CN100361569C - Method for preparing plant haploid embryo and haploid plant - Google Patents
Method for preparing plant haploid embryo and haploid plant Download PDFInfo
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- CN100361569C CN100361569C CNB200510022486XA CN200510022486A CN100361569C CN 100361569 C CN100361569 C CN 100361569C CN B200510022486X A CNB200510022486X A CN B200510022486XA CN 200510022486 A CN200510022486 A CN 200510022486A CN 100361569 C CN100361569 C CN 100361569C
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Abstract
The present invention provides a method for preparing haploid plant embryos and haploid plants, which belongs to the field of plant cultivation. The method for preparing haploid plant embryos comprises the following steps: a. one to three stigmas in a flower are cut off before a plant blooms; b. kept stigmas are artificially pollinated when petals bloom; c. haploid embryos are taken out from an unfertilized embryo sac when the fruits of the plant are mature. The method for preparing haploid plant embryos of the present invention comprises the following steps: a. the prepared haploid embryos are inoculated on a culture medium in order to culture into seedlings; b. the seedlings are trained, transplanted and cultured. The method of the present invention has the advantages of simple operation, low cost, high successful rate, wide popularization and favorable prospect of practical application without a special instrument.
Description
Technical field
The invention belongs to the plant cultivation field, be specifically related to a kind of method for preparing plant haploid embryo and haplobiont.
Background technology
The individuality, tissue or the cell that only have the gametophyte chromosome complement are cultivated under isolated condition, made it develop into complete plant corpus, this plant with single cover chromosome is called haplophyte (Haploid Plant).Monoploid is significant at plant genetics and breeding: the inbred line and the clone that 1. can obtain cross pollinated plant by monoploid cultivation and chromosome doubling fast.2. new germ plasm resource is created in the variation in cultivating by monoploid.3. mutagenesis monoploid can be found recessive mutation rapidly.4. the theoretical research in plant reproductive mechanism plays an important role, the aneuploid that obtains as monoploid and diploid hybrid helps to solve measures linkage group, diplontic chromosome set composition, the effect of gene dosage waits some problems, also provide good acceptor material (Zhang Li for modern genetic engineering research, Wang Jing. crop in cruciferae haploid breeding progress. Anhui agricultural science, 2,003 31 (2): 231～234).
Yet, occurring in nature, the frequency of the spontaneous generation of monoploid is extremely low, only is 0.001～0.01%, and this has limited haploid application widely.For high-frequency is induced monoploid, people attempted several different methods (Sun Zhidong, Wang Xuede, hair root richness etc., crop monoploid research and use progress. seed, 2000. (6): 37～39).For a long time, utilize flower pesticide, pollen to carry out tissue culture as explant, produce plant from microspore through callus or embryoid, be the important approach of wanting that produces haplophyte always.And utilize flower pesticide, the haploid etheogenesis method of pollen induction, and adopt flower pesticide, pollen to carry out tissue culture as explant, produce plant from microspore through callus or embryoid, the program complexity, it is harsh that condition of culture requires, and complex operation is costly.
