CN103283589A - Method for prolonging subculturing interval time of tissue culture seedlings of apple - Google Patents
Method for prolonging subculturing interval time of tissue culture seedlings of apple Download PDFInfo
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- CN103283589A CN103283589A CN2012100434961A CN201210043496A CN103283589A CN 103283589 A CN103283589 A CN 103283589A CN 2012100434961 A CN2012100434961 A CN 2012100434961A CN 201210043496 A CN201210043496 A CN 201210043496A CN 103283589 A CN103283589 A CN 103283589A
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Abstract
The invention discloses a method for prolonging the subculturing interval time of tissue culture seedlings of apple. A culture medium comprises the following components: 0.5-0.75 mg/L of MS+BA, 0.05 mg/L of NAA, 40.0 g/L of white granulated sugar and 6.0 g/L of agar; the packing quantity is 40-50% more than that in conventional breeding, and a bottleneck is sealed by using a plastic sealing film; tissue culture seedlings of apple are transferred to the culture medium and slowly grow about 2 weeks under the culture conditions that the temperature is 22-28 DEG C, the illumination intensity is 2000 lx, and the illumination time is 10-14 h/d, and then the tissue culture seedlings of apple are placed in a chemical agent storage box with a temperature of 2-8 DEG C so that the growth is slowed down; and when the culture medium is approximately dried, the tissue culture seedlings of apple are taken out of the refrigerated container to grow normally for 1-2 weeks or are directly transferred to a new culture medium. In such a way, the subculturing interval time of tissue culture seedlings of apple can be successfully prolonged from 2-3 months to 15-18 months, the survival rate of stem tips is more than 90%, the workload of subinoculation is greatly reduced, and the mutation probability is also reduced. The technique is applied to the germplasm preservation of plantlets of apples and other parts of orchardfruits and flowers, is simple, convenient, saving, and high in efficiency, and provides a new way for the preservation of germplasm resources.
Description
Technical field
The present invention relates to a kind of tissue culture technique that utilizes and carry out the method that apple germ plasm resource exsomatizes and preserves.
Background technology
Along with size of population sharp increase, unreasonable reclaiming wasteland, the large tracts of land deforestation, industrial pollution in addition, the environmental ecology balance is seriously damaged, and some preciousnesses, rare species face extinction or are on the brink of extinction.Simultaneously because the development of plant breeding technology, the popularization of artificial selection and breeding, kind constitutes the unification phenomenon and becomes clear day by day, and causes many useful precious germ plasm resources to be lost.Therefore, plant germplasm resource is preserved the problem that has become global concern.
The main means that plant germplasm resource is preserved have two kinds, and the one, preserve in former border, namely sets up the nature reserve and protect endangered plants on the spot; The 2nd, set up field germplasm gene pool in different border or the preservation etc. of exsomatizing.Preserve in former border and soil and the human resources that field germplasm gene pool need be a large amount of are set up in different border, the cost height, and the invasion and attack and the sick worm that subject to various natural calamities endanger, and also are not easy to germ plasm resource and exchange.
Exsomatize preserving germ plasm resource is plant explants to be organized to cultivate under gnotobasis preserve, and can overcome the shortcoming of conventional field preservation method, not land occupation, save storage space, remove daily management from, be convenient to transportation and exchange etc.From Henshaw in 1975 and Morel propose first to exsomatize preserve the strategy of plant germplasm since, this technology is subjected to international botanic extreme and payes attention to, nineteen eighty-two, IBPGR has set up stripped preservation Advisory Board specially, and many countries have set up the plant germplasm in vitro gene pool in succession.
Cultivate in the process of preserving germplasm at tissue, because medium nutrition exhausts, water evaporates, at set intervals, culture in time be shifted being inoculated in the new medium, to guarantee the culture normal growth.General about 2 months subcultures of apple group training seedling once need the cost human and material resources, because culture materials is subjected to the stimulation of exogenous hormone for a long time, and subculture number is more many, the probability that morphs also can be more big simultaneously.Along with being in full swing of cultured in vitro technology, based on the limiting growth method preservation technology that exsomatizes also obtained application.The limiting growth preservation method, namely in group training seedling subculture preservation process, external condition by the growth of regulation and control culture, make material growth be down to Min., nutrient consumption reduces, thereby prolongs subculture blanking time, reduce subculture number, both save the workload of test-tube plantlet subinoculation greatly, reduced the variation probability again, kept the genetic stability of germplasm.
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Summary of the invention
The present invention has set up a kind of apple group training seedling subculture method of blanking time that prolongs, for the stripped preservation that utilizes tissue culture technique to carry out apple germ plasm resource provides technical support.
