CN1723770A - Tissue culture tech. for hybrid chinaberry - Google Patents
Tissue culture tech. for hybrid chinaberry Download PDFInfo
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- CN1723770A CN1723770A CN 200510041096 CN200510041096A CN1723770A CN 1723770 A CN1723770 A CN 1723770A CN 200510041096 CN200510041096 CN 200510041096 CN 200510041096 A CN200510041096 A CN 200510041096A CN 1723770 A CN1723770 A CN 1723770A
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Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A cross and tissue culture technology for chinaberry includes such steps as fusing the organs, tissue, cells, embryo and protoplasm of chinaberry, margosa and Melia toosendan, artificial inducing and under lighting and temp to realize cross protoplasm fusion, generating the cell wall of cross chinaberry, splitting to become cell factor, secondary culture and induced growth.
Description
One, technical field:
The present invention relates to the hybridization tissue culture technology of a plant species, is a kind of chinaberry hybrid tissue culture technique.
Two, background technology:
Seal chinaberry (Neem) belongs to Meliaceae, aiphyllium, extensively plant in the torrid zone, subtropical zone, seal chinaberry comprehensive utilization value is very high, now developed 20 surplus in kind of the purposes, still the chinaberry element that utilizes of most worthy is done insecticide, is classic efficient, nontoxic, the environmental-pollution insecticide of generally acknowledging in the world at present.
Seal in the chinaberry the effective active composition---the chinaberry element mainly concentrates in the fruit, and the chinaberry element of local margosa and melia toosendan tree mainly concentrates on skin, leaf and root, and the chinaberry cellulose content in the seal chinaberry will be higher than China tree and melia toosendan far away.But the existence of seal chinaberry requires latitude and is strict, can't make it cross over north latitude 30 degree existence for a long time.
The seal chinaberry is moved northward reach the area plantation of crossing over more than north latitude 30 degree, and still can keep original merit, traditional cuttage, graft technology or seeds cultivation technology can't be accomplished.Though and traditional cuttage, graft technology can shorten the growth cycle of chinaberry, keeps the merit of chinaberry parent itself, because long-term vegetative propagation meeting makes viral the accumulation, harm increases the weight of.Though the seeds cultivation technology can be avoided above-mentioned shortcoming, proterties very easily takes place separate, and prolonged the growing-seedling period of chinaberry.At present, find to adopt the protoplast of seal chinaberry and local margosa, melia toosendan tree to merge as yet and cultivate the new varieties chinaberry that keeps the original proterties of seal chinaberry to adapt to local natural environment growth again.
CN1296239 has provided a kind of method for quickly breeding that prints the chinaberry tissue culture, is explant by explant children shoot, blade, and by cultivation, differentiation adventitious buds and the plant regeneration of callus, but the callus Growth abductive approach has weak point.Also fail simultaneously fundamentally to solve the problem that strain improves.
Three, summary of the invention:
The object of the invention provides and a kind ofly the seal chinaberry is moved northward reach the above area of north latitude 30 degree, still can keep the new product hybridization chinaberry of original seal chinaberry merit, the invention provides a kind of chinaberry hybrid tissue culture technique.
The present invention seeks to realize like this: chinaberry hybrid tissue culture technique is meant under hand control, the organ that seal chinaberry and margosa or melia toosendan tree or three are exsomatized is (as the tip of a root, stem apex, leaf, green fruit etc.), tissue is (as cambium, endosperm, cortex etc.), cell is (as somatic cell, reproductive cell etc.), embryo's (as ripe and immature embryo), protoplast (as take off wall after still viable protoplast) merges, in aseptic and suitable synthetic medium and illumination, under the conditions such as temperature, the artificial induction, realize the fusion of chinaberry hybridization protoplast, produce the cell wall of hybridization chinaberry and split into cell because of, change the cultivation of going down to posterity in the medium that goes down to posterity again over to, induced growth breeding hybridization chinaberry.More briefly be exactly, cut the growing point that seal chinaberry and margosa or melia toosendan tree has manufacturing, stem, leaf ability, its organ, tissue, cell, embryo and protoplast are merged, be placed on then in the artificial deployed medium, promote the formation of whole plant by the operation of plant hormone.
Improvement of the present invention is: the tissue culture technique of hybridization chinaberry is to utilize chinaberry stem apex, leaf or its hetero-organization through strict sterilization, sterilization treatment, be seeded in then on the suitable synthetic medium, at preference temperature (general 20-50 degree, down with), induced growth breeding under the humidity, illumination condition, obtain root, stem, the complete complete plantlet seedling of leaf at last.
