CN1376381A - Method for micro-propagating tea trees - Google Patents
Method for micro-propagating tea trees Download PDFInfo
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- CN1376381A CN1376381A CN 01109920 CN01109920A CN1376381A CN 1376381 A CN1376381 A CN 1376381A CN 01109920 CN01109920 CN 01109920 CN 01109920 A CN01109920 A CN 01109920A CN 1376381 A CN1376381 A CN 1376381A
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Abstract
The present invention relating to a noval microbody reproduction method for the tea tree coming from explant features that said explant is cultured in different culture media. Said explant is come from the fully close, semi-open or fully open callus.
Description
The present invention relates to the explant that a kind of employing cuts, the effective ways that are used for tea tree plant (tea (Camelia Sinensis)) micropropagation from leaf.
Tea be a kind of welcome have anticancer property contain the caffeine beverage (Jankun etc., why drinking tea can prophylaxis of cancer, " nature " 5:561; 1997).Though Camellia has many kinds, only the different cultivars of C.sinensis (L.) O.Kuntze or tea with it commercial be that very important (Braua D.N. edits, " science in the tealeaves cultivation and put into practice ", Calcutta tea research association; 53-68; 1989).
Important occupation is not only in the tealeaves cultivation provides the source, and be that the main path (Wilson that gets foreign exchange is earned in all tea growth areas, the world, R.C. the Wilson in Bian Ji " coffee, cocoa and the tea ", K.C. " botany and plant improvement ", CABI publishes, Wallingford, UK:167-173; 1999).But the gross yield of tealeaves is not enough to satisfy needs (Kabra, G.D. " the tealeaves statistics of phase tea time in 1999 ", VolVIII, No.3 9-11,99, the 30-31 of this country and world market; 1999).Because (fungi, insect and the virus) of different life and lifeless (freezing, hail, cold, arid, malnutrition etc.) stress (Wilson, R.C. the Wilson in Bian Ji " coffee, cocoa and the tea ", K.C. " botany and plant improvement ", CABI publishes, Wallingford, UK:167-173; 1999) productive rate of tealeaves and quality are further reduced.
The life cycle that the actual tea tree that belongs to woody seeds has the length that is accompanied by height self incompatibility and inbreeding decline, (Braua D.N. edited, Braua D.N. " tea tree trade " in " science in the tealeaves cultivation and put into practice ", Calcutta tea research association; 53-68; 1989), this has limited usually by conventional breeding method production high yield and excellent and anti-stress tea tree.Therefore, the application of biological technique method is a kind of effective and selective method.But a kind of efficient and reproducible renovation process is most important essential in advance for any biotechnology applications.
Serious problems in the tealeaves are blister fusarium wilts, because its infringement is used for the leaf and the young shoot of tea making, productive rate has descended 50% as a result.Therefore, suppress the blister fusarium wilt is badly in need of for remedying these losses very much.Determined that some have high yield and high-quality but the clone that easily suffers from the blister fusarium wilt, therefore need by bio-technology improvement, because can cause Gene Isolation and the required high yield and the forfeiture of high-quality characteristic such as the heterogeneous tissue of cotyledon explant such as the same source tissue of leaf explant.Therefore, existing method is not have above-mentioned relevant purposes such as heterogeneous the organizing of cotyledon explant, is badly in need of the method for development employing with the micropropagation of source tissue.The regeneration of leaf explant is best preferred, because:
(i) leaf explant is a homology;
(ii) leaf contains huge amount and duplicates several chloroplast DNAs, if therefore use leaf in the development of genetic manipulation process such as transgenosis or somatic hybridization, then leaf is expressed the several times that can increase;
(iii) leaf provides exhibiting high surface for any gene manipulation techniques;
(iv) leaf is the primary commercial source that commercially available one-tenth is sampled tea;
(v) leaf provides abundant raw material supply;
(vi) adopt the leaf can general good characteristic and the growth of overslaugh plant as explant.
If somaclonal variation in regenerative process, do not occur, so by somatic hybridization or usually need be through the regeneration of callus phase by the biotechnology crop improvement of transgenic technology.Because the tea tree life-span is long, to compare with the herbaceous plant of quick growth, the possibility of the chromosomal variation of callus in mutually is low.Therefore, after deliberation be used to use effective indirect method through the micropropagation of the tea tree plant of the leaf explant of callus phase.
