CN105052738A - Tamarix chinensis tissue culture and rapid propagation method - Google Patents
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Abstract
The invention relates to the technical field of wood tissue culture seedling raising in forest science, in particular to a Tamarix chinensis tissue culture and rapid propagation method. The method includes: acquisition of aseptic seedlings, with an involved medium containing 6-BA, IBA, GA, sucrose and agar; subculture: inoculating the aseptic seedlings into a subculture medium to conduct culture so as to obtain clustered shoots, with the medium containing ZT, IBA, sucrose and agar; rooting culture: inoculating the clustered shoots into a rooting medium containing IBA, NAA, GA, sucrose and agar to obtain rooted tissue cultured seedlings; and acclimatization and transplantation. The propagation method has high propagation coefficient and short propagation cycle, the obtained seedlings are strong and have a lot of root systems, and the survival rate of transplantation is over 96%. The method provided by the invention omits the strong seedling culture step in conventional methods, improves propagation efficiency, makes the Tamarix chinensis tissue culture and rapid propagation technology move towards production from laboratories, realizes large-scale production of Tamarix chinensis tissue culture seedlings, and provides technical support for promotion of Tamarix chinensis rapid propagation.
Description
Technical field
The Tree Organization that the present invention relates in forest-science cultivates seedling growing process field, relates to a kind of woody plant tissure and cultivates method for quickly breeding, be specifically related to a kind of method of Chinese tamarisk tissue-culturing quick-propagation.
Background technology
Chinese tamarisk (
tamarixchinensislour.), also known as Chinese Chinese tamarisk, for suspend (Tamaricaceae) Tamarix (
tamarixl.) typically perennially in secrete the woody halophytes of salt, happiness is born in river flat, beach, beachhead, moist saline land and sandy bare land.Its, leaf, flower there is very high ornamental value, and there are drought resisting, anti-saline and alkaline, the water-fast characteristic (Zhang Shengli etc. such as wet, 2015), strong adaptability, be not only excellent plant of checking winds and fixing drifting sand, simultaneously or water and soil conservation seeds, alkaline land improving and afforestation seeds, No.1 salt tolerant seeds acknowledged in Ye Shi China reproducting tree species, plant can resistance to 2.4% salt water irrigation, at soil saliferous be 25g/kg beach salty soil on can normal growth (Zhang Libin etc.,, but also be good firewood charcoal, establishment and construction timber 2008); Spray and leaf medicinal, diuresis of inducing sweat, wines used as antirheumatic, treatment measles, its social economy and ecological benefits have a high potential, development prospect is very wide.But its current modes of reproduction is mainly based on cuttage, breeding scale is less, reproduction coefficient is lower, and by seasonal restriction, seedling growth is uneven; Although cultivate the relevant report of breeding in a organized way at present, majority is only limitted to scientific research and is not applied to production, have impact on applying of Chinese tamarisk.
A small amount of paper and patent is had to be studied Chinese tamarisk tissue cultures correlation technique link at present, Chinese tamarisk disclosed in it and sibling species Tamarix laxa Willd (
t.laxawilld.) in tissue culture technique, nearly all documents and materials (Cheng Lei etc. 2001; Li Lihong etc. 2005; The 2006ab such as Wei little Min; Wang Peng etc. 2007; Zhang Yongcai etc. 2007; Qiao Mengji etc. 2007; Sun Xuxu etc. 2009) all Chinese tamarisk and sibling species tissue culture propagating technology are divided into the acquisition of aseptic seedling, Primary culture (being combined with aseptic seedling acquisition process of having is called Initial culture), breed that (subculture) is cultivated, five links such as culture of rootage and transplanting.Only indivedual documents and materials (Han Linna etc. 2010, Liu Baiyan etc. 2015) conceptually inquire into the forming seedling through one step culture tissue culture method shortening cultivation cycle, but its method is when without prejudice to plant growth rule, aseptic seedling cannot be avoided and obtain (Initial culture), propagation (subculture), and the link such as culture of rootage, its incubation time shortened is limited, cost-saving limited, if shorten the time in a large number, seedling growth amount, expand numerous amount inevitable less, and then cause its reproduction coefficient low, seedling growth is limited, the seedling propagation efficiency coordinated under each stage condition also cannot reach best, comprehensive benefit cannot maximize.Dispense propagation (subculture) and cultivate link, significantly can reduce reproduction coefficient.Current existing patent (Liu Baiyan etc. 2015) lacks acclimatization and transplants link, cannot Instructing manufacture well.
