CN100420370C - Culture method for obtaining embryoid by inducing microspore of hot pepper - Google Patents
Culture method for obtaining embryoid by inducing microspore of hot pepper Download PDFInfo
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Abstract
The invention relates to a culture method for obtaining embryoid by inducing capsicum microspore, belonging to the field of plant cell engineering. The method comprises the steps of determining a test material, selecting a flower bud, pretreating the flower bud, preparing two culture mediums of liquid and solid, sterilizing and subpackaging the two culture mediums, sterilizing the flower bud, aseptically stripping anthers, inoculating the anthers, culturing the anthers, naturally dissociating microspores, forming embryoids and the like, and the embryoids can normally grow and have high seedling rate. The present invention generally takes 8 weeks from the start of culturing to the obtention of embryoid bodies. If the invention is applied to breeding practice, the breeding process is greatly accelerated, the selection effect is obviously improved, a large amount of manpower and material resources are saved, and the invention is an effective method for accelerating the breeding of pure lines.
Description
Technical field
The invention belongs to the cell engineering field, relate to a kind of free, cultural method of chilli microspore, especially a kind of inducing chilli microspore obtains the cultural method of embryoid.
Background technology
Can obtain haplobiont by microspores culture.Then, double to obtain double haploid (dliploid pure lines), can directly obtain to be sheerly intermediate materials as parent or breeding through colchicine.It is a kind of method that obtains pure lines on unicellular level that Isolated microspore is cultivated, proterties neat and consistent in the pure lines system of acquisition, from generation to generation between stability strong, have a homozygosity of height.
Monoploid has important significance for theories and using value in heredity and breeding, the process of seed selection new varieties has been quickened in haploid breeding Study on Technology and application greatly.If in crossbreeding the haploid gamete of the reorganization of first-filial generation or two generations producer or plant chromosome through artificial doubling, just can obtain zygoid strain system (DH strain system), needn't carry out continuous selfed breeding pure lines of many generations again, thereby greatly shorten breeding cycle.In addition, the double haploid in the DH strain system also is the ideal material that carries out molecular labeling and genome research.For many years, isolated microspore culture technique is one of the important content in domestic and international cell engineering field always, more and more causes the attention of countries in the world researcher.Not only the experimental system because of it has important value for great theoretical questions such as understanding cell totipotency, phenogenesis mechanism, and has many important use aspect the plant genetics and breeding research.
Capsicum hybrid vigour is fairly obvious, and heterotic utilization has become the main path of pepper breeding.The comprehensive proterties of seed selection is good, and disease-resistant, high-quality, high yield, the pure lines that coordinate force is high are key technologies of capsicum heterosis utilization.The normal cross pollinated plant of Capsicum, natural hybrization rate height, generally needed for 4～6 generations selected and to obtain by selfing isolation and selection pure lines, not only the time is long, efficiency of selection is poor, and exist to show recessive relation between the gene, be difficult to select therefore that comprehensive proterties is good, coordinate force is high, suitable pure lines as strong advantage combined parent strain.Carry out the research of chilli microspore culture technique,, quicken breeding process, cultivate strong advantage capsicum and make up significant for the strong advantage combined parent strain pure lines of seed selection.
Utilize microspores culture seed selection parental inbred line (pure lines) to be widely used in crucifers such as Chinese cabbage, wild cabbage, induce the report that obtains embryoid and then regeneration plant in a large number yet have not yet to see from chilli microspore.As everyone knows, from the microspore inducing embryoid body, cultivating its bud into haplobiont again is microspores culture valid approach the most.Embryoid is to originate from a non-zygote cell, forms embryo shape structure through embryo development procedure, and it promptly shows as an obvious dipolar structure of tool when taking place the earliest.Through embryoid induction produce haplobiont by the callus approach have that quantity is many, speed fast, advantage such as structural integrity, genotype are stablized.Therefore, embryoid has special value as having the cultivation means of the individuality of specific good genetic character.
