CN106171986B - A kind of Pterospermi Heterophylli seed sprouts the tissue culture and rapid propagation method of approach - Google Patents

A kind of Pterospermi Heterophylli seed sprouts the tissue culture and rapid propagation method of approach Download PDF

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CN106171986B
CN106171986B CN201610546890.5A CN201610546890A CN106171986B CN 106171986 B CN106171986 B CN 106171986B CN 201610546890 A CN201610546890 A CN 201610546890A CN 106171986 B CN106171986 B CN 106171986B
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culture
medium
illumination
callus
culture medium
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CN106171986A (en
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黄肇宇
梁伟江
卢海啸
韦小红
陈柑宏
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Yulin Normal University
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Yulin Normal University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants

Abstract

The invention discloses the tissue culture and rapid propagation method that a kind of Pterospermi Heterophylli seed sprouts approach, comprise the following steps：The foundation of step 1, sterilizable material；Step 2, seed sprout culture；Step 3, evoked callus；Step 4, callus proliferation；Step 5, callus differentiation；Step 6, inducing clumping bud；After step 7, the Multiple Buds for the differentiation for obtaining step 6 cut off base portion, it is inoculated in culture medium, obtains strong sprout；Step 8：After the strong sprout that step 7 is obtained cuts off base portion, it is inoculated in culture medium, carries out culture of rootage.It is an object of the invention to provide a kind of complete method for tissue culture of Pterospermi Heterophylli quick, appreciation rate is high, survival rate is high.

Description

A kind of Pterospermi Heterophylli seed sprouts the tissue culture and rapid propagation method of approach
Technical field
The present invention relates to a kind of field of plant tissue culture technique, more particularly to a kind of Pterospermi Heterophylli seed to sprout the group of approach Tissue culture method for fast propagation.
Background technology
Pterospermi Heterophylli (Semiliquidambar cathayensis H.T.Chang) is Hamamelidaceae Pterospermi Heterophylli platymiscium, For the type sepecies of the new category Pterospermi Heterophylli category delivered in 1962, China endemic species.This kind is in academic research, traditional Chinese medicine is used and gardens Using etc. many aspects all there is high value.But because irrational utilization, its destruction Of resources are serious.Pterospermi Heterophylli exists The promulgations such as State Bueau of Environmental Protection in 1987《Chinese rare endangered plants register (first)》In, it is listed in country three Level emphasis level protection plant[2]；Edited in Fourier state in 1992《Chinese Plants Red Data Book-rare extinction plants》It is middle to be arranged For rare species[3]；Promulgated within 4th in State Council's August in 1999《National key protected wild plants register (first)》Middle lifting For II grade of protection plant of country.2003 Wang Song, Xie Yan chief editor's《Chinese species Red List》In, expert provides according to correlation Material, speculate that Pterospermi Heterophylli Threatening factors within 3 generations of past do not stop, population at least reduces 30%, therefore Pterospermi Heterophylli is arranged Enter easily danger grade, it is higher to face the probability died out for its population within following a period of time[5]。
Pterospermi Heterophylli medical value：Pterospermi Heterophylli is a kind of precious medicinal plant, and root, branch, bark can all be used as medicine, and can be invigorated blood circulation logical Network, dispelling wind and eliminating dampness, rheumatic arthritis can be treated, lumbar muscle strain, hemiplegia, bruise stasis of blood product, is swollen and ache, postpartum paralysis, outside Wound is usually used in stopping blooding, and is the Chinese medicine that the producing region masses commonly use.At present, Pterospermi Heterophylli has obtained extensive attention as important Chinese medicine And exploitation.Such as Banfenghe San, Chinese medicine patcher, Pterospermi Heterophylli class parenteral solution, seedling ridge bone strengthening capsules.