From calendar year 2001 so far, in all disclosed Chinese tamarisk tissue cultures documents and materials, except indivedual research example (Sun Xuxu etc. 2009) uses 1/2MS, basal medium is MS medium, hormone that each cultivation stage uses is limited to a-methyl α-naphthyl acetate (NAA), 6-benzyl aminoadenine (6-BA), kinetin (Kt), uses gibberellin (GA), heteroauxin (IAA) in indivedual research (Sun Xuxu etc. 2009).Wherein Primary culture (Initial culture) uses NAA, 6-BA etc. more, and propagation (subculture) is cultivated and used 6-BA, Kt etc. more, and in culture of rootage, NAA, 6-BA, Kt tri-kinds of hormones all have use.Somatotropin has multiple, and function is clear, and has a lot of practice reports.But in the more than ten years of Chinese tamarisk tissue culture technique research, only be confined in a small amount of hormone range, there are no the use of other hormones, define the repetitive research under fixed mode, there is no large innovation, and then may be the main cause causing cannot breaking through Chinese tamarisk tissue culture quick breeding bottleneck, production cannot being applied to.Other conventional Hormones, as indolebutyric acid (IBA) can promote Cell Differentiation and division, are conducive to the differentiation of the generation of new root and fibrovascular system, promote the formation of adventive root; Zeatin (ZT) can not only promote that lateral bud grows, irritation cell differentiation, and promote that callus germinates, excessive root can also be suppressed to be formed, and the zeatin of high concentration can also produce differentiation adventitious buds.
Report in documents and materials, use mercuric chloride sterilization more, not only easily cause aseptic seedling brownization, and easily cause health hazard, to environment to staff.Therefore, a kind of clean type disinfectant that uses of very necessary research and development is avoided causing Chinese tamarisk browning, innovative organization to cultivate the Chinese tamarisk tissue culture rapid propagation technique of formula, and promotion Chinese tamarisk factorial praluction, large-scale promotion are quoted.
Summary of the invention
In order to solve Chinese tamarisk tissue culture quick breeding bottleneck in above prior art, the problem of production cannot be applied to, the invention provides a kind of method of Chinese tamarisk tissue-culturing quick-propagation.
The present invention is obtained by following steps:
A method for Chinese tamarisk tissue-culturing quick-propagation, comprises the following steps:
(1) aseptic seedling obtains (Initial culture)
Fresh and tender bud is through process and sterilization, be connected in medium and cultivate, medium consists of in MS medium and adds 6-BA1.0 ~ 2.0mg/L, IBA0.05 ~ 0.15mg/L, GA0.05 ~ 0.15mg/L, sucrose 20 ~ 30g/L, agar 7g/L, pH value adjusts 5.8 ~ 6.0, condition of culture is: at 25 ± 2 DEG C, cultivate 15 ~ 20d under illumination 12 ~ 16h, light intensity 2000 ~ 3000lx condition, obtain aseptic seedling;
(2) squamous subculture
Aseptically the aseptic seedling of acquisition is inoculated on subculture medium and cultivates, culture medium prescription is add ZT0.3 ~ 0.8mg/L, IBA0.05 ~ 0.15mg/L, sucrose 20 ~ 30g/L, agar 7g/L in MS medium, pH value adjusts 5.8 ~ 6.0, at 25 ± 2 DEG C, illumination 12 ~ 16h, cultivate 20d under light intensity 3000lx condition, obtain Multiple Buds;
(3) culture of rootage
Aseptically Multiple Buds is inoculated on root media, culture medium prescription be 1 add IBA0.2 ~ 0.5mg/L, NAA0.05 ~ 0.1mg/L, GA0.05 ~ 0.1mg/L, sucrose 15 ~ 20g/L, agar 7g/L in 2MS, pH value adjusts 5.8 ~ 6.0, at 25 ± 2 DEG C, illumination 12 ~ 16h, cultivate 30 ~ 50d under light intensity 3000lx condition, obtain plantlet in vitro of taking root;
(4) acclimatization and transplants
The intact Chinese tamarisk that taken root by bottle is transplanted in shed, uses sunshade net sunshade, loosens bottle cap but do not open bottle cap to keep 2d after 2d, does not occur that bottle cap is just opened the seam of 1/5th by wilting, observes leaf growth situation afterwards; When bottle cap is opened completely and leaf growth is vigorous, Chinese tamarisk is shifted out in bottle, wash away root medium, be placed in carbendazim 50% wetting powder 5g/L solution and soak about 45s, being transplanted to waters every day in the cave dish installing matrix makes humidity reach 90%, buffering 7d, reform of nature temperature and humidity, 15d, is transplanted to land for growing field crops after new root grows.
