase, including N1~N26 inflorescence stages； II phase was the transitional period, and including N27~N37 inflorescence stages, (in the N27 stages, rachilla separate living tissue occurs；Rachilla point during N35 Raw tissue forms petal piece former base；Rachilla meristem development goes out petal piece during N36)；III phase was the Sex Differentiation phase, including In N38~N46 inflorescence stages, inflorescence development interval time overall length is about 4.5 months, is available cytohistology technology determination inflorescence In gender period, after inflorescence development, inflorescence follows petal piece former base growth (N38 stages) → petal piece and new on rachilla Petal piece former base growth (N39, N40) → flower primordium separate living tissue forms (N41~46 stage) this sequential development differentiation, male flower Related petal piece former base number on rachilla of sequence, female inflorescence, petal the piece number and new petal piece former base number, flower primordium are mitogenetic The difference that tissue number has the order of magnitude (according to statistics, is spent with 5~30 three bases on the ripe each rachilla of female inflorescence, is formed With 400~500 male flowers on the ripe each rachilla of male inflorescence), immature inflorescence gender can be judged accordingly.For example, spending original Base separate living tissue formation stages, the flower primordium separate living tissue number on a certain inflorescence rachilla are all far longer than female inflorescence rachilla flower Former base separate living tissue number (1 flower primordium separate living tissue will develop into 1 ripe flower), has the difference of the order of magnitude, and be in Male inflorescence is per rachilla flower primordium number destination region, and they are all also growing, and the number of flower primordium is still increasing, and can determine that The inflorescence is male inflorescence.

The present inventor carries out sections observation (as shown in Figure 5) by phase inflorescence development process not dominant to male and female：

In the N1 stages, there are cluster cells under phyllopodium, are inflorescences primordium (Fig. 5-1), size 0.3mm is in optics The earliest period for the inflorescence development seen under microscope.Cell contains 1 larger nucleus at this time.

N6 stages, inflorescences primordium initial development go out prophyll former base (Fig. 5-2), and the size of entire inflorescence is 0.85mm.This When, the center of inflorescences primordium is dome-shaped, and both sides are prophyll former base.Then, prophyll elongation (Fig. 5-3).

In the N10 stages, the fully wrapped around firmly inflorescences primordium of prophyll, the size of entire inflorescence is 1.15mm at this time；Moreover, inflorescence There is bennet bract former base (Fig. 5-4) in former base both sides.Bennet bract primordial cell continues to divide, is grown to serve as bennet bract (figure 5-5).At this point, there is dimension pipe in prophyll.Bennet bract continues to extend.N14 stages, the fully wrapped around firmly inflorescence point of bennet bract Raw tissue (Fig. 5-6), inflorescence length is 2.2mm at this time.

After prophyll and bennet bract wrap inflorescence meristem, it is original that rachilla bract occur in rachis both sides Cell is at this time N15 bud stages (Fig. 5-7), and inflorescence length is still 2.2mm.Rachilla bract initial cell continues to develop into Rachilla bract former base, rachilla bract (Fig. 5-8).There is dimension pipe in bennet bract at this time.

With the growth of rachilla bract and increasing for number, there is rachilla separate living tissue in the base portion of rachilla bract (Fig. 5-9:The N27 inflorescence stages), inflorescence length is 10mm at this time.The cell of rachilla separate living tissue has larger nucleocytoplasmic ratio.It is small The development mode of cob separate living tissue is that (Fig. 5-10 is shown for development from top to base portion：Rachilla bract at the top of rachis Has there is rachilla separate living tissue in base portion, and the rachilla bract of base portion does not occur rachilla separate living tissue also).Rachilla is mitogenetic Tissue arranges (Fig. 5-12) in the shape of a spiral in conical (Fig. 5-11) around rachis.

Rachilla separate living tissue continues to develop.During the N36 stages, rachilla meristem development goes out petal piece, at this time inflorescence Size is 26.5mm (Fig. 5-13).From Fig. 5-14 as can be seen that the rate of development of top rachilla is faster than the rachilla of base portion.

