CN102283128A - Method for overcoming vitrification phenomenon of sisal hemp tissue culture seedlings - Google Patents
Method for overcoming vitrification phenomenon of sisal hemp tissue culture seedlings Download PDFInfo
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- CN102283128A CN102283128A CN 201110204540 CN201110204540A CN102283128A CN 102283128 A CN102283128 A CN 102283128A CN 201110204540 CN201110204540 CN 201110204540 CN 201110204540 A CN201110204540 A CN 201110204540A CN 102283128 A CN102283128 A CN 102283128A
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Abstract
The invention relates to a method for overcoming a vitrification phenomenon of sisal hemp tissue culture seedlings. The method comprises the following steps of under an in vitro condition, after utilizing stem tips of sisal hemp to perform surface disinfection for explants, inoculating the explants to an improved SH culture medium which is added with 6-BA to perform primary culture for about 45 days, and then inoculating the aseptic explants to the improved SH culture medium which includes 6-BA to perform induced differentiation and culturing, performing subculture multiplication for the induced cluster bud in each 45-day by utilizing the culture medium with the same induced differentiation, so that a large amount of healthy seedlings can be obtained through rooting culture. The method can be used for controlling the vitrification phenomenon of the tissue culture seedlings by adjusting the effective components of the culture medium, thus, the method is simple, convenient, economic and highly efficient; producing the tissue culture seedlings by the common sisal hemp using the method can effectively overcome the vitrification phenomenon, therefore, the method has a wide application prospect.
Description
Technical field
The present invention relates to a kind of method of sisal hemp tissue culture, particularly relate to a kind of method that overcomes sisal hemp tissue culturing seedling vitrification phenomenon, belong to the tissue cultivating and seedling technical field.
Background technology
Sisal hemp is important tropical fiber crop, mainly is distributed in the part torrid zone, subtropical zone between the tropic of north and south, and planting area is very limited, and total resources is rare; Sisal fiber is shipping, fishing boat, the used cable of probing, netting, irreplaceable raw materials such as the steel cable rope heart of elevator, crane, the sisal hemp goods have been widely used in medal polish, extraordinary paper pulp, metallic article, transportation, mine, home decoration, carpet, technology, medicine and the auto industry at present.
H.11648, it is the improved seeds of the sixties in 20th century from external introduction that China sisal hemp produces the unique sisal hemp kind that goes up plantation at present, its fiber production height, cold resistance is strong, at China's large tracts of land popularizing planting, increased substantially sisal hemp output, China sisal hemp is leaped into the front ranks of the world, become the strong industry that to export goods and earn foreign currency.Yet, H.11648 3 life cycles have been experienced in plantation at present, because the random procreation of the long-term single and seedling of varieties of plant, cause present sisal hemp early blossoming early ageing phenomenon extremely outstanding, the fiber sucrose extraction has downward trend year by year, even the atavism of leaf margin spinosity appears, this is a kind of symbol of deterioration of variety; Because deterioration of variety, resistance weakens, and the sisal hemp disease is constantly grown, and has a strong impact on the stable high yield of numb garden.
The sisal hemp life cycle is long, only open once and spend all one's life, the distribution of its gene and the chance of reorganization are limited, and sisal hemp is the alien species of introducing, germ plasm resource all need be from external introduction, rearing new variety usually is subjected to the constraint of germ plasm resource, thereby the sexual hybridization breeding time is long, efficient is low, and the situation that varieties of plant is single also will exist over a long time.Fiber crops garden stable high yield, seed cultivation are crucial, and seedling is good, and the plant growing situation is energetic, resistance, bright leaf-making quantity height; The sisal hemp production practices of decades prove, H.11648 be good sisal hemp kind, its yielding ability is that at present other kind hardly matches, by to H.11648 purificating and rejuvenating, recover its kind property, can alleviate in a short time because the sisal hemp kind lacks the risk of bringing.By the screening of fine individual plant H.11648, utilize the tissue culture axillalry bud to become the seedling approach that fine individual plant is bred fast, use strain and be method of purification and be and realize the effective way of H.11648 purificating and rejuvenating.Therefore, the sisal hemp tissue culture is not only the active demand that present sisal hemp produces, and to alleviate sisal hemp varieties of plant long-term lacking important realistic meaning is arranged to utilizing gene engineering to carry out sisal hemp kind genetic improvement.
