CN103704136B - Tissue culture method for rapidly propagating wild lilies - Google Patents

Tissue culture method for rapidly propagating wild lilies Download PDF

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CN103704136B
CN103704136B CN201310722214.5A CN201310722214A CN103704136B CN 103704136 B CN103704136 B CN 103704136B CN 201310722214 A CN201310722214 A CN 201310722214A CN 103704136 B CN103704136 B CN 103704136B
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lily
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lilies
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明军
袁素霞
刘春�
徐雷锋
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Abstract

The invention relates to a tissue culture method for rapidly propagating wild lilies and belongs to the field of a flower culture technology. According to the method, bud-stage flower stems of the lilies are used as explants of tissue culture of the lilies and are completely disinfected before culture; besides that 6-BA (Benayl Aminopurine), NAA (Naphthalene Acetic Acid) and IBA (Indole Butyric Acid) are used as auxins or hormones in a culture medium, an MES (Methyl Ester Sulfonate) is added to be used as a buffering agent so as to effectively control the pH value in a tissue culture process, and the pH value is maintained in the optimal range of differentiation, propagation and growth of the lily explants; therefore, the induction efficiency and the propagation coefficient are improved, the induction rate is 98%-100%, the quantity of adventitious buds induced by each explant is not less than 15, the propagation coefficient is 5-7, the rooting rate is 100%, and the root system is healthy and strong. The tissue culture method is applicable to safe propagation of good single plants of filial generations and is an effective manner for protecting wild resources of lilies.

Description

The method for tissue culture of Lilium Germplasm Fast-propagation
Technical field
The present invention relates to the method for tissue culture being applicable to Lilium Germplasm Resources protectiveness and superior hybrid crosses offspring Fast-propagation, belong to flowers culture technique field.
Background technology
Lily is the general name of Liliaceae (Liliaceae) lilium (Lilium) plant, and its flower is very large, beautiful in colour, have very high to view and admire, edible and medical value.Whole world lily wild resource about has 100 kinds and mutation, and wherein China originates in about 55 kinds and mutation, kind nearly ten thousand.Collection and the protection of lily wild resource are the important foundations of Lilies breeding work; due to artificial and naturally impact; the primary border of natural resources goes to pot seriously on the one hand, and numerous species faces the danger of extinction, and the protection of propagation of Lilium Germplasm germ plasm resource is extremely urgent.On the other hand, modern society is in great demand to lily new varieties, needs constantly to release new varieties fast.And traditional lily resource is collected, breeding, all rely on to excavate Lilium Germplasm kind ball Planting in the different location or use kind of a ball to carry out tissue cultures, due to climatic environment condition difference, a large amount of wild species ball can not be survived in transplanting garden preserve, and extensive use based on kind of the tissue cultures of ball scale also usually because its each parasymbiosis carried or non-symbiotic effects, bacterium and occur that a large amount of just generation pollutes, cause material scrap.Thus, cause repetition field acquisition, very easily catastrophic collapse is caused to some important scarce resources.Kindred circumstances, also occurs in crossbreeding superior progeny choice breeding link.
The existing much research of being collected by the method for tissue cultures lily wild resource and protecting is all mainly for explant with kind of ball scale.Xiong Dan etc. (Xiong Dan, Chen Faju, beam is grand, etc. the Study on tissue culture Fujian Forestry science and technology of Yichang lily, 2007,34 (4): 91-94) be that explant has carried out Study on tissue culture to Yichang lily with scale.(Qiu Ninghong, Luo Linhui, the Wang Qin such as Qiu Ninghong, Deng. the tissue culture and rapid proliferation Chinese Wild plant resources of Lilium sulphureum Baker, 2004,23 (2): 64-65) choosing scale is explant, has filtered out the conditions of tissue culture of applicable Lilium sulphureum Baker.Zhao Qiang etc. (Zhao Qiang, Li Qingdian, Zhao Yuling. the tissue culture technique of Tsingtau lily studies northern gardening, 2007,6:213-214) with the scale of Tsingtau lily for explant, have studied the tissue culturing system of Tsingtau lily.(the Bakhshaie M such as Bakhshaie, Babalar M, Mirmasoumi M, et al. Somatic embryogenesis and plant regeneration of Lilium ledebourii (Baker) Boiss., an endangered species. Plant Cell, Tissue and Organ Culture (PCTOC), 2010, 102 (2): 229-235.) utilize scale for explant, by inducing embryonic type callus approach, establish the regenerating system of Li Daibai stamen lily (Lilium ledebourii (Baker) Boiss.).Chen Li waits quietly (Chen Lijing, Ge Ling, Zhang Lichao. fresh lily bulb bulb induce and Regeneration System China agronomy circular, 2011,27 (19): 121-124) with the bulb towards fresh lily bulb for explant, establish the Regeneration System towards fresh lily bulb.Ma Suxian etc. (Ma Suxian, Tian Zhiqiang, high elegant duckweed. Shanxi wild Lilium lancifo1ium Thunb bulb tissue cultures Shanxi Agricultural science, 2012,40 (4): 319-321) with Lilium lancifo1ium Thunb bulb-scale for explant, inquire into affect its break up principal element.(the Burun B such as Burun, Sahin O.Micropropagation of Lilium candidum L.:a rare and native bulbous flower of Turkey. Bangladesh Journal of Botany, 2013,42 (1): 185-187.) with the scale of madonna lily (Lilium candidum L.) for explant, establish madonna lily tissue expand traditional font system.