The present inventor carries out sections observation (such as Fig. 6 institutes by the female inflorescence growth course of phase dominant to male and female Show)：

In female inflorescence, each petal under piece can grow a three bases flower on rachilla.Three base flowers are active by intermediate one The female flower of energy and its adjoint male flower composition of both sides.Certain phase is developed, is come off with male flower meeting abortion.Three bases flower is scorpion tail Shape cyme.

There is floral meristem (Fig. 6-1) in about N47 stages, the three base petal piece oxters that rachilla separate living tissue is formed.This When floral meristem cell contain 1 larger core, in active state.Floral meristem is made of (Fig. 6-2) three parts： Outermost layer is apparent one layer of cells (L1), develops into epidermal cell in the future；It is several layers of active isodiametric cell under L1 layers, contains Larger nucleus；In region under isodiametric cell, cell caryoplasm is smaller, there is vacuole appearance in cell.Floral meristem is broken up Go out squamella 1 (B1), B1 oxters are developed with male flower 1 (asf1) (Fig. 6-3).There is cluster between the oxter of B1 and asf1 Cell, develops squamella 2 (B2) in the future, and the oxter of B2 is then developed close to the position of B1 with male flower 2 (asf2).Position It puts in order as B1, asf2, B2, asf1.It is nonsynchronous, the development of asf1 with male flower 1 and with the development between male flower 2 It is faster than asf2.

There is cluster meristematic cell (Fig. 6-4) between the oxter of B2 and asf2 in the N48 stages, these cells are then developed Go out squamella 3 (B3), the oxter of B3 develops female flower (ff), and ff is between B2 and B3.Asf1 has developed sepal and flower at this time Valve former base, the sepal former base of asf2 have also occurred.The characteristics of this development mode meets cincinnus.

All have there is (Fig. 6-5), and part has been developed with male flower with the sepal and petal of male flower in the N49 stages Stamen retrogressive (Fig. 6-6).The cell liquid alveolation of male flower sepal end.During this stage, the bennet elongation of male flower is female until being higher than Flower.The separate living tissue growth of female flower becomes larger, but still only develops squamella (B3), and female flower at this time is less than male flower.

In the N51 stages, the separate living tissue of female flower germinates, and keeps intact (Fig. 6-7) with male flower at this time.Then, it is female Colored reproductive organs continues to develop, while develops gynoecium and oecium (Fig. 6-8).At this point, the cell of female flower sepal end Vacuolization.Meanwhile develop dispensing (Fig. 6-9) with male flower.There is vacuolization in the side cell of anther, and the cell of opposite side There are one larger nucleus.

N52 stages, female flower development speed are faster than male flower.Female flower becomes larger rapidly, and remain unchanged with male flower size (Fig. 6- 10).Polyphenol, some Vacuole formations of petal are accumulated in sepal, petal and some cells of anther end in adjoint male flower. Also, occurs pollen bag (Fig. 6-11) in anther.In female flower, carpel base portion has developed ovary former base (Fig. 6-12).Female calyx Piece, petal end cell there is vacuolization phenomenon.

N53 stages, female flower continue to develop, and male flower still remains unchanged (Fig. 6-13).It can come off before flower maturity with male flower. Most female flowers have 3 sepals, 3 petals (Fig. 6-14, wherein 1,2 be squamella 2, squamella 3), but also have indivedual female flowers There are 4 sepals, 4 petals (Fig. 6-15, wherein 1,2 be B2, B3)；Its shape is identical with quality, is all faint yellow.Peel off petal And sepal, it is observed under stereomicroscope, it can be seen that female flower has 3 free carpels (Fig. 6-16).It can according to female flower transverse section To find out, ovary is 3 ventricles, and each ventricle has 1 ovule (Fig. 6-17).There is 1 to lead between the base portion of carpel and end Road is pistillar chord (Fig. 6-18), it is channel of the pollen tube before fertilization.

The present inventor carries out sections observation (such as Fig. 7 institutes by the male inflorescence growth course of phase dominant to male and female Show)：

About N47 stages, petal under piece grow floral meristem (Fig. 7-1).Floral meristem has 1 in cone, cell A larger core (Fig. 7-2).