Tissue culture and the plant regeneration of sisal hemp have also been done certain exploration both at home and abroad, abroad, have utilized seed, bulbil, walk stem and stem section etc. for explant, by the successful foundation of direct or indirect organ occurring mode the Asia Agave cantula Roxb (
A. cantalaRobx), the bastard indigo sisal hemp (
A. fourcroydesLem), common sisal hemp (
A. sisalanaPerrine), Agave amaniensis (X Agave angustifolia) (
A.Tequilana Weber cultivar azul),
A. parrasanaBerger,
A. tequilana,
A. crassispina,
A. duranguensis,
A.Vera-cruz Mill.,
A. atrovirensWith
A. arizonicaDeng the external regeneration system, and obtained complete regeneration plant, at home, the land-reclaimable Science ﹠ Technology Center in Guangdong Province as explant, obtains in a large number grow thickly bud by fast numerous mode with the unbearable bud stem apex of sisal hemp.Utilize H.11648 the bulbil of high yield plant to do explant at the beginning of the land-reclaimable scientific research institution 2005, Zhanjiang, Guangdong, successfully cultivated the seedling of strain more than 20,000 by fast numerous mode, Chinese Academy of Tropical Agricultural Sciences and University Of Hainan utilize respectively sisal hemp (
Agave sisalana Perrine ex Engelmann)Tender shoots and aseptic seedling blade are explant, obtain complete regenerated plant by callus of induce, Chinese Academy of Tropical Agricultural Sciences is an explant with straw point H.11648, obtained the H.11648 plant of some by fast numerous mode, but all there are vitrification phenomenon in various degree in clump bud that above method obtains or regeneration plant, this seminar has also carried out the sisal hemp regenerating system and has made up research, obtained the regeneration plant of some, but regeneration plant vitrifying rate reaches as high as more than 80%, had a strong impact on the external quick breeding of sisal hemp, to utilizing the biotechnology means to carry out the sisal hemp genetic improvement adverse effect has been arranged also simultaneously.
Tissue cultivating seedling vitrification phenomenon ubiquity in Plant Tissue Breeding, the vitrified plant shape that often is translucent, mode of appearance is often unusual.Usually the vitrifying plant is difficult to restore normal growth between follow-up culture period, still form the vitrifying seedling in successive transfer culture, and the differentiation capability of vitrifying seedling is low, is difficult to propagation and takes root into seedling.Because the physiological function of vitrifying seedling is unusual, is difficult to transplant survival, therefore numerous totally unfavorable to utilizing tissue culture technique to carry out the plant corpus extra income, tissue cultivating seedling vitrifying problem has also become problem demanding prompt solution in the tissue cultivating seedling production.Just carried out system research for the vitrified risk factor of tissue cultivating seedling as far back as Debergh in 1981 etc., but because the vitrified risk factor of different plants is very inequality, opinions vary for everybody, its vitrified pests occurrence rule and mechanism are also lacked real understanding so far, also do not have the blanket effective vitrifying generation technique measure that prevents.This method has not only overcome the vitrification phenomenon in the sisal hemp tissue-culturing rapid propagation to a great extent, simultaneously vitrified plant is restore normal growth, simultaneously can also induce vitrified callus regeneration to go out normal plant, lay a good foundation for utilizing biotechnology to carry out the sisal hemp correlative study.
Summary of the invention
The technical solution adopted in the present invention is for achieving the above object:
A kind of method that overcomes sisal hemp tissue culturing seedling vitrification phenomenon is characterized in that implementation step is as follows:
(1) explant surface sterilization and cultivation
Stem apex with sisal hemp bulbil seedling (5-10cm) or suction bud seedling (10-20cm) is that material carries out disinfection, earlier outer Lao Ye is peeled off, the flowing water flushing, handle 30min with 0.3% liquor potassic permanganate, follow on superclean bench with 75% alcohol-pickled 1min, soak 15-30 min with 0.1% mercuric chloride solution again, use aseptic water washing 3-5 time at last; Dry back cut-out explant, be inoculated in after the rip cutting on improvement SH+6-benzyl aminoadenine (6-BA) the 2.5-5.0mg/L medium, condition of culture is illumination every day 12-14 hour, secretly cultivates 10-12 hour, intensity of illumination 2000lux, cultivation temperature is 28 ± 2 ℃;
(2) the clump bud is induced and enrichment culture
