CN108739373A - A kind of method of fragrant shaddock stem apex numerous detoxification soon - Google Patents
A kind of method of fragrant shaddock stem apex numerous detoxification soon Download PDFInfo
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- CN108739373A CN108739373A CN201810366502.4A CN201810366502A CN108739373A CN 108739373 A CN108739373 A CN 108739373A CN 201810366502 A CN201810366502 A CN 201810366502A CN 108739373 A CN108739373 A CN 108739373A
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- stem apex
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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
- A01H4/008—Methods for regeneration to complete plants
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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
- A01H4/001—Culture apparatus for tissue culture
Abstract
The present invention relates to fruit tree detoxification reproduction technique field, the method for specifically a kind of fragrant shaddock stem apex numerous detoxification soon includes the following steps：(1) selection and disinfection of explant, fragrant shaddock stem tuber is taken to germinate indoors in spring or autumn, bud periodically sprinkles fungicide after growing up, after two weeks, bud is taken again, the liquor natrii hypochloritis that bud is placed on 5% after 35-40 DEG C of heat treatment handles 8-10min, and then 8-40 times of the anatomical lens on super-clean bench is lower carries out stem apex stripping, choose terminal bud stem apex or nucellar tissue as spare；(2) Initial culture after the stem apex of selection or nucellar tissue are placed again into 5% liquor natrii hypochloritis's processing 3-5min, are put into Initial culture base quickly breeding and grow up；By the selection of each stage culture medium and hormone in sterilizing to explant, tissue culture, the method for establishing a kind of numerous detoxification soon of fragrant shaddock stem apex provides theoretical foundation and technical support for fragrant shaddock tissue-cultured seedling factorial praluction, a new way is provided for breeding of new variety the present invention.
Description
Technical field
The present invention relates to fruit tree detoxification reproduction technique field, the method for specifically a kind of fragrant shaddock stem apex numerous detoxification soon.
Background technology
Fragrant shaddock fruit nutritive value is high, and taste is fragrant and sweet, is to have very much distinctive local characteristic agricultural product, and plantation fragrant shaddock is to increasing
Farmers' income, the living standard for improving the people have a very important significance.
Yellow twig is the representative disease of shaddock class, has resulted in the destruction in the millions of shaddock orchards in China.To improved seeds
It is to prevent the major measure of these diseases at present to carry out detoxification treatment.Stem apex gemmule grafts detoxification technology and mainly utilizes plant
Shoot apical meristem is virus-free, a kind of fast numerous detoxification technology that the fast principle of cell splitting rate grows up.
After Murashige etc. proposes micro-graft technology, the attention of various countries scientific research personnel is caused.Last century 80
Since age, developed country establishes the complete virus-free seedling-wood breeding of citrus breeding using stem apex gemmule grafting detoxification technology
System.Although the virus-free seedling-wood breeding System Construction of citrus breeding in China has certain basis, but not perfect, citrus breeding without
The production capacity of viral nursery stock is only capable of meeting 5% or so needed.Ma Fengtong etc. is transferred by the stem apex gemmule of several Citrus Cultivars
It connects, successfully obtains the gemmule grafting of sweet orange respectively, but identification is not detected to detoxification situation.Jiang Ling etc. is transferred by gemmule
It connects to obtain a large amount of graftings, then identifies, proves successfully to remove target pathogen.
Stem apex gemmule grafting detoxification technology is a kind of technology being combined grafting and tissue culture, and the sixth of the twelve Earthly Branches is proved that mandarin orange can be removed
The diseases such as tangerine yellow twig, bark cracking, decline disease, broken leaf disease.But currently, this technology answering in the nontoxic seedling proliferation of fragrant shaddock
With still blank out.The propagation method of fragrant shaddock is mainly that high pressure breeding and propagation by grafiting, both modess of reproduction make virus
It easily propagates, should be connect with stem apex gemmule grafting detoxification technology can remove virus, accelerate citrus reproduction speed, shorten life
The period is educated, high-quality introduces a collection is provided for production.
