CN102210266B - Culture medium for culturing lilium pumilum tissues - Google Patents
Culture medium for culturing lilium pumilum tissues Download PDFInfo
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- CN102210266B CN102210266B CN 201110071332 CN201110071332A CN102210266B CN 102210266 B CN102210266 B CN 102210266B CN 201110071332 CN201110071332 CN 201110071332 CN 201110071332 A CN201110071332 A CN 201110071332A CN 102210266 B CN102210266 B CN 102210266B
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- 241001634090 Lilium pumilum Species 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 title claims abstract description 13
- 238000012258 culturing Methods 0.000 title claims abstract 4
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims abstract description 23
- 230000001939 inductive effect Effects 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 239000005556 hormone Substances 0.000 abstract description 9
- 229940088597 hormone Drugs 0.000 abstract description 9
- 230000012010 growth Effects 0.000 abstract description 7
- 230000035755 proliferation Effects 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 3
- 230000006698 induction Effects 0.000 abstract description 3
- 241000131317 Capitulum Species 0.000 abstract 1
- 235000021048 nutrient requirements Nutrition 0.000 abstract 1
- 239000012883 rooting culture medium Substances 0.000 abstract 1
- 241001359444 Lilium tenuifolium Species 0.000 description 33
- 241000234435 Lilium Species 0.000 description 15
- 239000002609 medium Substances 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 8
- 230000004069 differentiation Effects 0.000 description 8
- 238000009395 breeding Methods 0.000 description 7
- 230000001488 breeding effect Effects 0.000 description 7
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 5
- 238000012549 training Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 230000003716 rejuvenation Effects 0.000 description 3
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000000384 rearing effect Effects 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 241001656767 Lilium columbianum Species 0.000 description 1
- 241001656752 Lilium philadelphicum Species 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000034373 developmental growth involved in morphogenesis Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000009400 out breeding Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000012879 subculture medium Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a culture medium for culturing lilium pumilum tissues. The best culture mediums for culturing the lilium pumilum tissues at different stages are screened; the proportion of various hormone in the culture medium is determined; and the culture mediums prepared according to the formula can meet the nutrient requirement and growth and development of the lilium pumilum at each stage. The culture mediums comprise a first generation induction culture mediums consisting of MS, 1.0 mg/L of 6-BA and 0.1 mg/L of NAA, a subgeneration proliferation culture medium consisting of MS, 0.2 mg/L of 6-BA and 0.01 mg/L of NAA, a rooting culture medium consisting of 1/2 MS and 0.5 mg/L of IBA and a capitulum culture medium consisting of 1/2 MS and 0.5 mg/L of IBA and 2 percent of sugar.
Description
Technical field
The invention belongs to plant biotechnology field, specifically, relate to the substratum of stages in the tissue culture of a kind of Lilium tenuifolium (Lilium pumilum).The present invention filters out the optimal medium of cultivating different times for lilium pumilum tissues, has determined the proportioning of each hormone in the substratum, can satisfy the nutritional need in each period of Lilium tenuifolium and grows according to the substratum of formulated of the present invention.
Background technology
Lilium tenuifolium (Lilium pumilum) has another name called Morningstar Lily, is the perennial bulb class of Liliaceae lilium flowers.Lilium tenuifolium is grown in arid, semiarid zone, indomitable vitality is arranged, can survive the winter smoothly in the open air, few at rainwater, without also can normal growth in the situation of pouring, performance and attractive in appearance on can both reach the requirement of greening, can in the drought resisting Afforestation Landscape, be used and promote.But because from the threat of the mankind or non-human two aspects, make its population fail under field conditions (factors) amount reproduction, quantity is few and exist the retrogression of nature problem.Solution is exactly the tissue culture that starts Lilium tenuifolium, and this is the effective ways of Fast-propagation and detoxification rejuvenation.