With the stem apex of initial culture after 45 days change over to clump bud induce with proliferated culture medium on carry out clump bud and induce and breed, the clump bud is induced with proliferated culture medium and is improvement SH medium, and adding exogenous hormone is 6-benzyl aminoadenine (6-BA), methyl (NAA) and indoles 3 butyric acid (IBA); The concentration of 6-BA is 1.0-5.0mg/L; The concentration of NAA is 0-1.5mg/L, and the concentration of IBA is 0-1.0mg/L, and pH is 5.8-6.0; Illumination 12-14h, dark 10-12h, illumination is 2000Lx, 28 ± 2 ℃ of temperature;
(3) culture of rootage
Through enrichment culture with after strong sprout, more than high 5cm, the seedling that has 4-5 sheet leaf changes root media over to and carries out culture of rootage, and the minimal medium of taking root is improvement SH, and the interpolation exogenous hormone is IBA, and hormone concentration is 0.5-2.5 mg/L, and pH is 5.8-6.0; Illumination 12-14h, dark 10-12h, 28 ± 2 ℃ of temperature;
(4) aseptic seedling is transplanted
Treat that plant takes root 2-3 during week, the bottle seedling is placed 1 week of natural conditions lower refining seedling, take out the medium of flush away base portion then, soak 3-5min with 1000 times of liquid of potassium permanganate, be transplanted to matrix and be in the plastics shading booth of coconut palm chaff and river sand (the coconut palm chaff: river sand is 1:1) and cultivate, obscurity is more than 75%, daytime, maximum temperature was no more than 32 ℃, night, minimum temperature was not less than 20 ℃, sooner or later respectively water permeable 1 time every day, and the survival rate of plant is more than 95%, and seedling begins to impose 2% composite fertilizer's liquid manure after growing young leaves, to use the little seedling leaf of clear water drip washing immediately after the fertilising, treat that seedling grows 3 of young leaves, can be transplanted to about height of seedling 10cm and continue under the natural environment to cultivate.
The a large amount of compositions of revising of above-mentioned improvement SH medium are potassium nitrate 2000-2520 mg/l, potassium dihydrogen phosphate 300-353 mg/l, nitric acid ammonia 100-300 mg/l, the FeSO in the SH molysite
4 .7H
2O is 15-27.8mg/l, Na
2-EDTA is 15-37.3mg/l, the ZnSO of SH trace
4 .7H
2O is 5-15mg/l.
The invention has the advantages that:
The present invention controls the tissue cultivating seedling vitrification phenomenon by the active principle of regulating medium first, utilize this law in sisal hemp stem apex clump bud is induced, can efficiently induce normal plant, vitrified regeneration plant is restore normal growth, be the tissue-culturing rapid propagation of sisal hemp, regenerating system foundation, genetic transformation etc. are had laid a good foundation.This method implementation step is simple, and fast, efficiently, implementation condition is loose, the effect highly significant.
Description of drawings
The clump bud that Fig. 1 induces for example 1 stem apex of the present invention
Fig. 2 is example 2 vitrified plant of the present invention
Fig. 3 is the recovery situation of example 2 vitrified plant cultivations of the present invention after 20 days
Fig. 4 is vitrified callus;
Fig. 5 is the indefinite bud (1 month) of vitrified callus induction
Fig. 6 cultivates (20 days) for adventitious bud rooting.
Embodiment
Below by embodiment the present invention is described in further details, these embodiment only are used for illustrating the present invention, do not limit the scope of the invention.
Embodiment 1
High 8-10cm bulbil seedling 15 strains of going bail for and being stored in south subtropics crop investigations institute sisal hemp field gene bank, remove root and Lao Ye, the flowing water flushing, handle 30min for 1000 times with liquor potassic permanganate, then with 75% alcohol-pickled 1min, soak 15-30 min with 0.1% mercuric chloride solution again, use aseptic water washing 3-5 time at last.Dry back cut-out explant, be inoculated in after the rip cutting on the improvement SH+6-BA3.0-5.0mg/L medium, condition of culture is illumination every day 12-14 hour, secretly cultivated 10-12 hour, and intensity of illumination 2000lux, cultivation temperature is 28 ± 2 ℃.Cultivate clump bud of transferring after 45 days and induce and proliferated culture medium, the clump bud is induced and is bred minimal medium and is improvement SH medium, and the interpolation exogenous hormone is 6-BA, NAA and IBA; The concentration of 6-BA is 1.0-5.0mg/L; The concentration of NAA is 0-1.5mg/L, and the concentration of IBA is 0-1.0mg/L, and pH is 5.8-6.0; Illumination 12-14h, dark 10-12h, illumination is 2000Lx, 28 ± 2 ℃ of temperature.Every 45-60 days subculture 1 time obtains 5347 strain whole plants altogether after half a year, the normal plant ratio is more than 98%.