If if therefore use leaf explant or plant is 50 years or longer very old tree, the regeneration capacity of woody plant is very poor.
In order to produce the plant of taking place or to form through the no-set young shoot of callus phase through somatic embryo, but transformation frequency arbitrary among both low (4-6%) or take root seldom, other ornamental plants kind that leaf explant has been used for Camellia, C.japonica and C.reticulata (Sanjose and Vieitez for example, the no-set young shoot regeneration of the external leaf of A.M. ripe Camellia (Camellia) reticulata, " horticultural science magazine " 67:677-683; 1992; Sanjose, M.C. and Vieitz, A.M. " derives from the regeneration of the Camellia plantlet of leaf explant culture " by embryo's generation and handle, " horticultural science ", 54:303-315; 1993; Pedroso, M.C. and Pais, M.S.C.japonica L. " the direct somatic embryogenesis the form of the foetus in the leaf becomes ", " plant cell is joined (Rep.) again " 12:639-643; 1993).And these all are ornamental plants kinds.But, not about the report of the method for the plant regeneration of the leaf explant that forms by the promptly commercial Camellia of C.sinensis or the callus in the tea tree, from no-set young shoot.
In order to study renovation process from leaf explant, Nakamura Y. has done in 1984 and has attempted (" effective ways of breeding outside the tea tree plant corpus " first, Proc.Internat.Symp. " latest developments of relevant tea yield ", China, Taiwan, the 1984:63-74 page or leaf), wherein callus is to be supplemented with auxin 2, the Nitsch of 4-dichlorphenoxyacetic acid and Nitsch medium (Nitsch, J.P. with Nitsch C., " derive from the haplophyte of pollen grain ", " science " (Washington), 163; 85-87; And Gamborg medium (Gamborg, O.L., Miller, R.A. and Ojima, K. " nutritional need of the suspension culture of soybean root cells ", " experimental cell research " 50:151-58 (1969)); 1968) obtain in.The shortcoming of this method is to fail to obtain form generation or the formation of no-set young shoot.In 1985, Nakamura (Naksmura, Y. " origin of explant is to the differentiation of the root in the tissue culture of tea tree plant and the influence of its different cultivars ", Shizuoka tea experiment centre 62:1-8; 1985) and Palni, Sood, Chand, Sharma, Rao and Jain (Palni, L.M.S.Sood, A., Chand, G., Sharma, M., Rao, D.V., Jain, N.K. " Study on tissue culture in the tea tree ", Proc.Internat.Sym. " relevant tea science ", Shizuoka, Japan, 395-399,1991) attempted plant regeneration again,, failed from this callus regeneration plant that takes root though wherein he has obtained to take root from the leaf callus through the leaf explant of callus phase.After this, until 1996 just relevant for the report of the plant regeneration of leaf explant, Kato (Kato, M. " somatic embryo of the prematurity leaf of the tea young shoot of growth in vitro takes place ", " plant cell is joined again " 15:920-926 wherein; 1996) from deriving from Murashige and Skoog medium (Murashige T. and Skoog F.A " the improvement medium that is used for the quick growth and the biologicall test of tobacco tissue culture ", " plant physiology " 15:473-497; The somatic embryo of the leaf explant of the growth in vitro plant 1962) has obtained some plants, and this culture media supplemented has 0.5mg/l 2,4 dichlorophenoxyacetic acid in the liquid and 0.8% agar to solidify 5mg/l in the medium.Yet the major defect of the method for Kato is as follows:
(i) the relevant somatic embryo explant of the inducing percentage very low (6%) of replying;
(ii) the donor plant is the sapling that causes the genetic mutation in the filial generation;
(iii) to be converted into the frequency of plant very low for somatic embryo, and promptly 7.1%;
(iv) derivative embryo is controlled in the specific region of leaf and does not provide unaccommodated transgenic research for them from all leaf surfaces;
(v) do not comprise the system that is used for cultivating leaf explant, and it comprises the exploitation from embryo's generation callus of the leaf explant of sapling from the selectivity dwarf thicket of maturation with good characteristic;
(vi) sapling is represented heterogeneous population, and the explant of collecting from the selectivity adult tree has good characteristic, because they are bred by clone or vegetative propagation mode.
With different medium be used for such as embryonic induction, for the second time the embryo is taken place and the different step process of embryonic development.
