CN104604676A - Culture media for tissue culture of red lily - Google Patents
Culture media for tissue culture of red lily Download PDFInfo
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- CN104604676A CN104604676A CN201410842988.6A CN201410842988A CN104604676A CN 104604676 A CN104604676 A CN 104604676A CN 201410842988 A CN201410842988 A CN 201410842988A CN 104604676 A CN104604676 A CN 104604676A
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Abstract
The invention discloses culture media for tissue culture of red lily. The best culture media for tissue culture of the red lily in different phases are screened out; hormone ratios in the culture media are determined; the culture media prepared according to the formula disclosed by the invention can meet nutritional demands and growth development of the red lily in various periods; the culture media include a primary induced culture medium prepared from MS, 1.0 mg/L of 6-BA, and 0.1 mg/L of NAA, a subculture proliferation culture medium prepared from MS, 2.5 mg/L of 6-BA, and 0.06 mg/L NAA, and a rooting culture medium prepared from 1/2 MS, 0.05 mg/L of IBA, and 0.5 g/L of AC.
Description
Technical field
The invention belongs to plant biotechnology field, specifically, relate to the medium in each stage in the tissue cultures of a kind of red lily.During the present invention filters out and cultivates for red Lilium Tissue, the optimal medium of different times, determines the proportioning of each hormone in medium, can meet the nutritional need in red lily each period and grow according to the medium of formulated of the present invention.
Background technology
Red lily is Liliaceae, perennial herb, is the xenogenesis of lily.Petal is red, the florescence 5-8 month.About there is kind more than 100 in the lily kind whole world, and China originates in about 55 kinds.Red lily just grows in Inner Mongol arid, semiarid zone, there is indomitable vitality, can survive the winter smoothly in the wild, few at rainwater, without also can normal growth when pouring, in performance and the requirement that aesthetically can reach greening, can be used in drought resisting Afforestation Landscape and promote.But due to the threat from the mankind or non-human two aspect, make its population quantity there is retrogression of nature problem and in tissue culture expanding propagation, occur that vitrification phenomenon causes factorial seedling growth limited amount.Solution is exactly the existing method for tissue culture to lily of improvement, and this is Fast-propagation, detoxification rejuvenation and reduce vitrified effective ways.
The tissue cultures of lily starts from the 1950's, carry out after tissue cultures succeeds first with lily bud scale from nineteen fifty-seven Robb, lily in-vitro propagate technology is increasingly mature, and the aspect development such as the Fast-propagation of improved seeds, rearing new variety and Germ-plasma resources protection are very fast.Up to now, domestic and international tissue cultures successful lily kind and kind have exceeded hundreds of, but what be more common in report is the cultivar produced for fresh cut-flowers, less to the Study on tissue culture of Wild lilies native to China.Sum up the data in literature over nearly 20 years, only more than ten plant the success of Lilium Germplasm tissue cultures, caning be counted on one's fingers especially of relevant red lily.The artificial propagation of red lily still mainly adopts bulb reproduction, point plants clove and scale cuttage, and reproductive number is limited and very easily cause disease infection and quality deterioration.The tissue culture method study on reproduction that can be used for solving degenerate problem and rejuvenating is at present less, and therefore carrying out red Lilium Tissue culture studies in a deep going way has become one of work that researcher urgently carries out.
Varieties distribution is carried out with the wild resource of China's abundant, apply diversified breeding technique, on the basis paying attention to conventional cross-breeding, breeding of new variety is carried out in conjunction with modern biotechnology means, carry out the batch production exploitation of kind of ball using detoxication and tissue culture seedling as yielding ability original seed, be the developing direction that Lilium Tissue is cultivated simultaneously.Tissue culture method breeding is drawn materials conveniently; not by the restriction of natural conditions; constantly can carry out amount reproduction; it is the most effectual way of red lily introducing and planting, Fast-propagation, detoxification rejuvenation and rearing new variety; a large amount of seedling can be obtained in a short time; solve the problem that provenance in cultivation is less, for the research from now on of red lily and scale, industrialization development utilize and provide theoretical foundation.
Tissue cultures is under isolated culture condition, and the histocyte of different plant and kindred plant different parts is different to nutritional requirement, only meets their respective particular/special requirements, could grow better.Preparation and the screening technique of grasping medium obtain one of successful key link of group training.Have studied the optimal medium of different phase in the training of red lily group present system.
Summary of the invention
The complete procedure of tissue cultures be generally divided into formulate culture scheme, explant select with process, inoculate, Initial culture, squamous subculture, strong sprout and culture of rootage, test-tube plantlet several sport technique segment such as rooting culture.Tissue cultures is under isolated culture condition, and the histocyte of different plant and kindred plant different parts is different to nutritional requirement, only meets their respective particular/special requirements, could grow better.And grasp the preparation of medium and screening technique obtains one of successful key link of group training.In laboratory conditions, reducing its vitrifying is a new problem need putting into practice to grope and inquire into simultaneously.When formulating this scheme can the data of reference few, all must be settleed one by one by test, key will solve explant and draw materials position, period, the selection of each cultivation period medium, fast breeding technique etc. after condition of culture and seedling.The present invention, to develop, reduces costs as target, to be formed for the purpose of batch production, industrialization production, formulates corresponding work plan and implementing method.