The tissue culture of lily starts from the 1950's, carry out with lily bud scale first after tissue culture succeeds from nineteen fifty-seven Robb, lily in-vitro propagate technology is increasingly mature, and the development of the aspects such as the Fast-propagation of improved seeds, rearing new variety and germ plasm resource preservation is very fast.Up to now, the lily kind of tissue culture success both at home and abroad and kind have surpassed hundreds of, but what be more common in report is the Cultivar that produces for fresh cutting flower, and be 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 culture, about caning be counted on one's fingers especially of Lilium tenuifolium.Under laboratory condition, the Growth and reproduction of control Lilium tenuifolium is one needs practice to go the new problem of groping and inquiring into.Prior art can reference data few, all must settle one by one by test, key will solve explant and draw materials position, period; The selection of each cultivation period substratum; Fast breeding technique etc. after culture condition and the seedling.Wild resource with China's abundant carries out the kind innovation, use diversified breeding technique, on the basis of paying attention to conventional cross-breeding, carry out breeding of new variety in conjunction with the modern biotechnology means, carrying out the batch production exploitation of kind of ball with the detoxication and tissue culture seedling as the productivity original seed simultaneously, is the developing direction that Lilium Tissue is cultivated.The artificial propagation of Lilium tenuifolium is still mainly adopted to divide and is planted bulblet, seeding method and scale cuttage, breeds limited amount and very easily causes disease to infect and quality deterioration.But the tissue culture method breeding research that can be used at present separating degeneration problem and rejuvenating is less, and therefore carrying out the lilium pumilum tissues culture studies in a deep going way has become researcher and demand one of work of carrying out urgently.
The tissue culture method breeding is drawn materials conveniently; be not subjected to the restriction of natural condition; can constantly carry out amount reproduction; it is the most effectual way of Lilium tenuifolium introducing culture, Fast-propagation, detoxification rejuvenation and rearing new variety; can obtain in a short time a large amount of seedlings; solved the less problem of provenance in the cultivation, for Lilium tenuifolium research and mass-producing, industrialization development utilization from now on provides theoretical foundation.Domestic and international many scholars also are studied the tissue culture of lily at present, be applied at present in the lily production, but what be more common in report is that Cultivar is used for fresh cutting flower production, substratum and method also are not suitable for Lilium tenuifolium, the present invention has systematically studied the optimal medium of different steps in the training of Lilium tenuifolium group, and this substratum is also cultivated lily applicable to wild tiger lily lily and part.
Summary of the invention
The complete procedure of tissue culture generally is divided into several sport technique segments such as rooting culture of formulating culture scheme, explant selection and processing, inoculation, first culture, succeeding transfer culture, strong sprout and root culture, test-tube plantlet.Tissue culture is under isolated culture condition, and the histocyte of different plants and kindred plant different sites is different to nutritional requirement, only has and has satisfied their particular requirements separately, could grow better.And preparation and the screening method of grasping substratum are to obtain one of successful key link of group training.
The objective of the invention is to filter out the optimal medium of cultivating different times for lilium pumilum tissues, determined the proportioning of each hormone in the substratum, can satisfy the nutritional need in each period of Lilium tenuifolium and grow according to the substratum of formulated of the present invention.
The present invention has formulated embodiment on the basis of carrying out information search, gather and analyzing, and comprehensive, multi-faceted organizes the training experiment, and the present invention has screened from first generation and induced → substratum of subculture increment → root culture → different times such as balling cultivation.Make first generation, breed, take root and the cultivation in balling each period reaches best effect.
Embodiment of the present invention comprise the optimal medium of each breeding time in the whole tissue culture procedures of Lilium tenuifolium, they draw through careful, repeated tests, are that the needs according to Lilium tenuifolium each breeding time have added dissimilar plant growth agents and different proportionings on the basis of minimum medium.
In embodiments of the invention, a kind of Lilium tenuifolium (Lilium pumilum) required substratum of each stage of tissue culture procedures is provided, it is characterized in that this substratum is by just forming for inducing culture, proliferated culture medium, root media, balling substratum.These Lilium tenuifolium group training stages optimal mediums filter out through comparison test.
One embodiment of the present invention has provided the first for inducing culture of a kind of Lilium tenuifolium (Lilium pumilum): MS+6-BA 1mg/L+NAA 0.1mg/L+Vc 5mg/L.Find that by research very little to the inducing action of bulblet without the substratum of hormone, Conventional Hormone has NAA, 6-BA etc.Wherein NAA and IAA are conducive to the formation of root; And 6-BA is indispensable composition for spore induction, but during the 6-BA excessive concentration, although the bulblet bulbous protrusion that differentiates is many, but very thin and delicate, final is vitrified invalid seedling, NAA/6-BA affects the differentiation degree of root in the first culture or bud, is MS+6-BA 1.0mg/L+NAA 0.1mg/L+Vc 5mg/L for the prescription of inducing culture just thereby filter out best.