Therefore, need to provide a kind of high efficiency micropropagation method of developing healthy and strong tea tree plant in the prior art.
Main purpose of the present invention is, is used for the high efficiency method through the micropropagation of the tea tree of callus phase, the healthy and strong tea tree of explant exploitation that obtains from leaf;
Another object of the present invention is to reconcile with relevant foreign gene introducing leaf explant and by direct biolistic rifle or indirect Agrobacterium and develop a large amount of genetically modified plants;
Still a further object of the present invention is that development derives from the renovation process of the leaf of protoplast and somatic hybridization;
Still a further object of the present invention is that related gene is introduced protoplast and studied this expression;
A further object of the present invention is to promote the absorption of virion.
Therefore, the invention provides a kind of use has the plant of vigor and stalwartness in a large number from the explant exploitation of leaf new micropropagation method.
Therefore, the invention provides the high efficiency method of the micropropagation of a kind of tea tree (tea (Cemallia sinensis)), described method comprises the following steps:
(a) excision is from the explant of that close up fully, the semi-open or complete open leaf of the tea tree plant of culture in vitro;
(b) be 20 ℃-40 ℃ in temperature, at least 52 μ g/molm
-2s
-1Cool white light exist and under 16 hour photoperiod of illumination, at first medium (Murashige T. and Skoog F.A " the correction medium that is used for the quick growth and the biologicall test of tobacco tissue culture ", " plant physiology " 15:473-497; 1962) cultivate the explant 4-6 week that is used for callus induction in, described medium is basic Murashige and the Skoog medium that is supplemented with the 0.8% agar curing of vitamin, 1-3mg/l glycine, 2.5-10.0mg/l 2,4 dichlorophenoxyacetic acid and 1.3% sucrose;
(c) described callus is transferred to second medium that is used for taking root 6-10 week at least, described medium is to be supplemented with the basic MS culture medium that 0.8% agar of cytokinin such as 0.5-8mg/l 6-benzylaminopurine and auxin such as 0.1-0.8mg/l indole-3-butyric acid or indole-3-acetic acid solidifies;
(d) the described callus of taking root is transferred to the 3rd medium 4-10 week that is used for causing young shoot, described medium is the free medium of auxin and contains 0.5-8mg/l 6-benzylaminopurine;
(e) described young shoot is transferred to cultivation 4-6 week in the 4th propagating culture medium, described medium is for being supplemented with 5 μ m phenyl thiadiazolyl group urea (Sandal I., Bhattacharya A. and Ahuja P.S.2001. " the efficient liquid culture systems that is used for the breeding of tea tree young shoot ", " plant cell tissue's organ culture " 00.1-6) liquid nutrient medium, the young shoot that obtains taking root;
(f) cut off long young shoot and obtain one and longly be the end of 3cm, it is carried out 20-30 minute processing with indole-3-butyric acid, and in the wide-mouth bottle of sand that contains 1: 1 ratio and native mixture this young shoot of cultivation 60-75 days; With
(g) young shoot of taking root is grown to obtain vigor and healthy and strong tea tree.
In one embodiment, explant is to be selected from tree plant cultivation kind for example Chinary, Assamica and Cambod.In the present invention, explant is to excise from fresh open leaf that close up fully, semi-open or complete.In fact, when leaf explant is leaf excision from the second and the 3rd position at young shoot tip, find that the whole or whole surface of leaf explant is responsive.This leaf can be selected from the tree plant cultivation kind of growing under in vivo any and/or the conditions in vitro.In other words, this explant be from first leaf or with the leaf that closes up fully of terminal bud utmost point close attachment excise.The leaf explant of any cultivar can use.By inducing the merismatic activity in the leaf explant, form by callus and can make indirect young shoot regeneration.
To in the solution that contains Bavistin (0.1%) and streptomycin (0.05%), clean from the leaf of tree plant cultivation kind, in polysorbas20 the washing and in containing 0.01% (w/v) mercuric chloride solution of a drop of liquid cleaning agent surface sterilizing, then in sterile distilled water thoroughly the washing.Then the explant of surface sterilizing is cultivated by method of the present invention.
In one embodiment, first medium, promptly callus inducing medium is supplemented with vitamin such as Cobastab
1-HCl 0.05-2.0mg/l, Cobastab
6-HCl 0.25-1.5mg/ml and nicotinic acid 0.25-1.5mg/l.At 52 μ g/molm
-2s
-1Cold fluorescence exist and under 16 hour photoperiod of illumination, place first medium to cultivate 6-10 week at least explant, preferred 6 weeks at 20-40 ℃.