Medicinal Pterospermi Heterophylli species differentiates：In China's different places, it is " Pterospermi Heterophylli " and makees medicinal plant and share 6 sections 8 to belong to 15 kinds, such as the Sassafras tsumu Hemsl (Dendvopanax dentigerus) of Araliaceae, the velutinous cinquefoil root tree (Pterospermum of Sterculiaceae Heterophyllum), and Lauraceae sassafras (Sassafras tzumu), effectiveness is identical with the Pterospermi Heterophylli of Hamamelidaceae, all It is to be used as medicine with root, stem or bark, but the Pterospermi Heterophylli (Semiliquidambar cathayensis H.T.Chang) of Hamamelidaceae Effect is optimal[9-10]。
2. the existing tissue culture technique situation of Pterospermi Heterophylli
In terms of Pterospermi Heterophylli tissue cultures, Chen Shihong etc.[11]Compared using single-factor and uniform design, have studied respectively Influence Pterospermi Heterophylli callus induction and the various factors of growth.Result of the test shows：Callus of Leaf inductivity is higher than stem Section；The most suitable minimal medium of callus induction is MS, and the optimum medium of growth is：MS+6-BA2.0mg/L+ NNA1.0mg/L+ sucrose 3%, light culture is extremely advantageous to callus growth, the root induction on root media, but repeatedly Experiment is showed no adventitious root.
Li Guozhen etc.[12]Using uniform design and single-factor comparison method, different minimal mediums, illumination condition, sugarcane are investigated Sugared concentration, and hormon species, concentration and combination are to Pterospermi Heterophylli callus induction, squamous subculture and the shadow broken up again Ring.Test result indicates that：Callus of Leaf inductivity is higher than stem section.The most suitable minimal medium of callus induction is MS, The optimum medium of growth is：MS+6-BA1.0mg/L+NAA1.0mg/L+ sucrose 3%.Dark culturing is to callus growth pole To be favourable.But root induction on root media, but it is showed no adventitious root through test of many times.
The endangered plants Pterospermi Heterophylli Semiliquidambar cathayensis tissue culture quick breeding technical research such as Hu Gang, To give birth to stem section of the Pterospermi Heterophylli with axillary bud and spire then as explant, carry out tissue cultures.Result of study shows：Heterophyllous wingseedtree leaf Piece is in Initial culture base：On MS+BA2.0mg/L+NNA0.5mg/L, callus induction rate 92%, bud induction rate is 16.7%, axillary bud is in Initial culture base：On MS+BA1.0mg/L+NNA0.1mg/L, axillary bud deriving rate is 58%, and stem section can be directly Induction budding is the fast numerous main explant of Pterospermi Heterophylli.Newly sprout in subculture multiplication medium：MS+BA0.5mg/L+ On NAA0.05mg/L, bud growth coefficient is 3.93, proliferated culture medium：MS+BA (1~0.5mg)/L+NAA (0.01~ 0.05mg)/L, preferable Multiple Buds root media are advisable with 1/2WPM+NAA2mg/L+ activated carbon 0.2%.Rooting rate can reach More than 90%.Pine forest fertile soil+perlite (4：1) relatively it is adapted to the growth transplanting survival rate 100% of Pterospermi Heterophylli, nursery stock growing way It is good, leaf green, it is possible to provide Pterospermi Heterophylli high quality seedling.
3. the deficiency of existing tissue culture technique
Requirement of the Pterospermi Heterophylli to ecological environment is harsher, and its natural propagation rate is relatively low, is adopted plus the long-term excess of people Dig, wild resource is extremely exhausted.In face of so severe form, it is seen that the research of the artificial propagation problem to Pterospermi Heterophylli is Extremely important.Plant tissue Sterile culture method is to seek to solve Pterospermi Heterophylli resource exhaustion, protect the effective of its improved seeds Approach.In recent years, people are increasing to the medicinal ingredient of Pterospermi Heterophylli and the research of value, and Pterospermi Heterophylli is sprouted from seed at present Hair, the sterile tissue culture studies to the entire flow of propagation strengthening seedling and rooting are had not been reported, and tissue culture sterile culture is quickly bred Aspect is more to be induced using stem section and blade as explant.
The content of the invention
It is an object of the invention to provide a kind of complete tissue training of Pterospermi Heterophylli quick, appreciation rate is high, survival rate is high The method of supporting.
