CN108148799A - The method of high frequency zone Rice Callus culture medium - Google Patents
The method of high frequency zone Rice Callus culture medium Download PDFInfo
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- CN108148799A CN108148799A CN201711498837.3A CN201711498837A CN108148799A CN 108148799 A CN108148799 A CN 108148799A CN 201711498837 A CN201711498837 A CN 201711498837A CN 108148799 A CN108148799 A CN 108148799A
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- callus
- culture medium
- high frequency
- frequency zone
- subculture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
Abstract
This application discloses a kind of methods of high frequency zone Rice Callus culture medium, include the following steps：1. callus induction；2. callus subculture；3. evoked callus bud, which breaks up and then the callus for breaking up budding is placed on root media, cultivates seedling, healing rate and planting percent are finally counted.The step 1. callus induction, further comprises：Mature embryo sterilizes：Rice paddy seed clear water is rinsed well, rinsed with sterile water 35 times；With 75% ethanol postincubation, 3 5min, rinsed with sterile water 35 times；Again with 5 10% 15 20min of hypochlorite disinfectant, rinsed with sterile water 45 times；Aseptic filter paper blots seed, is seeded to callus inducing medium.The method of the present invention establishes a kind of callus abductive approach that can be extensively suitable for most of rice material, once multiple kinds can also not only be screened with obtaining suitable culture medium prescription in the short time, time saving and energy saving, research progress is accelerated in help.
Description
Technical field
The present invention relates to molecular biology Biotechnology in Genetic Breeding fields, especially a kind of to utilize three step optimization high frequency zones
Suitable for cultivating the method for the culture medium of Rice Callus.
Background technology
Rice yield is high, and cultivated area is extensive, is the staple food crop of nearly 50% population in the whole world.Rice Production is to protecting
Hinder world food safety, solve regional problem of food and clothing and play an important roll with developing country's population below the poverty line quantity is reduced.
However, due to the imbalance of all parts of the world region development, the development of rice industry gradually produces many ask
Topic, first, there are many more the population below the poverty line and refugees in global range, it is big to the demand of grain, it needs to be strengthened to rice industry
Popularization and utilization；Second, some areas population is resourceful, natural environment is good, but rice yield is low, causes to soil
The wasting of resources；Third, with the raising of popular life level, to food flavor, the demand of good high-grade rice is also rice breeding
Another a major challenge of industry.
More than three major issues are solved, research energy must be concentrated on three aspects by we, carry on rice superelevation
The research of delivery and feed infant kind is cultivated the rice high yield kind for being suitable for regions of the world weather conditions and is increased to high-quality characteristic rice
Science research input.
At present, a variety of Sequencing of Rice Genome and genetic map drafting have been completed, deciphering to rice functional sequence,
The research of functional gene, the parsing of metabolic regulation network will greatly promote the progress of rice diversity breeding, meanwhile, it will pass
The conventional breeding methods of system with molecule, cell research technology be combined be also modern crop breeding development inexorable trend, and with
Genetic engineering based on tissue cultures will become the necessary means of genome times afterwards comprehensively gene function verification.
Therefore, how rapidly and efficiently filter out suitable callus tissue culture base become establish genetic engineering research body
The premise of system.
At present, the materials such as anther, young fringe, rataria, mature embryo are often used to the callus of inducing paddy rice, wherein ripe
The induction of embryo is relatively easy, and materials are not subject to seasonal restrictions, in genetic engineering utilization
The principal element for influencing rice callus formation and plant regeneration includes explant genotype, exogenous hormone, culture medium
The many aspects such as type and photoperiod and Subculture Time.Wherein the genotype of material and the type of exogenous hormone, concentration and match
Than being most important factor.The research of forefathers focuses mostly in single rice varieties or certain a kind of long-grained nonglutinous rice or the callus of japonica rice
In induction research, it can not extensively be suitable for most of rice varieties, when screening will be directed to different kinds, establish multiple optimization bodies
It is or even the time is wasted in above non-principal influence factor, it is time-consuming and laborious.
Invention content
The technical problems to be solved by the invention are, by many experiments early period, the present invention establishes one kind can be wide
It, not only can be to obtain suitable culture medium prescription in the short time suitable for the callus abductive approach of most of rice material, it can be with
Once multiple kinds are screened, time saving and energy saving, research progress is accelerated in help.