In one embodiment, in order to take root, the callus that step (b) is obtained is transferred in second medium, the basic MS culture medium that this medium solidifies for 0.8% agar that is supplemented with 0.5-8mg/l 6-benzylaminopurine and auxin such as indole-3-acetic acid or indole-3-butyric acid (0.1-0.8mg/l).This callus was placed for the second medium 4-6 week, preferred 6 weeks.The pH of medium is 5.6-7.6.At 52 μ g/molm
-2s
-1Cold fluorescence exist and under 16 hour photoperiod of illumination with this culture 24-30 ℃ of cultivation.
This callus is placed the 3rd medium 4-6 week that is used for taking root.
In one embodiment, the young shoot (about 3cm) that obtains from step (d) after 6 weeks of excision, and make it be grown in the 4th medium, this medium is the basic MS culture medium that is supplemented with the 0.8% agar curing of 3% sucrose and 5 μ M phenyl thiadiazolyl group ureas and 10 μ M methyls, grows to 2.0cm until them.This culture media supplemented has auxin and cytokinin.
The microbody young shoot that will so obtain then in one embodiment, is bred in the 20ml static liquid medium that contains 3% sucrose and 5 μ M phenyl thiadiazolyl group ureas.
In one embodiment, the auxin with the plant growth regulator that acts on callus formation and take root is selected from indole-3-butyric acid, indole-3-acetic acid, methyl and 2,4 dichlorophenoxyacetic acid.
In another embodiment, be selected from 6-benzylaminopurine, N with the cytokinin that acts on the plant growth regulator of taking root
6-furfuryladenine and phenyl thiadiazolyl group urea, but preferred 6-benzylaminopurine.
In another embodiment, at 4-10 after week after preferred 4 weeks, need will the responsive callus of taking root be transferred to and be used for auxin that young shoot the forms medium that dissociates.
In another embodiment, must in the 9.0cm Petri dish that contains 25ml medium (pH5.6), cultivate this material especially.
In another embodiment, medium maintenance pH is 5.6-6.6.
In another embodiment, the microbody young shoot is taken root, promptly by handle the end that cuts off with the 5.0mg/l indole-3-butyric acid, and its kind be coated with 1: 1 sand in the jar of reversing wide-mouth under the laboratory cultures condition: in 8 weeks in the aseptic mixture of soil, the young shoot that at room temperature will take root is transferred in the plastic tank then.
In another embodiment of the invention, the leaf explant of the selective vegetable of the external or indirect culture in vitro of above-mentioned used hybrid cultivar is carried out following steps:
I) basic MS culture medium that the open leaf explant that close up fully, semi-open or complete of the culture of any clone's culture in vitro is placed 0.8% agar that is supplemented with 3% sucrose and 6-benzylaminopurine (0.5-8.0mg/l) and indole-3-butyric acid (0.1-0.8mg/l) solidify;
Ii) explant is placed Petri dish be supplemented with 2-5% sucrose and 2.5,5.0,7.5 or 10.0mg/l 2,4-dichlorphenoxyacetic acid, indole-3-acetic acid, indole-3-butyric acid or the agar (0.8-1.0%) that combines with 6-benzylaminopurine or phenyl thiadiazolyl group urea (0,0.1,0.2mg/l) solidify in the basic MS culture medium, this medium contains the 25ml basic MS culture medium of agar gel, and it is being used for the cold fluorescence of callus induction (52 μ g/molm
-2s
-1) under in 25 ± 2 ℃ of cultivations;
Iii) in order to take root, the culture that top step is obtained is transferred in the basic MS culture medium that 0.8% agar that is supplemented with 3% sucrose that combines with indole-3-acetic acid, indole-3-butyric acid (0.1-0.8mg/l) and 6-benzylaminopurine or phenyl thiadiazolyl group urea (0.5-0.8mg/l) solidifies;
Iv) for unbodied young shoot forms, the callus culture thing of taking root that step is (iii) obtained is transferred in the basic MS culture medium of the 0.8% agar curing that is supplemented with 6-benzylaminopurine (0.5-0.8mg/l) and 3% sucrose;
V) the young shoot that (iv) obtains from step of 6 week back excisions and make it grow the basic MS culture medium that 0.8% agar that is supplemented with 3% sucrose and 5 μ M phenyl thiadiazolyl group ureas and 10 μ M methyls solidifies grows to 2.0cm until them;
The microbody young shoot that vi) will so obtain is then bred in the 20ml static liquid medium that contains 3% sucrose and 5 μ M phenyl thiadiazolyl group ureas;
The microbody young shoot is taken root, promptly by handle the end that cuts off with the 5.0mg/l indole-3-butyric acid, and its kind be coated with 1: 1 sand in the jar of reversing wide-mouth under the laboratory cultures condition: in 8 weeks in the aseptic mixture of soil, the young shoot that at room temperature will take root is transferred in the plastic tank then.
