CN104798685A - Establishment method of apple blade regeneration system - Google Patents
Establishment method of apple blade regeneration system Download PDFInfo
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- CN104798685A CN104798685A CN201510214752.2A CN201510214752A CN104798685A CN 104798685 A CN104798685 A CN 104798685A CN 201510214752 A CN201510214752 A CN 201510214752A CN 104798685 A CN104798685 A CN 104798685A
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Abstract
The invention discloses an establishment method of an apple blade regeneration system, which comprises the following steps of 1) performing sound seedling cultivation, 2) performing test tube seedling treatment and inoculation, 3) performing adventitious-bud sound seedling cultivation, and 4) performing rooting cultivation to form a test tube apple seedling. The method has the benefits that the establishment method of the apple blade regeneration system establishes the high-efficiency No. 2 Changfu apple blade regeneration system; an adventitious-bud regeneration rate of a blade reaches 100%; a regeneration coefficient reaches above 5; a rooting rate reaches above 86%; and a requirement of genetic transformation of an apple can be met.
Description
Technical field
The present invention relates to the method for building up of Apple Leaves regenerating system.
Background technology
Since James in 1989 etc. adopt the agriculture bacillus mediated mode of leaf disk method first passage to obtain the transfer-gen plant of apple, leaf disc transformation method has become the main method of carrying out Agrobacterium-mediated Genetic Transformation of Apple.Although Chinese scholars obtains marshal, imperial loud, high-pitched sound draws the transfer-gen plant with apple varieties such as Xin Qiaonajin, but due to the restriction of the problems such as Apple in Vitro regeneration capacity is poor, transformation system is perfect not, genetic transformation efficiency is not high, obtain apple transfer-gen plant difficulty larger.Therefore, the basis that efficient vitro Regeneration System is Agrobacterium-mediated Genetic Transformation of Apple is set up." Nagafu No.2 " is one of China's main cultivation apple improved seeds, but due to the regeneration rate of this kind lower, only have about 60%, never carry out the research of this kind genetic transformation aspect.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, studies, provide the method for building up of " Nagafu No.2 " Apple Leaves high-efficiency regeneration system from aspects such as Screening of Media, hormone combination, culture techniques.
To achieve these goals, technical scheme provided by the invention is: the method for building up of Apple Leaves regenerating system, comprises the following steps:
1) strong seedling culture: the strong seedling culture base of employing is MS medium+6-benzyl aminoadenine 0.5mg/L+ methyl α-naphthyl acetate 0.1mg/L+sucrose 30g/L+ agar 6.0g/L, pH6.0; Culture vessel is 150mL triangular flask, every bottle of packing strong seedling culture base 50ml; Condition of culture is intensity of illumination 3000lx, cultivation temperature 25-28 DEG C; The test-tube plantlet blade cultivating 40-50d is used as regrown material;
2) test-tube plantlet process and inoculation: the healthy and strong test-tube plantlet cultivating 40-50d in step 1), aseptically operate, get 2 ~ 3 pieces of well-grown blades in middle part, with scissors, blade both sides leaf margin is cut, be cut into the square of 0.8cm × 0.8cm, leaf faces up and is seeded on differentiation adventitious buds medium, inoculates 5 pieces of blades in every bottle; The differentiation adventitious buds medium adopted is MS medium+methyl α-naphthyl acetate 0.3 mg/L+6-benzyl aminoadenine 3.0 mg/L+ TDZ plant growth regulator 1.0 mg/L+polyvinylpyrrolidone 150mg/L+ sucrose 30g/L+ agar 6.0g/L, pH6.0; Culture vessel is 150mL triangular flask, every bottle of packing differentiation adventitious buds medium 50ml, 121 DEG C of sterilizing 20min; Condition of culture is: light culture 3 weeks after inoculation, and cultivation temperature is 20-25 DEG C, cultivates, intensity of illumination 1000-2000lx under being then placed in light, and the photoperiod is 16h/8h, cultivation temperature 25-28 DEG C;
3) until step 2) indefinite bud that produces of the blade cultivated is when growing into 0.5-1cm, and be transferred in the strong seedling culture base described in step 1) and cultivated, grow and obtain independently test-tube plantlet;
4) when the independently test-tube plantlet growth that step 3) obtains is to 10cm, be cut into the stem section of length 2cm, then be transferred on root media and cultivate, grow and obtain complete apple test-tube plantlet; Consisting of of described root media: 1/2MS medium+indolebutyric acid 0.2mg/L+ heteroauxin 0.5mg/L+ glucose 25g/L+ agar 6g/L, pH 6.0, condition of culture and step 2) identical.