The bulb of lily, under in vitro condition, under the induction of the suitable hormone of suitable concn, can be induced and produces indefinite bud or produce callus.The callus induced can differentiate indefinite bud through differentiation cultivation, after indefinite bud entered Multiplying culture, then carries out root induction, can obtain whole plant, thus reach the object organizing Fast-propagation.
Lily resource investigation, collection work carry out at the florescence usually because the florescence lily be easily found in numerous and diverse vegetation clump.And the fruit phase is difficult to find lily plant, the difficulty of seed of generally gathering in the wild is very large.So, normally excavate kind of a ball (bulb) at the florescence and carry out transplanting or tissue cultures.But florescence lily transplanting success is extremely low.
Usually, in the tissue culture procedures to lily wild resource, the scale of lily is all selected to do explant.Lily ball will be dug out like this and take away, destroy wild resource.In addition, lily bulb growth is in soil, and the microbe species in soil enriches, and with many types fungi or bacterium on scale, when carrying out Initial culture, pollution rate is high.
If using acrial part tissue if floral organ is as explant, that obviously can reduce collects resource material to the destruction of resource when florescence resource investigation to excavate kind of ball, meanwhile, also may reduce the amount that explant carries disease germs, thus reduce the pollution loss of Initial culture.Equally also be suitable for the breeding of superior hybrid crosses Progeny plants.
What reported is that the test of explant all concentrates on modern cultivar (as: ' Suo Bang ', ' Siberia ') and Moschus (cone drums) lily (lilium longiflorum) with floral organ, and differentiation rate is all below 90%, only there are (the clock Haifeng county such as clock Haifeng county, Huang Yuxiang, Zhong Huaiqin, Ye Xiuxian, Huang Minling. new PE curriculum floral organ Study on tissue culture. Fujian Journal of Agricultural Sciench, 2010,25 (2): 207 ~ 21, .) differentiation rate reach 90.8%, but the rate of increase of indefinite bud is the highest only has 4.8%, and growth potential is more weak.If be used in Lilium Germplasm Resources cultivation also to relate to just for pollution problem, but be showed no report.Liu Yali, open swordsman, Pan Xuejun (Liu Yali, open swordsman, Pan Xuejun. oriental hybrid lily ' floral organ culture of Suo Bang ' and Fast-propagation. northwest Botany Gazette, 2004,24(12): 2350 ~ 2354.) carry out cultured in vitro with the bennet of east lilium Sorbonne, holder, petal and filigree for explant and Fast-propagation is studied.Result shows: 4 kinds of floral organs all can directly induce generation indefinite bud, and wherein bennet inductivity on N6 minimal medium is 75.8%, MS is 72.2%.(the Lai Zhongxiong such as Lai Zhongxiong, Zheng Xianghong, Zhang Chun town, Chen Faxing, Lin Qingliang, Wu Jinshou. Plant regeneration from pedicel culture in vitro in Lilium longiflorum Thunb. University Of Agriculture and Forestry In Fujian's journal (natural science edition) .2004,33(2): 186 ~ 188.) with the bennet of Lilium longiflorum for explant has carried out the research of cultured in vitro regeneration plant. result shows at additional 2mg.L -1bA+0.1mg.L -1the inductivity of the MS medium pedicel culture generation clove of NAA can up to 86.67%.At additional 0.3mgL -1subculture on the MS medium of BA, growth coefficient is up to 5.1 at additional 0.5mg.L -1the 1/2MS medium of NAA is cultivated and takes root, rooting rate more than 90%.Lin Si specially waits (Lin Sizhuan, Zheng Xianghong, Chen Faxing. Lilium longiflorum lilium. Fujian fruit tree, 2004.128(1): 8 ~ 10.) think that bennet is the suitableeest organ of Lilium longiflorum on the ground all evoked callus, the optimal medium formula of its differentiation adventitious buds is MS+BA 2mg.L -1+ NAA 0.1mg.L -1; Be that the