CN103444523A - Method for quickly introducing embryonic callus through anther to regenerate plant - Google Patents

Method for quickly introducing embryonic callus through anther to regenerate plant Download PDF

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CN103444523A
CN103444523A CN2013100268415A CN201310026841A CN103444523A CN 103444523 A CN103444523 A CN 103444523A CN 2013100268415 A CN2013100268415 A CN 2013100268415A CN 201310026841 A CN201310026841 A CN 201310026841A CN 103444523 A CN103444523 A CN 103444523A
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callus
grape
culture
anther
root
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CN103444523B (en
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赵凤霞
高相彬
王正平
李海峰
宋学立
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Tobacco Research Institute Henan Academy Of Agricultural Sciences
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Tobacco Research Institute Henan Academy Of Agricultural Sciences
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Abstract

The invention provides a method for quickly introducing embryonic callus through anther to regenerate a plant, and belongs to the field of agricultural science. The method comprises the following steps: getting wine grape Riesling anther as a material; inducing the embryonic callus through the anther; performing differentiation culture on the callus; performing rooting culture on the differentiated seedlings; and acclimatizing and transplanting to accomplish the grape plant regeneration. The culture medium and the cultivation method have good effect, and have the advantages that the inductivity of the embryonic callus is high, the quality is high, and the cultivation cycle is relatively short; simultaneously, subculturing can be performed for a long term, and a constant loose embryonic callus state can be kept, therefore, sufficient materials are prepared for the study on production of secondary metabolite for grape cell culture, and the study on biotechnology. The culture medium and the anther cultivation method applied to grape genetic breeding can shorten the breeding cycle and provide an important technology platform for improving the grape varieties.