The fast development of Yongzhou fragrant shaddock planting industry so that the demand to detoxic seedling is increasing, and fragrant shaddock stem apex is fast numerous de-
The foundation that malicious gemmule transfers technology is to solve the effective way of detoxic seedling demand.Therefore, Hunan city fragrant shaddock germplasm is made full use of to provide
Source, carrying out fragrant shaddock stem apex in a deep going way, numerous detoxification gemmule transfers technical research soon, on the one hand for fragrant shaddock kind is seedling industrialized, large-scale production
Theoretical and technical foundation is provided, on the other hand or fragrant shaddock breeding, introduces a fine variety and preserving seed etc. provides foundation, has wide
Wealthy application prospect.The foundation of fragrant shaddock stem apex numerous detoxification technology soon is conducive to that Citrus Grandis traditional planting is transformed, improves ground
The competitiveness of area's featured agriculture product.
Invention content
Technical problem to be solved by the present invention lies in having overcome the deficiencies of the prior art and provide, a kind of fragrant shaddock stem apex is numerous soon
The method of detoxification establishes a kind of fragrant shaddock stem by the selection of each stage culture medium and hormone in sterilizing to explant, tissue culture
The method of sharp fast numerous detoxification provides theoretical foundation and technical support for fragrant shaddock tissue-cultured seedling factorial praluction, is carried for breeding of new variety
For a new way.
In order to solve the above technical problems, the present invention now proposes following technical scheme：A kind of side of fragrant shaddock stem apex numerous detoxification soon
Method includes the following steps：(1) selection and disinfection of explant takes fragrant shaddock stem tuber to germinate indoors, bud in spring or autumn
Fungicide is periodically sprinkled after growing up, and after two weeks, bud, bud is taken to be placed on 5% sodium hypochlorite after 35-40 DEG C of heat treatment again
Solution treatment 8-10min, then 8-40 times of the anatomical lens on super-clean bench is lower carries out stem apex stripping, choose terminal bud stem apex or
Nucellar tissue is as spare；(2) stem apex of selection or nucellar tissue are placed again into 5% liquor natrii hypochloritis by Initial culture
After handling 3-5min, it is put into Initial culture base quickly breeding and grows up；(3) second generation culture, by the stem apex after growing up or megarchidium group
It knits to be seeded on second generation culture medium and cultivate, be induced to differentiate into callus or adventitious bud；(4) basic culture, by callus
Or adventitious bud is seeded on minimal medium and cultivates, and develops into complete test tube seedling；(5) detoxification detection of test tube seedling.
Further, the fungicide in the step (1) is the mixed liquor of 0.1% carbendazim and 0.1% streptomysin.
Further, the stem apex or nucellar tissue are the part of the front end 0.3-0.5mm of terminal bud.
Further, the stem apex or nucellar tissue carry 1-2 phyllopodium.
Further, the group of the Initial culture base becomes：The NAA and/or 0.1- of MS culture mediums+0.1-0.5mg/L
The BA of 0.5mg/L, PH 5.6-6.0.
Further, the intensity of illumination at the initial stage of the Initial culture is 1000lx, increases to 2000lx after 4 weeks, after 6 weeks
Increase to 4000lx, cultivation temperature is 21-25 DEG C, light application time 14-18h/d.
Further, the group of the second generation culture medium becomes：The NAA and/or 0.1- of MS culture mediums+0.1-0.5mg/L
The GA of the BA+0.6-1.0mg/L of 0.5mg/L, PH 5.6-6.0.
Further, the group of the minimal medium becomes：MS culture medium+6-BA 0.2-0.5mg/L+NAA 0.2-
0.5mg/L+ sucrose 28-32g/L+ agar 6-8g/L, PH 5.6-7.0.
Further, the Initial culture base, second generation culture medium and the minimal medium are cold by Pasteur's antivirus
But it reuses afterwards.
Further, the test tube seedling is determined whether by PCR method for detecting virus containing fragrant shaddock virus.