San Noeum (San Noeum L H, Haploids d ' Hordeum vulgare L.par culture in vitro d ' ovaries non fecondes.Ann Amelior Plant, 1976.24:751～754) cultivate and to obtain haplobiont by barley (Hordeumvulgare) ovary of not pollinating first, and the term that adopts " parthenogenesis (in vitro gynogenesis) of exsomatizing " illustrates that under isolated condition unfertilized embryo-sac cell produces the phenomenon of haplobiont, opened up plant exsomatize parthenogenesis this obtain the approach of haplophyte.Subsequently, in succession from the not pollination ovary of various plants or (with) the ovule cultured in vitro obtains monoploid (Yang Jiangyi and Li Xufeng, 2002).The stripped parthenogenesis of plant mainly is the cultivation of ovule or ovary of not pollinating at first, but according to the statistics to this respect research, has more and more researchers to obtain female monoploid by cultivating bud even inflorescence in recent years.The parthenogenesis research of exsomatizing has in recent years obtained remarkable progress, on some plant varieties, filter out repeatability and cultivated program preferably, but induction frequency is all also on the low side on the whole, be necessary further to improve culture technique, improve induction frequency (Yang Jiangyi, Li Xufeng, the female haploid in-vitro inducing of 2002. plants. BULLETIN OF BOTANY Vol., 19 (5): 552～559).Studies show that, the factor that influences female monoploid in-vitro inducing frequency height is a lot, relates to a plurality of factors such as physiological status, blastular developmental stage, medium, vaccination ways, the influence of floral organ adjunct, condition of culture of genotype, the donor plant of donor plant.In addition, also exist between the above factor complex interactions (Yang Jiangyi, Li Xufeng, the female haploid in-vitro inducing of plant. BULLETIN OF BOTANY Vol., 2002.19 (5): 552～559).And because influence factor is many, so be difficult to find a blanket operating process, each plant wants successfully to carry out female monoploid in-vitro inducing all needs groping and a large amount of experiments repeatedly.
To nineteen ninety-five, cultivate 19 kind (Meng Jinling etc. that successful plant only has 7 sections by unfertilized ovary and ovule, plant reproductive genetics .1995. Beijing: Science Press), and correlative study focuses mostly on grass family, Solanaceae and composite family, account for that whole cultivations successfully plant 2/3, especially at most with gramineous plants research, account for whole kinds 1/3 (Ma Sanmei, Zhang Gaisheng, Liu Hongwei etc., haploid research of allogeneic cytoplasm inducing wheat and progress. Shaanxi agricultural science, 1998. (4): 3～9).The parthenogenesis of exsomatizing is at present used to such an extent that the most successful plant is a beet, secondly be non-woody plants such as onion, flameray gerbera, tobacco (Yang Jiangyi, Li Xufeng, the female haploid in-vitro inducing of 2002. plants. BULLETIN OF BOTANY Vol., 19 (5): 552～559).The stripped gynecogenic research of woody plant only has relevant report on walnut.
Currently used utilization do not pollinate ovary or (with) the ovule cultured in vitro obtains haploid stripped parthenogenesis method, has the program complexity, condition of culture requires harsh, complex operation, weak point such as costly.
Summary of the invention
First purpose of the present invention provides a kind of method for preparing the plant haploid embryo.
This method may further comprise the steps:
A, before flowering of plant, cut off 1～(n-1) individual column cap in spending;
B, when petal is open, carry out artificial pollination for the column cap that keeps;
C, when fruit is about to change maturation over to, from unfertilized blastular, take out haploid embryo;
N described in the step a is the column cap number in this plant flowers.
Further, in above-mentioned step a, when cutting off column cap, the column cap of reservation is evenly distributed.
Further: above-mentioned plant is a rose family Rosaceae plant.
Further: above-mentioned rosaceous plant is that loquat belongs to Eriobotrya Lindl plant.
Second purpose of the present invention provides uses the above-mentioned preparation-obtained plant haploid embryo of method.
The 3rd purpose of the present invention provides a kind of method for preparing the plant haploid plant.
This method may further comprise the steps:
A, directly above-mentioned plant haploid embryo is inoculated on the medium, cultivates into seedling;
B, seedling is carried out hardening, to adapt to external environment.
Further: the medium in the above-mentioned a step is: contain 6-BA 0.1～0.5mg/L, the MS medium of NAA 0.02～0.1mg/L and IBA 0.02～0.1mg/L.
Preferably: the medium in the above-mentioned a step is: contain 6-BA0.5mg/L, NAA0.02mg/L and IBA0.02mg/L.
Medium in the above-mentioned a step also can contain 0.1～0.5% active carbon.
Further, the medium in the above-mentioned a step contains 0.2% active carbon.