Description

A kind of method of flower pesticide rapid induction embryo callus regeneration plant
Technical field
The invention belongs to field of tissue culture, be specifically related to a kind of method of inducing grape flower pesticide formation embryo callus of cultivating by tissue.
Background technology
Set up stablize high frequency the grape regenerating system for optimizing the transgenosis condition, the raising transgene efficiency, carry out new grape variety seed selection and genetic improvement significant.The callus of take has a lot of advantages as acceptor material carries out the gene genetic Study on Transformation.The first, the cell broken up all is returned to the meristematic cell level of dedifferentiation, is easy to accept foreign gene, and transformation efficiency is higher; The second, expand numerous amount large, the transformed calli of acquisition expands numerous cultivation by subculture, can differentiate more transformed plant; The 3rd, but the equal evoked callus of Various Tissues, organ, and explant examination material is extensive.The acquisition of grape regenerating system at present is mainly by two approach: neomorph approach and somatic embryo Regeneration Ways.The neomorph approach is directly to form indefinite bud, adventive root on explant, the whole plant of regenerating, or explant induces the generation callus through dedifferentiation, thus callus break up the acquisition regeneration plant on differential medium.This approach regenerative process is simple, and the cycle is short, but easily produces chimera.The somatic embryo Regeneration Ways is directly to differentiate somatic embryo or plant tissue is first induced the generation callus by plant tissue, and callus is through inducing the organizator blast.Producing somatic embryo by organ induced embryonic callus such as flower pesticide, filigree, ovules is best, the most widely used induction mode of effect in the research of grape regenerating system.Induction Process exists embryonic callus induction efficiency low, is accompanied by the appearance of non-embryonic callus tissue, and cultivation period is long etc., has limited the application of grape anther culture in actual breeding.Therefore the inductivity that improves the grape embryo callus is a key issue in the research of grape anther culture technique.
Summary of the invention
The object of the invention is to overcome the low deficiency of existing grape flower pesticide induced embryonic callus inductivity, a kind of culture medium prescription and abductive approach of efficiently inducing grape flower pesticide to form embryo callus and then regeneration grapevine seedling is provided, improving the inductivity of embryo callus by the method, is to lay the foundation for setting up efficient grape anther culture plant regeneration.
The present invention is achieved through the following technical solutions:
The method of grape flower pesticide rapid induction embryo callus regeneration plant: utilizing wine grape Riesling flower pesticide is material, carries out the steps such as flower pesticide induced embryonic callus, Calli Differentiation cultivation, the cultivation of differentiation seedling rooting, acclimatization and transplants and completes grapevine seedling regeneration.According to the method, it is characterized in that grape is contained to spend front 12-14 days, pollen tetrad, to monokaryotic stage, is won the full bud of flower pesticide in the fine morning, and 4 ℃ of dark preservations 3 days can improve the efficiency of inducing of embryo callus; During from sterile bud, taking-up flower pesticide is inoculated into callus inducing medium under aseptic condition, the callus inducing medium formula is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.2mg L -1nicotinic acid, 150mgL -1inositol, 2.0mg L- 1p-CPA, 0.20mg L -1tDZ, 5mg L -1agNO 3, 10mg L -1gentamicin, 100mg L -1cysteine, 50mg L -1caseinhydrolysate, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.Embryo callus is inoculated into to organize on differential medium and carries out induction, organize differential medium to be: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.5mg L -1nicotinic acid, 150mg L -1inositol, 1.0mg L -1p-CPA, 0.20mg L -1tDZ, 0.5mg L -1nOA, 0.01mg L -1gA 3, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 10g L -1coconut palm breast, 5% sucrose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is 25 ℃ of temperature, humidity 70%, illumination 1000-1500lux, light application time 12 hours/day.To break up seedling and proceed to the root media root induction, the culture of rootage based component is 1/2MS macroelement, MS trace element, MS molysite, 0.1mg L -1thiamine hydrochloride, 0.8mg L -1puridoxine hydrochloride, 0.5mgL -1nicotinic acid, 100mg L -1inositol, 0.5mg L -1α-naphthaleneacidsodium, 0.5mg L -1nOA, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is first under light, to cultivate one week, more secretly cultivates one week, then is cultured to normally and takes root under light, 25 ℃ of temperature, humidity 70%, illumination 1500-2000lux, light application time 12 hours/day; When the seedling base portion grows 3-4 bar root, open bottle cap during the long 3cm of root, 25 ℃ of environmental temperatures, humidity 85%, intensity of illumination 2000lux, hardening 5 days; Then taking-up group training seedling, running water is cleaned the root medium, is transplanted in sterilized pearlite interstitial substance, after the 1/8MS nutrient solution irrigates, water weekly culture fluid one time, add a cover with the plastic sack of the upper end clip of nutritive cube formed objects and keep appropriateness, 25 ℃ of indoor cultivations 1 month, after be transplanted to land for growing field crops.
Further, the method is carried out according to following steps:
(1) flower pesticide collection and disinfecting: grape is contained and is spent front 12-14 days, and pollen tetrad, to monokaryotic stage, is won the full bud of flower pesticide in the fine morning, and 4 ℃ of dark preservations 3 days, then carry out surface sterilization to bud.
(2) anther callus is cultivated: during from sterile bud, taking-up flower pesticide is inoculated into callus inducing medium under aseptic condition.Described callus inducing medium formula is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.2mg L -1nicotinic acid, 150mg L -1inositol, 2.0mg L -1p-CPA, 0.20mg L -1tDZ, 5mg L -1agNO 3, 10mgL -1gentamicin, 100mg L -1cysteine, 50mg L -1caseinhydrolysate, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.
(3) Calli Differentiation is cultivated: embryo callus is inoculated into to organize on differential medium and carries out induction.The described differential medium formula of organizing is: MS macroelement, MS trace, MS molysite, 0.5mgL -1thiamine hydrochloride, 0.2mgL -1puridoxine hydrochloride, 0.5mgL -1nicotinic acid, 150mg L -1inositol, 1.0mg L -1p-CPA, 0.20mg L -1tDZ, 0.5mg L -1nOA, 0.01mg L -1gA 3, 50mgL -1caseinhydrolysate, 2.0mg L -1glycine, 10g L -1coconut palm breast, 5% sucrose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is 25 ℃ of temperature, humidity 70%, illumination 1000-1500lux, light application time 12 hours/day.