In order to solve the above technical problems, the present invention provides a kind of method of high frequency zone Rice Callus culture medium,
Include the following steps：
1. callus induction；
2. callus subculture；
3. evoked callus bud, which breaks up and then the callus for breaking up budding is placed on root media, cultivates seedling,
Finally count healing rate and planting percent.
The step 1. callus induction, further comprises：Mature embryo sterilizes：
Rice paddy seed clear water is rinsed well, rinsed with sterile water 3-5 times；
With 75% ethanol postincubation 3-5min, rinsed with sterile water 3-5 times；
Again with the hypochlorite disinfectant 15-20min of 5-10%, rinsed with sterile water 4-5 times；
Aseptic filter paper blots seed, is seeded to callus inducing medium.
The callus induction is comprising culture medium prescription：MS minimal mediums+hormone combinations+additional additive+sugarcane
Sugar+plant gel.
The hormone combinations are 2,4-D 1.5-2.5mg/L, 6-BA 0-0.5mg/L, KT 0-0.5mg/L.
1. the callus induction, further comprises：Mature embryo after disinfection is inoculated in culture medium, in 26-28
16h illumination 8h is dark at DEG C, light intensity 1000-2000lex cultures, and after callus is grown, the best embryo callus subculture of growing way is shifted
To subculture medium culture.
2. the callus subculture is comprising culture medium prescription：MS minimal mediums+hormone combinations+additional additive+
Sucrose+plant gel.
The hormone combinations are 2,4-D 1.0-2.0mg/L, 6-BA 0-0.5mg/L, KT 0-0.5mg/L.
2. the callus subculture, further comprises：The best embryo callus subculture of growing way is transferred to subculture medium
On, 16h illumination 8h is dark at 26-28 DEG C, and light intensity 1000-2000lex cultures are persistently cultivated 5-10 days.
The evoked callus bud breaks up comprising culture medium prescription：MS minimal mediums+hormone combinations+additionally add
Add object+sucrose+plant gel.
The hormone combinations are 6-BA 1.0-2.0mg/L, NAA 0-0.5mg/L, KT 1.0-2.0mg/L.
Advantageous effect of the present invention includes：The method of high frequency zone Rice Callus culture of the present invention, utilizes rice paddy seed
Ripe embryonal induction, subculture, bud break up three step optimizations and suitable callus tissue culture based formulas are obtained in relative short time,
The method of the present invention can also a variety of different cultivars rice of primary screening callus tissue culture formula, have eurytopicity.
Specific embodiment
The present invention is described in detail with reference to embodiment.To make the objectives, technical solutions, and advantages of the present invention clearer, bright
Really, the present invention is described in more detail by the following examples, but the invention is not limited in these embodiments.
In order to solve the above technical problems, the present invention provides a kind of method of high frequency zone Rice Callus culture medium,
Include the following steps：
1. callus induction；
2. callus subculture；
3. evoked callus bud, which breaks up and then the callus for breaking up budding is placed on root media, cultivates seedling,
Finally count healing rate and planting percent.
The step 1. callus induction, further comprises：Mature embryo sterilizes：
Rice paddy seed clear water is rinsed well, rinsed with sterile water 3-5 times；
With 75% ethanol postincubation 3-5min, rinsed with sterile water 3-5 times；
Again with the hypochlorite disinfectant 15-20min of 5-10%, rinsed with sterile water 4-5 times；
Aseptic filter paper blots seed, is seeded to callus inducing medium.
The callus induction is comprising culture medium prescription：MS minimal mediums+hormone combinations+additional additive+sugarcane
Sugar+plant gel.
The hormone combinations are 2,4-D 1.5-2.5mg/L, 6-BA 0-0.5mg/L, KT 0-0.5mg/L.
1. the callus induction, further comprises：Mature embryo after disinfection is inoculated in culture medium, in 26-28
16h illumination 8h is dark at DEG C, light intensity 1000-2000lex cultures, and after callus is grown, the best embryo callus subculture of growing way is shifted
To subculture medium culture.
2. the callus subculture is comprising culture medium prescription：MS minimal mediums+hormone combinations+additional additive+
Sucrose+plant gel.
The hormone combinations are 2,4-D 1.0-2.0mg/L, 6-BA 0-0.5mg/L, KT 0-0.5mg/L.
2. the callus subculture, further comprises：The best embryo callus subculture of growing way is transferred to subculture medium
On, 16h illumination 8h is dark at 26-28 DEG C, and light intensity 1000-2000lex cultures are persistently cultivated 5-10 days.