But the applicant find in first medium, to exist the 2,4 dichlorophenoxyacetic acid induced activity cell division of high concentration and not differential growth, to cause the callus formation in the plant tissue be known.In tea tree, also found this phenomenon.But these do not break up material and cause some to comprise growth such as the meristematic tissue bag of the vascular tissue of test-tube baby with the transfer of specific concentration in the medium that is supplemented with 6-benzylaminopurine and indole-3-acetic acid or indole-3-butyric acid.These meristematic tissue bags develop into unbodied at last, develop into amorphous young shoot then, are transferred in the free medium of the auxin that only contains the 6-benzylaminopurine.
Since 1984, it is the leaf explant generation young shoot through the callus phase of tea (Camellia Sinensis) that a few thing personnel have attempted from tea tree.But they fail to obtain the progress except that the callus of taking root so far.Novelty of the present invention is to recognize to have to the root limit when some concentration such as the auxin of 2,4 dichlorophenoxyacetic acid or indole-3-acetic acid or indole-3-butyric acid and causes morphogenetic potentiality.Therefore, suppose that this morphogenetic initator can be that the young shoot limit shifts to relative limit, just may obtain plant from this callus of taking root.Because known cytokinin can induce the young shoot form to take place, and has used the 6-benzylaminopurine, then almost can successfully obtain amorphous young shoot growth more than 30% in 100% explant.The present invention is a reported first can induce the growth of amorphous young shoot on the callus of taking root from leaf explant.
New characteristics of the present invention are that the method difference of the present invention and prior art is as follows:
(i) the percentage height of leaf explant reaction (almost 100%);
(ii) find to be selected from the leaf explant reaction efficiency height of ripe tea tree plant (external and external two kinds indirectly);
(iii) this method for optionally/clone's of elite clonal propagation is very effective;
(iv) this method relates to the callus formation of growing from all surface, and it is not limited to the special area of leaf surface;
(v) this method can produce amorphous young shoot formation more than 30%, and it grows into healthy and strong plant, is easy to after taking root breed or be transferred in the soil;
(vi) with failed to compare from the report that leaf explant obtains plant the high frequency plant regeneration that reported first of the present invention forms about the no-set young shoot by the callus that obtains from leaf explant in the past by no-set young shoot;
(vii) compare with the method that Kato (1996) proposes, this method provides the potential ability of higher-frequency base because of transforming.This is that this method provides possibility for individual cells because compare with induce the low-frequency genetic transformation somatic embryo that causes owing to the specific region, is about to this cell and carries out genetic transformation to have great breeding chance and to produce successful transformant.
By following accompanying drawing the present invention is further illustrated, wherein:
Fig. 1 represents the leaf explant of maximum reaction;
Fig. 2 is illustrated in the breeding of the callus of inducing of callus on the leaf explant and all leaf explants;
Fig. 3 A and Fig. 3 B represent taking root from all callus of leaf explant.The transition point of the no-set growth pathway of expression of taking root to form generation approach;
Fig. 4 represents to form from the no-set young shoot of the callus of taking root;
Fig. 5 represents from the microbody young shoot of no-set young shoot growth and the root that begins to form from the microbody young shoot;
Fig. 6 A-Fig. 6 D represents cell research, wherein healing tissue development (Fig. 6 A) occurs around all vascular bundles of leaf texture.
