CN103651143B - A kind of cultural method of white thorn plantlet in vitro - Google Patents
A kind of cultural method of white thorn plantlet in vitro Download PDFInfo
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- CN103651143B CN103651143B CN201310697008.3A CN201310697008A CN103651143B CN 103651143 B CN103651143 B CN 103651143B CN 201310697008 A CN201310697008 A CN 201310697008A CN 103651143 B CN103651143 B CN 103651143B
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Abstract
The invention discloses a kind of cultural method of white thorn plantlet in vitro, seedling mainly after dialogue thorn seed sprouting, suitable method is used to carry out disinfection and on easy medium, carry out stem-segment with single bud growth of directly taking root numerous soon, thus a set of simple and easy, economical, the thorn plantlet in vitro cultural method in vain fast set up.First the method finds medicament and the method for the tender white thorn seedling sterilization of suitable children; Next is bred in directly the take root mode of seedling of stem-segment with single bud, and namely Absorbable organic halogens obtains whole plant at short notice, and green of Plant Leaf is strengthened, healthy growth; Last medium running water replaces distilled water configuration, white sugar replaces sucrose to add as carbon source, avoid the use of too many extra plant growth regulator simultaneously, this obtains and saves greatly and simplify on cost and operation, for the Fast-propagation stung in vain provides a new practical approach, also lay a good foundation for the later stage realizes factorial praluction.
Description
Technical field
The present invention relates to group and cultivate kind of a field, be specifically related to a kind of cultural method of white thorn plantlet in vitro.
Background technology
White thorn another name western sand cherry, is commonly called as ground jujube, well developed root system, have anti-saline and alkaline, drought-resistant, fix the sand the good characteristic of improving the soil, be typical psammophyte, checking winds and fixing drifting sand, stablize desert, protect in Sha Qu oasis and play an important role.The cynomorium songaricum of white thorn root parasitism is the temperature compensation medicinal material that tradition is famous and precious.In recent years, new, potential medicinal and edible plant is found from wild plant resource, become the focus of Chinese scholars research, psammophyte stings in vain, is through long-term Natural Selection and one of winner remained, the vitality of its tanacity of Bai Ciyin and excellent gene and be subject to liking of the husky district people.White bur, containing multiple nutritional components and abundant trace element, has and regulates blood sugar, blood fat, hypotensive, significantly improve the effects such as body immunity, antifatigue, cold resistance, enhancement sleep, have high nutrition and medical value.Be the important natural pasture of the meadow that formed of sociales and constructive species or China's dust storm arid area domestic animal with it.But show according to investigations, white thorn has male sterile phenomenon, and interspecific cross is chaotic, variation is large, and seminal propagation quality deterioration is quite serious, and white thorn seed exists height dormancy problem simultaneously, cultivation degree is low, and this is that artificial commerial growing and fine-variety breeding bring very large difficulty.
Adopt tissue culture rapid propagation technique may be the effective way obtaining high-quality, neat white thorn nursery stock, this stings resource in vain for reasonable development and sustainable utilization positive effect.At present, more scholar He Zhenglun, Sun Xuexin, Guo Xiaohong etc. carried out the group training research of white thorn, and in the regenerating system formed, disinfectant is mercuric chloride.Mercuric chloride is comparatively large to the injury of explant, and the time, slightly improper will causing was sterilized unsuccessfully, or serious to explant lethality, and this causes difficulty to later stage work.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, provides a kind of economy, easy cultural method of suitable white thorn plantlet in vitro.
To achieve these goals, technical scheme provided by the invention is: a kind of cultural method of white thorn plantlet in vitro, comprises the following steps:
1) explant sterilization: by the seedling stinging seed sprouting in vain and obtain, clip tender stem segments, with aseptic water washing 4-5 time after sterilization, the white thorn stem section be sterilized;
2) the white thorn stem section disinfected in step 1) is inoculated on MS medium, is put in culturing room and cultivates, treat that its also jointing of taking root grows tall and obtain white thorn aseptic seedling;
3) stem Duan Kuaifan: by step 2) the white thorn aseptic seedling of growing tall to 4-5cm that obtains cuts stem with bud, and be connected on root media, be put in culturing room and cultivate, carry out root induction; Root induction process is: the white thorn aseptic seedling stem sections with terminal bud be directly connected on MS medium and carry out culture of rootage, and all the other sting aseptic seedling stem sections in vain and are connected on 1/2MS medium+0.5mg/L heteroauxin and carry out culture of rootage; Culturing room's condition of culture is temperature 25 ± 1 DEG C, intensity of illumination 2000Lx, illumination 16h/d.
Further, the cultural method of above-mentioned a kind of white thorn plantlet in vitro, in described step 1), disinfecting process is with 75% alcohol disinfecting 30s, then uses 10% hypochlorite disinfectant 15min-18min.