Summary of the invention
The objective of the invention is to, at the technical barrier that is difficult to obtain haplobiont in the chilli microspore cultivation by the embryoid approach, provide a kind of chilli microspore to cultivate the cultural method that obtains embryoid, this method can obtain a large amount of microspore embryoids in fast and stable ground.
In order to realize above-mentioned task, the present invention takes following technical solution:
A kind of inducing chilli microspore obtains the cultural method of embryoid, it is characterized in that may further comprise the steps:
(1) determine the examination material: choose have comprehensive merit the first-filial generation capsicum variety as test material;
(2) choose bud: coming into bloom or the Sheng phase, choose by aceto-camine pressed disc method microscopy and to be in the keep to the side bud of phase of monokaryon, the mode of appearance feature shows as corolla and calyx is isometric or corolla is longer than calyx slightly, regularly extracts open flower, allows plant be in bloom all the time;
(3) bud preliminary treatment: suitable bud is placed on 4 ℃ of low temperature refrigerators with self-styled plastic sack splendid attire and handled 1～3 day;
(4) preparation liquid nutrient medium and solid culture medium: two kinds of used minimal mediums of medium are Nitsch and Nitsch medium, and used carbon source is a maltose, and its concentration is 2%～3%; The coagulating agent that solid culture medium adds is an agar, and its concentration is 0.6%～0.8%;
The constituent of solid culture medium is: in Nitsch and Nitsch medium, add 0.6%～0.8% agar of medium weight, and 2%～3% maltose, 0.5%～1.0% active carbon, pH 6.0;
The constituent of liquid nutrient medium is: in Nitsch and Nitsch medium, add 2%～3% maltose, and the zeatin ZT of 0.4～0.8mg/L, the heteroauxin IAA of 0.5mg/L～1.0mg/L, pH 5.8;
(5) sterilization of medium and packing: solid culture is based on 121 ℃, and sterilization is 20 minutes under the 1.1Mpa condition, the liquid nutrient medium plastics filter filtration sterilization that two-layer miillpore filter is housed, the germ-free condition of assurance medium;
Each culture dish of sterilization back injects solid culture medium 6ml earlier, wait solid culture medium to solidify after, add liquid nutrient medium 3ml again, carry out packing;
(6) bud sterilization: earlier with flowing water flushing 30 minutes, use 30 seconds of alcohol surface sterilization of volume parts 70% again, sterilized 12～15 minutes with the clorox of volume fraction 5% afterwards, use aseptic water washing then 4 times, each 5 minutes;
(7) the aseptic flower pesticide that strips: on aseptic filter paper, strip flower pesticide, avoid damage, damage flower pesticide, and filigree is removed clean with sterile tweezers;
(8) flower pesticide inoculation: each culture dish is put into 12 pieces of flower pesticide, seals with Parafilm;
(9) anther culture: culture dish places incubator, the dark cultivation week under 9 ℃ of conditions, after change 25 ℃ of daytimes, night 20 ℃ tissue culture room over to, continue secretly to cultivate;
(10) the free naturally embryoid of microspore forms: under liquid-solid double-deck culture systems, flower pesticide swims on the liquid nutrient medium on upper strata, and through 2～3 weeks, flower pesticide normally splits, and microspore is released in the medium;
(11) growth of embryoid forms: after cultivating for 4 weeks, visible spheroidal embryo of naked eyes and heart type embryo, cultivated for 7 weeks after, as seen torpedo embryo and cotyledon type embryo, cultivated for 10 weeks after, the cotyledon type embryo is very obvious, afterwards the cotyledon type embryo is changed on the embryo germination medium, form whole plant.
Regeneration plant is identified the plant that is defined as microspore development through cytology and morphology.
The innovative point that the present invention gives prominence to is to carry out liquid-solid double-deck culture systems, adopt maltose as carbon source, low temperature treatment, continuous darkness is cultivated, thereby set up the new method that a kind of chilli microspore is cultivated embryoid, the embryoid that obtains by the method is up to 1 embryoid/flower pesticide, and embryoid grows normally the planting percent height.