Though when callus cell is being supplemented with 2, show there is not the blood vessel composition when cultivating in the CIM medium of 4-D, yet when callus is transferred to MS1 or MS2 medium, observe single or disperse the test-tube baby mass-sending to educate, show the beginning (Fig. 6 B) that similar meristematic tissue (meristemoid) is grown.But after 2 weeks of cultivation, these similar meristematic tissue are grown the back and are formed meristematic tissue structures in the MS1/MS2 medium.After final 4 weeks of cultivation, these meristematic tissue structural developments become bunch (Fig. 6 C) of 5-8 root.Be transferred in the MS3 medium after 4 weeks, some similar meristematic tissue develop into young shoot (Fig. 6 D).
In order further to understand the present invention, the following example is provided, but it should be considered as limiting the scope of the invention.
Embodiment 1
When at 25 ± 2 ℃ and 52 μ g/molm
-2s
-1Cold fluorescence exists and under 16 hour photoperiod of illumination reactive explant is placed and is supplemented with 3% sucrose and 2.5-10.0mg/l 2,0.8% agar of 4-D (pH5.6 ± 0.2) solidifies when 6-10 is all in the basic MS culture medium, and any leaf that close up fully, semi-open or complete open leaf plant of the extracorporeal culturing plant of important cultivar (Chinary, Assamica and Cambod) all is reactive explant.The callus that 6-10 was so grown after week is transferred to and is used for taking root or 0.8% agar that is supplemented with 3% sucrose and 0.5-8.0mg/l 6-benzylaminopurine and 0.1-0.8mg/l indole-3-butyric acid of no-set formation solidifies basic MS culture medium.4-10 is after week, and the callus of will taking root is transferred to and is used for the 0.8% agar curing basic MS culture medium that is supplemented with 3% sucrose and 0.5-8.0mg/l 6-benzylaminopurine that no-set young shoot forms.Excise no-set young shoot and make it solidify the microbody young shoot that grows into 1.5-2.0cm length in the basic MS culture medium, be placed on 6 weeks of breeding in the 20ml static liquid MS medium that is supplemented with 3% sucrose and 5 μ M phenyl thiadiazolyl group ureas then at 0.8% agar that is supplemented with 3% sucrose and 0.5-8.0mg/l6-benzylaminopurine and 0.1-0.8mg/l indole-3-butyric acid.Terminal 20-30 minute of the microbody young shoot that each 3.0cm that handle to cut off with indole-3-butyric acid (5.0mg/l) is long, and with its kind at sand: in the mixture of soil (1: 1) 60-75 days, the plantlet that will take root was transferred in the plastic tank at last.
Embodiment 2
Use from Himalayan living resources technology experiment farm research institute, Banuri, any leaf of that close up fully, the semi-open or complete open leaf explant of the 50 age selective vegetables of the important cultivar (Chinary, Assamica and Cambod) of Palampur (36 ° of N and 78.18 ° of E and the above sea level of 1290m) is as explant.Hairbrush and liquid cleaner with black carefully clean this leaf, washing and containing in 0.01% mercuric chloride solution of a drop of liquid cleaning agent and carry out surface sterilizing, thoroughly clean in distilled water then in containing the polysorbas20 of (0.1%) carbendazim and (0.05%) streptomycin.Explant each details in as stated above of sterilization is carried out similar cultivation.
Embodiment 3
When at 25 ± 2 ℃ and 52 μ g/molm
-2s
-1Cold fluorescence exists and under 16 hour photoperiod of illumination reactive explant is placed and is supplemented with 3% sucrose and 2.5-10.0mg/l 2,0.8% agar of 4-D (pH5.6 ± 0.2) solidifies when 6-10 is all in the basic MS culture medium, and other hybridization cultivar for example any leaf that close up fully, semi-open or complete open leaf explant of the extracorporeal culturing plant of Tocklai Variety 1 all is reactive explant.The callus that 6-10 was so grown after week is transferred to and is used for taking root or 0.8% agar that is supplemented with 3% sucrose and 0.5-8.0mg/l6-benzylaminopurine and 0.1-0.8mg/l indole-3-butyric acid of no-set formation solidifies basic MS culture medium.6-10 is after week, and the callus of will taking root is transferred to and is used for the 0.8% agar curing basic MS culture medium that is supplemented with 3% sucrose and 0.5-8.0mg/l 6-benzylaminopurine that no-set young shoot forms.To place 20ml static liquid MS medium 6 weeks of breeding that are supplemented with 3% sucrose and 5 μ M phenyl thiadiazolyl group ureas from the young shoot that no-set young shoot obtains.Terminal 20-30 minute of the microbody young shoot that each 3.0cm that handle to cut off with indole-3-butyric acid (5.0mg/l) is long, and with its kind at sand: in the mixture of soil (1: 1) 60-75 days, the plantlet that will take root was transferred in the plastic tank at last.