Described method, in preferred steps (1), culture medium prescription is add 6-BA1.0mg/L, IBA0.1mg/L, GA0.1mg/L, sucrose 30g/L, agar 7g/L in MS medium, condition of culture is: at 25 ± 2 DEG C, cultivates 18d under illumination 14h, light intensity 3000lx condition.
Described method, in preferred steps (2), culture medium prescription is add ZT0.5mg/L, IBA0.1mg/L, sucrose 30g/L, agar 7g/L in MS medium, at 25 ± 2 DEG C, cultivates 20d under illumination 14h, light intensity 3000lx condition.
Described method, in preferred steps (3) culture medium prescription be 1 add IBA0.5mg/L, NAA0.1mg/L, GA0.1mg/L, sucrose 15g/L, agar 7g/L in 2MS, at 25 ± 2 DEG C, under illumination 14h, light intensity 3000lx condition, cultivate 45d.
Described method, in preferred steps (1), fresh and tender bud is as follows through the step of process and sterilization:
Fresh and tender bud is placed on inside distilled water and soaks 30min, a small amount of washing powder and two tweens are added during immersion, with tap water 1 ~ 2h after 30min, explant is put on inoculation platform and inoculates, with 75% alcohol wipe inoculation platform before inoculation, open ultraviolet 30min, by Chinese tamarisk explant aseptic water washing 3 ~ 5 times in inoculation platform, use 75% alcohol-pickled 30S afterwards, then use aseptic water washing 3 times, use 2% clorox sterilization 3 ~ 9min again, blot explant surface moisture with aseptic filter paper.
Described method, preferred steps (4) mesostroma is the peat composed of rotten mosses: vermiculite volume ratio 3:1.
Beneficial effect of the present invention:
1) utilize environment-friendly hypochlorite disinfectant, overcome explant and to sterilize brownization problem;
2) and develop innovative quick squamous subculture and culture of rootage culture medium prescription; formulate simple acclimatization and transplants scheme; eliminate the strong seedling culture step in conventional method; improve reproductive efficiency; Chinese tamarisk tissue culture rapid propagation technique is made to move towards from laboratory to produce; achieve the large-scale production of Chinese tamarisk tissue cultures nursery stock, for fast numerous popularization of Chinese tamarisk provides technical support.
3) this propagation method, reproduction coefficient is high, and the breeding cycle is shorter, and the nursery stock of acquisition is strong, root system is more, and transplanting survival rate reaches more than 96%.