The method step that the present invention takes chilli microspore cultivation embryoid is simple, practical, and the efficient height is specially adapted to aspect, capsicum cell engineering field and uses.
Description of drawings
Fig. 1 is the embryoid of 4 week of microspores culture back formation.
Fig. 2 is the embryoid of 7 week of microspores culture back formation.
Fig. 3 is the embryoid of 10 week of microspores culture back formation.
Fig. 4 is the successive transfer culture situation of embryoid that microspore forms.
Fig. 5 is the embryoid regeneration seedling plant that microspore is induced.
Fig. 6 is that the monoploid transplantation of seedlings that microspore is induced survives (left side) and normal seed dliploid seedling (right side) growing state.
Fig. 7 is the chromosome (n=12) of the microspore haplobiont of inducing.
Fig. 8 is the chromosome (n=24) of the microspore double haploid of inducing.
The specific embodiment that provides below in conjunction with accompanying drawing and inventor further describes in detail the present invention.
Embodiment
According to technical scheme of the present invention, chilli microspore is cultivated the method for embryoid, and concrete implementation step is:
(1) determine the examination material: get have comprehensive merit first-filial generation capsicum variety P51 as test material.
(2) choose bud: come into bloom or the Sheng phase at P51, [draw from Dudu by the aceto-camine pressed disc method
Cinek, Rukiye Tipirdamam.The effect of cold treatment andcharcoal on the in vitro androgenesis of pepper (Capsicum annuum L.) .TurkJ Bot, 2002,26:131-139] microscopy chooses and is in the keep to the side bud of phase of monokaryon, bud mode of appearance feature shows as corolla and calyx is isometric or corolla is longer than calyx slightly, bud diameter 5mm, length 7mm.Regularly extract flower open on the plant, allow it be in bloom all the time.
(3) bud preliminary treatment: suitable bud is placed on 4 ℃ of low temperature refrigerators with self-styled plastic sack splendid attire and handled 1 day.
(4) preparation liquid nutrient medium and solid culture medium: two kinds of used minimal mediums of medium are Nitsch and Nitsch medium and (draw the Collin from H A, G S Edwards.Plant Cell Culture.BIOS Scientific Publishers Limited, 1998, pp20.Concrete constituent is for details see attached table), used carbon source is maltose, and its concentration is 2%.The coagulating agent that solid culture medium adds is an agar, and its concentration is 0.8%.
The constituent of solid culture medium is Nitsch and Nitsch medium (Nitsch and Nitsch prescription is seen attached list)+0.8% agar+2% maltose+0.5% active carbon, and pH 6.0; The constituent of liquid nutrient medium is Nitsch and Nitsch medium+2% maltose+0.5mg/L ZT (zeatin)+0.8mg/LIAA (heteroauxin), and pH 5.8.
The sterilization and the packing of (5) two kinds of medium: solid culture is based on 121 ℃, and sterilization is 20 minutes under the 1.1Mpa condition; Liquid nutrient medium is the plastics filter filtration sterilization of 25mm by the diameter that 0.45 μ m and the two-layer miillpore filter of 0.22 μ m are housed, and needs through autoclaving before wherein filter membrane and plastics filter use.
Each culture dish (diameter 60mm) injects earlier solid culture medium 6ml, treat that solid culture medium solidifies after, add liquid nutrient medium 3ml with graduated pipette again.
Packing, filtration sterilization are all carried out on superclean bench.
(6) bud sterilization: earlier with flowing water flushing 30 minutes, use 30 seconds of alcohol surface sterilization of volume fraction 70% again, sterilized 15 minutes with the clorox of volume fraction 5% afterwards, use aseptic water washing then 4 times, each 5 minutes.
(7) the aseptic flower pesticide that strips: on aseptic filter paper, carefully strip flower pesticide, avoid damage, damage flower pesticide, and filigree is removed clean with sterile tweezers.