Further, the method for building up of above-mentioned Apple Leaves regenerating system, described apple variety is " Nagafu No.2 " apple.
Regeneration rate (%)=regeneration bud leaf explant number/inoculation leaf explant number × 100;
The number of blade of the Bud Differentiation of gain factor=Bud Differentiation number/.
Beneficial effect of the present invention is: the method for building up of Apple Leaves regenerating system provided by the invention, establish " Nagafu No.2 " Apple Leaves high-efficiency regeneration system, blade adventitious shoot regeneration rate reaches 100%, gain factor reaches more than 5, rooting rate reaches more than 86%, can meet the requirement that genetic transformation carried out by apple.
Accompanying drawing explanation
Fig. 1 is " Nagafu No.2 " Apple Leaves adventitious shoot regeneration situation.
Fig. 2 is " Nagafu No.2 " apple rooting of vitro seedling situation.
Embodiment
embodiment 1:
The method for building up of " Nagafu No.2 " Apple Leaves regenerating system, comprises the following steps:
1) strong seedling culture: the strong seedling culture base of employing is MS medium+6-benzyl aminoadenine 0.5mg/L+ methyl α-naphthyl acetate 0.1mg/L+sucrose 30g/L+ agar 6.0g/L, pH6.0; Culture vessel is 150mL triangular flask, every bottle of packing strong seedling culture base 50ml; Condition of culture is intensity of illumination 3000lx, cultivation temperature 25-28 DEG C; The test-tube plantlet blade cultivating 40-50d is used as regrown material;
2) test-tube plantlet process and inoculation: the healthy and strong test-tube plantlet cultivating 40-50d in step 1), aseptically operate, get 2 ~ 3 pieces of well-grown blades in middle part, with scissors, blade both sides leaf margin is cut, be cut into the square of 0.8cm × 0.8cm, leaf faces up and is seeded on differentiation adventitious buds medium, inoculates 5 pieces of blades, as shown in Figure 1 in every bottle; The differentiation adventitious buds medium adopted is MS medium+methyl α-naphthyl acetate 0.3 mg/L+6-benzyl aminoadenine 3.0 mg/L+ TDZ plant growth regulator 1.0 mg/L+polyvinylpyrrolidone 150mg/L+ sucrose 30g/L+ agar 6.0g/L, pH6.0; Culture vessel is 150mL triangular flask, every bottle of packing differentiation adventitious buds medium 50ml, 121 DEG C of sterilizing 20min; Condition of culture is: light culture 3 weeks after inoculation, and cultivation temperature is 20-25 DEG C, cultivates, intensity of illumination 1000-2000lx under being then placed in light, and the photoperiod is 16h/8h, cultivation temperature 25-28 DEG C;
3) until step 2) indefinite bud that produces of the blade cultivated is when growing into 0.5-1cm, and be transferred in the strong seedling culture base described in step 1) and cultivated, grow and obtain independently test-tube plantlet;
4) when the independently test-tube plantlet growth that step 3) obtains is to 10cm, be cut into the stem section of length 2cm, then be transferred on root media and cultivate, grow and obtain complete apple test-tube plantlet, as shown in Figure 2; Consisting of of described root media: 1/2MS medium+indolebutyric acid 0.2mg/L+ heteroauxin 0.5mg/L+ glucose 25g/L+ agar 6g/L, pH 6.0, condition of culture and step 2) identical.