Further: the condition of culture in the above-mentioned a step is: 25 ± 1 ℃ of temperature, dark condition is cultivated after 5～15 days down, under illumination 2000 ± 500lux, 14～18h/d, cultivates one month.
The method for preparing the plant haploid embryo that first purpose of the present invention provided, its scope of application is extensive.In the operating process of this method, to note following item:
1, step a will choose and grow healthy and strong inflorescence and flower thinning, and every inflorescence stays 2～5 flowers, so that fruit can obtain the prescribing adequate nutrition supply in the future.
2, go the column cap must be before blooming, refer to that just the still unopened lantern florescence of general said flower (promptly flower is ripe, after the same day or 1～2 day with open period) carries out, to guarantee the fertilization process of pollinating.
3, in step a, when cutting off column cap, the column cap of reservation is evenly distributed as far as possible.Cut off at interval such as under the more situation of reservation column cap, trying one's best; And should be in the equally distributed while of the column cap that makes reservation under the situation that keeps less column cap, at least one column cap is left in assurance central area (center in column cap zone).
4, award with capacity pollen for remaining column cap, make column cap stick pollen, guarantee that it finishes fertilization process, form the normal seed of capacity, to create suitable hormone and nutrient environment, to bring out the ovule parthenogenesis of removing column cap.
Be meant when 5, the fruit among the step c is about to change maturation over to that fruit is about to change over to maturation but not full ripe period as yet, can draw in common prudence, as fruit tree for Citrus of loquat Eriobotrya japonica Lindl., peach Prunuspersica (L.) Batsch, apple Malus pumila Mill and Rutaceae Rutaceae or the like, be meant the period that pericarp is greener and pulp is harder.
The ovule that links to each other with the column cap that is removed in this method, owing to do not finish the double fertilization process, can not form normal 2 times of body seeds, but under the hormone that other normal development seeds in same is spent are created and the environment of nutritional condition, the energy induced parthenogenesis, form the haploid embryo of certain frequency, and then develop into complete monoploid seed (frequency is about more than 1%, much larger than abiogenous frequency).Because the complete monoploid seed volume that grows is like this named " seedlet " again much smaller than normal seed, should be haploid embryo by " seedlet " interior embryo, therefore very easy and normal seed is distinguished mutually.In this method, remove column cap, artificial pollination, from the monoploid seed, take out single step such as haploid embryo and all can finish with the routine operation method.Wherein, artificial pollination should be authorized the pollen of kindred plant, and the pollen of described kindred plant both can be the pollen of same breed, also can be the pollen of different cultivars, also can be the pollen mixture of several kinds.
Second plant haploid embryo that purpose provided of the present invention, be from grow complete monoploid seed, to obtain, these haploid embryos can use existing cultural method to cultivate becomes plant, and haplobiont wherein can be differentiated by various conventional monoploid detection methods.
The 3rd method for preparing the plant haploid plant that order provided of the present invention, be used for the haploid embryo that the method for preparing the plant haploid embryo provided by the present invention is prepared cultivate become haplobiont in, also the plant haploid embryo culture that additive method can be prepared becomes plant, especially is suitable for the cultivation to the haploid embryo that generates naturally.