(8) flower pesticide inoculation: each culture dish is put into 12 pieces of flower pesticide (flower pesticide is from different buds, in order to avoid the position of bud, size, age etc. cause error), and (Parafilm) seals with Parafilm.
(9) anther culture: culture dish places incubator, the dark cultivation week under 9 ℃ of conditions, after change 25 ℃ of daytimes, night 20 ℃ tissue culture room over to, continue secretly to cultivate.
(10) the free naturally and embryoid of microspore forms: under liquid-solid double-deck culture systems, flower pesticide swims on the liquid nutrient medium on upper strata, and through 2 weeks, flower pesticide normally splits, and microspore is released in the medium.
(11) growth of embryoid forms: after cultivating for 4 weeks, and visible spheroidal embryo of naked eyes and heart type embryo (Fig. 1).After cultivating for 7 weeks, visible torpedo embryo and cotyledon type embryo (Fig. 2).After cultivating for 10 weeks, the cotyledon type embryo is very obvious, afterwards the cotyledon type embryo is changed over to successive transfer culture on the embryo germination medium, and visible cotyledon changes green, and cotyledon flattens, and true leaf occurs, and root system forms, and forms whole plant (seeing Fig. 3,4,5).The monoploid transplantation of seedlings that microspore is induced survives (left side) and seed dliploid seedling (right side) growing state is referring to Fig. 6 normally, and regeneration seedling plant is identified to determine its ploidy through cytology and morphology, promptly identifies it is monoploid or dliploid.The chromosome of the haplobiont that microspore is induced (n=12) picture is seen Fig. 7, and the chromosome of the double haploid that microspore is induced (n=24) picture is seen Fig. 8.By tip of a root pressed disc method (Li Maoxue, Zhang Zanping. crop chromosome and investigative technique thereof. Beijing: the .1996 of Chinese agriculture publishing house, pp231-233), the haplobiont chromosome number is n=12.Monoploid shows as blade with respect to dliploid on morphology narrow and small, and internode shortens, and a little less than the vitality, bud is shaky.
Need to prove that the foregoing description is chosen first-filial generation capsicum variety P51 as experiment material, but is not limited to this material, this method can realize purpose of the present invention for the first-filial generation capsicum variety of other merits.
Subordinate list Nitsch and Nitsch medium constituent list (mg/L)
| KNO 3 | 950 | FeSO 4·7H 2O | 27.8 |
| NH 4NO 3 | 720 | Na 2EDTA | 37.3 |
| MgSO 4·7H 2O | 185 | Nicotinic acid (nicotinic acid) | 5 |
| CaCl 2·2H 2O | 220 | Pyridoxin-HCl (puridoxine hydrochloride) | 0.5 |
| KH 2PO 4 | 68 | Thiamine-HCl (thiamine hydrochloride) | 0.5 |
| MnSO 4·4H 2O H 3BO 4 | 25 10 | D-Biotin (D-vitamin h) Folic acid (folic acid) | 0.05 0.5 |
| ZnSO 4·7H 2O | 10 | Myo-Inositol (inositol) | 100 |
| CuSO 4·5H 2O | 0.025 | Glycine (glycine) | 2 |
| Na 2MoO 4·2H 2O | 0.25 |
Claims (1)
1. an inducing chilli microspore obtains the cultural method of embryoid, it is characterized in that may further comprise the steps:
(1) determines the examination material: choose the first-filial generation capsicum variety as test material;
(2) choose bud: coming into bloom or the Sheng phase, choose by aceto-camine pressed disc method microscopy and to be in the keep to the side bud of phase of monokaryon, its mode of appearance feature shows as corolla and calyx is isometric or corolla is longer than calyx slightly, regularly extracts open flower, allows plant be in bloom all the time;
(3) bud preliminary treatment: suitable bud is placed on 4 ℃ of low temperature refrigerators with self-styled plastic sack splendid attire and handled 1～3 day;