The technical scheme is that：A kind of Pterospermi Heterophylli seed sprouts the tissue culture and rapid propagation method of approach, comprises the following steps：
The foundation of step 1, sterilizable material：Pterospermi Heterophylli capsule is taken to take seed to carry out aseptic process after drying；
Step 2, seed sprout culture：It is inoculated in culture medium and cultivates after seed disinfection after step 1 is handled, is sprouted The seed of hair；
Step 3, evoked callus：The hypocotyl of the seed of sprouting after step 2 is handled is as induced material, shearing Into after stem section, it is inoculated in culture medium, culture obtains callus；
Step 4, callus proliferation：The callus that step 3 is obtained is transferred in culture medium, carries out Multiplying culture；
Step 5, callus differentiation, the callus after step 4 is bred are cut into block and are transferred to medium culture, obtain To the tissue with true leaf；
Step 6, inducing clumping bud, it is inoculated into after the true leaf in step 5 is cut in culture medium, that is broken up grows thickly Bud；
After step 7, the Multiple Buds for the differentiation for obtaining step 6 cut off base portion, it is inoculated in culture medium, obtains strong sprout；
Step 8：After the strong sprout that step 7 is obtained cuts off base portion, it is inoculated in culture medium, carries out culture of rootage.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, the culture medium in described step 2 includes Following components：Modified MS medium, GA30.2~0.8mg/L, VB21.0~5.0mg/L, white sugar 30g/L, agar 3.6g/L；
The cultivation temperature of described step 2 is 25~27 DEG C, and intensity of illumination is 1500~2000lx, light application time is 10 small When/day.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, the culture medium in described step 3 includes Following components：Modified MS medium, 6-BA0.5mg/L, 2,4-D0.2~0.5mg/L, VB21.0~6.0mg/L, white sugar 30g/ L, agar 3.6g/L；
The cultivation temperature of described step 3 is 25~27 DEG C, and intensity of illumination is 1500~2000lx, light application time is 10 small When/day.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, the culture medium in described step 4 includes Following components：Modified MS medium, 6-BA0.3~0.5mg/L, NAA0.05~0.4mg/L, VB21.0~3.0mg/L, white sugar 30g/L, agar 3.6g/L；
The cultivation temperature of described step 4 is 25~27 DEG C, and intensity of illumination is 1500~2000lx, light application time is 10 small When/day.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, the culture medium in described step 5 includes Following components：Modified MS medium, 6-BA0.03~0.08mg/L, NAA0.05~0.3mg/L, native white sugar 30g/L, agar 3.6g/L；
The cultivation temperature of described step 5 is 24~28 DEG C, intensity of illumination is 2000~3000lx, light application time is 10 small When/day.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, the culture medium in described step 6 includes Following components：Modified MS medium, 6-BA0.5~0.8mg/L, NAA0.1~0.3mg/L, VB21.0~5.0mg/L, white sugar 30g/L, agar 3.6g/L；
The intensity of illumination of described step 6 is 1500~2000lx, daily illumination cultivation 12 hours.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, the culture medium in described step 7 includes Following components：Modified MS medium, NAA1.0~2.0mg/L, 6-BA0.1~1.0mg/L, VB22.0mg/L、GA31.0mg/L、 Sucrose 30g/L, agar 3.6g/L；
The intensity of illumination of described step 7 is 2000~3000lx, daily illumination cultivation 12 hours.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, the culture medium in described step 8 includes Following components：1/2 modified MS medium, NAA1.0mg/L, IBA1.0mg/L, GA32.0mg/L, sucrose 30g/L, agar 3.6g/ L；
The intensity of illumination of described step 8 is 2000~3000lx, illumination cultivation 12 hours.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, NH in described modified MS medium4NO3 Content is 340mg/L, KNO3Content is 1318mg/L.
