CN102726296A - Method for establishing tissue culture regeneration system of tea tree - Google Patents
Method for establishing tissue culture regeneration system of tea tree Download PDFInfo
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- CN102726296A CN102726296A CN2012102279424A CN201210227942A CN102726296A CN 102726296 A CN102726296 A CN 102726296A CN 2012102279424 A CN2012102279424 A CN 2012102279424A CN 201210227942 A CN201210227942 A CN 201210227942A CN 102726296 A CN102726296 A CN 102726296A
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Abstract
The invention relates to a method for establishing tissue culture regeneration system of tea tree. The method comprises the steps of inducing callus; performing callus subculture and proliferation; inducing adventitious bud; proliferating adventitious bud; hardening seedlings; rooting and transplantation. The method includes two proliferation stages, i.e., callus proliferation and adventitious bud proliferation. The callus proliferation manner has the characteristics of simple proliferation manner and stable inheritance. After subculture for 4 years, cell activity and differentiation ability have no obvious change. The callus with low phenols content can be taken as genetic transformation object. The technology establishes solid experimental foundation for establishment of genetic transformation system of tea tree.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to the method that a kind of tea tree tissue culture regenerating system is set up.
Background technology
Plant Tissue Breeding is to be a kind of cultured in vitro technology of theoretical foundation foundation with the cell totipotency.Since 20 beginnings of the century set up, the aspects such as quick breeding, breed improvement, genetic engineering breeding of plant have been widely used in
[1]Tea tree regenerating system or tissue culture technique are the bases of setting up the tea tree genetic conversion system, and the latter is the important technological platform of carrying out gene function checking, the breeding of tea tree cydorge gene, the research of tea tree secondary metabolism.Regrettably, owing to lack suitable regenerating system, the tea tree genetic conversion system is not set up so far, has limited the development of tea tree gene engineering, metabolic engineering.1969, Fo Lisite just began the research of tea tree tissue culture as the pioneer
[2]If do not consider genetic transformation, in fact the tissue culture of tea tree is comparatively ripe.Between the spray of tea tree, stipes and the tea seed be the explant of using always, and shoot proliferation is main propagation mode.Ban Naji
[3], Ba Ge
[4], Meng Daer
[5]And Sun Jun
[6]Between the stipes Deng employing ripe land for growing field crops tea tree spray or seedling is explant; Carry out regeneration tests through axillalry bud or adventitious bud proliferation form; This mode has inheritance stability, characteristics that the rate of increase is high, but because the aldehydes matter content of explant is high, is difficult to carry out the tea tree genetic transformation.Seed also is the desirable explant of tea tree regeneration, Wu Zhenduo
[7], add rattan
[8-9], and Meng Daer
[10]Utilize seed to induce somatic embryo Deng all, and further utilize the somatic embryo plant that regenerates.Experiment showed, that somatic embryo is the desirable object of tea tree genetic transformation, Meng Daer
[11]Even utilize this mode successfully to obtain first transfer-gen plant of tea tree, but in this mode, somatic embryo induce and propagation is technological difficulties.Though inducing of somatic embryo is comparatively difficult, inducing of embryo callus is then comparatively simple.
Summary of the invention
In order to lay the foundation for the tea tree genetic transformation, the present invention provides a kind of method of tea-tree tissue culture regenerating system foundation.