differentiation rate of the indefinite bud of explant reaches 86.67% with bennet, proliferated culture medium is with MS+BA 0.3mg.L -1be advisable, reproduction coefficient reaches 5.1.Bud seedling growth potential is strong.Wang Hui and Jia Guixia (Wang Hui, Jia Guixia. the Study on tissue culture of the multiple explant of ' Siberia ' lily. Chinese ornamental horticulture progress 2007.185 ~ 190.) using the bennet in ' Siberia ', filigree, ovary as explant, ovary differentiation capability is the strongest, wherein bennet best result rate 50%, ovary 58.3%.The optimal medium of inducing it to break up is MS+6-BA 1mg.L -1+ NAA 0.lmg.L -1+ 2,4-D 0.lmg.L -1.(the Xu Jieting such as Xu Jieting, Wang Yue, Tang Kexuan, Tang Dongqin. Lilium longiflorum flower portion organizes cultured in vitro and plant regeneration. Shanghai Communications University's journal (agricultural science version), 2008,26(1): 13 ~ 16.) be organized as explant with the tender colored portion of Lilium longiflorum children and carry out cultured in vitro.Have studied the induction of its callus and the impact of plant regeneration when different parts explant and plant growth regulating substance.Result shows.The ability of different explants evoked callus is different.By being followed successively by filigree > holder > bennet > style > petal to weak by force.In During Callus Induction.Filigree, holder and bennet are at medium MS+NAA 1.0mg.L -1+ BA 0.5mg.L -1on inductivity all up to 80%.Alternately at MS+NAA 1.0mg.L -1+ BA 0.5mg.L -1with MS+NAA 0.5mg.L -1+ BA 0.25mg.L -1two kinds of propagation of cultivating upper cultivation and being conducive to callus most.The differentiation of indefinite bud is with MS+KT 1.0mg.L -1+ NAA 0.5mg.L -1best.Root media is with MS+NAA 0.2mg.L -1effect as well.Wherein bennet inductivity 84.17%.(the clock Haifeng county such as clock Haifeng county, Huang Yuxiang, Zhong Huaiqin, Ye Xiuxian, Huang Minling. new PE curriculum floral organ Study on tissue culture. Fujian Journal of Agricultural Sciench, 2010,25 (2): 207 ~ 21, .) with the bennet of new PE curriculum, holder, petal, filigree and style (removal column cap) for explant, carry out cultured in vitro and Fast-propagation research.Result shows that 5 kinds of floral organs all can directly induce generation indefinite bud, and its inducibility size is holder > bennet > filigree > petal, style.Wherein bennet inductivity is 90.8%.The appropriate media of evoking adventive bud is: MS+2.0mg.L -16-BA+0.2mg.L -1nAA.But the rate of increase of indefinite bud is the highest only has 4.8%, and growth potential is more weak.
Summary of the invention
Technical problem to be solved by this invention provides lily infarcted tissue cultural method for above-mentioned prior art present situation; to reach when not destroying lily wild resource; ensure safety, reliably obtain Lilium Germplasm germ plasm resource plantlet in vitro, thus realize carrying out available protecting to Lilium Germplasm Resources.
The method for tissue culture of Lilium Germplasm Fast-propagation, comprises the steps:
(1) get flower bud phase children tender pedicel, sterilization removes miscellaneous bacteria,
(2) by young tender pedicel rip cutting into two, then cut into chunks, vertical section be positioned over and inducing culture carries out Fiber differentiation go out indefinite bud,
(3) indefinite bud induced received on proliferated culture medium carry out Multiplying culture, obtain Multiple Buds,
(4) Multiple Buds is received on root media and carry out culture of rootage, obtain the aseptic plantlet in vitro of root system stalwartness.
Described inducing culture is: MS+6-BA 0.5mg.L -1+ NAA 1.5mg.L -1+ sucrose 30.0 g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0; Proliferated culture medium is MS+IBA 0.1mg.L -1+ sucrose 60.0 g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0; Root media is: 1/2MS+IBA 0.1mg.L -1+ NAA 0.1mg.L -1+ MES 2g.L- 1+ sucrose 60.0g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0.