According to the 3rd purpose of the present invention, this method for preparing the plant haploid plant can take following concrete grammar to carry out: above-mentioned plant haploid embryo directly is inoculated in contains 6-BA0.1～0.5mg/L, NAA0.02～0.1mg/L, on the sterilization MS medium of IBA 0.02～0.1mg/L and 0.1～0.5% active carbon, place 25 ± 1 ℃, (it is green that cotyledon turns in 5～15 days in cultivation under the dark condition, embryo begins to sprout), then under the condition of illumination 2000 ± 500lux, 14～18h/d, cultivated about one month, and treated that it grew up to complete seedling strain.Subsequently, hardening is carried out in strain to monoploid loquat seedling, to adapt to external environment.Hardening off method is: blake bottle was moved on under room temperature (15～25 ℃) condition 2～5 days, allow the monoploid seedling cultivated adapt to gradually ambient light according to and variation of temperature; Throw off the aseptic film that seals of blake bottle then, outside air is entered in the blake bottle, make the variation of monoploid seedling acquisition, after 3～7 days, the loquat seedling is taken out in blake bottle resistance and the adaptation outside air humidity of microorganism under the normal natural conditions.Clean the medium on the loquat shoot root, transplant in the basin of the perlite of sterilizing and 1: 1 mixed-matrix of peat, place under the sealed environment condition, incubation keeps natural lighting, 100% relative moisture, room temperature, and per two weeks with MS nutrient solution (being the MS medium) fertilising once.Remove SLD after 1 month gradually, the relative moisture that reduces loquat seedling growing environment gradually removes SLD at last to natural level.
Adopt the normal dyeing body detecting method from seedling, to identify haplobiont.
The method that the present invention prepares the plant haploid embryo with directly compare by the existing method of unfertilized blastular of method for tissue culture cultured in vitro or ovule, advantage is: simple to operate, expense is cheap, do not need special instrument and equipment and reagent, and widely applicable, success rate is high, can overcome the influence of plant species difference well, is applicable to that the land for growing field crops promotes.The method that the present invention prepares the plant haploid embryo can make plant monoploid seed generation frequency bring up to 1%～2%, is higher than abiogenous frequency out and away.Haploid embryo of the present invention requires low to condition of culture, the easier seedling of cultivating into, and the growth of seedling is also more healthy, and the transplanting survival rate of cultivating the monoploid seedling strain that obtains can reach more than 96%.The present invention prepares the method for plant haploid plant, when guaranteeing culture effect, to instrument and equipment require lower, step is easy, expense is cheap, has good actual application prospect.
Description of drawings
Fig. 1 is the outside drawing of the loquat haploid embryo of the present invention's preparation, the circle mark be the loquat haploid embryo, the minimum scale of scale is 1mm among the figure.
Fig. 2 is the chromosome map (amplifying 1000 times) of the loquat plant of the present invention's preparation, and wherein Fig. 2 A is the chromosome map of loquat haplobiont, and Fig. 2 B is the chromosome map of loquat liploid plant.
Embodiment:
The present invention will be described below by the detailed description to better embodiment of the present invention.But it is limitation of the present invention that this explanation should not be construed as, and those skilled in the art can make various changes or modification according to the present invention, only otherwise break away from technological thought of the present invention, all belongs to the defined scope of claim of the present invention.
The preparation of embodiment 1 haploid embryo
1, chooses well-grown loquat plant 20 strains (kind is big five-pointed star), be divided into 2 groups at random: 4 strains are that control group, 16 strains are experimental group, before loquat is about to bloom 1-2 days, every inflorescence is chosen 1～4 ball-type flower (flower that is in this period is open as yet on the inflorescence that grows fine from each plant, pollen is prematurity still, so the pollination fertilization process does not take place), all the other flowers are all extractd.Push the ball-type flower of selecting and remain aside, cut off 1～4 (cut off 1 at random, or 1 column cap is cut 2～3 at interval, or only keep one of the flower center, cut off 4) in 5 column caps with eye scissors.
When 2, treating the petal opening (removing column cap after 2～3 days), award with capacity pollen mixture (big five-pointed star: dragon's fountain No. 1=1: 1) for 1～4 column cap that keeps.
3, extract the bud that does not carry out artificial removal's column cap, pollination.Allow the flower that has carried out artificial treatment continue to grow, grow fruit, dredge in batches and remove to grow undesired and unnecessary fruit, each fruit ear stays 2～5 fruits at last, so that fruit can reach full growth.