The concrete operations step that a kind of tea-tree tissue culture regenerating system is set up is following:
1.1 inducing of callus
Pluck the raw material of the tea seed of late September physiological ripening, i.e. explant as the tea tree regenerating system; Get 5～10 of tea seeds, the shell of green is peelled off, with the alcohol immersion of concentration 75% 30 seconds, with aseptic washing 3～4 times; Mercuric chloride solution with concentration 0.1% soaked 10 minutes again, with aseptic washing 3～4 times; Under the ultra-clean condition, peel off kind of a skin, choose the tea tree seed of peelling off kind of skin complete, that have embryo and be inoculated in common B
5On the medium, inoculum concentration is per 40 milliliters and connects 3～4; Cultivated for 2 weeks, have the tea tree callus to grow; Said common B
5Added dichlorophenoxyacetic acid and 6-chaff aminopurine in the medium, wherein the addition of dichlorophenoxyacetic acid is 2.5mg/L, and the addition of 6-chaff aminopurine is 0.5mg/L;
1.2 the subculture of callus and propagation
The tea tree callus is downcut, and be cut into 6～10 millimeters callus lines of diameter, be incubated at common B
5On the medium, inoculum concentration connects 5～10 for per 40 milliliters; Condition of culture is 25～28 degree for dark, temperature; Changed once fresh common B in per 21 days
5Medium; Cultivated for 3 weeks, obtain fresh weight and can breed and be 1.8～2.2 times original propagation callus; With this understanding, this callus can be stablized subculture 4 years;
1.3 inducing of indefinite bud,
To breed callus and be inoculated on the adventitious bud induction culture base, 5～10 of the per 40 milliliters of callus that connect 6～10 millimeters sizes of diameter of inoculum concentration; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 25～28 degree, and light intensity is 3000～6000 luxs; Per 4 weeks are changed an adventitious bud induction culture base; Cultivated for the 3rd week, have the part callus to change green; Cultivated for the 4th week, the inductivity of indefinite bud is 15.71%, cultivates 2 months, and inductivity rises to 24.29%, cultivates 4 months, and inductivity is up to 65.71%; Said adventitious bud induction culture base is the basis with the MS medium, adds indolebutyric acid and 6-benzyladenine, and wherein the addition of indolebutyric acid is 0.1mg/L, and the addition of 6-benzyladenine is 2.5mg/L;
1.4 the propagation of indefinite bud
The indefinite bud that newly induces is downcut, plant on the adventitious bud proliferation medium, the amount of planting connects 1～3 for per 100 milliliters; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 27 degree, and light intensity is 4000 luxs; Cultivated for 3 weeks, a large amount of buds of growing thickly grow; With this understanding, can constantly breed indefinite bud; Said adventitious bud proliferation medium is the basis with the MS medium, adds indolebutyric acid and phenyl thiadiazolyl group urea, and wherein the addition of indolebutyric acid is that the addition of 0.1mg/L, phenyl thiadiazolyl group urea is 0.015mg/L;
1.5 strong sprout
The bud of will growing thickly downcuts, and plants on the strong seedling culture base, and the amount of planting connects 1～3 for per 100 milliliters; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 27 degree, and light intensity is 4000 luxs; Cultivated for 3 weeks, the bud of growing thickly on average can be grown 2～3 centimetres, obtains tea shoot; Said strong seedling culture base is the basis with the MS medium, adds indolebutyric acid, and the addition of indolebutyric acid is 0.3mg/L;
1.6 take root with transplant
Choose the tea shoot that height of seedling reaches the sturdy unrooted more than 5 centimetres, directly plant in the soil of taking root, condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 25 degree, and light intensity is 3000～6000 luxs; At the transplanting initial stage, carry out the blade face water spray every day for three times; Cultivated for 8 weeks, survival rate is 41.64%, and the tea shoot of all survivals is all taken root, and the said soil prescription of taking root is a Denmark Pin Shi Top peat soil: vermiculite evenly mixes by mass ratio 1:1.
The useful technique effect of the inventive method embodies in the following areas:
1. the present invention has set up a kind of embryonic callus induction--the external tea-tree tissue culture regenerating system of taking root of embryo callus propagation-adventitious bud inducing--adventitious bud proliferation--.This regenerating system not only has callus induction rate height, simple, the inheritance stability characteristics of propagation mode, and callus can become the object of genetic transformation because aldehydes matter content is lower.Therefore present technique is that solid experiment basis has been established in the foundation of tea tree genetic conversion system;
2. had for two multiplicative stages in step, i.e. callus propagation and adventitious bud proliferation.Callus propagation mode has that the propagation mode is simple, the characteristics of inheritance stability, and experiment finds that at subculture the vigor and the differentiation capability of cell do not have significant change after 4 years.