Accompanying drawing explanation
Fig. 1 is Bud polarization,
Fig. 2 is Shoot propagation,
Fig. 3-1 sees for side takes root situation, and Fig. 3-2 to be taken root situation for sight at the bottom of bottle,
Fig. 4 for closing a bottle hardening,
Fig. 5-1 is for transplanting, and Fig. 5-2 is transplant survival,
Fig. 6 is scale quick propagation.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described:
embodiment 1the selection of Initial culture base
[1] draw materials: Chinese tamarisk explant has drawn from the harsh longer fresh and tender twig of large Tanaka, after drawing materials, selects fresh and tender bud as test material,
[2] test material pre-treatment: be placed on inside distilled water and soak 0.5h, adds a small amount of washing powder and two tweens, uses tap water 2h subsequently during immersion, after flushing, explant is put on inoculation platform and inoculates,
[3] prepare before inoculation: before inoculation, with 75% alcohol wipe inoculation platform, open ultraviolet 0.5h.By Chinese tamarisk explant aseptic water washing 3 times, use 75% alcohol-pickled 30S afterwards, then use aseptic water washing 3 times,
[4] clorox sterilization: with 2% clorox sterilization processing 3min, 5min, 7min, 9min, be connected on the various medium based on MS medium subsequently, find, in above-mentioned various medium, in sterilization 9min situation, the variable color a little of Chinese tamarisk explant, all the other are all normal, explant are put into group training room and cultivate, observe growing way and long bacterium situation every day, more than 7min fungistatic effect is all better, is treated to best with 7min
[5] Screening of Media: with clorox sterilization 7min, is connected to the 10 kinds of medium (A-J) upper (see table 1) based on MS medium, each medium pH 5.8 subsequently by the explant handled well.Explant is put into group training room to cultivate, every day observes growing way.Table 1 specific as follows.Chinese tamarisk explant all starts in most of medium, but its start-up time, axillary bud growth speed, growth potential there are differences, and certain variation appears in department's axillalry bud.Best with Chinese tamarisk explant growing way in (D) MS+6-BA (1.0mg/L)+IBA (0.1mg/L)+GA (0.1mg/L)+sucrose (30g/L)+agar (7g/L) medium.
The growth perfonnance of table 1 Chinese tamarisk explant on Initial culture base
Conclusion: be just commissioned to train in educating, medium is MS+6-BA (1.0mg/L)+IBA (0.1mg/L)+GA (0.1mg/L)+sucrose (30g/L)+agar (7g/L), alcohol disinfecting 30min, hypochlorite disinfectant 7min, the growth of explant is best.Chinese tamarisk has 4700 bottles in present laboratory, about 19000 seedlings.The first of Chinese tamarisk to be filled a prescription full maturity for subculture, and just formula is MS+6-BA (1.0mg/L)+IBA (0.1mg/L)+GA (0.1mg/L)+sucrose (30g/L)+agar (7g/L) generation.
Embodiment 2: the selection of subculture medium
After obtaining aseptic explant, use instead on subculture medium (see table 2) and carry out subculture cultivation, 20d turns seedling once, and the situations such as Chinese tamarisk Bud polarization, branch internode length, growing way all there are differences, in table 2.
The performance of table 2 Chinese tamarisk aseptic seedling on subculture medium
Conclusion: in subculture, E medium is the Growth and Differentiation of MS+ zeatin ZT (0.5mg/L)+IBA (0.1mg/L)+sucrose (30g/L)+more applicable Chinese tamarisk of agar (7g/L).Subculture medium is MS+ zeatin ZT (0.5mg/L)+IBA (0.1mg/L) ++ sucrose (30g/L)+agar (7g/L).
Embodiment 3: the selection of root media
Chinese tamarisk tissue cultures most critical be exactly touch formula of taking root, in culture of rootage, have employed the formula of taking root based on MS, 1/2MS medium, there is very big-difference, in table 3 in the situations of taking root such as its main root, side root.With P medium 1 2MS+IBA (0.5mg/L)+NAA (0.1mg/L)+GA (0.1mg/L)+sucrose (15g/L)+agar (7g/L), pH=5.8 rooting efficiency is best.