Embodiment 2
Get 85 bunches of (every bunch of 5-8 strain) partially or completely vitrified regeneration plants and be inoculated on the improvement SH medium, the interpolation exogenous hormone is 6-BA, NAA and IBA; The concentration of 6-BA is 1.0-5.0mg/L; The concentration of NAA is 0-1.5mg/L, and the concentration of IBA is 0-1.0mg/L, and pH is 5.8-6.0; Illumination 12-14h, dark 10-12h, intensity of illumination is 2000Lx, 28 ± 2 ℃ of temperature.Cultivate after 1 month 23 bunches and restore normal growth fully, 34 bunches of parts are recovered, and vitrifying seedling recovery ratio is 67.06%.
[0014] embodiment 3
Get 62 of the callus agglomerates of vitrified band bud, remove vitrified bud and tender leaf, be inoculated on the improvement SH medium, the interpolation exogenous hormone is 6-BA, NAA and IBA; The concentration of 6-BA is 1.0-5.0mg/L; The concentration of NAA is 0-1.5mg/L, and the concentration of IBA is 0-1.0mg/L, and pH is 5.8-6.0; Illumination 12-14h, dark 10-12h, illumination is 2000Lx, 28 ± 2 ℃ of temperature.The back observation of 2 weeks has 55 callus agglomerates that adventitious bud inducing is arranged, the indefinite bud that wherein has 47 callus agglomerates to induce is normal substantially, the indefinite bud that has 8 callus agglomerates to induce has vitrifying in various degree, and the adventitious bud inducing ratio is 88.71%, and wherein normal indefinite bud ratio is 85.45%.
Claims (5)
1. a method that overcomes sisal hemp tissue culturing seedling vitrification phenomenon is characterized in that: comprise the steps:
(1) explant surface sterilization and cultivation
Stem apex with sisal hemp bulbil seedling or suction bud seedling is that material carries out disinfection, earlier outer Lao Ye is peeled off, the flowing water flushing, handle 30min with 0.3% liquor potassic permanganate, then on superclean bench, handle 1min, handle 15-30 min with 0.1% mercuric chloride solution again, use aseptic water washing 3-5 time at last with 75% alcohol, dry the back rip cutting, be inoculated on the improvement SH+6-benzyl aminoadenine 6-BA2.5-5.0mg/L medium and carried out initial culture 40-50 days; Condition of culture is illumination every day 12-14 hour, secretly cultivated 10-12 hour, and intensity of illumination 2000lux, cultivation temperature is 28 ± 2 ℃;
(2) the clump bud is induced and enrichment culture
To change over to through the stem apex of initial culture clump bud induce with proliferated culture medium on carry out clump bud and induce and breed, the clump bud is induced with proliferated culture medium and is improvement SH medium, adding exogenous hormone is 6-benzyl aminoadenine 6-BA, methyl NAA and indoles 3 butyric acid IBA; The concentration of 6-BA is 1.0-5.0mg/L; The concentration of NAA is 0-1.5mg/L, and the concentration of IBA is 0-1.0mg/L, and pH is 5.8-6.0; Illumination 12-14h, dark 10-12h, illumination is 2000Lx, 28 ± 2 ℃ of temperature;
(3) culture of rootage
Through the clump bud induce, enrichment culture and after strong sprout, more than high 5cm, the seedling that has 4-5 sheet leaf changes root media over to and carries out culture of rootage, the minimal medium of taking root is improvement SH, adding exogenous hormone is indoles 3 butyric acid IBA, and hormone concentration is 0.5-2.5 mg/L, and pH is 5.8-6.0; Illumination 12-14h, dark 10-12h, 28 ± 2 ℃ of temperature;
(4) aseptic seedling is transplanted
Treat that plant takes root 2-3 week, when seedling grows the strong root of 3-5 bar, the bottle seedling is placed 1 week of natural conditions lower refining seedling, take out the medium of flush away base portion then, soak 3min with 1000 times of liquid of potassium permanganate, be transplanted to matrix and be in the plastics shading booth of coconut palm chaff and river sand and cultivate, obscurity is more than 75%, and daytime, maximum temperature was no more than 32 ℃, and night, minimum temperature was not less than 20 ℃, sooner or later respectively water permeable 1 time every day, and the survival rate of plant is more than 95%; Seedling begins to impose 2% composite fertilizer's liquid manure after growing young leaves, will use the little seedling leaf of clear water drip washing immediately after the fertilising, treats that seedling grows can be transplanted to about 3 of young leaves, height of seedling 10cm under the natural environment to continue to cultivate.