The object of the invention is to filter out the optimal medium of different times in cultivating for red Lilium Tissue, determine the proportioning of each hormone in medium, the nutritional need in red lily each period can be met according to the medium of formulated of the present invention and grow.
This application study being a novelty and displaying great creativity, having carried out information search, gather with the basis analyzed on formulated embodiment, comprehensively, multi-faceted carrying out group training experiment, this mainly introduce the screening relevant with the application just generation the different times such as induction → subculture increment → culture of rootage medium.Make first generation, breed, the cultivation in each period of taking root reaches optimum efficiency.
Embodiment of the present invention comprise the optimal medium of each breeding time in the whole tissue culture procedures of red lily, they are through carefully, repeated tests draws, be on the basis of minimal medium according to red lily each breeding time need with the addition of dissimilar plant growth substance and different proportionings.
In embodiments of the invention, provide the medium needed for a kind of red Lilium Tissue incubation each stage, it is characterized in that, this medium is by just forming for inducing culture, proliferated culture medium, root media, balling medium.These each stage optimal mediums of red lily groups training are through that comparative test filters out.
One embodiment of the present invention there is provided the first for inducing culture of a kind of red lily: MS+6-BA 1.0mg/L+NAA 0.1mg/L, this inducing culture not only inductivity is high, and each explant to increase bud number newly many, differentiation rate is the highest, and its differentiation rate can reach 82.6%.
One embodiment of the present invention there is provided the proliferated culture medium MS+6-BA 2.5mg/L+NAA0.06mg/L of a kind of red lily.
One embodiment of the present invention there is provided a kind of red lily root media: 1/2MS+IBA 0.05mg/L+AC0.5g/L.This root media rooting efficiency is better, and rooting rate reaches 92.9%.Base portion of taking root can increase the Multiple Buds of band root newly, on average can reach 3-4.The growth of visible low salt concn to red lily root is favourable.
In the present embodiment, in scope as well known to those skilled in the art, NAA, 6-BA, IBA are the abbreviation of methyl α-naphthyl acetate, 6-benzyl aminopurine, indolebutyric acid respectively.
In the whole tissue culture procedures of red lily that the present invention selects, the optimal medium of each breeding time fully can meet the nutritional need in red lily each period and grow; ensure that red Lilium Tissue cultivates successful implementation; reach key and core that procedure produces, for the foundation of red lily regenerating system and large-scale production provide strong support.
Embodiment
The material of the red lily of embodiment 1. and screening statistical method
1.1, material
The red lily of experiment picks up from Daqunshan Mountains, suburb nearby, Huhehaote City, the bulb collected is planted the test nursery in our company, and according to test progress, different times samples and sampling supplements at any time.Adopt red lily bud scale, earth is removed in flushing, sterile chamber put into by super-clean bench, drip in the detergent solution of " Tween-20 " at often liter of dropping 2-3 and soak 60s, proceed in the alcohol of 75% and soak 30s, then use the mercuric chloride solution process 25min of 0.2%, sterile water drip washing 3-4 time, suck dry moisture on aseptic filter paper, in the medium that direct access prepares in advance.
1.2, statistical computation formula
Survival rate=(the explant sum of viable explant number/inoculation) × 100%
Just for bud induction rate=(the explant sum of the explant number/inoculation of sprouting) × 100%
Shoot proliferation multiple=(sum of the Multiple Buds number induced/access individual plant bud) × 100%
Rooting rate=(the explant sum of the explant number/inoculation of differentiation root) × 100%
1.3, method, results and analysis
Take MS as minimal medium, make curing agent with the carragheen of 3.5g/L, the edible sugar of 3% replaces sucrose as carbon source, replaces distilled water with running water, adds the screening that the basic element of cell division of different ratio and growth hormone carry out medium respectively.
The screening of each stage optimal medium of the red lily group training of embodiment 2.
The selection of 2.1 inductive differentiation mediums
The concentration of 6-BA is established 0.2mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.0mg/L five concentration gradients, the concentration of NAA establishes 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.5mg/L tetra-concentration gradients, carry out different assembly, every bottle graft a slice bulb during inoculation, observe scale on different culture media indefinite bud a situation arises.
As seen from Table 1,7 kinds of hormone combinations all can induce red lily to break up clove, but the differentiation rate of each combination is different, No. 3 MS+6-BA 1.0mg/L+NAA0.1mg/L are inducing cultures of red lily bud scale medium the best, not only inductivity is high, and each explant to increase bud number newly many, differentiation rate is the highest, and its differentiation rate can reach 82.6%.Result is as shown in table 1.