Compared with prior art, the beneficial effects of the present invention are：1, the present invention to explant selection by using stem
Point or nucellar tissue, and carry out disinfection to it, survival rate is improved, viral infection rate is reduced, realizes the mesh of detoxification
's；
2, by being cultivated on culture medium, suitable nutrient is provided, survival rate is improved, avoids extraneous factor
It influences, realizes the purpose quickly bred；
3, entire incubation strictly controls, and is conducive to the further research to fragrant shaddock tissue culture technology；
4, viral diagnosis has been carried out to test tube seedling, it is ensured that the virus elimination rate of fragrant shaddock, thus high-quality, safe for fragrant shaddock seedling,
High yield and being widely applied is laid a good foundation.
Specific implementation mode
, there are further understanding and understanding in the effect of to make to structure feature of the invention and being reached, to preferable
Embodiment illustrates, is described as follows：
(1) preparation of culture medium
Embodiment 1
1) Initial culture base：The BA of the NAA and/or 0.1mg/L of MS culture mediums+0.1mg/L, PH 5.6.
2) second generation culture medium：The GA of the BA+0.6mg/L of the NAA and/or 0.1mg/L of MS culture mediums+0.1mg/L, PH are
5.6。
3) minimal medium：MS culture medium+6-BA 0.2mg/L+NAA 0.2mg/L+ sucrose 28g/L+ agar 6g/L, PH
5.6。
By taking the Initial culture base for configuring 1L as an example, specific process for preparation is：Weigh the MS culture mediums needed for 1L culture mediums
Each mass parts dissolve, mix successively；Then the NAA0.1mg and/or BA0.1mg in Initial culture based formulas are weighed successively,
It is added separately to dissolve successively in MS solution, constant volume, it is 5.6 to adjust pH value；It is finally dispensed, in 121 DEG C of high temperature, high pressure
0.1MPa sterilizes 15-20 minutes, is used after culture medium cooling.
Embodiment 2
1) Initial culture base：The BA of the NAA and/or 0.3mg/L of MS culture mediums+0.3mg/L, PH 5.8.
2) second generation culture medium：The GA of the BA+0.8mg/L of the NAA and/or 0.3mg/L of MS culture mediums+0.3mg/L, PH are
5.8。
3) minimal medium：MS culture medium+6-BA 0.3mg/L+NAA 0.3mg/L+ sucrose 30g/L+ agar 7g/L, PH
5.8。
By taking the Initial culture base for configuring 1L as an example, specific process for preparation is：Weigh the MS culture mediums needed for 1L culture mediums
Each mass parts dissolve, mix successively；Then the NAA0.3mg and/or BA0.3mg in Initial culture based formulas are weighed successively,
It is added separately to dissolve successively in MS solution, constant volume, it is 5.8 to adjust pH value；It is finally dispensed, in 121 DEG C of high temperature, high pressure
0.1MPa sterilizes 15-20 minutes, is used after culture medium cooling.
Embodiment 3
1) Initial culture base：The BA of the NAA and/or 0.5mg/L of MS culture mediums+0.5mg/L, PH 6.0.
2) second generation culture medium：The GA of the BA+1.0mg/L of the NAA and/or 0.5mg/L of MS culture mediums+0.5mg/L, PH are
6.0。
3) minimal medium：MS culture medium+6-BA 0.5mg/L+NAA 0.5mg/L+ sucrose 32g/L+ agar 8g/L, PH
6.0。
By taking the Initial culture base for configuring 1L as an example, specific process for preparation is：Weigh the MS culture mediums needed for 1L culture mediums
Each mass parts dissolve, mix successively；Then the NAA0.5mg and/or BA0.5mg in Initial culture based formulas are weighed successively,
It is added separately to dissolve successively in MS solution, constant volume, it is 6.0 to adjust pH value；It is finally dispensed, in 121 DEG C of high temperature, high pressure
0.1MPa sterilizes 15-20 minutes, is used after culture medium cooling.
Table 1
As shown in Table 1, influence of the selection of medium component to tissue culture is smaller.