Further, the cultural method of above-mentioned a kind of white thorn plantlet in vitro, described step 2) and step 3) in, medium MS and 1/2MS, carbon source uses white sugar, and medium is used is conventional running water in configuration.
Further, the cultural method of above-mentioned a kind of white thorn plantlet in vitro, white thorn used is wild white thorn.
Further, the cultural method of above-mentioned a kind of white thorn plantlet in vitro, the configuration of described MS medium and 1/2MS medium consists of:
MS medium: MS mother liquor composition+30g white sugar+0.1g inositol+4.5g agar powder, running water constant volume configures;
1/2MS medium: MS mother liquor composition dosage reduces by half+30g white sugar+0.1 inositol+4.5g agar powder, running water constant volume configures;
The constituent of described MS mother liquor is counted with mass concentration:
Macroelement: NH
4nO
31650mg/L, KNO
31900 mg/L, MgSO
47H
2o 370 mg/L, KH
2pO
4170 mg/L;
Trace element: KI 0.83 mg/L, H
3bO
36.2 mg/L, MnSO
44H
2o 22.3 mg/L, ZnSO
47H
2o 8.6 mg/L, Na
2mnO
42H
2o 0.25 mg/L, CuSO
45H
2o 0.025 mg/L, CoCl
26H
2o 0.025 mg/L;
Vitamin: nicotinic acid 10 mg/L, puridoxine hydrochloride 1 mg/L, thiamine hydrochloride 1 mg/L;
Calcium salt: CaCl
22H
2o 440 mg/L;
Molysite: FeSO
47H
2o 27.8 mg/L, Na
2-EDTA2H
2o 37.3 mg/L.
It is the first step regenerated that explant is cultivated, and the growth conditions of aseptic seedling is directly connected to carrying out of later stage work.Therefore, the impact test that this method has carried out different disinfectant repeatedly, different disinfecting time is sterilized on explant, during to solving 0.1% mercuric chloride sterilization, it is not thorough that disinfecting time short-range missile causes explant sterilization, or disinfecting time is slightly long, explant just brownization death, or the problem such as sterilization rate is too low.This method finally determines disinfectant 10% clorox adopted, Nature comparison is gentle, little to explant injury power, disinfecting time is determined at 15-18min simultaneously, not only explant still can be kept fit green and vigor, sterilization rate also reaches 90%-100%, and this greatly saves explant and to draw materials work, also for later stage test provides sufficient aseptic seedling to do reliable guarantee.
White thorn stem-segment with single bud provided by the invention is directly taken root seedling on medium, clip is as the group training approach of hyperplasia section by section, whole process does not experience the Induction and differentiation stage of callus, and this proliferating way is very favourable to maintenance white thorn plantlet in vitro stabilization characteristics of genetics.The medium running water configuration simultaneously provided and white sugar replace sucrose to add this method as carbon source, not only do not have influence on the growth of normally taking root of plantlet in vitro, and also decrease on cost.
Beneficial effect of the present invention is: the cultural method of a kind of white thorn plantlet in vitro provided by the invention, economical, easy, for the Fast-propagation stung in vain provides a practicable new way.First the method works out the scheme being suitable for the tender white thorn explant sterilization of children, proposes suitable disinfectant and disinfecting time; Next is tested out at short notice i.e. Absorbable organic halogens and obtains the series of measures of whole plant, and whole process is bred in directly the take root mode of seedling of stem-segment with single bud; It is also proposed used medium is replace distilled water configuration with running water simultaneously, white sugar replaces sucrose to add as carbon source, overcome the use of too many plant growth regulator in prior art, obtain the maturation method that is suitable for the easy cultivation of white thorn plantlet in vitro, cost and operation obtain and saves greatly and simplify, dialogue thorn Biology Breeding and factorial praluction have practical application and are worth.