One embodiment of the present invention has provided the proliferated culture medium of a kind of Lilium tenuifolium (Lilium pumilum): MS+6-BA 0.2mg/L+NAA 0.01mg/L.Find that by research the 6-BA of same high density has restraining effect to lily bulb propagation, excessive concentration can suppress the growth of bud, along with increasing of 6-BA concentration, when 6-BA concentration surpasses 2.0mg/L, grows thickly and downgrades obviously, easily produces the vitrifying seedling.The optimum multiplication medium prescription that filters out is MS+6-BA 0.2mg/L+NAA0.01mg/L.
One embodiment of the present invention has provided a kind of Lilium tenuifolium (Lilium pumilum) root media: 1/2MS+IBA 0.5mg/L.From 20 days test-results of root culture, low salt concn is favourable to the growth of Lilium Germplasm root, and the prescription of suitable substratum of taking root is 1/2MS+IBA 0.5mg/L.
One embodiment of the present invention has provided a kind of Lilium tenuifolium (Lilium pumilum) balling substratum: 1/2MS+IBA 0.5mg/L+ white sugar 2%.Find by research, no matter Lilium tenuifolium is at MS or in the 1/2MS substratum, the mass/volume degree of edible sugar increases, balling there is promoter action, it is even more ideal to compare 1/2MS+IBA 0.5mg/L+ white sugar 2%, balling quantity is many, and bulb is larger, transplants the seedling rate best results.
In embodiment of the present invention, in scope as well known to those skilled in the art, NAA, 6-BA, IBA, V
cRespectively naphthylacetic acid, 6-benzyl aminopurine, indolebutyric acid, ascorbic abbreviation.
In the whole tissue culture procedures of the Lilium tenuifolium that the present invention selects the optimal medium of each breeding time can fully satisfy each period of Lilium tenuifolium nutritional need and grow; to guarantee lilium pumilum tissues cultivation successful implementation; reach key and core that procedure is produced, for Lilium tenuifolium Establish of regeneration and large-scale production provide strong support.
Embodiment
The material of embodiment 1. Lilium tenuifoliums and screening statistical method
The Lilium tenuifolium of experiment usefulness is in picking up from Daqunshan Mountains, suburb nearby, Huhehaote City, and the bulb that collects is planted test nursery in our company, replenishes according to test progress, different times sampling and at any time sampling.Explant is the bulb of Lilium tenuifolium.
Take MS as minimum medium, make solidifying agent with the carrageenin of 3.5g/L, 3% edible sugar replaces sucrose as carbon source, replaces distilled water with tap water, and the phytokinin and the growth hormone that add respectively different proportionings carry out the screening of substratum.
The screening of embodiment 2. Lilium tenuifolium groups training stages optimal medium
1, the selection of inductive differentiation medium
The concentration of 6-BA is established 0.2mg/L, 0.5mg/L, five concentration gradients of 1.0mg/L, 2.0mg/L, the concentration of NAA is established 0.1mg/L, 0.2mg/L, four concentration gradients of 0.3mg/L, 0.5mg/L, 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 be induced lily bud scale differentiation bulblet, but the differentiation rate of each combination is different, No. 1 MS+6-BA 0.1mg/L+NAA 0.1mg/L is that Lilium tenuifolium scale culture base is best inducing culture, not only inductivity is high, and the newly-increased bud number of each explant is many, and differentiation rate is the highest, and its differentiation rate can reach 60.0%.