Table 3 Chinese tamarisk culture of rootage situation
To sum up can draw the formula that Chinese tamarisk tissue cultures is complete:
First generation: MS+6-BA (1.0mg/L)+IBA (0.1mg/L)+GA (0.1mg/L)+sucrose (30g/L)+agar (7g/L), pH=5.8
Subculture: MS+ (0.5mg/L)+IBA (0.1mg/L) ++ sucrose (30g/L)+agar (7g/L), pH=5.8
Take root: 1 2MS+ indolebutyric acid IBA (0.5mg/L)+NAA (0.1mg/L)+GA (0.1mg/L)+sucrose (15g/L)+agar (7g/L), pH=5.8
Embodiment 4: acclimatization and transplants
(1) be transplanted in homemade Small plastic shed 3.21 100 bottles are taken root intact Chinese tamarisks, use sunshade net sunshade,
(2) loosen bottle cap gently two days later but do not open bottle cap and keep about two days, if two days later blade do not occur wilting just bottle cap is opened 1/5th crack, observation leaf growth situation afterwards,
(3) when bottle cap is opened completely and leaf growth is vigorous (this process approximately needs about one week), Chinese tamarisk is shifted out in bottle, wash away root medium, be placed in carbendazim 50% wetting powder 5g/L solution and soak about 45s, be transplanted in the cave dish installing matrix, move on in the dish of cave and will note keeping humidity, water every day and make humidity reach 90%, note, if to water on blade and root is excessively not wet.Chinese tamarisk plantlet in vitro transplants growing state in table 4,
(4) cushion about 7d, allow Chinese tamarisk seedling reform of nature temperature and humidity, about 15d, treats that new root grows and just can move on in the soil of land for growing field crops, and carry out seedling management, even if whole group is trained process and complete.
Table 4 Chinese tamarisk plantlet in vitro transplants growing state
Embodiment 5:
(1) aseptic seedling obtains (Initial culture)
Select fresh and tender bud as experiment material, be placed on inside distilled water and soak 30min, a small amount of washing powder and two tweens are added during immersion, tap water 2h is used after 30min, explant is put on inoculation platform and inoculates, with 75% alcohol wipe inoculation platform before inoculation, open ultraviolet 30min, by Chinese tamarisk explant aseptic water washing 3 times in inoculation platform, use 75% alcohol-pickled 30S afterwards, then aseptic water washing is used 3 times, use 2% clorox sterilization 7min again, explant surface moisture is blotted with aseptic filter paper, finally be connected to MS+6-BA (1.0mg/L)+IBA (0.1mg/L)+GA (0.1mg/L)+sucrose (30g/L)+agar (7g/L) medium to cultivate, pH value adjusts 5.8, condition of culture is: at 25 ± 2 DEG C, illumination 14h, 18d is cultivated under light intensity 3000lx condition, obtain aseptic seedling,
(2) breed (subculture) to cultivate
Aseptically the aseptic seedling of acquisition is inoculated on MS+ZT (0.5mg/L)+IBA (0.1mg/L)+sucrose (30g/L)+agar (7g/L) fast breeding culture medium, pH value adjusts 5.8, at 25 ± 2 DEG C, illumination 14h, cultivate 20d under light intensity 3000lx condition, obtain a large amount of Multiple Buds;
(3) culture of rootage
Aseptically Multiple Buds segmentation is inoculated in 1 on 2MS+IBA (0.5mg/L)+NAA (0.1mg/L)+GA (0.1mg/L)+sucrose (15g/L)+agar (7g/L) root media, pH value adjusts 5.8, at 25 ± 2 DEG C, illumination 14h, cultivate 45d under light intensity 3000lx condition, obtain plantlet in vitro of taking root;
(4) acclimatization and transplants
The intact Chinese tamarisk that taken root by bottle is transplanted in shed, uses sunshade net sunshade, loosens bottle cap gently but do not open bottle cap to keep about 2d after 2d, if now do not occur, bottle cap is just opened the crack of 1/5th by wilting, observes leaf growth situation afterwards; Until bottle cap open completely cut leaf growth vigorous time (about 7d), Chinese tamarisk is shifted out in bottle, wash away root medium, be placed in carbendazim 50% wetting powder 5g/L solution and soak about 45s, be transplanted in the cave dish installing matrix (matrix is the peat composed of rotten mosses: vermiculite volume ratio 3:1), move on in the dish of cave and will note keeping humidity, water every day and make humidity reach 90%, buffering 7d, progressively reform of nature temperature and humidity, 15d, is transplanted to land for growing field crops after new root grows.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not by the restriction of embodiment; other is any do not deviate from Spirit Essence of the present invention and principle under make change, modification, combination, substitute, simplify and all should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (6)