4, the beginning of April is to mid-April, when loquat diameter 2～3cm, surperficial fine hair are sloughed off, when fruit is about to change over to but prematurity still of maturation (pericarp is that green pulp is hard), experimental group is divided into A, B, C, D group again, each plucks 100 fruits at random on the fruit ear that every strain loquat of control group and 4 experimental group was handled, pluck 400 fruits altogether for every group, in flowing water, pericarp is cleaned.Under the aseptic condition on the superclean bench,, take out unfertilized blastular (unfertilized blastular diameter 2～3mm, flat with aseptic tweezers with the fruit strip off.And normal seed diameter is 1～3cm), places sterile petri dish.With aseptic operation cutter strip off blastular, take out haploid embryo.
Use formula: haploid embryo pick-up rate=acquisition haploid embryo number/(obtaining normal seed number+acquisition haploid embryo number)
*100% calculates the haploid embryo pick-up rate, the results are shown in Table 1.The profile and the size of haploid embryo are seen Fig. 1.
The preparation result of table 1 loquat haploid embryo
| Grouping | Handle No. of inflorescences/flower number | Results fruit number | Obtain normal seed number | Obtain " seedlet " (haploid embryo) number | The haploid embryo pick-up rate |
| Control group (being untreated) A B C D | 160/400 160/400 160/400 160/400 160/400 | 400 400 400 400 400 | 1480 1452 1652 1472 1588 | 0 20 28 16 32 | 0 1.35％ 1.67％ 1.08％ 1.98％ |
The preparation of embodiment 2 haplobionts
To organize prepared haploid embryo by A among the embodiment 1, directly be inoculated in respectively on the sterilization MS medium, directly be inoculated in and add 6-BA 0.5mg/L, NAA 0.1mg/L on the sterilization MS medium of IBA 0.1mg/L and 0.5% active carbon, places under 25 ± 1 ℃ of dark conditions and cultivated 15 days, 25 ± 1 ℃, illumination 18h, night, 6h cultivated into seedling (after cultivating month approximately) under the 1500lux light intensity.
After waiting to grow up to complete seedling strain, the monoploid loquat is carried out hardening, to adapt to external environment.Hardening off method is: blake bottle was moved on to room temperature (15～25 ℃) condition following 2 days, allow the monoploid seedling of cultivating adapt to ambient light photograph and variation of temperature gradually; Throw off the aseptic film that seals of blake bottle then, outside air is entered in the blake bottle, make the variation of monoploid seedling acquisition, after 5 days, the loquat seedling is taken out in blake bottle resistance and the adaptation outside air humidity of microorganism under the normal natural conditions.Clean the medium on the loquat shoot root, transplant in the basin of the perlite of sterilization and 1: 1 mixed-matrix of peat, place under the enclosed environment condition and cultivate, incubation maintenance natural lighting, 100% relative moisture, 25 ± 1 ℃.Per two weeks with MS nutrient solution (being the MS medium) fertilising once.Remove SLD after 1 month gradually, the relative moisture that reduces loquat seedling growing environment gradually removes SLD at last to natural level.
Adopt the normal dyeing body detecting method to differentiate monoploid seedling strain and haplobiont (the results are shown in Table 2).The chromosome map of monoploid loquat is seen Fig. 2 A, and the chromosome map of dliploid loquat is seen Fig. 2 B.
The preparation of embodiment 3 haplobionts
To organize prepared haploid embryo by B among the embodiment 1, directly be inoculated on the sterilization MS medium, directly be inoculated in and add 6-BA 0.3mg/L, NAA0.05mg/L on the sterilization MS medium of IBA0.05mg/L and 0.3% active carbon, places and cultivates after 7 days under 25 ± 1 ℃ of dark conditions, in 25 ± 1 ℃, illumination 18h, night, 6h cultivated into seedling (after cultivating month approximately) under the 2000lux light intensity.
