CN103461138B - A kind of lamiophlomis rotata isolated culture one-step seedling medium and method thereof - Google Patents
A kind of lamiophlomis rotata isolated culture one-step seedling medium and method thereof Download PDFInfo
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- CN103461138B CN103461138B CN201310437034.2A CN201310437034A CN103461138B CN 103461138 B CN103461138 B CN 103461138B CN 201310437034 A CN201310437034 A CN 201310437034A CN 103461138 B CN103461138 B CN 103461138B
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Abstract
The invention discloses a kind of lamiophlomis rotata isolated culture one-step seedling medium and method thereof, this medium makes lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling.Described one-step culture base medium based on MS medium, add the 6-benzyl aminopurine (6-BA) of variable concentrations and combination, methyl α-naphthyl acetate (NAA) and 2,4-dichlorphenoxyacetic acid (2,4-D), test-tube plantlet blade directly breaks up indefinite bud and adventive root as after explant inoculation through callus induction, and a step obtains lamiophlomis rotata regeneration plant.The present invention without the need to regenerating seedling by inducing the callus of acquisition to transfer in Initial culture base in differential medium, and compared with cultured in vitro more regeneration seedling, its step simplifies, and cultivation cycle is short, reproducible, and planting percent is high.
Description
Technical field
The invention belongs to biological technical field, particularly, relate to a kind of lamiophlomis rotata isolated culture one-step seedling medium, apply the method that this medium makes lamiophlomis rotata test-tube plantlet blade forming seedling through one step culture, correspondingly, present invention provides a kind of method optimizing this medium.
Background technology
Lamiophlomis rotata Lamiophlomis rotata (Benth.) Kudo is Labiatae, lamiophlomis rotata platymiscium, and this genus only has lamiophlomis rotata kind.Main product Tibet, Qinghai, Gansu, western Sichuan and northwestern Yunnan Province; Be born in the rubble beach of intensity weathering on plateau or high mountain or stone matter alpine meadow, flood land, height above sea level 2700 ~ 4500m.Have typical alpine plant feature, as plant is short, stem is short, well developed root system, and blade is comparatively large, pastes ground and launches.These features and its growing environment are severe cold region, dry wind is large, radiation is strong and day and night temperature is large etc. relevant.
Lamiophlomis rotata belongs to traditional Tibetan medicine, all herbal medicine among the people, controls traumatic injury, arthralgia and myalgia, the stagnation of the circulation of vital energy are sprained one's back, flows yellow water after edema, yellow water is amassed in joint, cancellous bone inflammation.In addition according to Qinghai, big white mouse femoral artery, arteria brachialis, arteria carotis crosscut are broken hemostasis trial, think that this kind has good haemostatic effect.Along with the memory space of wild lamiophlomis rotata progressively reduces, the research of lamiophlomis rotata is also more and more come into one's own.Also part is had to carry out to the research work of lamiophlomis rotata being introduced a fine variety to low altitude area.As Jin Lan etc. by by lamiophlomis rotata rhizome bud from height above sea level 4300m be wildly transplanted to the Experimental Base of height above sea level 2366m and 3100m after, to the Pollen Activity of transplanting ground lamiophlomis rotata, pollen germination and the rate that naturally terminates are added up, found that lamiophlomis rotata be transplanted to comparatively after low altitude area Pollen Activity low, without pollen germination on column cap, Natural seed setting rate is 0.Therefore, in low altitude area, the seedling of lamiophlomis rotata is obtained, more feasible in Plant Tissue Breeding mode.But, by the research to medium component, adopt the technology of once group training seedling, have not been reported.
Summary of the invention
For the problems referred to above, main purpose of the present invention is to provide a kind of medium and the cultural method of optimizing lamiophlomis rotata isolated culture one-step seedling, the lamiophlomis rotata test-tube plantlet blade one-step culture base obtained by this optimization method and the cultural method making its forming seedling through one step culture.Apply this lamiophlomis rotata one-step culture base and this cultural method, lamiophlomis rotata test-tube plantlet blade is after formation callus, do not need to transfer on differential medium, but direct seedling differentiation on former medium, improve the regeneration efficiency of lamiophlomis rotata test-tube plantlet blade callus, shorten incubation time, simplify operating procedure, provide an effective way for the rejuvenation of lamiophlomis rotata good seed is numerous soon with breed improvement.
The present invention is achieved through the following technical solutions:
A kind of lamiophlomis rotata isolated culture one-step seedling medium, make lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, described medium is medium based on MS medium, and in 1L MS medium, also need to add following component, and make the final concentration of each component as follows:
6-BA(6-benayl aminopurine) 1.0 ~ 2.0mg/L;
NAA(methyl α-naphthyl acetate) 0.5 ~ 1.5mg/L;
2,4-D(2,4-dichlorphenoxyacetic acid) 0 ~ 0.1mg/L.