(2) fast numerous detoxification of fragrant shaddock stem apex
A kind of method of fragrant shaddock stem apex numerous detoxification soon, includes the following steps：(1) selection and disinfection of explant, in spring
Or autumn takes fragrant shaddock stem tuber to germinate indoors, bud periodically sprinkles fungicide after growing up, after two weeks, bud, bud is taken to pass through again
The liquor natrii hypochloritis that 5% is placed on after 35-40 DEG C of heat treatment handles 8-10min, then 8-40 times of the anatomical lens on super-clean bench
Lower progress stem apex stripping, choose terminal bud stem apex or nucellar tissue as spare；(2) Initial culture, by the stem apex of selection or pearl
After heart tissue is placed again into 5% liquor natrii hypochloritis's processing 3-5min, it is put into Initial culture base quickly breeding and grows up；(3)
Stem apex after growing up or nucellar tissue are seeded on second generation culture medium and cultivate by second generation culture, be induced to differentiate into callus or
Person's adventitious bud；(4) basic culture, callus or adventitious bud are seeded on minimal medium and cultivated, and are developed into complete
Test tube seedling；(5) detoxification detection of test tube seedling.
Preferably, the fungicide in step (1) is the mixed liquor of 0.1% carbendazim and 0.1% streptomysin.
Preferably, stem apex or nucellar tissue are the part of the front end 0.4mm of terminal bud；Stem apex or nucellar tissue are with 1-2
Phyllopodium.
Preferably, the intensity of illumination at the initial stage of Initial culture is 1000lx, increases to 2000lx after 4 weeks, increases to after 6 weeks
4000lx, cultivation temperature are 25 DEG C, light application time 16h/d.
Table 2
| Survival rate (50) | |
| Explant is sterilized | 90% |
| Explant is not sterilized | 45% |
As seen from the above table, explant is sterilized before tissue culture, in case virus infection, substantially increases survival rate.
(3) viral diagnosis of fragrant shaddock test tube seedling
1) preparation of fragrant shaddock RNA：Selection has infected blade of the fragrant shaddock without syndrome virus, cucumber mosaic virus, Huanglong's virus and has been
Material extracts geneome RNA using Omega E.Z.N.A plant RNA extraction kits, is stored in -20 DEG C, spare.
2) cDNA is synthesized：Fragrant shaddock is designed without syndrome virus, cucumber mosaic virus, yellow twig according to fragrant shaddock virus gene sequence
The specific primer pair of poison；The each reaction total volume of cDNA synthesis is 20ul, and reaction reagent includes total serum IgE 2ul, 10umol
Oligo (dT) 151ul, ddH2O 7.5ul, 70 DEG C of water-bath 5min, ice bath 5min；Add 2.5mM/l dNTP 4ul, 5 ×
Buffer 4ul, 40U/ul RNase inhibitor 0.5ul, 200U/ul M-MLV reverse transcriptase 1ul, response procedures are 42 DEG C anti-
Transcribe 60min, 70 DEG C of inactivation 15min.
3) PCR amplification：It is 25ul that PCR, which reacts total volume, and taking the cDNA that 1ul reverse transcriptions synthesize, PCR is anti-as pcr template
The system is answered to include：The forward and reverse primer of 10 × buffer 2.5ul, 25mM/l Mg2+2ul, 2.5mM/l dNTP 2ul, 10uM/l
Each 1ul, 5U/ul Taq archaeal dna polymerases 0.2ul, 15.3ul ddH2O；Amplification program is：94 DEG C of pre-degeneration 5min；94 DEG C of changes
Property 30s, 56 DEG C annealing 30s, 72 DEG C extension 30s, totally 35 cycle；72 DEG C of extension 10min；4 DEG C terminate reaction.
4) electrophoretic band is observed：It is compareed with electrophoretic band, establishes whether fragrant shaddock test tube seedling still infects corresponding virus.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than to limit it；
Although the present invention is described in detail referring to the foregoing embodiments, it will be understood by those of skill in the art that it still may be used
To modify to the technical solution that previous embodiment is recorded or equivalent replacement of some of the technical features；And this
A little modifications or substitutions, the spirit and scope for various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution.