After waiting to grow up to complete seedling strain, the monoploid loquat is carried out hardening, to adapt to external environment.Hardening off method is: blake bottle was moved on to room temperature (15～25 ℃) condition following 3 days, allow the monoploid seedling of cultivating adapt to ambient light photograph and variation of temperature gradually; Throw off the aseptic film that seals of blake bottle then, outside air is entered in the blake bottle, make the variation of monoploid seedling acquisition, after 3 days, the loquat seedling is taken out in blake bottle resistance and the adaptation outside air humidity of microorganism under the normal natural conditions.Clean the medium on the loquat shoot root, transplant in the basin of the perlite of sterilization and 1: 1 mixed-matrix of peat, place under the sealed environment condition and cultivate, incubation maintenance natural lighting, 100% relative moisture, 25 ± 1 ℃.Per two weeks with MS nutrient solution (being the MS medium) fertilising once.Remove SLD after 1 month gradually, the relative moisture that reduces loquat seedling growing environment gradually removes SLD at last to natural level.
Adopt the normal dyeing body detecting method to differentiate monoploid seedling strain and haplobiont (the results are shown in Table 2).
The preparation of embodiment 4 haplobionts
To organize prepared haploid embryo by plant C among the embodiment 1, directly be inoculated in and add 6-BA 0.1mg/L, NAA 0.01mg/L, on the sterilization MS medium of IBA 0.02mg/L and 0.1% active carbon, place under 25 ± 1 ℃ of dark conditions and cultivated 5 days, in 25 ± 1 ℃, illumination 14h, night, 10h cultivated into seedling (after cultivating month approximately) under the 2500lux light intensity.
After waiting to grow up to complete seedling strain, the monoploid loquat is carried out hardening, to adapt to external environment.Hardening off method is: blake bottle was moved on to room temperature (15～25 ℃) condition following 3 days, allow the monoploid seedling of cultivating adapt to ambient light photograph and variation of temperature gradually; Throw off the aseptic film that seals of blake bottle then, outside air is entered in the blake bottle, make the variation of monoploid seedling acquisition, after 4 days, the loquat seedling is taken out in blake bottle resistance and the adaptation outside air humidity of microorganism under the normal natural conditions.Clean the medium on the loquat shoot root, transplant in the basin of the perlite of sterilization and 1: 1 mixed-matrix of peat, place under the sealed environment condition and cultivate, incubation maintenance natural lighting, 100% relative moisture, 25 ± 1 ℃.Per two weeks with MS nutrient solution (being the MS medium) fertilising once.Remove SLD after 1 month gradually, the relative moisture that reduces loquat seedling growing environment gradually removes SLD at last to natural level.
Adopt the normal dyeing body detecting method to differentiate monoploid seedling strain and haplobiont (the results are shown in Table 2).
The preparation of embodiment 5 haplobionts
To organize prepared haploid embryo by D among the embodiment 1, directly be inoculated in and add 6-BA0.5mg/L, NAA0.02mg/L, on the sterilization MS medium of IBA0.02mg/L and 0.2% active carbon, place and cultivate (it is green that cotyledon turns, and embryo begins to sprout) after 7 days under 25 ± 1 ℃ of dark conditions, in 25 ± 1 ℃, illumination 16h, night, 8h cultivated into seedling (after cultivating month approximately) under the 2000lux light intensity.
After waiting to grow up to complete seedling strain, the monoploid loquat is carried out hardening, to adapt to external environment.Hardening off method is: blake bottle was moved on to room temperature (15～25 ℃) condition following 3 days, allow the monoploid seedling of cultivating adapt to ambient light photograph and variation of temperature gradually; Throw off the aseptic film that seals of blake bottle then, outside air is entered in the blake bottle, make the variation of monoploid seedling acquisition, after 5 days, the loquat seedling is taken out in blake bottle resistance and the adaptation outside air humidity of microorganism under the normal natural conditions.Clean the medium on the loquat shoot root, transplant in the basin of the perlite of sterilization and 1: 1 mixed-matrix of peat, place under the sealed environment condition and cultivate, incubation maintenance natural lighting, 100% relative moisture, 25 ± 1 ℃.Per two weeks with MS nutrient solution (being the MS medium) fertilising once.Remove SLD after 1 month gradually, the relative moisture that reduces loquat seedling growing environment gradually removes SLD at last to natural level.