(4) the differentiation seedling rooting is cultivated: will break up seedling and proceed to the root media root induction, described culture of rootage based component is 1/2MS macroelement, MS trace element, MS molysite, 0.1mg L -1thiamine hydrochloride, 0.8mgL -1puridoxine hydrochloride, 0.5mgL -1nicotinic acid, 100mgL -1inositol, 0.5mg L -1α-naphthaleneacidsodium, 0.5mg L -1nOA, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is first under light, to cultivate one week, more secretly cultivates one week, then is cultured to normally and takes root under light, 25 ℃ of temperature, humidity 70%, illumination 1500-2000lux, light application time 12 hours/day.
(5) hardening, heel in, transplant: when the seedling base portion grows 3-4 bar root, open bottle cap during the long 3cm of root, 25 ℃ of environmental temperatures, humidity 85%, intensity of illumination 2000lux, hardening 5 days; Then taking-up group training seedling, running water is cleaned the root medium, is transplanted in sterilized pearlite interstitial substance, after the 1/8MS nutrient solution irrigates, water weekly culture fluid one time, add a cover with the plastic sack of the upper end clip of nutritive cube formed objects and keep appropriateness, 25 ℃ of indoor cultivations 1 month, after be transplanted to land for growing field crops.
Effect of the present invention:
Compared with the prior art, the present invention adopts and contains the grape bud of spending front 12-14 days, before inoculation, bud is carried out to 4 ℃ of dark preservations 3 days, is conducive to the raising of embryonic callus induction efficiency.Callus inducing medium is compared with common MS medium, contains higher thiamine hydrochloride and inositol, has improved the efficiency of inducing of embryo callus; The growth hormone of higher concentration is conducive to evoked callus and forms; Gentamicin has alleviated the pollution that manual operation causes to a certain extent; AgNO 3alleviated the callus browning necrosis with cysteine; Organize differential medium to compare with common MS medium, the thiamine hydrochloride of higher concentration and inositol and additional coconut palm breast are conducive to the differentiation of callus and the formation of embryoid, the sucrose of high concentration (5%) is conducive to the growth of embryoid, the gibberellin GA of trace 3be conducive to the growth of embryoid; Root media is compared the puridoxine hydrochloride (8%) that contains higher concentration with common MS medium, be conducive to the growth of root system; The present invention adopts 0.28% de-acetyl gellan gum as curing agent, with agar, compares, and de-acetyl gellan gum transparency is high, good permeability, solidifying low with point, few containing impurity components such as Ca, Mg, be conducive to contained nutriment in medium and be absorbed by plants, there is higher value.
Utilize medium of the present invention to carry out the anther cultural method of grape and have advantages of that frequency of embryonic callus induction is high, cultivation cycle is short, the inductivity of embryo callus is between 40%-60%, and cultivation cycle is about 7 months.Flower pesticide is drawn materials conveniently, simple in structure, and genotype is abundant, and method is easy, easy operating and application.
The accompanying drawing explanation:
Fig. 1: flower pesticide evoked callus figure;
Fig. 2: Calli Differentiation figure;
Fig. 3: culture of rootage seedling.
Embodiment:
1, flower pesticide collection and disinfecting:
(1) flower pesticide collection: grape is contained and is spent front 12-14 days, and pollen tetrad, to monokaryotic stage, is won the full bud of flower pesticide in the fine morning, 4 ℃ of dark preservations 3 days.
(2) flower pesticide is disinfected: bud is carried out to surface sterilization, i.e. first immersion treatment 1 minute in the alcohol of 75% (v/v), use again aseptic water washing 3 times, then use the clorox of 2% (v/v) to disinfect 8 minutes, finally use aseptic water washing 5 times.
(3) inoculation: get sterile bud, in super-clean bench under aseptic condition, remove petal by microscope with dissecting needle, clamp filigree with tweezers, be inoculated in diameter with taking out the flower pesticide Direct Uniform and be that in the culture dish that 9cm contains callus inducing medium, (callus inducing medium is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.2mg L -1nicotinic acid, 150mg L -1inositol, 2.0mg L -1p-CPA, 0.20mg L -1tDZ, 5mg L -1agNO 3, 10mg L -1gentamicin, 100mg L -1cysteine, 50mg L -1caseinhydrolysate, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2).
2, anther callus is cultivated: culture dish is inverted, and 25 ℃ of dark culturing, and a situation arises with callus to observe the flower pesticide situation of change, can observe the callus appearance after 20 days.Induction Process produces embryo and non-embryo two class callus, and embryo callus mostly is the yellow particle shape, and quality is tight, and non-embryonic callus is organized as translucent pasty state, short texture, and quality is soft amorphous.Distinguish under microscope and identify embryo callus and non-embryonic callus tissue, by the embryo callus subculture amplification of identifying, every 4 weeks subcultures once.
3, Calli Differentiation is cultivated: embryo callus is inoculated into to organize on differential medium and breaks up cultivation (the tissue differential medium is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.5mg L -1nicotinic acid, 150mg L -1inositol, 1.0mg L -1p-CPA, 0.20mg L -1tDZ, 0.5mg L -1nOA, 0.01mg L -1gA 3, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 10g L -1coconut palm breast, 5% sucrose and 0.28% de-acetyl gellan gum, pH 6.2).Produce loose, scleroid cells,primordial group after 2 weeks, these cell masses are easy to break away from maternal tissue, develop into gradually spherical, heart-shaped, cotyledon shape embryoid, break up seedling.Condition of culture is 25 ℃ of temperature, humidity 70%, illumination 1000-1500lux, light application time 12 hours/day.
4, the differentiation seedling rooting is cultivated:
To break up seedling proceeds to the root media root induction (the culture of rootage based component is 1/2MS macroelement, MS trace element, MS molysite, 0.1mg L -1thiamine hydrochloride, 0.8mg L -1puridoxine hydrochloride, 0.5mg L -1nicotinic acid, 100mg L -1inositol, 0.5mg L -1α-naphthaleneacidsodium, 0.5mg L -1nOA, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2).Condition of culture is to cultivate one week under first light, more secretly cultivates one week, then is cultured to normally and takes root under light, 25 ℃ of temperature, humidity 70%, illumination 1500-2000lux, light application time 12 hours/day.
5, hardening, heel in, transplant:
When the seedling base portion grows 3-4 bar root, open bottle cap during the long 3cm of root, 25 ℃ of environmental temperatures, humidity 85%, intensity of illumination 2000lux, hardening 5 days; Then taking-up group training seedling, running water is cleaned the root medium, be transplanted in sterilized pearlite interstitial substance, after the 1/8MS nutrient solution irrigates, water weekly culture fluid one time, add a cover with the plastic sack of the upper end clip of nutritive cube formed objects and keep appropriateness, 25 ℃ of indoor cultivations 1 month, after be transplanted to land for growing field crops and get final product.
Utilize said method, the present invention is by Riesling grape flower pesticide induced embryonic callus and obtain regeneration plant, and embryonic callus induction efficiency is higher, and between 40%-60%, cultivation cycle is short, about 7 months.