Embodiment
embodiment 1:
(1) for examination material: wild white stings;
(2) explant sterilization: the seedling that seed sprouting is obtained, clip tender stem segments, with 75% alcohol disinfecting 30s, compares the process that stem section carries out different time with two kinds of disinfectants subsequently respectively, 0.1% mercury chloride process 4,6,8,10min; The clorox process 7 of 10%, 10,12,15,18min, aseptic water washing 4-5 time, be connected on MS medium (running water configuration), often process 10 bottles, every bottle of 3 strain explants, are put in temperature 25 ± 1 DEG C, intensity of illumination 2000Lx, cultivate under the condition of illumination 16h/d, within several days, add up sterilization situation afterwards;
(3) stem section induction: good white thorn stem section to be sterilized is taken root on MS medium (running water configuration) and jointing grows tall to 4-5cm, stem-segment with single bud can be cut, two kinds of modes are divided to cultivate: one is connected on differential medium to carry out proliferation-inducing, and differential medium arranges two kinds: MS+0.5mg/LBA+1.0mg/LNAA+2.0mg/LGA and MS+2.0mg/LBA+0.5mg/LIBA; One directly carries out root induction, and often process 10 bottles, every bottle of 3 strain explants, are put in temperature 25 ± 1 DEG C, intensity of illumination 2000Lx, cultivate under the condition of illumination 16h/d, add up respective result;
(4) root induction: the stem-segment with single bud cut, is connected on root media, is put in culturing room and cultivates, carry out root induction.Root induction medium arranges two kinds: MS medium (running water configuration) and 1/2MS+0.5mg/LIBA medium.Often process 10 bottles, every bottle of 3 strain explants, are put in temperature 25 ± 1 DEG C, intensity of illumination 2000Lx, cultivate under the condition of illumination 16h/d, add up situation of taking root;
Result shows, 0.1% mercuric chloride is as disinfectant, and its lethality is very big, and when processing 8min and 10min, whole brownization of explant is dead; When processing 4min, though explant is hurt gently, but mycelia will grow gradually, sterilizes not thorough; When processing 6min, the status and appearance of explant is different, has brownization dead, has sterilization halfway, also have sterilization clean, sterilization rate only 13.33%, and finds that stem section late growing stage slowly or stagnation.10% clorox is as disinfectant, and its Nature comparison is gentle, should all not find the brownization phenomena of mortality by middle explant throughout.When processing 7min, miscellaneous bacteria has a lot, sterilizes not thorough; When processing 10min and 12min, sterilization rate can reach more than 60%, and when processing 15min and 18min, sterilization rate can reach 90% to 100%, and explant is all healthy, and growth of can taking root on appropriate media.
Show from the differentiation of stem section and the result of root induction, the stem-segment with single bud proliferation-inducing approach of employing, on the disclosed wild white thorn differentiation and proliferation medium selected, have no bud point differentiation and proliferation, only expanding slightly, yellowing leaf death subsequently.This may be the problem that white thorn genotype relies on, and this approach can carry out hormone adjustment again and continue research.The direct root induction approach of stem-segment with single bud adopted, in MS medium (running water configuration), only have the stem section of band terminal bud to take root, in 1/2MS+0.5mg/LIBA medium, young tender stem with bud is taken root well, and aging stem with bud is taken root more difficult.1/2MS+0.5mg/LIBA medium is also used instead running water configuration below, difference is not had with comparing before to root induction.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (1)
1. a cultural method for white thorn plantlet in vitro, is characterized in that, comprise the following steps:
1) explant sterilization: by the seedling stinging seed sprouting in vain and obtain, clip tender stem segments, with aseptic water washing 4-5 time after sterilization, the white thorn stem section be sterilized; Disinfecting process is with 75% alcohol disinfecting 30s, then uses 10% hypochlorite disinfectant 15min-18min; White thorn used is wild white thorn;
2) the white thorn stem section disinfected in step 1) is inoculated on MS medium, is put in culturing room and cultivates, treat that its also jointing of taking root grows tall and obtain white thorn aseptic seedling; Medium MS, carbon source uses white sugar, and medium is used is conventional running water in configuration;
3) stem Duan Kuaifan: by step 2) the white thorn aseptic seedling of growing tall to 4-5cm that obtains cuts stem with bud, and be connected on root media, be put in culturing room and cultivate, carry out root induction; Root induction process is: the white thorn aseptic seedling stem sections with terminal bud be directly connected on MS medium and carry out culture of rootage, and all the other sting aseptic seedling stem sections in vain and are connected on 1/2MS medium+0.5mg/L heteroauxin and carry out culture of rootage; Culturing room's condition of culture is temperature 25 ± 1 DEG C, intensity of illumination 2000Lx, illumination 16h/d; Medium MS and 1/2MS, carbon source uses white sugar, and medium is used is conventional running water in configuration.
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| CN103238517A (en) * | 2013-04-08 | 2013-08-14 | 天津农学院 | Induction method of nitraria loose embryonic callus |
| CN103250643A (en) * | 2013-05-15 | 2013-08-21 | 中国林业科学研究院 | Tangut white spine clone in-vitro rooting culture method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103238517A (en) * | 2013-04-08 | 2013-08-14 | 天津农学院 | Induction method of nitraria loose embryonic callus |
| CN103250643A (en) * | 2013-05-15 | 2013-08-21 | 中国林业科学研究院 | Tangut white spine clone in-vitro rooting culture method |
Non-Patent Citations (3)
| Title |
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| "白刺的离体繁殖";何正伦;《甘肃林业科技》;19891231(第2期);第52-53页 * |
| "白刺组培快繁的研究";张红晓等;《经济林研究》;20031231;第21卷(第4期);第60-63页 * |
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