The evoked callus bud breaks up comprising culture medium prescription：MS minimal mediums+hormone combinations+additionally add
Add object+sucrose+plant gel.
The method of high frequency zone Rice Callus culture medium of the embodiment of the present invention, step include：
The seed maturity embryo of disinfection is placed on culture medium, by three crucial incubations 1. callus induction, 2.
Callus subculture and 3. evoked callus bud, which break up and then the callus for breaking up budding are placed on root media, to be trained
Seedling is formed, finally counts healing rate and planting percent.
In above-mentioned steps, mature embryo sterilization method is：Rice paddy seed full, of the same size is selected, clear water rinses dry
Only, rinsed with sterile water 3-5 times；With 75% ethanol postincubation 3-5min, rinsed with sterile water 3 times；Disappeared again with the sodium hypochlorite of 5-10%
Malicious 15-20min, rinsed with sterile water 4-5 times；Aseptic filter paper blots seed, is seeded to callus inducing medium.
1. callus induction is wherein crucial incubation comprising culture medium prescription：MS minimal mediums+hormone combinations
+ additional additive+sucrose+plant gel.Wherein hormone combinations are 2,4-D 1.5-2.5 mg/L, 6-BA 0-0.5mg/L, KT
0-0.5mg/L, 3 kinds of hormones set 3 gradients respectively in the above range, carry out L9 (34) and combine, are combined into 9 kinds of formulas altogether.
Mature embryo after disinfection is inoculated in this 9 kinds of culture mediums, 16h illumination 8h is dark at 26-28 DEG C, light intensity 1000-
2000lex is cultivated, and after callus is grown, the best embryo callus subculture of growing way is transferred to subculture medium culture.
2. callus subculture is wherein crucial incubation comprising culture medium prescription：MS minimal mediums+hormone combinations
+ additional additive+sucrose+plant gel.Wherein hormone combinations are 2,4-D 1.0-2.0 mg/L, 6-BA 0-0.5mg/L, KT
0-0.5mg/L, 3 kinds of hormones set 3 gradients respectively in the above range, carry out L9 (34) and combine, are combined into 9 kinds of formulas altogether.
The best embryo callus subculture of growing way is transferred on this 9 kinds of subculture mediums, 16h illumination 8h is dark at 26-28 DEG C, light intensity
1000-2000lex is cultivated, and is persistently cultivated 5-10 days or so.
3. the differentiation of evoked callus bud is wherein crucial incubation comprising culture medium prescription：MS minimal mediums+swash
Element combination+additional additive+sucrose+plant gel.Wherein hormone combinations are 6-BA 1.0-2.0mg/L, NAA 0-0.5mg/L,
KT 1.0-2.0mg/L, 3 kinds of hormones set 3 gradients respectively in the above range, carry out L9 (34) and combine, are combined into 9 kinds altogether
Formula.Embryo callus subculture after the best subculture of growing way is transferred on this 9 seed bud inducing culture, the 16h light at 26-28 DEG C
According to 8h dark, light intensity 1000-2000lex cultures, lasting culture is broken up to callus sprouts.
Above-mentioned Furcation defects culture medium prescription is：1/2MS culture mediums+paclobutrazol+activated carbon+sucrose+agar.It will induce
The callus of bud is transferred to Furcation defects culture medium, and 16h illumination 8h is dark at 26-28 DEG C, light intensity 1000-2000lex trainings
It supports, lasting culture to seedling of taking root.
In above-mentioned key incubation, MS minimal mediums are most basic, most collective media in Plant Tissue Breeding,
Its formula is as follows：
Above-mentioned key culture medium is also containing additional additive, respectively caseinhydrolysate, proline, glutamine and mountain
Pears alcohol, corresponding concentration 0.3-0.5g/L, 0.3-1.0g/L, 0.2-1.0g/L and 10-40g/L, preferably 0.3g/L,
0.5g/L, 0.5g/L and 10g/L.
Above-mentioned key culture medium is also containing sucrose and agar, and wherein sucrose concentration is 20-30g/L, and plant gel is a concentration of
3-5g/L, preferably 30g/L and 3g/L.
Above-mentioned 1/2MS culture mediums halve for MS culture medium a great number of elements, and other constituent concentrations are constant.
Wherein a concentration of 1.0-2.0mg/L of paclobutrazol contained by Furcation defects culture medium, concentration of activated carbon 0.01-0.05%,
Sucrose concentration is 20-30g/L, agar concentration 7-8g/L, preferably respectively 2.0mg/L, 0.01%, 30g/L and 8g/L.