The technical solution adopted for the present invention to solve the technical problems is: with the retarding of growing adjustment slightly of apple group training seedling medium, inoculation is by the slow seedling growth of a period of time, change cryogenic conditions about 8 ± 1 ℃ into by conventional 25 ± 3 ℃ condition of culture, effectively suppress test-tube plantlet growth; By test, filter out suitable group training seedling joint filling material, solve the problem of high humidity in the refrigerating box, easy mildew fungi pollution, prolong the holding time of apple group training seedling.
Particular content of the present invention, the concrete grammar step that namely prolongs apple group training seedling subculture blanking time is as follows:
(1) preparation subculture medium: MS+BA0.5 ~ 0.75mg/L+NAA0.05mg/L+ white granulated sugar 40.0g/L+ agar 6.0g/L regulates pH6.0 before the medium sterilization; For saving takes up room, culture vessel adopts the 50ml triangular flask, and every bottle of medium divides the branch loading amount of loading amount medium will be more than conventional tissue culture propagation the time, generally grasps about 30 ~ 35 bottles of 1L medium packing; Because refrigerator seals tight, high humidity, the joint filling material that tissue cultivation tampon commonly used adds brown paper is easy to mouldy long bacterium, the use group train the special-purpose sealing film, can effectively solve pollution problem, can also suppress the medium water evaporates better than tampon.
(2) apple group training seedling is inserted in the medium, at 25 ± 3 ℃, intensity of illumination 2000lx, slow seedling was grown about 2 weeks under the condition of culture of light application time 10-14h/d, can enter healthy and strong growth conditions by assurance group training seedling like this.
(3) the group training seedling that will delay behind the seedling is put into the retarding of growing of 2-8 ℃ of medicine container, uses this refrigerating box, and following advantage is arranged: capacity is big, and the multilayer shelf is arranged, and can the adjusting play, and the group that can store training seedling quantity is many; The transparent vacuum glass door can provide the growth of group training seedling required scattered light, if full dark condition, group training seedling does not almost have blade, stem thin and delicate, is unfavorable for next subinoculation and recovers growth that glass door also is convenient to the growing state of observation group's training seedling simultaneously; Case has the giant-screen digital temperature to show outward, and the overtemperature alarm function is arranged, and the high-performance circulating fan makes the temperature inside the box stable, even.Observe during practical application, temperature was generally at 8 ± 1 ℃ after refrigerating box was piled group training seedling.
(4) group of retarding of growing training seedling generally can keep 15-18 month, and the stem apex survival rate is more than 90%, and it is withered to wait to find that medium approaches, and can take out from refrigerating box, recovers growth 1-2 week in culturing room's normal condition, or directly inserts new medium.
The beneficial effect of patent of the present invention is, by changing the joint filling material of culture vessel, high humidity, easily pollution problem have been solved, apple group training seedling can be put into refrigerating box, effectively slowed down the growth of group training seedling, reduced nutrient consumption and medium water evaporates, successfully the subculture with apple group training seedling extended to 15-18 month by 2 months blanking time, significantly reduce the workload of subinoculation, also reduced the variation probability.This technology is applied to apple and other parts orchard fruit and the quality guarantee of flowers group training seed is deposited, and is simple, convenient, save, effectively, and preserving for germ plasm resource provides new way.
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Description of drawings
Fig. 1 is about to put into refrigerating box for the apple group training seedling that delays after seedling is grown.
Fig. 2 trains seedling for the apple group of storing at refrigerating box 18 months.
Embodiment
This technology has been groped for many years, and just how simply the growth of effectively inhibition group of low temperature training seedling is apparent, create suitable storage requirement, efficiently.Once tested with 0-50 ℃ of illumination box, found that the incubator internal portioning layer was few, the space waste; If long-term control 5-8 ℃ low temperature, the incubator compressor is perishable, and multi-functional illumination box only is used for germplasm retain costs height.Organized the quality guarantee of training seed from 2008 with 2-8 ℃ of medicine container and deposit, because high humidity in the refrigerator, after group training seedling was put into 4 months, conventional joint filling material tampon and the brown paper of group training was mouldy long bacterium, causes tissue culture seedling pollution; Used group training special-purpose sealing film in 2009 instead, because its counterdie is the polyacrylic film of humidity, solved pollution problem, suppress the medium water evaporates better than tampon simultaneously, make its long preservation group training seedling simple, feasible.Verification experimental verification through 3 years, successfully preserved Fuji, Qiao Najin, gold hat, golden short life, loud, high-pitched sound, tens apple varieties such as Wang Lin, Malus spectabilis, M system and stock germplasm and part flowers group train seedling, these germplasms all can be placed the 1-1.5 subculture once in refrigerating box, the stem apex survival rate is more than 90%.Be apple variety Qiao Najin and stock M
26Sometimes browning phenomenon occurs, can suitably produce in advance.
