CN103461138A - One-step plantlet formation medium and method for isolated culture of lamiophlomis rotata - Google Patents
One-step plantlet formation medium and method for isolated culture of lamiophlomis rotata Download PDFInfo
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- CN103461138A CN103461138A CN2013104370342A CN201310437034A CN103461138A CN 103461138 A CN103461138 A CN 103461138A CN 2013104370342 A CN2013104370342 A CN 2013104370342A CN 201310437034 A CN201310437034 A CN 201310437034A CN 103461138 A CN103461138 A CN 103461138A
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Abstract
The invention discloses a one-step plantlet formation medium and method for the isolated culture of lamiophlomis rotata. By adopting the one-step plantlet formation medium, a lamiophlomis rotata test-tube plantlet leaf forms a plantlet in one step through isolated culture. The one-step plantlet formation culture medium is characterized in that a MS (Murashige and Skoog) medium is used as a basic medium, and 6-benzylaminopurine (6-BA), naphthylacetic acid (NAA) and 2,4-dichlorphenoxyacetic acid (2,4-D) which are different in concentrations and combinations are added to the base medium. According to the one-step plantlet formation method, the test-tube plantlet leaf serving as an explant is inoculated, then is induced by a callus and is directly differentiated into an adventitious bud and an adventitious root, so that the lamiophlomis rotata regenerated plantlet is obtained by one step. By adopting the one-step plantlet formation method, the callus obtained by induction in an initial medium does not need to be transferred into a differential medium for regenerating the plantlet. Compared with an isolated culture multi-step regeneration plantlet formation method, the one-step plantlet formation method has the advantages that the steps are simplified, the culture period is short, the repeatability is good, and the 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, the present invention also provides a kind of method of optimizing this medium.
Background technology
Lamiophlomis rotata Lamiophlomis rotata (Benth.) Kudo, be Labiatae, the lamiophlomis rotata platymiscium, and this genus only has kind of lamiophlomis rotata.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 short as plant, stem is short, well developed root system, and blade is larger, pastes ground and launches.It is large etc. relevant that these features and its growing environment are that high and cold anoxic, dry wind are large, radiation reaches by force day and night temperature.
Lamiophlomis rotata belongs to traditional Tibetan medicine, and all herbal medicine of using among the people is controlled that traumatic injury, arthralgia and myalgia, the stagnation of the circulation of vital energy are sprained one's back, flowed yellow water, the long-pending yellow water in joint, cancellous bone inflammation after edema.In addition according to Qinghai to the disconnected hemostasis trial of big white mouse femoral artery, arteria brachialis, arteria carotis crosscut, think that this kind has haemostatic effect preferably.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.To the research work of lamiophlomis rotata being introduced a fine variety to low altitude area, also there is part to carry out.As Jin Lan etc. by by lamiophlomis rotata rhizome bud from the Experimental Base that wildly is transplanted to height above sea level 2366m and 3100m of height above sea level 4300m, to transplanting the Pollen Activity of ground lamiophlomis rotata, pollen germination and natural end rate are added up, found that lamiophlomis rotata be transplanted to low height above sea level after Pollen Activity low, on column cap, without pollen germination, Natural seed setting rate is 0.Therefore, the seedling in low altitude area to lamiophlomis rotata obtains, more feasible in the Plant Tissue Breeding mode.But, by the research to medium component, adopt the once technology of 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 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 that makes its forming seedling through one step culture.Apply this lamiophlomis rotata one-step culture base and this cultural method, the lamiophlomis rotata test-tube plantlet blade is after forming callus, do not need to transfer on differential medium, but direct seedling differentiation on former medium, improved lamiophlomis rotata test-tube plantlet blade callus regeneration efficiency, shortened incubation time, simplified operating procedure, for the rejuvenation of lamiophlomis rotata good seed is fast numerous and breed improvement provides an effective way.
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 be take the MS medium as basal medium, and in 1L MS medium, also need to add following component, and make the final concentration of each component as follows:
The 6-BA(6-benayl aminopurine) 1.0～2.0mg/L;
The NAA(methyl α-naphthyl acetate) 0.5～1.5mg/L;
2,4-D(2, the 4-dichlorphenoxyacetic acid) 0～0.1mg/L.
Described lamiophlomis rotata isolated culture one-step seedling medium, take MS as basal medium, and the final concentration of each component of hormone added is:
6-BA 2.0mg/L；
NAA 1.0mg/L。
A kind of cultural method of lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, comprise the steps:
(1) according to described formulated lamiophlomis rotata isolated culture one-step seedling medium, and carry out sterilization treatment;
(2) choose the consistent lamiophlomis rotata in vitro cuttings in source, under aseptic condition, cut the about 0.5cm * 0.5cm of the blade launched fully on top, be affixed on the described medium of step 1 with vacuum side of blade;
(3) medium after the inoculation of step (2) gained being placed in to temperature is under 18～20 ℃ of conditions, full dark culturing, after 7～10d, carry out again illumination 12～16h every day, 1000～1200Lx, after cultivating 30～40d, callus forms, after cultivating 50～60d, the statistics callus differentiates indefinite bud and adventive root gradually, forms test-tube plantlet;
(4) by test-tube plantlet through conventional hardening technology, carry out test-tube seedling transplanting, after 30d, statistics is transplanted 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 forming 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 are high, easy to operate, the advantages such as the cultivation program is simple, for lamiophlomis rotata provides good method at the low altitude area seedling fostering.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
A kind of method of optimizing 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:
With the MS medium that do not add any hormone in contrast, adopt the L of Three factors-levels
9(3
4) Orthogonal Experiment and Design, in the MS medium, 6-BA, NAA and 2, the 4-D of interpolation variable concentrations and combination, be mixed with 10 groups of different lamiophlomis rotata one-step culture bases.In each group, the concentration of the 6-BA contained, NAA and 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)
By after medium sterilization, choose the consistent lamiophlomis rotata in vitro cuttings in source, under aseptic condition, cut the blade that launch fully on top, and cut about 0.5cm * 0.5cm big and small blade, with vacuum side of blade, be affixed on the medium of research; 6 explants of every bottle graft kind, 10 bottles of each combination inoculations; It is under 20 ℃ of conditions that the rear medium of inoculation is placed in temperature, and full dark culturing, carry out 16h illumination cultivation every day after 7～10d, and light intensity is 1200Lx; After illumination cultivation 20d, callus induction rate as seen from the results in Table 2, produce the explant number of callus/inoculation explant number * lO0%, and the formational situation of callus.Continue to add up the Calli Differentiation result after illumination cultivation 50d in former medium, participate in table 2, be i.e. the callus piece number of Bud Differentiation/total callus piece number * 100%, and seedling situation.