Culture medium of the present invention is configured to mother liquor respectively by each ingredient, first prepares MS fluid nutrient mediums and boils, then according to
It is secondary to add in various hormonal components, additional additive, sucrose and plant gel, pH to 5.8-6.0, packing are adjusted after melting completely
Into culture dish, 121 DEG C of saturated vapors sterilizing 20min.After the disinfection of seed maturity embryo, 1. it is cured by three crucial incubations
Injured tissue induce, 2. callus subculture and 3. evoked callus bud differentiation and then by break up sprout callus be placed in life
Seedling is cultivated on root culture medium, finally counts healing rate and plantlet differentiation rate.
Healing rate (%)=(the embryo number for generating embryo number/inoculation of callus) × 100%
Plantlet differentiation rate (%)=(the callus block number of callus block number/transfer of Differentiation From Calli) × 100%
Embodiment 1
A kind of culture medium of high frequency zone Rice Callus induction is to add 3 kinds respectively in MS minimal mediums to swash
Element, additional additive (caseinhydrolysate 0.3g/L, proline 0.5g/L, glutamine 0.5g/L and sorbierite 10g/L), sugarcane
Sugared 30g/L and plant gel 3g/L.3 kinds of hormone according to the form below (units：Mg/L), carry out L9 (34) to combine, be combined into 9 kinds altogether and match
Side.
Mature embryo after disinfection is inoculated in this 9 kinds of culture mediums, 16h illumination 8h is dark at 26-28 DEG C, light intensity
1000-2000lex is cultivated, and after callus is grown, counts healing rate.
Embodiment 2
A kind of culture medium of high frequency zone Rice Callus subculture is to add 3 kinds respectively in MS minimal mediums to swash
Element, additional additive (caseinhydrolysate 0.3g/L, proline 0.5g/L, glutamine 0.5g/L and sorbierite 10g/L), sugarcane
Sugared 30g/L and plant gel 3g/L.3 kinds of hormone according to the form below (units：Mg/L), carry out L9 (34) to combine, be combined into 9 kinds altogether and match
Side.
The best embryo callus subculture of growing way is transferred on this 9 kinds of subculture mediums, 16h illumination 8h is black at 26-28 DEG C
Secretly, light intensity 1000-2000lex is cultivated, and is persistently cultivated 7 days or so.
Embodiment 3
A kind of culture medium of high frequency zone Rice Callus bud differentiation, is to add 3 kinds respectively in MS minimal mediums
Hormone, additional additive (caseinhydrolysate 0.3g/L, proline 0.5g/L, glutamine 0.5g/L and sorbierite 10g/L),
Sucrose 30g/L and plant gel 3g/L.3 kinds of hormone according to the form below (units：Mg/L), carry out L9 (34) to combine, be combined into 9 kinds altogether
Formula.
Embryo callus subculture after the best subculture of growing way is transferred on this 9 kinds of subculture mediums, the 16h light at 26-28 DEG C
According to 8h dark, light intensity 1000-2000lex cultures, lasting culture is broken up to callus sprouts, and counts plantlet differentiation rate.
Embodiment 4
Choose 4 kinds of long-grained nonglutinous rices (beautiful sesame oil accounts for, Hunan morning Xian 17, in praise early 17 and own first familiar generation-miscellaneous Yc17D5) and 4 kinds
Japonica rice (Nipponbare, beautiful sesame oil account for, peaceful salt 1 and even round-grained rice 4) seed maturity embryo selects plumpness as donor, in the same size
Seed, screen the mature embryo callus culture medium prescription of each rice variety respectively using above-mentioned Screening of Media method,
Healing rate and plantlet differentiation rate are counted, result is summarized as follows：
Using the method for the present invention to the callus screening of medium formula of 4 kinds of long-grained nonglutinous rices, healing rate can be made to reach 60%-
80%, plantlet differentiation rate reaches more than 80%-90%, since Different Rice Varieties deposit the sensibility of hormon and concentration
In difference, so the optimum medium formula of different rice varieties is clearly different, hormone optimization provided by the present invention
Concentration range can meet the callus tissue culture demand of most of rice varieties, but be not meant to be optimal selection, profit
Significantly the use scope of hormone can be set more gradients with the method, optimal medium formula is obtained with this.So
On the basis of the present invention, those skilled in the art make some modifications or improvements it, are easily.Therefore, without departing from
These modifications or improvements on the basis of spirit of the invention, belong to the scope of protection of present invention.