Claims (2)

1. the method for a grape flower pesticide rapid induction embryo callus regeneration plant, it is characterized in that: utilizing wine grape Riesling flower pesticide is material, carries out the steps such as flower pesticide induced embryonic callus, Calli Differentiation cultivation, the cultivation of differentiation seedling rooting, acclimatization and transplants and completes grapevine seedling regeneration.
2. method according to claim 1, is characterized in that, described method is carried out according to following steps:
(1) flower pesticide collection and disinfecting: grape is contained and is spent front 12-14 days, and pollen tetrad, to monokaryotic stage, is won the full bud of flower pesticide in the fine morning, and 4 ℃ of dark preservations 3 days, then carry out surface sterilization to bud.
(2) anther callus is cultivated: during from sterile bud, taking-up flower pesticide is inoculated into callus inducing medium under aseptic condition.Described callus inducing medium formula is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mgL -1puridoxine hydrochloride, 0.2mg L -1nicotinic acid, 150mg L -1inositol, 2.0mg L -1p-CPA, 0.20mg L -1tDZ, 5mg L -1agNO 3, 10mgL -1gentamicin, 100mg L -1cysteine, 50mg L -1caseinhydrolysate, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.
(3) Calli Differentiation is cultivated: embryo callus is inoculated into to organize on differential medium and carries out induction.The described differential medium formula of organizing is: MS macroelement, MS trace, MS molysite, 0.5mgL -1thiamine hydrochloride, 0.2mgL -1puridoxine hydrochloride, 0.5mgL -1nicotinic acid, 150mg L -1inositol, 1.0mg L -1p-CPA, 0.20mg L -1tDZ, 0.5mg L -1nOA, 0.01mg L -1gA 3, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 10g L -1coconut palm breast, 5% sucrose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is 25 ℃ of temperature, humidity 70%, illumination 1000-1500lux, light application time 12 hours/day.
(4) the differentiation seedling rooting is cultivated: will break up seedling and proceed to the root media root induction, described culture of rootage based component is 1/2MS macroelement, MS trace element, MS molysite, 0.1mg L -1thiamine hydrochloride, 0.8mg L -1puridoxine hydrochloride, 0.5mg L -1nicotinic acid, 100mg L -1inositol, 0.5mg L -1α-naphthaleneacidsodium, 0.5mg L -1nOA, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is first under light, to cultivate one week, more secretly cultivates one week, then is cultured to normally and takes root under light, 25 ℃ of temperature, humidity 70%, illumination 1500-2000lux, light application time 12 hours/day.
(5) hardening, heel in, transplant: when the seedling base portion grows 3-4 bar root, open bottle cap during the long 3cm of root, 25 ℃ of environmental temperatures, humidity 85%, intensity of illumination 2000lux, hardening 5 days; Then taking-up group training seedling, running water is cleaned the root medium, is transplanted in sterilized pearlite interstitial substance, after the 1/8MS nutrient solution irrigates, water weekly culture fluid one time, add a cover with the plastic sack of the upper end clip of nutritive cube formed objects and keep appropriateness, 25 ℃ of indoor cultivations 1 month, after be transplanted to land for growing field crops.
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Cited By (7)