The technology of the present invention also comprises: to producing the continuous selfing of plantlet seedling.Produce offspring's diversity and develop to pure type zygote direction.The plantlet seedling that produces is selected.Select satisfactory pure type zygote.Tissue cultivating to above-mentioned plantlet seedling: in the selection of explant type, preferentially select the mode of blastogenesis bud for use, and avoid mode through callus regeneration as far as possible, and to reduce the variation that takes place in the incubation, stability, the uniformity of the genetic character of guaranteeing to raise up seed; Coordinating the relation between growth rate and bud seedling quality, reaching under the prerequisite of certain growth rate the raising of attention bud seedling quality and survival rate in the selection of culture medium prescription; The selection of condition of culture then is tending towards nature as far as possible, to reduce the input of artificial adjusting, reduces seedling cost.
Characteristics of the present invention are: the present invention is in long-term, a large amount of experiment of process, successfully cultivated the Hybrid of seal chinaberry and margosa, melia toosendan tree, utilize the hybrid tissue culture technique, adopt margosa, the melia toosendan tree of the high seal chinaberry of chinaberry cellulose content and this area to be the parent, comprehensive merit separately, through for many years hybrid experiment, constantly screening in the numerous types of filial generation, the chinaberry new varieties that the high chinaberry cellulose content that successfully cultivating finally needs can be grown in the above area of north latitude 30 degree again.The method still belongs to initiative at home, and has filled up the blank of plant Melia Meliaceae Hybrid.The meaning of protoplast separation and Culture is 1, removed cell wall is that obstacle has been put down in fusion between the plant cell, has also opened up road for making new varieties simultaneously.2, protoplast can be taken in foreign DNA, organelle, bacterium or virion, and the hereditary variety that these characteristics and plant totipotency are integrated as higher plant lays the first stone.3, obtain the good starting material of cell clone and seed selection mutant.
By Plant Tissue Breeding, can go out a large amount of plant identical from a part of somatic cell quickly breeding of plant corpus with maternal plant.The present invention is a comprehensive high-new biotechnology.Its know-why relates to the totipotency principle of the genetic variation principle of plant breeding and hybrid vigour principle, plant cell, by somatic hybridization: fully without sexual process, only merge by somatic cell and make hybrid.By the utilization of present technique, the hybridization chinaberry can all can be grown seedlings throughout the year, has aseptic, high-quality, reproduction rate height, produces in batches, and year-round supply is convenient to advantages such as transportation.Utilize two kinds or three kinds of protoplasts fusions, make the protoplast regeneration that is merging go out cell wall, thereby generate the cell of hybrid chinaberry, plant hybridizes behind cell division or callus.
Four, embodiment:
Protoplast merges: 1, the separation preparation of the protoplast of hybridization chinaberry: will print chinaberry and margosa, three kinds of blades of melia toosendan tree or seal chinaberry and margosa, surface sterilization → fragmentation of two kinds of blades of seal chinaberry and melia toosendan tree also removed PH7-8 (or cellulase solution in the solution of the epidermis → blade immersion of fragmentation is contained HEL, macerozyme solution) and dissolving broken wall → filtration of carrying out plant cell in the solution of homeo-osmosis agent (as isopropyl alcohol etc.) dezymotize → clean → centrifugal, protoplast is put into a small amount of PEG of matrix → add, make the adventitia instability of protoplast, placement 10min → adding protoplast culture medium dilution PEG every 5min merges to promote protoplast, slightly shake after each dilution, protoplast will resuspension → mixture centrifugal 10min under 100g, centrifugal back cleaning in the medium that does not contain PEG → centrifugal again, resuspension is standby on identical medium.Again good protoplast is moved into the MS of volume and contain in the culture medium solution of agarose cultivation → protoplast hybridize again cell wall and split into cell mass after the osmotic pressure in cultivation → adjusting medium that in the agarose culture matrix, goes down to posterity, induce hybrid cell group to be divided into root, the stem → change the cultivation of going down to posterity in the medium that goes down to posterity over to of plant, induced growth is bred.Or adopt other biology commonly used to promote fusion method, as viral method.
Typical medium: adopt MS+NAAI-3.0ppm+BA 0.5-1ppm+3% sucrose+9% mannitol.
Attention: after 1. protoplast separates, a little less than being highly brittle, need the protectant protection of osmotic pressure to form up to cell wall.2. at different research objects, the level of the growth hormone and the basic element of cell division will be done suitable adjustment in the above-mentioned medium.
Two kinds of blade protoplasts of seal chinaberry and margosa or melia toosendan merge the cell that obtains does not have remarkable difference.
Cultural method: the fluid matrix cultivation, semiliquid matrix cultivation, the solid matrix cultivation, nurse is cultivated.
The step of solid culture: protoplast move into medium → 1 volume contain the medium of protoplast mixed with the medium that 1 volume contains agarose → the reversing culture dish descends cultivation → protoplasts to produce cell wall again and split into the cultivation of going down to posterity of cell mass → cell mass in agarose matrix at 25 ℃, should reduce the formation that osmoticum is beneficial to callus → the induce root that is divided into plant, stem in the medium.