Major advantage of the present invention is:
(1) healthy and strong plant can regenerate from the leaf texture of real homology;
(2) the present invention can be used for producing anti-blister droop plant;
(3) this method also can be used for protoplast cultivation and somatic hybridization;
(4) this method can be used for the chloroplaset conversion by direct TRANSFER METHOD;
(5) compare with the Direct Regeneration of the leaf texture that does not have middle callus, by the callus phase Therefore can produce the Fast-propagation of individual cells transformant, develop the high-frequency transgene method and be that have can Can;
(6) with former report in the somatic embryo of specific region induce and compare because callus Grow from all leaf surfaces, therefore the frequency of the transformant by the method is much higher than existing side Method;
(7) the present invention can be used for related gene is introduced the development method of protoplast and is used for studying it Express;
(8) the present invention can be used for promoting the absorption of virion;
(9) the present invention can be used for producing the matrilinear inheritance feature expressed such as cytoplasmic male sterility, anti-Plant such as the herbicide of atrazine;
(10) with in being pre-existing in method use the 50-100ml culture medium to compare, accompanying Ti Shi to cultivate Using the 25ml culture medium in the ware is a kind of cost effective method.
Claims (23)
1. method that is used for the micropropagation of tea tree plant, described method comprises the following steps:
(a) excision is from the explant of that close up fully, the semi-open or complete open leaf of the tea tree plant of culture in vitro;
(b) be 20 ℃-40 ℃ in temperature, at least 52 μ g/molm
-2s
-1Cool white light exist and under 16 hour photoperiod of illumination, in first medium, cultivate the explant 6-10 week that is used for callus induction, described medium is basic Murashige and the Skoog medium that is supplemented with the 0.8% agar curing of vitamin, 1-3mg/l glycine, 2.5-10.0mg/l 2,4 dichlorophenoxyacetic acid;
(c) described callus is transferred to second medium that is used for taking root 6-10 week at least, described medium is to be supplemented with the basic MS culture medium that 0.8% agar of cytokinin such as 0.5-8mg/l 6-benzylaminopurine and auxin such as 0.1-0.8mg/l indole-3-butyric acid or indole-3-acetic acid solidifies;
(d) the described callus of taking root is transferred to the 3rd medium 4-10 week that is used for causing young shoot, described medium is the free medium of auxin and contains 0.5-8mg/l 6-benzylaminopurine;
(e) described young shoot is transferred to cultivation 4-6 week in the 4th propagating culture medium, the young shoot that obtains taking root, described medium are the liquid agar basic MS culture medium that is supplemented with 5 μ M phenyl thiadiazolyl group ureas;
(f) cut off long young shoot and obtain the long end of 3cm, it is carried out 20-30 minute processing, and in the wide-mouth bottle of the sand that contains 1: 1 ratio and the mixture of soil, cultivated young shoot 60-75 days with indole-3-butyric acid; With
(g) young shoot of taking root is grown to obtain vigor and healthy and strong tea tree plant.
2. the method for claim 1, wherein said explant is the tree plant cultivation kind that is selected from such as Chinary, Assamica and Cambod.
3. the method for claim 1, wherein said explant be from the open leaf that close up fully, semi-open or complete of terminal bud utmost point close attachment excise.
4. the method for claim 1, wherein by inducing the merismatic activity in the leaf explant, leaf explant forms by callus and is used for indirect young shoot regeneration.
5. the method for claim 1, wherein the leaf explant of the arbitrary tree plant cultivation kind by the selective vegetable of inducing merismatic activity in the leaf explant, derive from vivo or growing under the conditions in vitro can form by callus and be used for indirect young shoot regeneration.
6. the method for claim 1, wherein said explant are from any part excision of leaf surface.
7. the method for claim 1, wherein said explant are from the second and the 3rd leaf excision at young shoot tip.
8. the method for claim 1, wherein said leaf explant is to clean in the solution that contains 0.1% carbendazim and 0.05% streptomycin, in polysorbas20, wash, and in containing 0.01% (w/v) mercuric chloride solution of a drop of liquid cleaning agent, carry out surface sterilizing, thoroughly washing in sterile distilled water then.