Described inducing culturing condition is condition of culture is temperature (25 ± 2) DEG C, shading, and Multiplying culture condition is condition of culture is temperature (25 ± 2) DEG C, shading, culture of rootage condition is condition of culture is temperature (25 ± 2) DEG C, illumination (intensity of illumination 2000lx, light/dark cycle 16h/8h).
Following sequential steps is taked in described sterilization: be first placed in by young tender pedicel containing 50% carbendazim 500 times of liquid, 50mg.L -1streptomycin and 50mg.L -1in the aqueous solution of penicillin, 150rpm vibrates before 3 ~ 5h carries out and disinfects; Then bennet is placed in 75% alcohol and soaks concussion 20 ~ 30s, finally put into 10% liquor natrii hypochloritis and soak concussion 13min, with aseptic water washing 3 times, rinse 1 ~ 2min at every turn.
The length of described segment is 1cm.
The explant that this method selects the flower bud phase bennet of lily to train as lily group, avoid the destruction to lily wild resource, and bennet is aerial tissues, and carry disease germs few, Initial culture pollution rate during using bennet as explant is lower, and differentiation rate is high.Add MES buffer in the medium, be conducive to the acquisition of high inductivity and growth coefficient, the protection for lily wild resource provides effective means.
Bennet selected by the present invention is flower bud phase young tender material, and comparatively ageing tissues is strong for the tender organization material regeneration capacity of children.
The thorough disinfection of explant is the key ensureing to obtain aseptic seedling.Front conditions for sterilization is placed in by bennet containing 50% carbendazim 500 times of liquid, 50mg.L -1agricultural streptomycin and 50mg.L -1in the aqueous solution of agricultural penicillin, 150rpm vibrates 3 ~ 5h.Carbendazim is a kind of broad spectrum activity fungicide, and its action principle is the formation of spindle in the mitosis of interference pathogen, affects cell division, thus plays the effect of antifungal.Penicillin is that one mainly has powerful antibacterial action to gram-positive bacteria, some Gram-negative bacterias are also had to the beta-lactam antibiotic comparatively pretended, agricultural streptomycin is that one mainly has stronger antibacterial action aminoglycoside antibiotics to some Gram-negative bacterias.Comprehensive sterilization can be carried out for dissimilar bacterium and fungi simultaneously by used in combination for three kinds of dissimilar bactericide thus reduce the pollution rate of explant in Initial culture; After explant, conditions for sterilization soaks concussion 20 ~ 30s in 75% alcohol, 10% liquor natrii hypochloritis, soaks concussion 13min.It is a kind of oxidizing bactericide that hypochlorous acid is received, but also can damage plant cell while kill bacteria and fungi, too short with the time of hypochlorite disinfectant, then do not reach the effect of thorough sterilizing; Sterilization time is long, can damage explant.The time that preferred lily bennet carries out hypochlorite disinfectant is 13min, during clorox concentration 10%.Bennet has gone out after bacterium, clean up residual clorox, in order to avoid damage explant.Preferred mode of washing is aseptic water washing 3 times, washing time 1 ~ 2min.
When carrying out Initial culture using bennet as explant, by bennet rip cutting into two, vertical section contact medium.Be conducive to the generation of indefinite bud like this.Preferred Peduncle Length is about 1cm.
PH value is the key factor affecting explant differential growth, and large quantity research shows that pH is conducive to differentiation and the growth of lily bennet explant 5.8 ~ 6.0 time.Explant is in the process of cultivating, can secrete in many metabolites and medium, thus change the pH of medium, be unfavorable for differentiation and the growth of explant, the name of MES is called 2-(N-morpholine) ethyl sulfonic acid, be a kind of acidic buffer, when medium adds finite concentration MES, the pH value of medium can be made to maintain within limits.Preferred MES concentration is 2g.L-1.
The explant that the inventive method adopts the flower bud phase bennet of lily to train as lily group, simultaneously before cultivation, do to sterilize thoroughly to bennet, in the medium except adopting 6-BA, NAA, IBA is as outside growth hormone or hormone, also add MES as buffer, effectively control the acid-base value in tissue culture procedures, pH value long term maintenance is broken up at lily explant, in the suitableeest scope of propagation and growth, improve induced efficiency and growth coefficient, inductivity is 98 ~ 100%, the indefinite bud number of each explant induction is not less than 15, growth coefficient is 5 ~ 7, rooting rate is 100% and root system is healthy and strong, be applicable to the safety breeding of filial generation fine individual plant, protection for lily wild resource provides effective means.