It is as follows that hybridization chinaberry group is cultivated seedling-growing method: good healthy and strong maternal plant selections → explant collection and surface sterilizing → inoculation, explant cultivations → clump bud induce → and successive transfer culture (little cuttage breed combine) → clump bud propagation → terminal bud hardening of taking root → transplant with clump bud.
Embodiment:
Method for tissue culture: 1, explant collection and surface sterilizing: in the selection of explant type, preferentially select the mode of blastogenesis bud for use, and avoid mode through callus regeneration as far as possible, and to reduce the variation that takes place in the incubation, stability, the uniformity of the genetic character of guaranteeing to raise up seed; 2, inoculation: coordinating the relation between growth rate and bud seedling quality, reaching under the prerequisite of certain growth rate the raising of attention bud seedling quality and survival rate in the selection of culture medium prescription; 3, successive transfer culture is with combine method successive transfer culture 4-7 time of little cuttage and clump bud propagation: 4, terminal bud is taken root, the transplanting hardening.The selection of condition of culture then is tending towards nature as far as possible, to reduce the input of artificial adjusting, reduces seedling cost.
Group training embodiment: in the selection of explant type, preferentially select the mode (mode through callus regeneration easily produces variation) of blastogenesis bud for use, stability, the uniformity of the genetic character of guaranteeing to raise up seed; Coordinating the relation between growth rate and bud seedling quality, reaching under the prerequisite of certain growth rate the raising of attention bud seedling quality and survival rate in the selection of culture medium prescription; The selection of condition of culture then is tending towards nature as far as possible, to reduce the input of artificial adjusting, reduces seedling cost.
Specific as follows: adopting the stem with bud of annual root turion seedling germinating is explant, be cut into the stem with bud of long 1.0cm behind the defoliation, every stem section contains 1 axillalry bud, handle back (alcoholic solution with 70% soaks the several seconds) through routine disinfection, take out the back and in time receive immersion 6-15 minute → with the aseptic water washing 3 times → stem section the is cut into segment that has a pair of lateral bud of 1-1.5cm with 10% hypochlorous acid, be seeded in the inducing culture under the gnotobasis, every bottle graft 4-10 → seal → place in the culturing room about 25 ℃, under certain illumination condition, cultivate → will sprout and cut into stem section with 12 buds, change over to and carry out successive transfer culture in the proliferation and subculture medium, make it differentiate the bud of growing thickly; The base portion bud of growing thickly directly changes proliferated culture medium over to and cultivates.Subculture cycle is 25-30 days.Breed after some, make the shunting of part culture.The 2-3cm terminal bud that enrichment culture is obtained downcuts in the access root media, and it is neat to obtain taking root, and size is even, the complete seedling of robust growth.Enter strong sprout and take root, bottle outlet, in the culture of rootage experiment, IBA and IAA can directly lead root of hair, the seedling base portion does not produce callus, but IBA more helps root induction than IAA, when IBA concentration between 0.5-1, root of hair is fast, rooting rate can reach 4-7cm up to 100% height of seedling, and blade is big, and seedling is sturdy; If IBA and NAA are used, then can reach good root of hair effect, the root of hair initial time is 6d, the formation main root is sturdy behind the 12-15d, fibrous root is flourishing.Height of seedling 5-8cm, the big and dark green sturdy seedling of leaf.But use NAA separately, though well developed root system, it is slow to take root, and rooting rate is low.The transplantation of seedlings front opening bottle stopper of taking root is placed on sunny, and temperature is taken exercise near the place of natural temperature and can be transplanted in 1-5 days.
Claims (7)
1, the culture technique of hybridization chinaberry, it is characterized in that: chinaberry hybrid tissue culture technique is meant under hand control, the tip of a root that chinaberry and margosa or melia toosendan tree or three exsomatize will be printed, stem apex, leaf, organs such as green fruit, cambium, endosperm, tissues such as cortex, somatic cell, cells such as reproductive cell, embryos such as ripe and immature embryo, protoplast, comprise and take off that still viable protoplast merges behind the wall, in aseptic and synthetic medium and illumination, under the 20-50 degree temperature condition, carry out the artificial induction, realize the fusion of chinaberry hybridization protoplast, produce the cell wall of hybridization chinaberry and split into cell because of, change the cultivation of going down to posterity in the medium that goes down to posterity again over to, induced growth breeding hybridization chinaberry.
2, by the described culture technique of hybridizing chinaberry of claim 1, it is characterized in that by cutting the growing point that seal chinaberry and margosa or melia toosendan tree has manufacturing, stem, leaf ability, its organ, tissue, cell, embryo and protoplast are merged, be placed on then in the artificial deployed medium, promote the formation of whole plant by the operation of plant hormone.