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Publication number Priority date Publication date Assignee Title
CN104686361A (en) * 2015-03-20 2015-06-10 浙江万里学院 Induction and culture method of embryonic callus of grape
CN111149674A (en) * 2020-02-28 2020-05-15 科稷达隆生物技术有限公司 Culture method for improving transplanting survival rate of plant tissue culture seedlings
CN111758575A (en) * 2020-08-01 2020-10-13 江苏高航农业科技有限公司 Grape anther embryonic callus differentiation culture medium
CN111758574A (en) * 2020-08-01 2020-10-13 江苏高航农业科技有限公司 Grape anther induction culture medium for improving embryogenic callus induction rate
CN115211370A (en) * 2022-08-18 2022-10-21 西北农林科技大学 Cabernet sauvignon flower organ callus induction culture medium and culture method
CN115316271A (en) * 2022-07-26 2022-11-11 江苏省农业科学院宿迁农科所 Tissue culture method of muscadine
CN115843690A (en) * 2022-12-15 2023-03-28 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） Regeneration method using honeysuckle anther as explant

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104686361A (en) * 2015-03-20 2015-06-10 浙江万里学院 Induction and culture method of embryonic callus of grape
CN111149674A (en) * 2020-02-28 2020-05-15 科稷达隆生物技术有限公司 Culture method for improving transplanting survival rate of plant tissue culture seedlings
CN111758575A (en) * 2020-08-01 2020-10-13 江苏高航农业科技有限公司 Grape anther embryonic callus differentiation culture medium
CN111758574A (en) * 2020-08-01 2020-10-13 江苏高航农业科技有限公司 Grape anther induction culture medium for improving embryogenic callus induction rate
CN115316271A (en) * 2022-07-26 2022-11-11 江苏省农业科学院宿迁农科所 Tissue culture method of muscadine
CN115211370A (en) * 2022-08-18 2022-10-21 西北农林科技大学 Cabernet sauvignon flower organ callus induction culture medium and culture method
CN115211370B (en) * 2022-08-18 2023-09-01 西北农林科技大学 Cabernet sauvignon flower organ callus induction medium and culture method
CN115843690A (en) * 2022-12-15 2023-03-28 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） Regeneration method using honeysuckle anther as explant
CN115843690B (en) * 2022-12-15 2023-08-08 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） Regeneration method using honeysuckle anther as explant

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Inventor after: Zhao Fengxia

Inventor after: Gao Xiangbin

Inventor after: Wang Zhengping

Inventor after: Li Yaoyu

Inventor after: Song Pengfei

Inventor after: Li Haifeng

Inventor after: Song Xueli

Inventor after: Meng Zhiyong

Inventor before: Zhao Fengxia

Inventor before: Gao Xiangbin

Inventor before: Wang Zhengping

Inventor before: Li Haifeng

Inventor before: Song Xueli

COR Change of bibliographic data

Free format text: CORRECT: INVENTOR; FROM: ZHAO FENGXIA GAO XIANGBIN WANG ZHENGPING LI HAIFENG SONG XUELI TO: ZHAO FENGXIA GAO XIANGBIN WANG ZHENGPING LI YAOYU SONG PENGFEI LI HAIFENG SONG XUELI MENG ZHIYONG

C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20141112

Termination date: 20160108