1. a method for Chinese tamarisk tissue-culturing quick-propagation, is characterized in that comprising the following steps:
(1) aseptic seedling obtains
Fresh and tender bud is through process and sterilization, be connected in medium and cultivate, culture medium prescription is add 6-BA1.0 ~ 2.0mg/L, IBA0.05 ~ 0.15mg/L, GA0.05 ~ 0.15mg/L, sucrose 20 ~ 30g/L, agar 7g/L in MS medium, pH value adjusts 5.8 ~ 6.0, condition of culture is: at 25 ± 2 DEG C, cultivate 15 ~ 20d under illumination 12 ~ 16h, light intensity 2000 ~ 3000lx condition, obtain aseptic seedling;
(2) squamous subculture
Aseptically the aseptic seedling of acquisition is inoculated on subculture medium and cultivates, culture medium prescription is add ZT0.3 ~ 0.8mg/L, IBA0.05 ~ 0.15mg/L, sucrose 20 ~ 30g/L, agar 7g/L in MS medium, pH value adjusts 5.8 ~ 6.0, at 25 ± 2 DEG C, illumination 12 ~ 16h, cultivate 20d under light intensity 3000lx condition, obtain Multiple Buds;
(3) culture of rootage
Aseptically Multiple Buds is inoculated on root media, culture medium prescription be 1 add IBA0.2 ~ 0.5mg/L, NAA0.05 ~ 0.1mg/L, GA0.05 ~ 0.1mg/L, sucrose 15 ~ 20g/L, agar 7g/L in 2MS, pH value adjusts 5.8 ~ 6.0, at 25 ± 2 DEG C, illumination 12 ~ 16h, cultivate 30 ~ 50d under light intensity 3000lx condition, obtain plantlet in vitro of taking root;
(4) acclimatization and transplants
The intact Chinese tamarisk that taken root by bottle is transplanted in shed, uses sunshade net sunshade, loosens bottle cap but do not open bottle cap to keep 2d after 2d, does not occur that bottle cap is just opened the seam of 1/5th by wilting, observes leaf growth situation afterwards; When bottle cap is opened completely and leaf growth is vigorous, Chinese tamarisk is shifted out in bottle, wash away root medium, be placed in carbendazim 50% wetting powder 5g/L solution and soak about 45s, being transplanted to waters every day in the cave dish installing matrix makes humidity reach 90%, buffering 7d, reform of nature temperature and humidity, 15d, is transplanted to land for growing field crops after new root grows, and obtains Chinese tamarisk nursery stock.
2. method according to claim 1, it is characterized in that in step (1), culture medium prescription is add 6-BA1.0mg/L, IBA0.1mg/L, GA0.1mg/L, sucrose 30g/L, agar 7g/L in MS medium, condition of culture is: at 25 ± 2 DEG C, 18d is cultivated under illumination 14h, light intensity 3000lx condition.
3. method according to claim 1, is characterized in that in step (2), culture medium prescription is add ZT0.5mg/L, IBA0.1mg/L, sucrose 30g/L, agar 7g/L in MS medium, at 25 ± 2 DEG C, cultivates 20d under illumination 14h, light intensity 3000lx condition.
4. method according to claim 1, to it is characterized in that in step (3) culture medium prescription be 1 add IBA0.5mg/L, NAA0.1mg/L, GA0.1mg/L, sucrose 15g/L, agar 7g/L in 2MS, at 25 ± 2 DEG C, under illumination 14h, light intensity 3000lx condition, cultivate 45d.
5. method according to claim 1, is characterized in that in step (1), fresh and tender bud is as follows through the step of process and sterilization:
Fresh and tender bud is placed on inside distilled water and soaks 30min, washing powder and two tweens are added during immersion, with tap water 1 ~ 2h after 30min, explant is put on inoculation platform and inoculates, with 75% alcohol wipe inoculation platform before inoculation, open ultraviolet 30min, by Chinese tamarisk explant aseptic water washing 3 ~ 5 times in inoculation platform, use 75% alcohol-pickled 30S afterwards, then use aseptic water washing 3 times, use 2% clorox sterilization 3 ~ 9min again, blot explant surface moisture with aseptic filter paper.
6. method according to claim 1 and 2, is characterized in that step (4) mesostroma is the peat composed of rotten mosses: vermiculite volume ratio 3:1.
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