Described lamiophlomis rotata isolated culture one-step seedling medium, medium based on MS, the final concentration of each component of the hormone added is:
6-BA 2.0mg/L；
NAA 1.0mg/L。
A cultural method for lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, comprises the steps:
(1) according to described formulated lamiophlomis rotata isolated culture one-step seedling medium, and sterilization treatment is carried out;
(2) choose the consistent lamiophlomis rotata in vitro cuttings in source, under aseptic condition, cut the blade launched completely on top and be about 0.5cm × 0.5cm, be affixed on the medium described in step 1 with vacuum side of blade;
(3) the inoculation wild Oryza species of step (2) gained being placed in temperature is under 18 ~ 20 DEG C of conditions, full dark culturing, after 7 ~ 10d, carry out illumination 12 ~ 16h every day again, 1000 ~ 1200Lx, after cultivating 30 ~ 40d, Callus formation, add up callus after cultivating 50 ~ 60d and differentiate indefinite bud and adventive root gradually, form test-tube plantlet;
(4) by test-tube plantlet through conventional hardening technology, carry out test-tube seedling transplanting, after 30d statistics transplant survival rate.
Beneficial effect of the present invention is: described lamiophlomis rotata isolated culture one-step seedling medium and this medium of application make the method for lamiophlomis rotata Tube leaves isolated culture one-step seedling, leaf explant is after formation callus, do not need to transfer on differential medium, but direct seedling differentiation on former medium, it is short that the method has the regeneration period, reproduction coefficient and regeneration frequency high, easy to operate, the advantages such as cultivation program is simple, for lamiophlomis rotata provides good method at low altitude area seedling fostering.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Optimize a method for lamiophlomis rotata isolated culture one-step seedling medium, this medium makes lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, comprises the following steps:
Not add the MS medium of any hormone in contrast, adopt the L of Three factors-levels
9(3
4) Orthogonal Experiment and Design, in MS medium, add 6-BA, NAA and 2, the 4-D of variable concentrations and combination, be mixed with 10 groups of different lamiophlomis rotata one-step culture bases.In each group, 6-BA, NAA of containing and the concentration of 2,4-D and combination are in table 1
Table 1 has the lamiophlomis rotata one-step culture base orthogonal L of different hormone combinations thing
9(3
4)
After medium sterilization, choose the lamiophlomis rotata in vitro cuttings that source is consistent, under aseptic condition, cut the blade that top is launched completely, and cut about 0.5cm × 0.5cm big and small blade, be affixed on the medium of research with vacuum side of blade; Every bottle graft kind 6 explants, each combination inoculates 10 bottles; It is under 20 DEG C of conditions that inoculation wild Oryza species is placed in temperature, and full dark culturing, carries out 16h illumination cultivation every day after 7 ~ 10d, light intensity is 1200Lx; After illumination cultivation 20d, callus induction rate as seen from the results in Table 2, namely produces the explant number/inoculation explant number × lO0% of callus, and calli induction situation.Add up Calli Differentiation result continue illumination cultivation 50d in former medium after, participate in table 2, be i.e. callus block number/total callus block number × 100% of Bud Differentiation, and seedling situation.
The impact that table 2 different hormone combinations is induced Callus of Leaf
Table 3 different hormone combinations is on the impact of blade differentiation adventitious buds
The test-tube plantlet hardening survival rate that table 4 different hormone combinations obtains
The experimental data of analytical table 2 gained is known, and except control group, each group lamiophlomis rotata one-step culture base all can induce lamiophlomis rotata blade to form callus preferably, and its inductivity all reaches more than 80%, and the 3rd, 6,7,8,9 groups, inductivity reaches more than 90%; And the 2nd, 3,4,5,6,8,9 groups all have good callus quality, wherein, the callus of the 3rd, 8,9 groups is best in quality.In analytical table 3, data are known again, and the 3rd, 5, callus in 9 groups fails differentiation and bud formation, and the 2nd, 4, in 6,8 groups, the differentiation rate of the callus in the 2nd group and the 4th group is poor, the number of days that sprouts is the longest, and Multiple Buds quantity is few and growing way is poor, and the differentiation rate of the 6th group and the 8th group is better, the number of days that sprouts is shorter, best with the 8th component rate, the number of days that sprouts is the shortest, and Multiple Buds quantity is many and growing way is better.
Associative list 1 analyzes the 2nd again, and 4,6, the concentration of each hormone in the medium corresponding to 8 groups and combination, wherein, the concentration of 6-BA in the the the 2nd, 4,6,8 group is followed successively by 1.0mg/L, 1.5mg/L, 1.5mg/L, 2.0mg/L; The concentration of NAA in the the the 2nd, 4,6,8 group is followed successively by 1.0mg/L, 0.5mg/L, 1.5mg/L, 1.0mg/L; The concentration of 2,4-D in the the the 2nd, 4,6,8 group is followed successively by 0.1mg/L, 0.1mg/L, 0mg/L, 0mg/L.Table 4 is that test-tube plantlet hardening survives number and survival rate, it is preferred that the 8th group.
In summary, the lamiophlomis rotata nutrient media components of lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling can be made to be: medium based on MS medium, and in 1L MS medium, also need the final concentration of component and each component added to be:
6-BA 2.0mg/L；
NAA 1.0mg/L。
Embodiment 2
In order to avoid the error of the experimental result that human factor causes, the applicant asks different researchers under the same conditions, every bottle graft kind 6, inoculate 8 bottles altogether, utilize the lamiophlomis rotata one-step culture base identical with embodiment, carried out the experimental implementation identical with embodiment 1, its experiment the results are shown in Table 5 and table 6.