9. the method for claim 1, wherein first medium comprises and being supplemented with such as Cobastab
1-HCl (0.05-2.0mg/l), Cobastab
6The basic Murashige and the Skoog medium of the standard of the 2,4 dichlorophenoxyacetic acid of the vitamin of-HCl (0.25-1.5mg/ml) and nicotinic acid (0.25-1.5mg/l) and glycine (1.0-3.0mg/l) and the preferred 5.0mg/l of 2.5-10mg/l.
10. the medium that the method for claim 1, wherein said explant must place the above-mentioned 2.5-10mg/l of being supplemented with 2,4 dichlorophenoxyacetic acid is 6-10 week at least, but preferred 6 weeks.
11. the method for claim 1, wherein second medium comprises and is supplemented with 0.5-8mg/l 6-benzylaminopurine and 0.1-0.8mg/l indole-3-butyric acid or indole-3-acetic acid, but the basic MS culture medium of preferred 2.0mg/l 6-benzylaminopurine and 0.2mg/l indole-3-butyric acid or indole-3-acetic acid.。
12. the method for claim 1, the easy induction explant that wherein will contain callus is placed 6-10 week at least in second medium, but after preferred 6 weeks.
13. the method for claim 1, wherein the 3rd medium comprises the free basic MS culture medium of auxin of the 6-benzylaminopurine that is supplemented with the preferred 2.0mg/l of 0.5-8.0mg/l.
14. the method for claim 1 is wherein placed the responsive explant of taking root 4-10 week at least in the 3rd medium, but after preferred 4 weeks.
15. the method for claim 1, wherein the auxin with the plant growth regulator that acts on callus formation and take root is selected from indole-3-butyric acid, indole-3-acetic acid, methyl and 2,4 dichlorophenoxyacetic acid.
16. the method for claim 1 is wherein taken root and the cytokinin of the plant growth regulator that young shoot forms is selected from 6-benzylaminopurine, N with acting on
6-furfuryladenine and phenyl thiadiazolyl group urea, but preferred 6-benzylaminopurine.
17. the method for claim 1, wherein 6-10 is after week but need after preferred 6 weeks responsive leaf explant is transferred to and contain the auxin that is used for taking root and the medium of cytokinin.
18. being transferred to, the method for claim 1, the callus that wherein needs to take root contain the above-mentioned free medium of auxin that only is used for the cytokinin that young shoot forms, but preferred 6-benzylaminopurine.
19. the method for claim 1, wherein 4-10 is after week but need after preferred 4 weeks the responsive callus of taking root is transferred to and be used for the free medium of auxin that young shoot forms.
20. the method for claim 1 wherein must contain 25ml medium (this material of cultivation in the 9.0cm Petri dish of pH5.6 especially.
21. the method for claim 1 is wherein at 52 μ g/molm
-2s
-1Cold fluorescence under 16 hour photoperiod of illumination can be used for all cultures.
22. the method for claim 1 was wherein used the end of 3.0cm at least 20-30 minute of the long young shoot that indole-3-butyric acid (5.0mg/l) handle to cut off before being transferred to the mixture of described soil.
23. the method for claim 1, wherein said sand: the mixture of soil uses in 1: 1 ratio.
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| CN 01109920 CN1205857C (en) | 2001-03-23 | 2001-03-23 | Method for micro-propagating tea trees |
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|---|---|---|---|
| CN 01109920 CN1205857C (en) | 2001-03-23 | 2001-03-23 | Method for micro-propagating tea trees |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561086A (en) * | 2014-12-26 | 2015-04-29 | 安徽农业大学 | Method and application for silencing CsPSP1 gene in plant by using RNAi(ribose nucleic acid interfere) |
| CN105494107A (en) * | 2016-02-25 | 2016-04-20 | 湖南农业大学 | Tea tree tissue culture method |
-
2001
- 2001-03-23 CN CN 01109920 patent/CN1205857C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561086A (en) * | 2014-12-26 | 2015-04-29 | 安徽农业大学 | Method and application for silencing CsPSP1 gene in plant by using RNAi(ribose nucleic acid interfere) |
| CN105494107A (en) * | 2016-02-25 | 2016-04-20 | 湖南农业大学 | Tea tree tissue culture method |
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