Accompanying drawing explanation
Situation when Fig. 1 is Lilium sulphureum Baker in embodiment 1 (Lilium sulphureum Baker) bennet explant induction cultivation 20d.
Situation when Fig. 2 is Lilium sulphureum Baker in embodiment 1 (Lilium sulphureum Baker) bennet explant induction cultivation 35d.
Fig. 3 is that Lilium sulphureum Baker in embodiment 1 (Lilium sulphureum Baker) adventitious bud proliferation cultivates 23d situation.
Fig. 4 is Lilium sulphureum Baker in embodiment 1 (Lilium sulphureum Baker) inducing clumping bud culture of rootage 30d situation.
Situation when Fig. 5 is Yichang lily in embodiment 2 (Lilium leucanthum (Baker) Baker) bennet explant induction cultivation 34d.Fig. 6 is that Yichang lily in embodiment 2 (Lilium leucanthum (Baker) Baker) adventitious bud proliferation cultivates 20d situation.
Fig. 7 is Yichang lily in embodiment 2 (Lilium leucanthum (Baker) Baker) inducing clumping bud culture of rootage 32d situation.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described in further detail.
The method for tissue culture of Lilium Germplasm Fast-propagation, comprises the steps:
1) get flower bud phase children tender pedicel to be placed in containing 50% carbendazim 500 times of liquid, 50mg.L -1agricultural streptomycin and 50mg.L -1in the aqueous solution of agricultural penicillin, 150rpm vibrates before 3 ~ 5h carries out and disinfects;
2) bennet of pre-treatment is placed in 75% alcohol and soaks concussion 20 ~ 30s, then put into 10% liquor natrii hypochloritis and soak concussion 13min, with aseptic water washing 3 times, rinse 1 ~ 2min at every turn;
3) cut young tender pedicel, rip cutting into two, is cut into a cm, is positioned over by the vertical section of bennet on inducing culture and carries out Fiber differentiation, and condition of culture is temperature (25 ± 2) DEG C, shading;
4) indefinite bud induced is transferred on proliferated culture medium and carries out Multiplying culture, and obtain Multiple Buds, condition of culture is temperature (25 ± 2) DEG C, shading;
5) Multiple Buds is moved receive on root media and take root, obtain aseptic seedling.Condition of culture is temperature (25 ± 2) DEG C, illumination (intensity of illumination 2000lx, light/dark cycle 16h/8h).
Wherein, the inducing culture of described bennet is: MS+6-BA 0.5mg.L -1+ NAA 1.5mg.L -1+ sucrose 30.0g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0; The proliferated culture medium of indefinite bud is MS+IBA 0.1mg.L -1+ sucrose 60.0g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0; Root media is: 1/2MS+IBA 0.1mg.L -1+ NAA 0.1mg.L -1+ MES 2g.L- 1+ sucrose 60.0g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0.
Embodiment 1
1) the young tender pedicel of the Lilium sulphureum Baker gathered from Yunnan (Lilium sulphureum Baker) is placed in containing 50% carbendazim 500 times of liquid, 50mg.L -1agricultural streptomycin and 50mg.L -1in the aqueous solution of agricultural penicillin, 150rpm vibrates before 3h carries out and disinfects;
2) bennet of pre-treatment is placed in 75% alcohol and soaks concussion 20s, then put into 10% liquor natrii hypochloritis and soak concussion 13min, with aseptic water washing 3 times, rinse 1min at every turn;
3) cut young tender pedicel, rip cutting into two, is cut into a cm, the vertical section of bennet is positioned over inducing culture (MS+6-BA 0.5mg.L -1+ NAA 1.5mg.L -1+ sucrose 30.0g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0) on carry out Fiber differentiation, condition of culture is temperature (25 ± 2) DEG C, shading; As shown in Figure 1 and Figure 2
4) after 5d, induce indefinite bud, the indefinite bud induced is transferred to proliferated culture medium (MS+IBA 0.1mg.L -1+ sucrose 60.0g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0) on carry out Multiplying culture, 34 d obtain Multiple Buds.Condition of culture is temperature (25 ± 2) DEG C, shading; As shown in Figure 3,
5) Multiple Buds is moved receive root media (1/2MS+IBA 0.1mg.L -1+ NAA 0.1mg.L -1+ sucrose 60.0g.L -1+ MES2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0) on carry out culture of rootage, the condition of culture of culture of rootage: temperature (25 ± 2) DEG C, illumination (intensity of illumination 2000lx, light/dark cycle 16h/8h); As shown in Figure 4.