The impact that table 5 different hormone combinations is induced Callus of Leaf
Table 6 different hormone combinations is on the impact of blade differentiation adventitious buds
The test-tube plantlet hardening survival rate that table 7 different hormone combinations obtains
The experimental data of analytical table 5 gained is known, and except control group, each group lamiophlomis rotata one-step culture base all can induce lamiophlomis rotata blade preferably, and its inductivity all reaches more than 80%, and the 1st, 3,4,5,6,8 and 9 group, inductivity reaches more than 90%.In analytical table 5, data are known again, and the callus in the 3rd group fails differentiation and bud formation; And in the 4th, 5,6,8,9 groups, the 4th and the 5th component rate is too low, Multiple Buds quantity is few and growing way is poor; The 9th group of number of days that sprouts is oversize, and Multiple Buds quantity is few; Better with the differentiation rate of the 6th, 8 group, the number of days that sprouts is shorter, and Multiple Buds quantity is many and growing way better, and best with the 8th component rate, the sky of sprouting is the shortest, and Multiple Buds quantity is maximum, and the growing way of bud is best.
Associative list 6, the final hardening survival rate of table 7 test-tube plantlet again, the concentration of each hormone in the medium corresponding to known 8th group and combination, known, the component of best lamiophlomis rotata one-step culture base is, in 1L MS medium:
6-BA 2.0mg/L；
NAA 1.0mg/L。
In summary, embodiment 1 is substantially identical with the result of embodiment 2, has good repeatability.
Embodiment 3
A method for lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, comprises the steps:
(1) lamiophlomis rotata one-step culture base is prepared: in 1LMS medium, add 2.0mg6-benayl aminopurine and 1.0mg methyl α-naphthyl acetate, carry out sterilization treatment, stand-by;
(2) choose the consistent lamiophlomis rotata in vitro cuttings in source, under aseptic condition, cut the blade launched completely on top and be about 0.5cm × 0.5cm, be affixed on the medium described in step 1 with vacuum side of blade;
(3) the inoculation wild Oryza species of step (2) gained being placed in temperature is 20 DEG C of incubators, and full dark culturing, carries out 16h/d illumination cultivation after 7 ~ 10d, and light intensity is 1200Lx;
(4) in Initial culture base, after illumination cultivation 55d, lamiophlomis rotata regeneration plant is obtained.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (1)
1. a cultural method for lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, is characterized in that, comprise the steps:
(1) according to the formulated lamiophlomis rotata isolated culture one-step seedling medium of the following stated, and sterilization treatment is carried out;
Medium based on MS, the final concentration of each component of the hormone added is:
6-BA 2.0mg/L；
NAA 1.0mg/L；
(2) choose the consistent lamiophlomis rotata in vitro cuttings in source, under aseptic condition, cut the blade 0.5cm × 0.5cm launched completely on top, be affixed on the medium described in step (1) with vacuum side of blade;
(3) the inoculation wild Oryza species of step (2) gained being placed in temperature is under 18 ~ 20 DEG C of conditions, full dark culturing, after 7 ~ 10d, carry out illumination 12 ~ 16h every day again, 1000 ~ 1200Lx, after cultivating 30 ~ 40d, Callus formation, add up callus after cultivating 50 ~ 60d and differentiate indefinite bud and adventive root gradually, form test-tube plantlet.
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| CN106612705A (en) * | 2016-08-31 | 2017-05-10 | 中国农业科学院兰州畜牧与兽药研究所 | Technology for improving wild lamiophlomis rotata seed germination |
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| CN102177850A (en) * | 2011-04-01 | 2011-09-14 | 上海植物园 | Quick tissue culture breeding and seedling raising method for narcissus tazetta var. chinensis |
| CN102657086A (en) * | 2012-05-10 | 2012-09-12 | 中国科学院西北高原生物研究所 | Method for tissue culture and in-vitro rapid propagation of lamiophlomis rotata |
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Patent Citations (4)
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|---|---|---|---|---|
| US5908771A (en) * | 1997-01-31 | 1999-06-01 | R. J. Reynolds Tobacco Company | Method for regeneration of salvia species |
| CN102119664A (en) * | 2011-01-21 | 2011-07-13 | 江苏九久环境科技有限公司 | Method for culturing muscari botryoides mill with plant tissues |
| CN102177850A (en) * | 2011-04-01 | 2011-09-14 | 上海植物园 | Quick tissue culture breeding and seedling raising method for narcissus tazetta var. chinensis |
| CN102657086A (en) * | 2012-05-10 | 2012-09-12 | 中国科学院西北高原生物研究所 | Method for tissue culture and in-vitro rapid propagation of lamiophlomis rotata |
Non-Patent Citations (1)
| Title |
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| 独一味试管苗快繁技术研究;刘正玉等;《西藏农业科技》;20071231;第29卷(第3期);第20页第6-7段，第21页第1-3段 * |
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