Description of drawings
Fig. 1 is common B
5The tea callus that medium is induced.
Fig. 2 is common B
5The tea callus of cultivating in the medium dark.
Fig. 3 is the tea callus of cultivating under the illumination of bud inducing culture.
The tea shoot that Fig. 4 induces for the bud inducing culture.
The bud of living again that Fig. 5 induces for the bud proliferated culture medium.
Fig. 6 is tea shoot elongation on the strong seedling culture base.
Fig. 7 is the tea shoot after 9 weeks of transplanting.
Embodiment
Below in conjunction with embodiment the present invention is done explanation further.
Embodiment 1:
Capital equipment: superclean bench; The constant temperature illumination box; The autoclave sterilization pot; PH meter
Reagent and material:
1. explant material: the seed of common tea tree
2. main agents:
(1) 75% ethanol, medical type 75% alcohol of buying on the market is mainly used in the sterilization of explant;
(2) 1% mercury chloride (HgCl
2), take by weighing 1 gram mercury chloride and be dissolved in 1 premium on currency, be used for the explant sterilization;
(3) plant hormone class: the dichlorophenoxyacetic acid of buying on the market (2,4-D), indolebutyric acid (IBA), 6-benzyladenine (6-BA), 6-chaff aminopurine (KT), phenyl thiadiazolyl group urea (TDZ);
(4) vermiculite and Denmark Pin Shi Top peat soil are bought from market.
The concrete operations step that a kind of tea-tree tissue culture regenerating system is set up is following:
1.1 inducing of callus
Pluck the raw material of the tea seed of late September physiological ripening, i.e. explant as the tea tree regenerating system; Get 5～10 of tea seeds, the shell of green is peelled off, with the alcohol immersion of concentration 75% 30 seconds, with aseptic washing 3～4 times; Mercuric chloride solution with concentration 0.1% soaked 10 minutes again, with aseptic washing 3～4 times; Under the ultra-clean condition, peel off kind of a skin, choose the tea tree seed of peelling off kind of skin complete, that have embryo and be inoculated in common B
5On the medium, inoculum concentration is per 40 milliliters and connects 3～4; Cultivated for 2 weeks, have the tea tree callus to grow, see Fig. 1; Above-mentioned common B
5Hormone dosage is dichlorophenoxyacetic acid 2.5mg/L, 6-chaff aminopurine 0.5mg/L in the medium, and other element consumptions are B
5The known concentration of medium.
1.2 the subculture of callus and propagation
The tea tree callus is downcut, and be cut into 6～10 millimeters callus lines of diameter, be incubated at common B
5On the medium, inoculum concentration connects 5～10 for per 40 milliliters; Condition of culture is 25～28 degree for dark, temperature; Changed once fresh common B in per 21 days
5Medium; Cultivated for 3 weeks, obtain fresh weight and can breed, see Fig. 2 to about 2 times original propagation callus; With this understanding, this callus can be stablized subculture 4 years.
1.3 inducing of indefinite bud
To breed callus and be inoculated on the adventitious bud induction culture base, 5～10 of the per 40 milliliters of callus that connect 6～10 millimeters sizes of diameter of inoculum concentration; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 25～28 degree, and light intensity is 3000～6000 luxs; Per 4 weeks are changed an adventitious bud induction culture base; Cultivated for the 3rd week, have the part callus to change green, see Fig. 3; Cultivated for the 4th week, the inductivity of indefinite bud is 15.71%, sees Fig. 4, cultivates 2 months, and inductivity rises to 24.29%, cultivates 4 months, and inductivity is up to 65.71%; Hormone dosage is indolebutyric acid 0.1mg/L, 6-benzyladenine 2.5mg/L in the above-mentioned adventitious bud induction culture base, and other element consumptions are the known concentration of MS medium.