Adopt the normal dyeing body detecting method to differentiate monoploid seedling strain and haplobiont (the results are shown in Table 2).
Use formula: monoploid seedling strain pick-up rate=haploid embryo cultivation gained seedling strain number/acquisition haploid embryo is counted * 100% and is calculated A, B, C, D group monoploid seedling strain pick-up rate respectively, the results are shown in Table 2.
Use formula: the haplobiont number/haploid embryo of monoploid seedling strain survival rate=transplanting survival is cultivated gained seedling strain number
*100% calculates A, B, C, D group haplobiont survival rate respectively, the results are shown in Table 2.
The preparation result of table 2 loquat haplobiont
| Grouping | Obtain the haploid embryo number | Haploid embryo is cultivated gained seedling strain number | Monoploid seedling strain pick-up rate | Transplant the haplobiont number of survival | Monoploid seedling strain survival rate |
| Control group (being untreated) A B C D | 0 20 28 16 32 | 0 16 20 12 28 | 80％ 71.4％ 75％ 87.5％ | 0 14 17 11 27 | 87.5％ 85％ 91.7％ 96.2％ |
Above-mentioned example shows: the method that the present invention prepares the plant haploid embryo can make plant monoploid seed generation frequency bring up to 1%～2%.Be higher than abiogenous frequency out and away.Simultaneously the inventive method with directly cultivate the advantage that unfertilized blastular or ovule compare with the method that obtains haploid embryo and be by method for tissue culture, adopt the method simple to operate, do not need special instrument and equipment, expense is cheap and the success rate height, is applicable to that the land for growing field crops promotes.The easier seedling of cultivating into of the haploid embryo that the inventive method prepares, monoploid seedling strain pick-up rate can reach more than 87%, and the growth of seedling is also very healthy, and the survival rate of monoploid seedling strain can reach more than 96%.The preparation method of haplobiont of the present invention has only used a kind of medium, and is less demanding to instrument and equipment, step is easy, and expense is cheap, and has good actual application prospect.
Attached:
MS medium (Murashige and Skoog medium) prescription:
Macroelement: two hydration calcium chloride (CaCl
2) 440.0mg/L, potassium dihydrogen phosphate (KH
2PO
4) 170.0mg/L, potassium nitrate (KNO
3) 1900.0mg/L, bitter salt (MgSO
4.7H
2O) 370.0mg/L, ammonium nitrate (NH
4NO
3) 1650.0mg/L;
Trace element: Salzburg vitriol (CuSO
4.5H
2O) 0.025mg/L, molysite (FeNaEDTA) 36.7mg/L, boric acid (H
3BO
3) 6.2mg/L, Manganous sulfate monohydrate (MnSO
4.H
2O) 16.9mg/L, Zinc vitriol (ZnSO
4.7H
2O) 8.6mg/L, four molybdic acid hydrate sodium (Na
2Mo
4.4H
2O) 0.25mg/L, potassium iodide (KI) 0.83mg/L.Organic matter: glycine (Glycine) 2.0mg/L, inositol (Myo-inositol) 100mg/L, nicotinic acid (Nicotinic acid) 0.5mg/L, VB
60.5mg/L, VB1 0.1mg/L.