N49 stages, floral meristem side grow sepal (Fig. 7-3).It can be seen that the separate living tissue of male flower from this figure 3 layers can be divided into：Outermost layer is apparent one layer of cells (L1), develops into epidermal cell in the future；It is several layers active etc. under L1 layers Diameter cell contains larger nucleus (L2)；In region under isodiametric cell, cell caryoplasm is smaller, there is vacuole appearance in cell (L3)。

N50 stages, floral meristem both sides initial development go out petal former base (Fig. 7-4).At this point, the cell of sepal end goes out Existing vacuolization.

N51 stages, the stamen retrogressive of male flower germinate (Fig. 7-5).At this point, sepal and the cell liquid of petal terminal region Alveolation.There is (Fig. 7-6) in N52 stages, stamen.In the N53 stages, there is (Fig. 7-7) in anther in male flower.At this point, the cell of anther is all Containing there are one larger nucleus, show that the anther of early development is made of mitotically active cell.It can be with by anther rip cutting figure Find out, anther is made of (Fig. 7-8) outermost one layer of epidermis and internal homocellular.Then with the development of anther, table Subcutaneously there are 4 groups of archesporiums, the nucleus of archesporium is more than the nucleus (Fig. 7-9) of other cells inside epidermis.By hero Flower cross section can be seen that：Every male flower has 3 sepals, 3 petals, 6 stamens (Fig. 7-10).Archesporium passes through 1 flat week Two layers inside and outside being divided into, internal layer is sporogenous cell, develops into microsporocyte in the future, and outer layer is primary wall cell.

There is microsporocyte in pollen bag and proceed by meiosis in the N55 stages.Microsporocyte compared with Greatly, cytoplasm is dense (Fig. 7-11).N56 stages, microsporocyte start to accumulate callose wall, and microsporocyte is thin Karyon is big (Fig. 7-12).At this point, its cross section can be seen like butterfly (Fig. 7-13) in observation anther cross section.Then, microspore is female Cell proceeds by meiosis, and the 1st postmeiotic generates a microspore diad (Fig. 7-14), the 2nd subtrahend point 4 haploid cells, i.e. 4 microspores are formed after splitting.When meiosis is just completed, 4 microspores are still wrapped in callosity In matter, it is arranged in tetrahedral (Fig. 7-15).Later with the disappearance of callose wall, 4 microspores be released (Fig. 7- 16).The microspore just released is in irregular shape.Microspore continues to develop, ultimately form like triangle pollen grain (Fig. 7- 17)。

Inventor's research shows that, oil palm female flower in Sex Differentiation early period there are one stage both sexes phase, by 1 in center It functional female flower and forms with male flowers at 2 of its both sides, with the development of inflorescence, is selectively lost with male flower respectively It educates, comes off before flowering.And male flower gynoecium after Sex Differentiation is not grown, and only remains a pistillode, there is no two In property stage phase, it is directly entered the unisexuality phase.This difference explanation is difficult to once entering after the gender idiophase to female, male flower Gender is converted, and is improved female flower ratio to regulation and control, should be taken measures before Sex Differentiation, i.e. II in the male and female not dominant phase Oil palm inflorescence sex phenotype was carried out before phase (transitional period) N35 stages.

The technical solution adopted in the present invention：

It is a kind of to use cytohistology technology determination oil palm immature inflorescence method for distinguishing, it is as follows：

1st, it draws materials

The inflorescence in not dominant III phase of phase (Sex Differentiation phase) of male and female is chosen on oil palm tree, first removes prophyll and flower Obstruct bract, then put into again in FAA fixers.

2nd, dehydration with it is transparent

The inflorescence sample fixed is placed to 2~3h in 30%, 50%, 70%, 100% ethyl alcohol successively.Then it uses N-butanol places 40~50h, a clarifier is changed per 8h wherein as clarifier.

3rd, waxdip and embedding

It is 1 in volume ratio at 55~65 DEG C:10~15h is placed in 1 n-butanol and the mixed liquor of paraffin, then pure 10~15h of waxdip again in wax changes primary pure wax per 4h in the meantime.Then with KEDEE KD-M biological tissue embedding machines to male and female The rachilla of the immature inflorescence of not dominant III phase of phase is embedded.

4th, slice and roasting piece

Embedded sample is carried out to repair block, then carries out longitudinal section (small ear with Leica RM2235 rotary microtomes The longitudinal direction of axis), thickness is 5~10 μm.It is dried on piece machine in KD-H, 40~50 DEG C carry out roasting piece, and the time is 6~10h.