Claims (10)
1. a kind of method of fragrant shaddock stem apex numerous detoxification soon, which is characterized in that include the following steps：(1) selection of explant with disappear
Poison takes fragrant shaddock stem tuber to germinate indoors in spring or autumn, and bud periodically sprinkles fungicide after growing up, after two weeks, take bud again,
The liquor natrii hypochloritis that bud is placed on 5% after 35-40 DEG C of heat treatment handles 8-10min, then the anatomical lens on super-clean bench
8-40 times lower to carry out stem apex stripping, choose terminal bud stem apex or nucellar tissue as spare；(2) Initial culture, by the stem of selection
After point or nucellar tissue are placed again into 5% liquor natrii hypochloritis's processing 3-5min, quickly breeding length is put into Initial culture base
Greatly；(3) stem apex after growing up or nucellar tissue are seeded on second generation culture medium and cultivate, be induced to differentiate into callus by second generation culture
Tissue or adventitious bud；(4) basic culture, callus or adventitious bud are seeded on minimal medium and cultivated, is developed into
Complete test tube seedling；(5) detoxification detection of test tube seedling.
2. a kind of method of fragrant shaddock stem apex according to claim 1 numerous detoxification soon, which is characterized in that in the step (1)
Fungicide be 0.1% carbendazim and 0.1% streptomysin mixed liquor.
3. a kind of method of fragrant shaddock stem apex according to claim 1 numerous detoxification soon, which is characterized in that the stem apex or megarchidium
It is organized as the part of the front end 0.3-0.5mm of terminal bud.
4. a kind of method of fragrant shaddock stem apex according to claim 1 or 3 numerous detoxification soon, which is characterized in that the stem apex or
Nucellar tissue carries 1-2 phyllopodium.
5. a kind of method of fragrant shaddock stem apex according to claim 1 numerous detoxification soon, which is characterized in that the Initial culture base
Group become：The BA of the NAA and/or 0.1-0.5mg/L of MS culture mediums+0.1-0.5mg/L, PH 5.6-6.0.
6. a kind of method of fragrant shaddock stem apex according to claim 1 numerous detoxification soon, which is characterized in that the Initial culture
The intensity of illumination at initial stage is 1000lx, increases to 2000lx after 4 weeks, increases to 4000lx after 6 weeks, and cultivation temperature is 21-25 DEG C,
Light application time is 14-18h/d.
7. a kind of method of fragrant shaddock stem apex according to claim 1 numerous detoxification soon, which is characterized in that the second generation culture medium
Group become：The GA of the BA+0.6-1.0mg/L of the NAA and/or 0.1-0.5mg/L of MS culture mediums+0.1-0.5mg/L, PH are
5.6-6.0。
8. a kind of method of fragrant shaddock stem apex according to claim 1 numerous detoxification soon, which is characterized in that the minimal medium
Group become：MS culture medium+6-BA 0.2-0.5mg/L+NAA 0.2-0.5mg/L+ sucrose 28-32g/L+ agar 6-8g/L, PH
5.6-7.0。
9. a kind of method of fragrant shaddock stem apex according to claim 1 numerous detoxification soon, which is characterized in that the Initial culture
Base, second generation culture medium and the minimal medium reuse after Pasteur kills virus cooling.
10. a kind of method of fragrant shaddock stem apex according to claim 1 numerous detoxification soon, which is characterized in that the test tube seedling is logical
PCR method for detecting virus is crossed to determine whether containing fragrant shaddock virus.
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Cited By (2)
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| CN114176006A (en) * | 2021-12-23 | 2022-03-15 | 廉江市茗皇红橙果业有限公司 | Biological detoxification breeding method for red orange seedlings |
| CN114885837A (en) * | 2022-04-07 | 2022-08-12 | 南充市农业科学院 | Hormone-free culturing method for citrus stem tips |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114176006A (en) * | 2021-12-23 | 2022-03-15 | 廉江市茗皇红橙果业有限公司 | Biological detoxification breeding method for red orange seedlings |
| CN114885837A (en) * | 2022-04-07 | 2022-08-12 | 南充市农业科学院 | Hormone-free culturing method for citrus stem tips |
| CN114885837B (en) * | 2022-04-07 | 2023-04-25 | 南充市农业科学院 | Hormone-free culture method for citrus stem tip |
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