This experimental pollution rate is 0%, and adventitious bud induction frequency is 100%, and value-added coefficient is 5.5, and rooting rate is 100%(table 1).After the bennet Initial culture of about 1cm, average energy obtains 25 indefinite buds.
Table 1 Lilium sulphureum Baker bennet adventitious bud inducing differentiation and proliferation situation
Embodiment 2
1) the young tender pedicel of the Yichang lily gathered from Yichang (Lilium leucanthum (Baker) Baker) is placed in containing 50% carbendazim 500 times of liquid, 50mg.L -1agricultural streptomycin and 50mg.L -1in the aqueous solution of agricultural penicillin, 150rpm vibrates before 4h carries out and disinfects;
2) bennet of pre-treatment is placed in 75% alcohol and soaks concussion 20s, then put into 10% liquor natrii hypochloritis and soak concussion 13min, with aseptic water washing 3 times, rinse 1min at every turn;
3) cut young tender pedicel, rip cutting into two, is cut into a cm, the vertical section of bennet is positioned over inducing culture (MS+6-BA 0.5mg.L -1+ NAA 1.5mg.L -1+ sucrose 30.0g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0) on carry out Fiber differentiation, condition of culture is temperature (25 ± 2) DEG C, shading; As shown in Figure 5,
4) after 5d, induce indefinite bud, the indefinite bud induced is transferred to proliferated culture medium (MS+IBA 0.1mg.L -1+ sucrose 60.0g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0) on carry out Multiplying culture, 34d obtains Multiple Buds.Condition of culture is temperature (25 ± 2) DEG C, shading; As shown in Figure 6,
5) Multiple Buds is moved receive root media (1/2MS+IBA 0.1mg.L -1+ NAA 0.1mg.L -1+ sucrose 60.0g.L -1+ MES2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0) on carry out culture of rootage, the condition of culture of culture of rootage: temperature (25 ± 2) DEG C, illumination (intensity of illumination 2000lx, light/dark cycle 16h/8h); As shown in Figure 7.
This experimental pollution rate is 2.0%, and inductivity is 98.3%, and growth coefficient is 6.0, and rooting rate is 100%(table 2).After the bennet Initial culture of about 1cm, average energy obtains 24 indefinite buds.
Table 2 Yichang lily bennet adventitious bud inducing differentiation and proliferation situation

Claims (2)

1. the method for tissue culture of Lilium Germplasm Fast-propagation, comprises the steps:
(1) get flower bud phase children tender pedicel, sterilization removes miscellaneous bacteria,
(2) by young tender pedicel rip cutting into two, then cut into chunks, vertical section be positioned over and inducing culture carries out Fiber differentiation go out indefinite bud,
(3) indefinite bud induced received on proliferated culture medium carry out Multiplying culture, obtain Multiple Buds,
(4) Multiple Buds is received on root media and carry out culture of rootage, obtain the aseptic plantlet in vitro of root system stalwartness;
Described inducing culture is: MS+6-BA 0.5mg.L -1+ NAA 1.5mg.L -1+ sucrose 30.0g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0; Proliferated culture medium is MS+IBA 0.1mg.L -1+ sucrose 60.0g.L -1+ MES 2g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0; Root media is: 1/2MS+IBA 0.1mg.L -1+ NAA 0.1mg.L -1+ MES 2g.L -1+ sucrose 60.0g.L -1+ agar 6.0g.L -1, pH=5.8 ~ 6.0;
Described inducing culturing condition is condition of culture is temperature (25 ± 2) DEG C, shading, Multiplying culture condition is condition of culture is temperature (25 ± 2) DEG C, shading, culture of rootage condition is condition of culture is temperature (25 ± 2) DEG C, intensity of illumination 2000lx, light/dark cycle 16h/8h;
Following sequential steps is taked in described sterilization: be first placed in by young tender pedicel containing 50% carbendazim 500 times of liquid, 50mg.L -1streptomycin and 50mg.L -1in the aqueous solution of penicillin, 150rpm vibrates before 3 ~ 5h carries out and disinfects; Then bennet is placed in 75% alcohol and soaks concussion 20 ~ 30s, finally put into 10% liquor natrii hypochloritis and soak concussion 13min, with aseptic water washing 3 times, rinse 1 ~ 2min at every turn.
2. method according to claim 1, the length of described segment is 1cm.
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