1.4 the propagation of indefinite bud
The indefinite bud that newly induces is downcut, plant on the adventitious bud proliferation medium, the amount of planting connects 1～3 for per 100 milliliters; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 27 degree, and light intensity is 4000 luxs; Cultivated for 3 weeks, a large amount of buds of growing thickly grow, and see Fig. 5; With this understanding, can constantly breed indefinite bud; Hormone dosage is indolebutyric acid 0.1mg/L, phenyl thiadiazolyl group urea 0.015mg/L in the above-mentioned adventitious bud proliferation medium, and other element consumptions are the known concentration of MS medium.
1.5 strong sprout
The bud of will growing thickly downcuts, and plants on the strong seedling culture base, and the amount of planting connects 1～3 for per 100 milliliters; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 27 degree, and light intensity is 4000 luxs; Cultivated for 3 weeks, the bud of growing thickly on average can be grown 2～3 centimetres, obtains tea shoot, sees Fig. 6; Hormone dosage is indolebutyric acid 0.3mg/L in the above-mentioned strong seedling culture base, and other element consumptions are the known concentration of MS medium.
1.6 take root with transplant
Choose the tea shoot that height of seedling reaches the sturdy unrooted more than 5 centimetres, directly plant in the soil of taking root, condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 25 degree, and light intensity is 3000～6000 luxs; At the transplanting initial stage, carry out the blade face water spray every day for three times; Cultivated for 8 weeks, survival rate is 41.64%, and the tea shoot of all survivals is all taken root, and sees Fig. 7.The above-mentioned soil prescription of taking root is a Denmark Pin Shi Top peat soil: vermiculite evenly mixes by quality 1:1.
List of references
[1] Xiao Zheli, Liu Jinfeng. the progress of Plant Tissue Breeding and new technology are used [J]. the Ningxia agriculture and forestry science and technology. 2011, (01) .13-15
[2] Fo Lisite, the research [J] of polyphenol metabolism in the tea tree tissue culture that the G. tea tree is induced. journal of biological chemistry. 1969,113 (5): 765.
（Forrest?G.?Studies?on?the?polyphenol?metabolism?of?tissue?cultures?derived?from?the?tea?plant?(Camellia?sinensis?L.)[J].?Biochemical?Journal.?1969,?113(5):?765.）
[3] Ban Naji M, external the taking root of Ah lattice Wal B. tea tree [J]. India's experimental biology magazine. 1990,28:936-939
（Banerjee?M,?Agarwal?B.?(Camellia?sinensis?L.)?O.?Kuntze)[J].?Indian?Journal?of?Experimental?Biology.?1990,?28:?936-939.）
[4] crust lattice N, Bern L, Nan Di S. utilize method for tissue culture to breed tea tree [J] in a large number. plant physiology and molecular biology. and 1997,3:99-103.
（Bag?N,?Palni?L,?Nandi?S.?Mass?propagation?of?tea?using?tissue?culture?methods[J].?Physiol.?Mol.?Biol.?Plants.?1997,?3:?99-103.）
[5] Meng Daer TK clings to the inferior A of proper tower, people such as Su De A. utilize the microbody tea tree breeding [J] of Thidiazuron. and plant growth regulating. 1998,26 (1): 57-61.
（?Mondal?TK,?Bhattacharya?A,?Sood?A
?et?al.?Micropropagation?of?tea?(Camellia?sinensis?(L.)?O.?Kuntze)?using?Thidiazuron[J].?Plant?growth?regulation.?1998,?26(1):?57-61.）
[6] Sun Jun, as if Zhang Zhengzhu is spring dawn etc.; A kind of tea tree clone Regeneration in Vitro cultured method (P). 2011.09.02.
[7] Wu Zhenduo. the research of tea tree tissue culture [J]. Chinese agronomy committee newspaper. 1976,93:30-42.
[8] add the Study on tissue culture [J] of rattan M. camellia and Cordyceps sinensis. Japanese thremmatology .1982,32:267-277
(Kato?M.?Results?of?organ?culture?on?Camellia?japonica?and?C.?sinensis[J].?Jpn.?J.?Breed.?1982,?32(Supplement?2):?267-277.)