The above is only several embodiments of the present invention, any type of limitation is not done to the present invention, although this
Invention is disclosed as above with preferred embodiment, however not to limit the present invention, any person skilled in the art,
In the range of not departing from technical solution of the present invention, make a little variation using the technology contents of the disclosure above or modification is impartial
Equivalence enforcement case is same as, is belonged in technical solution of the present invention protection domain.
Claims (10)
- A kind of 1. method of high frequency zone Rice Callus culture medium, which is characterized in that include the following steps：1. callus induction；2. callus subculture；3. evoked callus bud, which breaks up and then the callus for breaking up budding is placed on root media, cultivates seedling, finally Count healing rate and planting percent.
- 2. the method for high frequency zone Rice Callus culture medium according to claim 1, which is characterized in that the step is 1. Callus induction further comprises：Mature embryo sterilizes：Rice paddy seed clear water is rinsed well, rinsed with sterile water 3-5 times；With 75% ethanol postincubation 3-5min, rinsed with sterile water 3-5 times；Again with the hypochlorite disinfectant 15-20min of 5-10%, rinsed with sterile water 4-5 times；Aseptic filter paper blots seed, is seeded to callus inducing medium.
- 3. the method for high frequency zone Rice Callus culture medium according to claim 1, which is characterized in that the callus group Knit induction is comprising culture medium prescription：MS minimal mediums+hormone combinations+additional additive+sucrose+plant gel.
- 4. the method for high frequency zone Rice Callus culture medium according to claim 3, which is characterized in that the hormone group It is combined into 2,4-D 1.5-2.5mg/L, 6-BA 0-0.5mg/L, KT 0-0.5mg/L.
- 5. the method for high frequency zone Rice Callus culture medium according to claim 1, which is characterized in that the 1. callus Tissue induction, further comprises：Mature embryo after disinfection is inoculated in culture medium, 16h illumination 8h is dark at 26-28 DEG C, Light intensity 1000-20001ex is cultivated, and after callus is grown, the best embryo callus subculture of growing way is transferred to subculture medium culture.
- 6. the method for high frequency zone Rice Callus culture medium according to claim 1, which is characterized in that the 2. callus Tissue subculture be comprising culture medium prescription：MS minimal mediums+hormone combinations+additional additive+sucrose+plant gel.
- 7. the method for high frequency zone Rice Callus culture medium according to claim 6, which is characterized in that the hormone group It is combined into 2,4-D 1.0-2.0mg/L, 6-BA 0-0.5mg/L, KT 0-0.5mg/L.
- 8. the method for high frequency zone Rice Callus culture medium according to claim 1, which is characterized in that the 2. callus Subculture is organized, is further comprised：The best embryo callus subculture of growing way is transferred on subculture medium, the 16h illumination at 26-28 DEG C 8h is dark, and light intensity 1000-20001ex cultures are persistently cultivated 5-10 days.
- 9. the method for high frequency zone Rice Callus culture medium according to claim 1, which is characterized in that the induction is cured Injured tissue bud breaks up comprising culture medium prescription：MS minimal mediums+hormone combinations+additional additive+sucrose+plant gel.
- 10. the method for high frequency zone Rice Callus culture medium according to claim 9, which is characterized in that the hormone It is combined as 6-BA 1.0-2.0mg/L, NAA 0-0.5mg/L, KT 1.0-2.0mg/L.
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| CN108739401A (en) * | 2018-06-24 | 2018-11-06 | 合肥扬扬农业科技有限公司 | A kind of production method of rice paddy seed |
| CN110169357A (en) * | 2019-06-18 | 2019-08-27 | 天津市农作物研究所(天津市水稻研究所) | A kind of production method of rice paddy seed |
| CN111004817A (en) * | 2019-12-30 | 2020-04-14 | 北京市农林科学院 | Agrobacterium-mediated rice genetic transformation method |
| CN111657272A (en) * | 2020-06-23 | 2020-09-15 | 上海市农业生物基因中心 | Ultralow-temperature preservation method for rice callus |
| CN113016614A (en) * | 2021-03-29 | 2021-06-25 | 广东海洋大学 | Method for culturing and propagating calluses of mature embryos of long hair grains of seawater rice |
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| CN113016614A (en) * | 2021-03-29 | 2021-06-25 | 广东海洋大学 | Method for culturing and propagating calluses of mature embryos of long hair grains of seawater rice |
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Application publication date: 20180612 |