5th, dewaxing and dyeing

(1) wax band slice turpentine oil is impregnated 20min；(2) turpentine oil more renewed impregnates wax band slice 20min； (3) with volume ratio position 1:1 turpentine oil and the mixed liquid dipping 3min of absolute alcohol；(4) absolute alcohol impregnates 3min；(5) 95% wine Essence impregnates 2min；(6) 85% alcohol impregnate 2min；70% alcohol impregnates 2min；(7) 50% alcohol impregnate 2min；(8) 1% Periodic acid impregnate (when environment temperature be no more than 20 DEG C, aoxidize 5~10min；When then suitably reducing oxidation when environment temperature is high Between)；(9) flowing water rinses 5~10min, is then embathed with distilled water；(10) Schiff reagents are protected from light 10~30min of dyeing；(11) 0.5% Sodium Metabisulfite embathes twice, and 1~2min has reached color separation purpose every time；(12) flowing water rinses 5~10min, steams Distilled water embathes 10~20min；In the case of (13) 50~60 DEG C, 1% aniline blue black dyeing 10min；(14) 7% acetic acid quickly soak It steeps and removes excess dyestuff, then air drying is washed with distilled water；(15) absolute alcohol embathes 3min；(16) more renew Absolute alcohol, embathe 3min；(17) absolute alcohol more renewed, embathes 3min；(18) it is 1 with volume ratio:1 absolute alcohol and turpentine Oily mixed liquid dipping 3min；(19) turpentine oil impregnates 5min；(20) turpentine oil more renewed impregnates 5min；(21) neutral tree is used Glue carries out mounting.The LEICA DMLB microscopes for being finally equipped with LEICA DFC500 camera lenses are observed and are clapped to slice According to calculating petal piece former base number, petal the piece number and new petal piece former base number, flower primordium separate living tissue number.

6th, the judgement of oil palm immature inflorescence gender

The decision rule of oil palm immature inflorescence gender：If A>50 or B>50 or C>50, then the inflorescence is male inflorescence；If A< 30 or B<30 or C<30, then the inflorescence is female inflorescence.

A：Petal piece former base number on each rachilla；

B：Petal the piece number and new petal piece former base number on each rachilla；

C：Flower primordium separate living tissue number on each rachilla.

The present invention in morphology and the difference of cytohistology level, utilizes cell tissue according to the female of oil palm, male inflorescence Technology judges female, male inflorescence gender in the male and female not dominant phase ahead of time, effectively shortens search time, accelerates flow of research, be The relationship for further studying oil palm inflorescence Sex Differentiation mechanism and external factor provides basis, this in being produced in oil palm for realizing Florescence and sex ratio regulation and control, raising yield etc. are of great significance.

Description of the drawings

Fig. 1：Oil palm inflorescence development complete procedure length changing rule

Fig. 2：Oil palm inflorescence (removal bract) development complete procedure length changing rule

Fig. 3：The quality change rule of oil palm inflorescence in entire growth course

Fig. 4：The quality change rule of oil palm inflorescence (removal bract) in entire growth course

Fig. 5：The Cytohistological Observation of the not dominant phase oil palm inflorescence development of male and female and analysis

In figure：1：Inflorescences primordium initial development.2：Prophyll initial development.3：Prophyll grows.4：Bennet bract former base Initial development.5：Bennet bract is grown.6：The fully wrapped around firmly inflorescence meristem of bennet bract.7：Rachilla bract former base rises Begin.8：Rachilla bract occurs.9：Rachilla separate living tissue initial development.10：Rachilla separate living tissue number increases.11：It is small Cob separate living tissue is grown.12：Rachilla arranges in the shape of a spiral.13：Petal piece initial development.14：Petal piece is grown.

Initialism：Fb, petal piece；Fpb, bennet bract；Im, inflorescence meristem；Ip, inflorescences primordium；P, prophyll； Pbp, bennet bract former base；Pp, prophyll former base；Pv, desmogen band；Rab, rachilla bract；Rabi, rachilla bract are former Beginning cell；Rabp, rachilla bract former base；Ram, rachilla separate living tissue.