[9] add rattan M. through the tea tree of cotyledon cultivation and fast numerous research [J] of Cordyceps sinensis. Japanese thremmatology .1986,31-38
(Kato?M.?Micropropagation?through?cotyledon?culture?in?Camellia?japonica?L.?and?C.?sinensis?L[J].?Japanese?Journal?of?Breeding?(Japan).?1986,31-38)
[10] Meng Daer TK clings to the inferior A of proper tower, people such as Su De A. the inducing of the secondary embryo of tea tree [J]. and plant physiology .2001,158 (7): 945-951.
(Mondal?TK,?Bhattacharya?A,?Ahuja?PS.?Induction?of?synchronous?secondary?somatic?embryogenesis?in?Camellia?sinensis?(L.)?O.?Kuntze[J].?Journal?of?plant?physiology.?2001,?158(7):?945-951.)
[11] Meng Daer TK clings to the inferior A of proper tower, people such as Su De A. agriculture bacillus mediated tea tree somatic embryo transgenic research [J]. and plant cell report .2001,20 (8): 712-720.
(Mondal?T,?Bhattacharya?A,?Ahuja?P
?et?al.?Transgenic?tea?[Camellia?sinensis?(L.)?O.?Kuntze?cv.?Kangra?Jat]?plants?obtained?by?Agrobacterium-mediated?transformation?of?somatic?embryos[J].?Plant?Cell?Reports.?2001,?20(8):712-720.)。
Claims (1)
1. the method set up of a tea-tree tissue culture regenerating system is characterized in that comprising following operating procedure:
1.1 inducing of callus
Pluck the raw material of the tea seed of late September physiological ripening, i.e. explant as the tea tree regenerating system; Get 5～10 of tea seeds, the shell of green is peelled off, with the alcohol immersion of concentration 75% 30 seconds, with aseptic washing 3～4 times; Mercuric chloride solution with concentration 0.1% soaked 10 minutes again, with aseptic washing 3～4 times; Under the ultra-clean condition, peel off kind of a skin, choose the tea tree seed of peelling off kind of skin complete, that have embryo and be inoculated in common B
5On the medium, inoculum concentration is per 40 milliliters and connects 3～4; Cultivated for 2 weeks, have the tea tree callus to grow; Said common B
5Added dichlorophenoxyacetic acid and 6-chaff aminopurine in the medium, wherein the addition of dichlorophenoxyacetic acid is 2.5mg/L, and the addition of 6-chaff aminopurine is 0.5mg/L;
1.2 the subculture of callus and propagation
The tea tree callus is downcut, and be cut into 6～10 millimeters callus lines of diameter, be incubated at common B
5On the medium, inoculum concentration connects 5～10 for per 40 milliliters; Condition of culture is 25～28 degree for dark, temperature; Changed once fresh common B in per 21 days
5Medium; Cultivated for 3 weeks, obtain fresh weight and can breed and be 1.8～2.2 times original propagation callus; With this understanding, this callus can be stablized subculture 4 years;
1.3 inducing of indefinite bud,
To breed callus and be inoculated on the adventitious bud induction culture base, 5～10 of the per 40 milliliters of callus that connect 6～10 millimeters sizes of diameter of inoculum concentration; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 25～28 degree, and light intensity is 3000～6000 luxs; Per 4 weeks are changed an adventitious bud induction culture base; Cultivated for the 3rd week, have the part callus to change green; Cultivated for the 4th week, the inductivity of indefinite bud is 15.71%, cultivates 2 months, and inductivity rises to 24.29%, cultivates 4 months, and inductivity is up to 65.71%; Said adventitious bud induction culture base is the basis with the MS medium, adds indolebutyric acid and 6-benzyladenine, and wherein the addition of indolebutyric acid is 0.1mg/L, and the addition of 6-benzyladenine is 2.5mg/L;
1.4 the propagation of indefinite bud
The indefinite bud that newly induces is downcut, plant on the adventitious bud proliferation medium, the amount of planting connects 1～3 for per 100 milliliters; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 27 degree, and light intensity is 4000 luxs; Cultivated for 3 weeks, a large amount of buds of growing thickly grow; With this understanding, can constantly breed indefinite bud; Said adventitious bud proliferation medium is the basis with the MS medium, adds indolebutyric acid and phenyl thiadiazolyl group urea, and wherein the addition of indolebutyric acid is that the addition of 0.1mg/L, phenyl thiadiazolyl group urea is 0.015mg/L;