Table 1 hormone concentration and the impact of combination on red lily bud scale adventitious bud inducing thereof
The selection of 2.2 subculture multiplication medium
The red lily seedling that 3-4cm has tied clove is about by what differentiate, be inoculated on MS and 1/2MS medium respectively, additional IBA0.5mg/L, food white granulated sugar 30g/L, carragheen 3.5g/L, directly carry out root induction, 6-BA establishes 2.5mg/L, 2.0mg/L, 1.5mg/L, 0.5mg/L tetra-concentration gradients, NAA establishes 0.06mg/L, 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.5mg/L five concentration gradients totally seven kinds of medium, every bottle graft 4 individual plants, the differential growth situation of observed and recorded regeneration clove, observes the rate of increase under different hormone combinations condition on the 20th day.
As seen from Table 2,7 kinds of hormone combinations all can induce red lily to break up clove, but the indefinite bud upgrowth situation of each combination is different, and No. 1 MS+6-BA 2.5mg/L+NAA 0.06mg/L is red lily adventitious bud proliferation upgrowth situation optimal medium.
Table 2 hormone concentration and the impact of combination on red lily adventitious bud proliferation thereof
2.3 the selection of root media
By good for growing way in subculture medium seedling, be divided into individual plant and proceed in root media, every bottle graft enters 4 strains, and on observed and recorded different culture media, the situation of taking root of seedling, added up rooting rate after 10 days.
From the culture of rootage result of the test of 10 days, two formation of medium to root all have inducing action, and wherein No. 2 root media rooting efficiencies are better, and rooting rate reaches 92.9%.Base portion of taking root can increase the Multiple Buds of band root newly, on average can reach 3-4.The growth of visible low salt concn to red lily root is favourable, and suitable medium of taking root is 1/2MS+IBA 0.05mg/L+AC0.5g/L (table 3).
The impact that table 3 mineral salt are taken root on seedling
Embodiment 3. red lily group each stage of training cultivates flowchart illustrations
3.1 get explant
Bulb → excision injury → sterilization → the aseptic explant of red lily
3.2 just for Fiber differentiation
Red lily explant → 1/2MS medium cultivate → is tied the red lily seedling of clove
3.3 shoot proliferations are cultivated
The MS medium of the bulb → improvement of red lily is cultivated for 3 generations → the upper cultivation of MS 2 generation → improvement MS medium on cultivate 1 generation → tied the red lily seedling of clove
3.4 culture of rootage
The seedling of to have tied on the red lily seedling → root media of clove → having taken root
3.5 bottle outlets are transplanted
The seedling taken root → be transplanted in small flower
Above embodiment object is for illustrating content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
Claims (4)
1. the medium needed for red Lilium Tissue incubation each stage, is characterized in that, this medium is by just for inducing culture, and proliferated culture medium, root media forms.
2. first for inducing culture described in claim 1, it consists of the MS medium containing 1mg/L 6-BA, 0.1mg/LNAA.
3. the proliferated culture medium described in claim 1, it consists of the MS medium containing 2.5mg/L 6-BA, 0.06mg/LNAA.
4. the root media described in claim 1, it consists of the 1/2MS medium containing 0.05mg/L IBA, 0.5g/LAC.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106171347A (en) * | 2016-06-07 | 2016-12-07 | 中国农业科学院蔬菜花卉研究所 | A kind of lily bud scale cottage method |
| CN112251528A (en) * | 2020-10-19 | 2021-01-22 | 北京林业大学 | Microsatellite DNA molecular marker for identifying ploidy of lily and application thereof |
| CN113317206A (en) * | 2021-07-19 | 2021-08-31 | 中国科学院昆明植物研究所 | Culture medium combination for tissue culture and rapid propagation of lilium martagon, tissue culture and rapid propagation method and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106171347A (en) * | 2016-06-07 | 2016-12-07 | 中国农业科学院蔬菜花卉研究所 | A kind of lily bud scale cottage method |
| CN112251528A (en) * | 2020-10-19 | 2021-01-22 | 北京林业大学 | Microsatellite DNA molecular marker for identifying ploidy of lily and application thereof |
| CN112251528B (en) * | 2020-10-19 | 2021-07-06 | 北京林业大学 | Microsatellite DNA molecular marker for identifying ploidy of lily and application thereof |
| CN113317206A (en) * | 2021-07-19 | 2021-08-31 | 中国科学院昆明植物研究所 | Culture medium combination for tissue culture and rapid propagation of lilium martagon, tissue culture and rapid propagation method and application |
| CN113317206B (en) * | 2021-07-19 | 2021-11-05 | 中国科学院昆明植物研究所 | Culture medium combination for tissue culture and rapid propagation of lilium martagon, tissue culture and rapid propagation method and application |
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