IBA: indolebutyric acid (indolebutyric acid)
NAA: methyl (α-naphthaleneacetic acid)
6-BA:6-benzyl purine (6-benzyladenine)
Claims (5)
1. the preparation method of a plant haploid plant, it may further comprise the steps:
A, before flowering of plant, every inflorescence is chosen 1～4 ball-type flower from the inflorescence that grows fine, all the other flowers are all extractd, and cut off 1～(n-1) individual column cap in selected the spending then, n is the column cap number in this plant flowers;
B, when petal is open, carry out artificial pollination for the column cap that keeps, extract the bud that does not carry out artificial removal's column cap and do not carry out artificial pollination, after waiting to grow fruit, dredge in batches and remove to grow abnormal fruit and unnecessary fruit;
C, when fruit is about to change maturation over to, from unfertilized blastular, take out haploid embryo;
D, the haploid embryo that takes out is inoculated in contains 6-BA 0.1～0.5mg/L, on the MS medium of NAA 0.01～0.1mg/L and IBA 0.02～0.1mg/L, cultivate into seedling;
E, hardening, transplant cultivate plant;
Wherein, described plant is that rose family Rosaceae loquat belongs to Eriobotrya Lindl plant.
2. the preparation method of plant haploid plant according to claim 1 is characterized in that:
Among the described step a, when cutting off column cap, the column cap of reservation is evenly distributed.
3. the preparation method of plant haploid plant according to claim 1 is characterized in that: the described medium of steps d is: contain 6-BA 0.5mg/L, the MS medium of NAA 0.02mg/L and IBA 0.02mg/L.
4. according to the preparation method of each described plant haploid plant of claim 1～3, it is characterized in that: the described medium of steps d also contains 0.1～0.5% active carbon.
5. according to the preparation method of each described plant haploid plant of claim 1～3, it is characterized in that: the condition of culture of steps d is: 25 ± 1 ℃ of temperature, and dark condition was cultivated 5～15 days down, then at light intensity 2000 ± 500lux, under the condition of illumination 14～18h/d, cultivated one month.
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| CN103238610A (en) * | 2013-04-23 | 2013-08-14 | 内蒙古农业大学 | Chemical composition for promoting gladiolus corm to quickly grow as well as preparation method and application of chemical composition |
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| CN101690461B (en) * | 2009-11-02 | 2012-07-04 | 四川农业大学 | Method for preparing triploid plants |
| CN104663345B (en) * | 2014-11-27 | 2017-04-05 | 山东省果树研究所 | For the softwood cutting propagation method and promoter compositionss of Dwarf Stocks For Apple Trees |
| CN105532406A (en) * | 2016-01-13 | 2016-05-04 | 四川农业大学 | White-pulp loquat new strain preparing method based on red-pulp loquat variety |
| CN116369202B (en) * | 2023-03-15 | 2024-04-19 | 新疆农业科学院哈密瓜研究中心 | Method for inducing muskmelon haploid material by mechanically damaged stigmas and application |
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| CN1418950A (en) * | 2002-12-26 | 2003-05-21 | 北京市海淀区植物组织培养技术实验室 | Method for obtaining haploid plant strain by culturing anther or pollen |
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| CN1418950A (en) * | 2002-12-26 | 2003-05-21 | 北京市海淀区植物组织培养技术实验室 | Method for obtaining haploid plant strain by culturing anther or pollen |
Non-Patent Citations (2)
| Title |
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| 从玉米传粉子房培养出单倍体植株. 汤飞宇，丁菲，王国英.福建农林大学学报(自然科学版)，第33卷第4期. 2004 * |
| 枇杷胚乳愈伤组织诱导和不定芽发生的研究. 彭晓军，王永清.四川农业大学学报，第20卷第3期. 2002 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103238610A (en) * | 2013-04-23 | 2013-08-14 | 内蒙古农业大学 | Chemical composition for promoting gladiolus corm to quickly grow as well as preparation method and application of chemical composition |
| CN103238610B (en) * | 2013-04-23 | 2015-03-25 | 内蒙古农业大学 | Chemical composition for promoting gladiolus corm to quickly grow as well as preparation method and application of chemical composition |
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