3, by the culture technique of the described hybridization of claim 1 chinaberry, it is characterized in that producing the continuous selfing of hybridization plant seedling, produce offspring's diversity and develop to pure type zygote direction.The plantlet seedling that produces is selected.Select satisfactory pure type zygote.
4, by the culture technique of the described hybridization of claim 1 chinaberry, it is characterized in that tissue cultivating: in the selection of explant type, preferentially select the mode of blastogenesis bud for use to above-mentioned plantlet seedling
5, culture technique by the described hybridization of claim 1 chinaberry, the stem with bud that it is characterized in that the germinating of tiller seedling is an explant, be cut into the stem with bud of long 1.0cm behind the defoliation, every stem section contains 1 axillalry bud, after the routine disinfection processing, the stem section is cut into the segment that has a pair of lateral bud of 1-1.5cm, be seeded in the inducing culture under the gnotobasis, every bottle graft 4-10 → seal → place in the culturing room about 25 ℃, light application time 12 hours, intensity of illumination 3000lux cuts into the stem section of 1-2 bud of band with sprouting after the propagation, change over to and carry out successive transfer culture in the proliferation and subculture medium, make it differentiate the bud of growing thickly; The base portion bud of growing thickly directly changes proliferated culture medium over to and cultivates.Subculture cycle is 25-30 days.Breed after some, make the shunting of part culture.
6,, it is characterized in that successive transfer culture breeds the method successive transfer culture that combines with little cuttage and clump bud by the culture technique of the described hybridization of claim 1 chinaberry.
7, culture technique by claim 1 or 2 described hybridization chinaberrys, its feature merges from the separation preparation of the protoplast of hybridization chinaberry: will print chinaberry and margosa, melia toosendan tree three kinds of blades or stem eye surface sterilization → fragmentation are also removed the epidermis → blade immersion of fragmentation the is contained solution or the cellulase solution of HEL, in the macerozyme solution, carry out the dissolving broken wall of plant cell in the solution of pH value 7-8 and homeo-osmosis agent, filtration is dezymotized, clean, centrifugal, protoplast is put into matrix, induce with PEG, centrifugal back is cleaned in the medium that does not contain PEG, centrifugal again, again good protoplast is moved into the MS of volume and contain in the culture medium solution of agarose and cultivate, protoplast hybridize again cell wall and split into cell mass after the cultivation of in the agarose culture matrix, going down to posterity, induce hybrid cell group to be divided into the root of plant, stem.
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| CN 200510041096 CN1723770A (en) | 2005-07-19 | 2005-07-19 | Tissue culture tech. for hybrid chinaberry |
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| CN 200510041096 CN1723770A (en) | 2005-07-19 | 2005-07-19 | Tissue culture tech. for hybrid chinaberry |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870889A (en) * | 2009-04-21 | 2010-10-27 | 露西娅·阿特霍尔图亚加尔塞斯 | Method for expanding cell tissue from Jatropha curcas |
| CN102273409A (en) * | 2011-06-28 | 2011-12-14 | 蔡明� | Tissue culturing method for sugarcane seeds |
| CN103598088A (en) * | 2013-09-05 | 2014-02-26 | 南京大渊美容保健有限公司 | Chinaberry chromosome doubling method |
| CN109717077A (en) * | 2019-01-24 | 2019-05-07 | 安徽农业大学 | A kind of cultural method of China tree callus |
| CN110622864A (en) * | 2019-10-23 | 2019-12-31 | 国家林业和草原局泡桐研究开发中心 | Chromosome Doubling Rapid Propagation Method of Neem Tissue Culture |
-
2005
- 2005-07-19 CN CN 200510041096 patent/CN1723770A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870889A (en) * | 2009-04-21 | 2010-10-27 | 露西娅·阿特霍尔图亚加尔塞斯 | Method for expanding cell tissue from Jatropha curcas |
| CN102273409A (en) * | 2011-06-28 | 2011-12-14 | 蔡明� | Tissue culturing method for sugarcane seeds |
| CN103598088A (en) * | 2013-09-05 | 2014-02-26 | 南京大渊美容保健有限公司 | Chinaberry chromosome doubling method |
| CN103598088B (en) * | 2013-09-05 | 2016-08-10 | 南京大渊美容保健有限公司 | Chinaberry chromosome doubling method |
| CN109717077A (en) * | 2019-01-24 | 2019-05-07 | 安徽农业大学 | A kind of cultural method of China tree callus |
| CN110622864A (en) * | 2019-10-23 | 2019-12-31 | 国家林业和草原局泡桐研究开发中心 | Chromosome Doubling Rapid Propagation Method of Neem Tissue Culture |
| CN110622864B (en) * | 2019-10-23 | 2021-05-28 | 国家林业和草原局泡桐研究开发中心 | A method for rapid multiplication of chromosomes in tissue culture of Neem |
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