Claims (5)
1. one kind prolongs the apple group training seedling subculture method of blanking time, it is characterized in that apple group training seedling is inserted in the medium of MS+BA0.5 ~ 0.75mg/L+NAA0.05mg/L+ white granulated sugar 40.0g/L+ agar 6.0g/L, slow seedling growth through a period of time, change cryogenic conditions about 8 ± 1 ℃ into by conventional 25 ± 3 ℃ condition of culture, effectively suppressed test-tube plantlet growth; Select suitable group training seedling joint filling material for use, solved the problem of high humidity in the refrigerating box, easy mildew fungi pollution, prolonged the holding time of apple group training seedling.
2. train the seedling subculture method of blanking time according to right 1 described prolongation apple group, it is characterized in that the 1.0mg/L that BA concentration was bred by routine in the medium when quality guarantee of apple group training seed was deposited reduces to 0.5 ~ 0.75mg/L, white granulated sugar concentration is brought up to 40 g/L by 30 ~ 35g/L.
3. train the seedling subculture method of blanking time according to right 1 described prolongation apple group, it is characterized in that culture vessel adopts the 50ml triangular flask, increase by 40 ~ 50% when medium can amount is bred than routine.
4. according to the described prolongation apple group training of the right 1 seedling subculture method of blanking time, it is characterized in that the culture vessel joint filling material selects group training special-purpose sealing film for use, do not use tampon and brown paper.
5. train the seedling subculture method of blanking time according to right 1 described prolongation apple group, elder generation is at 25 ± 3 ℃ after it is characterized in that tissue culture plant inoculation, intensity of illumination 2000lx, slow seedling was grown about 2 weeks under the condition of culture of light application time 10-14h/d, put into the medium-term and long-term preservation of 2-8 ℃ of medicine container then.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104186313A (en) * | 2014-08-04 | 2014-12-10 | 江苏农林职业技术学院 | Inducing culture medium of malus domestica borkh pulp callus tissue and inducing multiplication culture method of inducing culture medium |
| CN107950392A (en) * | 2017-11-09 | 2018-04-24 | 沈阳静冶生物科技有限公司 | A kind of method for extending Chinese cabbage microspore embryoid regeneration plant subculture interval time |
| CN108739393A (en) * | 2018-06-13 | 2018-11-06 | 河北农业大学 | A method of extending the Tissue culture the seedling of grape holding time |
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| EP1131405A2 (en) * | 1998-10-23 | 2001-09-12 | Exelixis Plant Sciences, Inc. | Rapid recovery of shoots through thin stem slices after preconditioning of micropropagated fruit tree shoots |
| CN101019503A (en) * | 2007-02-02 | 2007-08-22 | 宁波大学 | Germ plasm preserving method for kelp |
| CN102217537A (en) * | 2011-04-20 | 2011-10-19 | 中国科学院华南植物园 | Low-temperature conservation method of banana tissue culture plantlet provenances |
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2012
- 2012-02-24 CN CN2012100434961A patent/CN103283589A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1131405A2 (en) * | 1998-10-23 | 2001-09-12 | Exelixis Plant Sciences, Inc. | Rapid recovery of shoots through thin stem slices after preconditioning of micropropagated fruit tree shoots |
| CN101019503A (en) * | 2007-02-02 | 2007-08-22 | 宁波大学 | Germ plasm preserving method for kelp |
| CN102217537A (en) * | 2011-04-20 | 2011-10-19 | 中国科学院华南植物园 | Low-temperature conservation method of banana tissue culture plantlet provenances |
Non-Patent Citations (2)
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| IRINA KOVALCHUK, ET AL.: "Medium, container and genotype all influence in vitro cold storage of apple germplasm", 《PLANT CELL TISS ORGAN CULT》, vol. 96, 31 December 2009 (2009-12-31), pages 127 - 136, XP019649790 * |
| 王晨等: "温度和渗透压对苹果试管苗延缓生长法种质保存的效应", 《中国农学通报》, vol. 24, no. 12, 31 December 2008 (2008-12-31), pages 335 - 338 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104186313A (en) * | 2014-08-04 | 2014-12-10 | 江苏农林职业技术学院 | Inducing culture medium of malus domestica borkh pulp callus tissue and inducing multiplication culture method of inducing culture medium |
| CN104186313B (en) * | 2014-08-04 | 2016-06-15 | 江苏农林职业技术学院 | The inducing culture of apple pulp callus and proliferative induction cultural method thereof |
| CN107950392A (en) * | 2017-11-09 | 2018-04-24 | 沈阳静冶生物科技有限公司 | A kind of method for extending Chinese cabbage microspore embryoid regeneration plant subculture interval time |
| CN108739393A (en) * | 2018-06-13 | 2018-11-06 | 河北农业大学 | A method of extending the Tissue culture the seedling of grape holding time |
| CN108739393B (en) * | 2018-06-13 | 2021-10-08 | 河北农业大学 | Method for prolonging preservation time of grape tissue culture seedlings |
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Application publication date: 20130911 |