key problem in technology point:
1, getting blade as explant from the test-tube plantlet cultivating stalwartness is the basis of improving blade adventitious shoot regeneration, grows thin and delicate test-tube plantlet blade of poor quality, and when cultivating as explant, easy brownization is dead;
2, during inoculation, the rear-face contact medium of the facing up of blade, blade, otherwise, easily produce callus, be unfavorable for the generation of indefinite bud;
3, when the Elongation of adventitious bud produced is to 0.5-1cm, is transferred in time on strong seedling culture base and cultivates, make it grow for independently test-tube plantlet as early as possible;
4, when test-tube plantlet grows to 10cm, being cut into length is that the stem of 2cm is short, is transferred on root media and cultivates, make it grow for complete apple test-tube plantlet.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. the method for building up of Apple Leaves regenerating system, is characterized in that, comprises the following steps:
1) strong seedling culture: the strong seedling culture base of employing is MS medium+6-benzyl aminoadenine 0.5mg/L+ methyl α-naphthyl acetate 0.1mg/L+sucrose 30g/L+ agar 6.0g/L, pH6.0; Culture vessel is 150mL triangular flask, every bottle of packing strong seedling culture base 50ml; Condition of culture is intensity of illumination 3000lx, cultivation temperature 25-28 DEG C; The test-tube plantlet blade cultivating 40-50d is used as regrown material;
2) test-tube plantlet process and inoculation: the healthy and strong test-tube plantlet cultivating 40-50d in step 1), aseptically operate, get 2 ~ 3 pieces of well-grown blades in middle part, with scissors, blade both sides leaf margin is cut, be cut into the square of 0.8cm × 0.8cm, leaf faces up and is seeded on differentiation adventitious buds medium, inoculates 5 pieces of blades in every bottle; The differentiation adventitious buds medium adopted is MS medium+methyl α-naphthyl acetate 0.3 mg/L+6-benzyl aminoadenine 3.0 mg/L+ TDZ plant growth regulator 1.0 mg/L+polyvinylpyrrolidone 150mg/L+ sucrose 30g/L+ agar 6.0g/L, pH6.0; Culture vessel is 150mL triangular flask, every bottle of packing differentiation adventitious buds medium 50ml, 121 DEG C of sterilizing 20min; Condition of culture is: light culture 3 weeks after inoculation, and cultivation temperature is 20-25 DEG C, cultivates, intensity of illumination 1000-2000lx under being then placed in light, and the photoperiod is 16h/8h, cultivation temperature 25-28 DEG C;
3) until step 2) indefinite bud that produces of the blade cultivated is when growing into 0.5-1cm, and be transferred in the strong seedling culture base described in step 1) and cultivated, grow and obtain independently test-tube plantlet;
4) when the independently test-tube plantlet growth that step 3) obtains is to 10cm, be cut into the stem section of length 2cm, then be transferred on root media and cultivate, grow and obtain complete apple test-tube plantlet; Consisting of of described root media: 1/2MS medium+indolebutyric acid 0.2mg/L+ heteroauxin 0.5mg/L+ glucose 25g/L+ agar 6g/L, pH 6.0, condition of culture and step 2) identical.
2. the method for building up of Apple Leaves regenerating system according to claim 1, is characterized in that, described apple variety is " Nagafu No.2 " apple.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105230488A (en) * | 2015-10-29 | 2016-01-13 | 广西大学 | Cymbidium lancifolium leaf tissue culture rapid propagation method |
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|---|---|---|---|---|
| CN103931492A (en) * | 2013-01-23 | 2014-07-23 | 天水市果树研究所 | Tissue-culture rapid seedling growing method for apple rootstock M9 |
| CN104054581A (en) * | 2014-07-14 | 2014-09-24 | 西北农林科技大学 | Rapid reproduction method for apple variety Yuhua-Early-Fuji |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103931492A (en) * | 2013-01-23 | 2014-07-23 | 天水市果树研究所 | Tissue-culture rapid seedling growing method for apple rootstock M9 |
| CN104054581A (en) * | 2014-07-14 | 2014-09-24 | 西北农林科技大学 | Rapid reproduction method for apple variety Yuhua-Early-Fuji |
Non-Patent Citations (2)
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| 吴雅琴等: "昌红苹果离体叶片不定芽的诱导及植株再生", 《云南农业大学学报》 * |
| 田春英等: "长富2号红富士苹果离体叶片高效再生不定芽技术研究", 《河北农业大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105230488A (en) * | 2015-10-29 | 2016-01-13 | 广西大学 | Cymbidium lancifolium leaf tissue culture rapid propagation method |
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Application publication date: 20150729 |