Fig. 6：The Cytohistological Observation of female flower development and analysis

In figure：1：Female floral meristem initial development.2：Floral meristem is grown.3：Squamella 1 (B1) occurs.4：Female flower Separate living tissue occurs.5：Female floral meristem growth.6：There is stamen retrogressive with male flower 1.7：Female flower sepal is developed.8：Carpel Initial development.9：With anther development in male flower.10：The arrangement mode of three bases flower.11：There is pollen bag with male flower.12：Son Room former base occurs.13：Female flower growth, and male flower stops development.14,15：Perianth arranges.16：It is observed under stereomicroscope Carpel.17：Ovule occurs.18：The rip cutting figure of pistillar chord.

Initialism：A, anther；Asf1, with male flower 1；Asf2, with male flower 2；B1, squamella 1；B2, squamella 2；B3, Squamella 3；C, carpel；Ff, female flower；Fm, floral meristem；O, ovule；Ovp, ovary former base；Pd, bennet；Pe, petal；Ps, Pollen bag；Pv, desmogen；S, sepal；Sta, staminodium；Stp, stamen retrogressive.

Fig. 7：The Cytohistological Observation of male flower development and analysis

In figure：1：Male flower separate living tissue occurs.2：Male flower separate living tissue is grown.3：Sepal initial development.4：Petal originates Development.5：Stamen retrogressive initial development.6：Stamen initial development.7：Anther initial development.8：The cross section of stamen.9：Archespore is thin Born of the same parents occur.10：The arrangement mode of stamen.11：Microsporocyte occurs.12：Microsporocyte starts to accumulate callose wall. 13：Anther cross section.14：Microspore diad occurs.15：Microspore tetrad occurs.16：4 microspores are released. 17：Pollen is in subtriangular.

Initialism：Arc, archesporium；Con, connective；Ep, epidermal cell；L1, L2, L3：First, second and third confluent monolayer cells； Msp, microsporocyte；St, stamen.

Specific embodiment

With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for Illustrate the present invention, but be not limited to the scope of the present invention.Test method without specific conditions in the following example, usually According to normal condition, or according to the normal condition proposed by manufacturer.

Oil palm inflorescence will undergo the time (Corley, 1976) of 2~3 years from germinating opening, and process is very long, this is Realize that florescence and sex ratio regulation and control, heterosis utilization, raising yield and breeding bring very big difficulty in oil palm production.Research Show that at least Sex Determination and differentiation may be participated in there are four types of different types of factor：Abiotic factor (such as water stress), generation Thank factor (such as organic C storage), hormonal readiness and inherent cause.Advantageously formed under water stress male inflorescence (Corley, 1976).Drought condition has the function of to make oil palm female flower hero, but its physiology and genetic mechanisms still need to be illustrated.There is morning Phase document thinks that oil palm has gender plasticity.

Embodiment

Step 1. carries out field planning and experimental design.

Cultivating oil palm test tube clone seedling, oil palm seedling age, kind etc. with 50 mu of ground must neat and consistent.According to the cultivation in production Training density is planned：Plant 10 plants of oil palms in 1 mu of ground.

Design abiotic factor (such as water stress), metabolic factor (such as organic C storage), fertilizer element type and level, hormone The factors such as level carry out experiment process to Oil Palm.

Step 2. is drawn materials

A, B, C are respectively labeled as in three places (distribution triangular in shape) in plantation at random, each place selects five plants Oil palm tree chooses the inflorescence that oil palm is in male and female not dominant III phase of phase, three phases is divided to choose：1. petal piece former base growth step Section；2. petal piece and new petal piece former base growth phase；3. flower primordium separate living tissue formation stages.Remove respective vanes, Take out inflorescence.

The measurement of step 3. inflorescence length and quality

The length of the inflorescence of selection is directly measured successively with graduated scale.Each inflorescence measures entire length, and measure removal Inflorescence length after bract.

The quality of larger inflorescence is in oil palm garden electronic balance (the permanent good board DT- of Changshu Jiaheng Balance Instruments Co., Ltd. 1000E types, maximum capacity 1000g, minimum weigh 0.01g, verification scale interval 0.01g) it weighs, smaller inflorescence (below N47) Quality one thousandth electronic balance (model：Precisa XS365M-SCS, maximum capacity 365g, minimum weigh 0.001g, inspection Calibration value 0.001g) it weighs.Each inflorescence weighs total quality, and weighs the inflorescence quality after removal bract.