The impact that table 2 different hormone combinations is induced Callus of Leaf
The impact of table 3 different hormone combinations on the 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 is organized lamiophlomis rotata one-step culture base and all can induce preferably the lamiophlomis rotata blade to form callus, 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 callus quality preferably, 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 is failed 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 is analyzed concentration and the combination of each hormone in the 2nd, 4,6,8 groups of corresponding medium again, and wherein, 6-BA is the 2nd, and the concentration in 4,6,8 groups is followed successively by 1.0mg/L, 1.5mg/L, 1.5mg/L, 2.0mg/L; NAA is the 2nd, and the concentration in 4,6,8 groups is followed successively by 1.0mg/L, 0.5mg/L, 1.5mg/L, 1.0mg/L; 2,4-D is the 2nd, and the concentration in 4,6,8 groups is followed successively by 0.1mg/L, 0.1mg/L, 0mg/L, 0mg/L.Table 4 is that the test-tube plantlet hardening survives number and survival rate, and best is the 8th group.
In summary, can make the lamiophlomis rotata nutrient media components of lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling be: to take the MS medium as basal medium, and in 1L MS medium, also need the component added and the final concentration of each component to be:
6-BA 2.0mg/L；
NAA 1.0mg/L。
Embodiment 2
The error of the experimental result caused for fear of human factor, the applicant asks different researchers under the same conditions, 6 of every bottle graft kinds, inoculate altogether 8 bottles, 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
The impact of table 6 different hormone combinations on the 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 organizes lamiophlomis rotata one-step culture base all can induce the lamiophlomis rotata blade preferably, and its inductivity all reaches more than 80%, and the 1st, 3,4,5,6,8 and 9 groups, inductivity reaches more than 90%.In analytical table 5, data are known again, and the callus in the 3rd group is failed 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; With the 6th, the differentiation rate of 8 groups is better, and the number of days that sprouts is shorter, and Multiple Buds quantity is many and growing way is 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, concentration and the combination of each hormone in known the 8th group of corresponding medium, 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, the result of embodiment 1 and embodiment 2 is basic identical, has repeatability preferably.
Embodiment 3
A kind of method of lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, comprise the steps:
(1) preparation lamiophlomis rotata one-step culture base: in the 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 about 0.5cm * 0.5cm of the blade launched fully on top, be affixed on the described medium of step 1 with vacuum side of blade;
(3) medium after the inoculation of step (2) gained being placed in to temperature is 20 ℃ of incubators, and full dark culturing, carry out the 16h/d illumination cultivation after 7～10d, and light intensity is 1200Lx;
(4) after illumination cultivation 55d, obtain the lamiophlomis rotata regeneration plant in first culture base.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (3)
1. a lamiophlomis rotata isolated culture one-step seedling medium, make lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, it is characterized in that, described medium be take the MS medium as basal medium, and, in 1L MS medium, also need to add following component, and make the final concentration of each component as follows:
6-BA 1.0～2.0mg/L；
NAA 0.5～1.5mg/L；
2，4-D 0～0.1mg/L。
2. lamiophlomis rotata isolated culture one-step seedling medium according to claim 1, is characterized in that, take MS as basal medium, the final concentration of each component of hormone added is:
6-BA 2.0mg/L；
NAA 1.0mg/L。
3. the cultural method of a lamiophlomis rotata test-tube plantlet blade isolated culture one-step seedling, is characterized in that, comprises the steps:
(1) according to the described formulated lamiophlomis rotata of claim 1 or 2 isolated culture one-step seedling medium, and carry out sterilization treatment;
(2) choose the consistent lamiophlomis rotata in vitro cuttings in source, under aseptic condition, cut the about 0.5cm * 0.5cm of the blade launched fully on top, be affixed on the described medium of step 1 with vacuum side of blade;
(3) medium after the inoculation of step (2) gained being placed in to temperature is under 18～20 ℃ of conditions, and full dark culturing, after 7～10d, then carry out illumination 12～16h, 1000～1200Lx every day.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106171992A (en) * | 2016-07-15 | 2016-12-07 | 四川禾木本业农林科技有限公司 | A kind of Gesneriaceae forming seedling through one step culture tissue culture and rapid propagation method |
| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2013
- 2013-09-23 CN CN201310437034.2A patent/CN103461138B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|
| 刘正玉等: "独一味试管苗快繁技术研究", 《西藏农业科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106171992A (en) * | 2016-07-15 | 2016-12-07 | 四川禾木本业农林科技有限公司 | A kind of Gesneriaceae forming seedling through one step culture tissue culture and rapid propagation method |
| CN106171992B (en) * | 2016-07-15 | 2019-03-15 | 四川七彩林业开发有限公司 | A kind of Gesneriaceae forming seedling through one step culture tissue culture and rapid propagation method |
| 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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