(4) preparation liquid nutrient medium and solid culture medium: two kinds of used minimal mediums of medium are Nitsch and Nitsch medium, and used carbon source is a maltose, and its concentration is 2%～3%; The coagulating agent that solid culture medium adds is an agar, and its concentration is 0.6%～0.8%;
The constituent of solid culture medium is: in Nitsch and Nitsch medium, add 0.6%～0.8% agar of medium weight, and 2%～3% maltose, 0.5%～1.0% active carbon, pH 6.0;
The constituent of liquid nutrient medium is: in Nitsch and Nitsch medium, add 2%～3% maltose, and the zeatin ZT of 0.4～0.8mg/L, the heteroauxin IAA of 0.5mg/L～1.0mg/L, pH 5.8;
(5) sterilization of medium and packing: solid culture is based on 121 ℃, and sterilization is 20 minutes under the 1.1Mpa condition, the liquid nutrient medium plastics filter filtration sterilization that two-layer miillpore filter is housed, the germ-free condition of assurance medium;
Each culture dish of sterilization back injects solid culture medium 6ml earlier, wait solid culture medium to solidify after, add liquid nutrient medium 3ml again, carry out packing;
(6) bud sterilization: earlier with flowing water flushing 30 minutes, use 30 seconds of alcohol surface sterilization of volume fraction 70% again, sterilized 12～15 minutes with the clorox of volume fraction 5% afterwards, use aseptic water washing then 4 times, each 5 minutes;
(7) the aseptic flower pesticide that strips: on aseptic filter paper, strip flower pesticide, avoid damage, damage flower pesticide, and filigree is removed clean with sterile tweezers;
(8) flower pesticide inoculation: each culture dish is put into 12 pieces of flower pesticide, seals with Parafilm;
(9) anther culture: culture dish places incubator, the dark cultivation week under 9 ℃ of conditions, after change 25 ℃ of daytimes, night 20 ℃ tissue culture room over to, continue secretly to cultivate;
(10) the free naturally embryoid of microspore forms: under liquid-solid double-deck culture systems, flower pesticide swims on the liquid nutrient medium on upper strata, and through 2～3 weeks, flower pesticide normally splits, and microspore is released in the medium;
(11) growth of embryoid forms: after cultivating for 4 weeks, visible spheroidal embryo of naked eyes and heart type embryo, cultivated for 7 weeks after, as seen torpedo embryo and cotyledon type embryo, cultivated for 10 weeks after, the cotyledon type embryo is very obvious, afterwards the cotyledon type embryo is changed on the embryo germination medium, form whole plant.
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| CN101822213B (en) * | 2009-12-21 | 2012-06-06 | 河北农业大学 | Method for improving embryoid induction rate of isolated microspore culture of hot (sweet) pepper |
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| CN101874473B (en) * | 2010-08-09 | 2012-03-28 | 武汉大学 | Method for inducing maize microspore to form embryoid |
| CN105638455B (en) * | 2014-11-14 | 2018-03-20 | 石河子大学 | A kind of method and culture medium that Drying pepper haplobiont is obtained by Anther Culture |
| CN104663430B (en) * | 2014-12-09 | 2016-07-06 | 云南农业大学 | A kind of can eat something rare and the breeding method of purple chili that anthocyanidin content is high |
| CN105028205A (en) * | 2015-08-03 | 2015-11-11 | 河南科技大学 | Method for directly cultivating capsicum annuum L. anthers into seedlings |
| CN106718895A (en) * | 2016-12-06 | 2017-05-31 | 重庆市农业科学院 | A kind of method of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm |
| CN111869566A (en) * | 2020-08-01 | 2020-11-03 | 梁江 | Ginseng free microspore induction culture method |
| CN115669544A (en) * | 2022-11-29 | 2023-02-03 | 安徽农业大学 | New method for culturing pepper anther based on embryoid approach and ploidy identification of plant |
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