By the entire length of taken inflorescence, removal bract after inflorescence length, total quality and removal bract after flower Sequence quality and the corresponding data of 1~table of table 2 are compared, and observe its degree of agreement, determine whether selected inflorescence is target Material.

Manufacturing for step 4. oil palm inflorescence paraffin tissue sections is analyzed with cytohistology

According to actual conditions, to male and female, the immature inflorescence of not dominant III phase of phase does longitudinal section and carries out microexamination.

4.1 oil palm inflorescence paraffin tissue sections are manufactured

It is 4.1.1 fixed

Sample inflorescence is first removed into prophyll and bennet bract, is then put into again in FAA fixers.In FAA fixers Middle placement at least for 24 hours, can also preserve for a long time.

4.1.2 dehydration with it is transparent

The material fixed is placed into 2h in 30%, 50%, 70%, 100% ethyl alcohol successively.Then made with n-butanol It for clarifier, places 2 days wherein, a clarifier is changed per 8h.

4.1.3 waxdip and embedding

It is 1 in volume ratio at 60 DEG C:12h is placed in 1 n-butanol and the mixed liquor of paraffin, is then soaked again in pure wax Wax 12h changes primary pure wax per 4h in the meantime.Then the rachilla of inflorescence is carried out with KEDEE KD-M biological tissue embeddings machines Embedding.

4.1.4 slice and roasting piece

Embedded sample is carried out to repair block, then with Leica RM2235 rotary microtomes along rachilla longitudinal direction into Row slice, thickness are 8 μm.It is dried on piece machine in KD-H, 45 DEG C carry out roasting piece, time 8h.

4.1.5 dewaxing and dyeing

(1) wax band slice turpentine oil is impregnated 20min；(2) turpentine oil more renewed impregnates wax band slice 20min； (3) it is 1 with volume ratio:1 turpentine oil and the mixed liquid dipping 3min of absolute alcohol；(4) absolute alcohol impregnates 3min；(5) 95% wine Essence impregnates 2min；(6) 85% alcohol impregnate 2min；70% alcohol impregnates 2min；(7) 50% alcohol impregnate 2min；(8) 1% Periodic acid impregnate (when environment temperature be no more than 20 DEG C, aoxidize 7.5min；Oxidization time is then suitably reduced when environment temperature is high)； (9) flowing water rinses 7.5min, is then embathed with distilled water；(10) Schiff reagents are protected from light dyeing 20min；(11) 0.5% it is inclined Sodium bisulfite embathes twice, and each 1.5min has reached color separation purpose；(12) flowing water rinses 7.5min, and distilled water embathes 15min；In the case of (13) 55 DEG C, 1% aniline blue black dyeing 10min；(14) 7% acetic acid rapid soakings once remove extra dye Then material, air drying are washed with distilled water；(15) absolute alcohol embathes 3min；(16) absolute alcohol more renewed, embathes 3min；(17) absolute alcohol more renewed, embathes 3min；(18) it is 1 with volume ratio:1 absolute alcohol and turpentine oil mixed liquid dipping 3min；(19) turpentine oil impregnates 5min；(20) turpentine oil more renewed impregnates 5min；(21) mounting is carried out with neutral gum.

The immature inflorescence histotomy analysis of step 5. male and female not dominant III phase of phase and Sexual discriminating

The LEICA DMLB microscopes for being equipped with LEICA DFC500 camera lenses are observed and are taken pictures to slice, calculate flower Bract former base number, petal the piece number and new petal piece former base number, flower primordium separate living tissue quantity, indices are less than 30 One group, number 1；It it is one group more than 50, number 2 calculates average, the results are shown in Table 3 respectively.