1.5 strong sprout
The bud of will growing thickly downcuts, and plants on the strong seedling culture base, and the amount of planting connects 1～3 for per 100 milliliters; Condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 27 degree, and light intensity is 4000 luxs; Cultivated for 3 weeks, the bud of growing thickly on average can be grown 2～3 centimetres, obtains tea shoot; Said strong seedling culture base is the basis with the MS medium, adds indolebutyric acid, and the addition of indolebutyric acid is 0.3mg/L;
1.6 take root with transplant
Choose the tea shoot that height of seedling reaches the sturdy unrooted more than 5 centimetres, directly plant in the soil of taking root, condition of culture is illumination in 12 hours and 12 hours dark replacing, and temperature is 25 degree, and light intensity is 3000～6000 luxs; At the transplanting initial stage, carry out the blade face water spray every day for three times; Cultivated for 8 weeks, survival rate is 41.64%, and the tea shoot of all survivals is all taken root, and the said soil prescription of taking root is a Denmark Pin Shi Top peat soil: vermiculite evenly mixes by mass ratio 1:1.
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| CN102934603A (en) * | 2012-10-30 | 2013-02-20 | 安徽农业大学 | Asexual rapid propagation method of tea trees and root taking culture solution |
| CN103931477A (en) * | 2014-04-29 | 2014-07-23 | 地缘（厦门）生物科技有限公司 | Novel acacia melanoxylon tissue culture seedling rooting method |
| CN105104194A (en) * | 2015-08-20 | 2015-12-02 | 中央民族大学 | Method for promoting Anji white tea callus proliferation and improving tea polyphenol content in Anji white tea calluses |
| CN105494107A (en) * | 2016-02-25 | 2016-04-20 | 湖南农业大学 | Tea tree tissue culture method |
| CN107022519A (en) * | 2017-05-16 | 2017-08-08 | 安徽农业大学 | The isolated culture method of tealeaves attachment independent single cells |
| CN108834900A (en) * | 2018-07-31 | 2018-11-20 | 安徽农业大学 | Inhibit the cultural method of explant oxidizing brown stain and endophytic bacterial contamination in tea-tree tissue culture |
| CN113331059A (en) * | 2021-07-21 | 2021-09-03 | 贵州大学 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
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| CN102934603A (en) * | 2012-10-30 | 2013-02-20 | 安徽农业大学 | Asexual rapid propagation method of tea trees and root taking culture solution |
| CN102934603B (en) * | 2012-10-30 | 2014-07-09 | 安徽农业大学 | Asexual rapid propagation method of tea trees and root taking culture solution |
| CN103931477A (en) * | 2014-04-29 | 2014-07-23 | 地缘（厦门）生物科技有限公司 | Novel acacia melanoxylon tissue culture seedling rooting method |
| CN105104194A (en) * | 2015-08-20 | 2015-12-02 | 中央民族大学 | Method for promoting Anji white tea callus proliferation and improving tea polyphenol content in Anji white tea calluses |
| CN105104194B (en) * | 2015-08-20 | 2017-06-23 | 中央民族大学 | Promote Anji white tea callus proliferation and the method for improving wherein polyphenol content |
| CN105494107A (en) * | 2016-02-25 | 2016-04-20 | 湖南农业大学 | Tea tree tissue culture method |
| CN107022519A (en) * | 2017-05-16 | 2017-08-08 | 安徽农业大学 | The isolated culture method of tealeaves attachment independent single cells |
| CN108834900A (en) * | 2018-07-31 | 2018-11-20 | 安徽农业大学 | Inhibit the cultural method of explant oxidizing brown stain and endophytic bacterial contamination in tea-tree tissue culture |
| CN113331059A (en) * | 2021-07-21 | 2021-09-03 | 贵州大学 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
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Application publication date: 20121017 |