2. a kind of method that overcomes sisal hemp tissue culturing seedling vitrification phenomenon according to claim 1 is characterized in that: described sisal hemp bulbil seedling is the high seedling of 5-10cm.
3. a kind of method that overcomes sisal hemp tissue culturing seedling vitrification phenomenon according to claim 1 is characterized in that: described suction bud seedling is the high seedling of 10-20cm.
4. a kind of method that overcomes sisal hemp tissue culturing seedling vitrification phenomenon according to claim 1, it is characterized in that: a large amount of compositions of revising of described improvement SH medium are potassium nitrate 2000-2520 mg/l, potassium dihydrogen phosphate 300-353 mg/l, nitric acid ammonia 100-300 mg/l, the FeSO in the SH molysite
4 .7H
2O is 15-27.8mg/l, Na
2-EDTA is 15-37.3mg/l, the ZnSO of SH trace
4 .7H
2O is 5-15mg/l.
5. a kind of method that overcomes sisal hemp tissue culturing seedling vitrification phenomenon according to claim 1 is characterized in that: the ratio of described coconut palm chaff and river sand is 1:1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102742506A (en) * | 2012-08-02 | 2012-10-24 | 广东海洋大学 | Method for utilizing chlorine dioxide to disinfect sisal hemp explant materials |
| CN102835312A (en) * | 2012-08-17 | 2012-12-26 | 中国热带农业科学院南亚热带作物研究所 | Method for overcoming adventitious buds vitrification of sisal hemp |
| CN103039370A (en) * | 2013-01-29 | 2013-04-17 | 中国热带农业科学院南亚热带作物研究所 | Method for establishing golden-edge arc-leaf maguey isolated cultivation and regeneration system |
| CN103250641A (en) * | 2013-05-13 | 2013-08-21 | 江苏省农业科学院 | Devitrification method for vitrified regeneration seedlings of cabbage type rape |
| CN108812307A (en) * | 2018-05-24 | 2018-11-16 | 广西壮族自治区亚热带作物研究所 | The narrow quick mating system of leaf American aloe tissue cultures |
| CN109197592A (en) * | 2018-10-24 | 2019-01-15 | 河南云帮农业科技有限公司 | A method of reducing albumen mulberry tissue culture Vitrification |
-
2011
- 2011-07-21 CN CN 201110204540 patent/CN102283128B/en not_active Expired - Fee Related
Non-Patent Citations (5)
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| 《中国麻业》 20051231 吕玲玲等 剑麻组培过程中玻璃化问题的探讨 第115～117页 1-5 第27卷, 第3期 * |
| 《中国麻业》 20051231 李愿平等 H.11648麻珠芽组织培养技术研究 第184～189页 1-5 第27卷, 第4期 * |
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| 《广西热带农业》 20071231 洪向平等 剑麻的组织培养快繁技术 第29页 1-5 , 第5期 * |
| 《植物生长调节剂在植物组织培养中的应用》 20030331 王小菁等 常用培养基的配方 化学工业出版社 第113页 1-5 , 第1版 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102742506A (en) * | 2012-08-02 | 2012-10-24 | 广东海洋大学 | Method for utilizing chlorine dioxide to disinfect sisal hemp explant materials |
| CN102835312A (en) * | 2012-08-17 | 2012-12-26 | 中国热带农业科学院南亚热带作物研究所 | Method for overcoming adventitious buds vitrification of sisal hemp |
| CN102835312B (en) * | 2012-08-17 | 2014-09-10 | 中国热带农业科学院南亚热带作物研究所 | Method for overcoming adventitious buds vitrification of sisal hemp |
| CN103039370A (en) * | 2013-01-29 | 2013-04-17 | 中国热带农业科学院南亚热带作物研究所 | Method for establishing golden-edge arc-leaf maguey isolated cultivation and regeneration system |
| CN103039370B (en) * | 2013-01-29 | 2014-03-19 | 中国热带农业科学院南亚热带作物研究所 | Method for establishing golden-edge arc-leaf maguey isolated cultivation and regeneration system |
| CN103250641A (en) * | 2013-05-13 | 2013-08-21 | 江苏省农业科学院 | Devitrification method for vitrified regeneration seedlings of cabbage type rape |
| CN108812307A (en) * | 2018-05-24 | 2018-11-16 | 广西壮族自治区亚热带作物研究所 | The narrow quick mating system of leaf American aloe tissue cultures |
| CN109197592A (en) * | 2018-10-24 | 2019-01-15 | 河南云帮农业科技有限公司 | A method of reducing albumen mulberry tissue culture Vitrification |
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