Every Judging index on not dominant III phase of the phase inflorescence rachilla of 3 oil palm male and female of table

The above results show related petal piece former base number on each rachilla of male inflorescence, female inflorescence, petal the piece number and New petal piece former base number, flower primordium separate living tissue number are dramatically different, have the difference of the order of magnitude, and inflorescence is all also being grown, When being transitioned into male and female dominant phase, the number of flower primordium is still increasing, and is can be determined that accordingly in petal piece former base number, petal the piece number And new petal piece former base number or flower primordium separate living tissue number be female when being less than 30, more than 50 when be male.Therefore, The present invention is other in the not dominant phase judgement male and female flowering sequence of male and female ahead of time using cytohistology technology, greatly shortens search time, Accelerate flow of research, provide basis for the influence of research different experiments factor and processing for oil palm inflorescence sex phenotype, have Significance.

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications Also it should be regarded as protection scope of the present invention.

Claims (1)

1. a kind of use cytohistology technology determination oil palm immature inflorescence method for distinguishing, which is characterized in that its specific steps is such as Under：

1) it, draws materials

The inflorescence in male and female not dominant III phase of phase is chosen on oil palm tree, first removes prophyll and bennet bract, then throws again Enter into FAA fixers；The male and female not dominant III phase of phase is inflorescence 36.5~129.5mm of overall length, inflorescence after removal bract Length is 7.8~21.4mm, and the quality of inflorescence is 0.429~11.305g, remove the quality of inflorescence after bract for 0.018~ 1.326g；

2), dehydration with it is transparent

The inflorescence sample fixed is placed to 2~3h in 30%, 50%, 70%, 100% ethyl alcohol successively；Then with positive fourth Alcohol places 40~50h, a clarifier is changed per 8h wherein as clarifier；

3), waxdip and embedding

It is 1 in volume ratio at 55~65 DEG C:10~15h is placed in 1 n-butanol and the mixed liquor of paraffin, then in pure wax 10~15h of waxdip again changes primary pure wax per 4h in the meantime；Then male and female are not shown with KEDEE KD-M biological tissue embeddings machines The rachilla of the immature inflorescence of property III phase of phase is embedded；

4), slice and roasting piece

Embedded sample is carried out to repair block, then carries out longitudinal section with Leica RM2235 rotary microtomes, thickness for 5~ 10μm；It is dried on piece machine in KD-H, 40~50 DEG C carry out roasting piece, and the time is 6~10h；

5), dewaxing and dyeing

(1) wax band slice turpentine oil is impregnated 20min；(2) turpentine oil more renewed impregnates wax band slice 20min；(3) it uses Volume ratio is 1:1 turpentine oil and the mixed liquid dipping 3min of absolute alcohol；(4) absolute alcohol impregnates 3min；(5) 95% alcohol impregnate 2min；(6) 85% alcohol impregnate 2min；70% alcohol impregnates 2min；(7) 50% alcohol impregnate 2min；(8) 1% periodic acid It impregnates；(9) flowing water rinses 5~10min, is then embathed with distilled water；(10) Schiff reagents are protected from light 10~30min of dyeing； (11) 0.5% Sodium Metabisulfite embathes twice, and 1~2min has reached color separation purpose every time；(12) flowing water rinse 5~ 10min, distilled water embathe 10~20min；In the case of (13) 50~60 DEG C, 1% aniline blue black dyeing 10min；(14) 7% vinegar Sour rapid soaking once removes excess dyestuff, then air drying is washed with distilled water；(15) absolute alcohol embathes 3min； (16) absolute alcohol more renewed, embathes 3min；(17) absolute alcohol more renewed, embathes 3min；(18) it is 1 with volume ratio:1 it is pure Alcohol and turpentine oil mixed liquid dipping 3min；(19) turpentine oil impregnates 5min；(20) turpentine oil more renewed impregnates 5min； (21) mounting is carried out with neutral gum；Finally be equipped with the LEICA DMLB microscopes of LEICA DFC500 camera lenses to be sliced into Row is observed and is taken pictures, and calculates petal piece former base number, petal the piece number and new petal piece former base number, flower primordium separate living tissue number Amount；

6), the judgement of oil palm immature inflorescence gender

The decision rule of oil palm immature inflorescence gender：If A>50 or B>50 or C>50, then the inflorescence is male inflorescence；If A<30, Or B<30 or C<30, then the inflorescence is female inflorescence；

A：Petal piece former base number on each rachilla；

B：Petal the piece number and new petal piece former base number on each rachilla；

C：Flower primordium separate living tissue number on each rachilla.