Concrete statistical formula is:
The explant sum of surviving rate=viable explant number/inoculation) * 100%
Just for bud induction rate=(the explant sum of the explant number that sprouts/inoculation) * 100%
Shoot proliferation multiple=(sum of the Multiple Buds number that induces/access individual plant bud) * 100%
Rooting rate=(the explant sum of the explant number of differentiation root/inoculation) * 100%
Bulblet inductivity=(the explant sum of the explant number of differentiation bulblet/inoculation) * 100%
Table 1 hormone concentration and combination thereof are on the impact of Lilium tenuifolium scale adventitious bud inducing
2, the selection of shoot proliferation substratum
To induce the indefinite bud that differentiates to be cut into individual plant, be inoculated on the proliferated culture medium, 6-BA establishes 0.2mg/L, 0.5mg/L, four concentration gradients of 1.0mg/L, 2.0mg/L, NAA establishes 0.01mg/L, 0.1mg/L, four concentration gradients of 0.2mg/L, 0.3mg/L totally seven kinds of substratum, 4 individual plants of every bottle graft, the differential growth situation of observed and recorded regenerated adventitious bud was added up the proliferation rate of bud under the different hormone combinations condition in 30 days afterwards.
Behind inducing culture, the 2.0-4.0cm budlet that will produce in inducing culture moves into respectively (referring to table 2) in the proliferated culture medium, and the bastem section of 15 days rear section materials begins observed and recorded, statistical results after 30 days when the bud clump occurring.Observation shows that the 6-BA of high density has restraining effect to lily bulb propagation, and excessive concentration can suppress the growth of bud, along with increasing of 6-BA concentration, when 6-BA concentration surpasses 2.0mg/L, grows thickly and downgrades obviously, easily produces the vitrifying seedling.The optimum multiplication medium prescription that filters out is MS+6-BA0.2mg/L+NAA0.01mg/L.
Table 2 hormone concentration and combination thereof are on the impact of Lilium tenuifolium adventitious bud proliferation
3, the selection of root media
With the seedling preferably of growing way in the subculture medium, be divided into individual plant and change in the root media, every bottle graft enters 4 strains, and the situation of taking root of seedling was added up rooting rate after 20 days on the observed and recorded different culture media.
From 20 days test-results of root culture, two substratum all have inducing action to the formation of root, and wherein No. 2 root media rooting efficiencies are better, and rooting rate reaches 88.89%.The base portion of taking root can increase the bulblet with root newly, on average can reach 3-4.As seen low salt concn is favourable to the growth of Lilium Germplasm root, and suitable substratum of taking root is 1/2MS+IBA0.5mg/L (table 3).
The impact that table 3 inorganic salt are taken root on seedling
4, the take root selection of balling substratum
Based on the basis of root media, the Multiple Buds of propagation is inoculated into edible white sugar content brings up on the substratum of 40-50g/L, observe the situation of inducing of test-tube bulblet, did once statistics in 30 days.
Our test shows no matter Lilium tenuifolium is to cultivate at MS or at 1/2MS to concentrate, and sugared content increases to 2%, balling is had promoter action, and it is even more ideal to compare 1/2MS+IBA 0.5mg/L+ white sugar 2%, and balling quantity is many, bulb is larger, transplants the seedling rate best results.
Above embodiment purpose is for specifying 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, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
Claims (1)
1. required substratum of each stage of lilium pumilum tissues culturing process is characterized in that, this substratum by just for inducing culture, proliferated culture medium, root media and balling substratum form, wherein,
Just for inducing culture for containing 1mg/L 6-BA, 0.1mg/LNAA, the MS substratum of 5mg/L Vc;
Proliferated culture medium is for containing 0.2mg/L 6-BA, the MS substratum of 0.01mg/LNAA;
Root media is the 1/2MS substratum that contains 0.5mg/L IBA;
The balling substratum is for containing 0.5mg/L IBA, the 1/2MS substratum of additional 2% white sugar.
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| CN103222427A (en) * | 2013-05-08 | 2013-07-31 | 四川农业大学 | Method for efficiently inducing Luding lily test tube bulbels |
| CN104604676A (en) * | 2014-12-30 | 2015-05-13 | 内蒙古和信园蒙草抗旱绿化股份有限公司 | Culture media for tissue culture of red lily |
| 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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| Title |
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| 傅玉兰，等.影响百合试管鳞茎增殖因素的研究.《安徽农业大学学报》.2001,第28卷(第2期), * |
| 葛蓓孛,等.细叶百合组织培养植株再生.《东北林业大学学报》.2010,第38卷(第5期), * |
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