In the tissue culture and rapid propagation method that above-mentioned Pterospermi Heterophylli seed sprouts approach, wrapped in 1/2 described modified MS medium Contain：
NH4NO3170mg/L；
KNO3659mg/L；
MgSO4·7H2O 185mg/L；
KH2PO485mg/L；
CaCl2·2H2O 220mg/L；
MnSO4·4H2O 22.3mg/L；
ZnSO4·7H2O 8.6mg/L；
H3BO3 6.2mg/L；
KI 0.83mg/L；
Na2MoO4·2H2O 0.25mg/L；
CuSO4·5H2O 0.025mg/L；
CoCl2·6H2O 0.025mg/L；
FeSO4·7H2O 27.8mg/L；
Na2-EDTA·2H2O 37.3mg/L；
Glycine 2.0mg/L；
Thiamine hydrochloride 0.1mg/L；
Puridoxine hydrochloride 0.5mg/L；
IV B nicotinic acid 0.5mg/L；
Inositol 100mg/L.
Beneficial effects of the present invention are as follows：
The present invention on the basis of studying Pterospermi Heterophylli, passes through a large amount of increasings to seed seedling using seed as explant The Multiple Buds to be formed are grown, Multiple Buds is carried out strengthening seedling and rooting under the influence of different factors, to strengthen test tube seedling vitality, with Shorten and adapt to the external environment time so as to improve survival rate.
The present invention complete technology path be：Using seed as explant evoked callus callus proliferation, grow thickly Bud induction, the complete rapid propagation system of Pterospermi Heterophylli is set up finally by strong sprout and culture of rootage.
The present invention inductivity be with seed sprout be seedling, inductivity reaches 93%.Seedling is induced to form diameter big The small light green color callus in 0.8~1.6cm, its growth coefficient is set to reach 10.8 through callus proliferation medium.Induction Multiple Buds simultaneously make its value-added coefficient reach 7.9.3.0~4.0cm of height of seedling, strong sprout rate 100% (strong sprout rate (%)=(strong sprout strain number/ It is inoculated with strain number) x100%)[14].Rooting rate is 94.6%.Substantial amounts of Pterospermi Heterophylli tissue culture can be rapidly cultivated using the method Seedling, so as to effectively protect and breed the species, meets the needs of market is to Pterospermi Heterophylli.
Brief description of the drawings
Fig. 1 and 2 is that the seed of embodiment 1 sprouts cultivation results；
Fig. 3 and 4 is the callus induction and increment result of embodiment 1；
Fig. 5 and 6 is the callus differentiated result of embodiment 1；
Fig. 7 and 8 is the inducing clumping bud result of embodiment 1；
Fig. 9,10 and 11 are the strong seedling culture result of embodiment 1；
Figure 12,13,14 and 15 are the culture of rootage result of embodiment 1.
Embodiment
With reference to embodiment, technical scheme is described in further detail, but not formed pair Any restrictions of the present invention.
Embodiment 1
A kind of Pterospermi Heterophylli seed sprouts the tissue cultures of approach
(1) the sprouting culture of Pterospermi Heterophylli seed：Ripe wild Pterospermi Heterophylli capsule of that duty of Guangxi Fangcheng Port will be taken at, will Ripe Pterospermi Heterophylli capsule, dry to ninety percent dry rear taking-up seed, as explant raw material；The seed chosen is clean with washing Seminal plasma is washed, and is placed on superclean bench, then soaks 5~7min with 0.1% sodium hypochlorite stoste, then with aseptic water washing 5 ~8 times；Be inoculated in after explant is sterilized in sterile inducing culture, temperature be 25~27 DEG C, intensity of illumination be 1500~ 2000lx, light application time is cultivate under conditions of 10 hours/day, 15 days seed germination rates reach 77%, and germination rate reaches after 30 days To 93%.See accompanying drawing 1 and Fig. 2.
Seed germination medium is：
B1：Improve MS+GA30.2mg/L+VB21.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B2：Improve MS+GA30.5mg/L+VB21.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B3：Improve MS+GA30.8mg/L+VB22.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B4：Improve MS+GA30.8mg/L+VB23.5mg/L+ white sugar 30g/L+ agar 3.6g/L；
(2) induction and increment of Pterospermi Heterophylli callus：The hypocotyl after Pterospermi Heterophylli seed is sprouted is chosen as induction material Material, after cutting into 1.5~2.0cm stem section, be inoculated in sterile inducing culture temperature be 25~27 DEG C, intensity of illumination be 1500~2000lx, light application time form diameter in 0.8~1.6cm to be cultivated 20~30 days under conditions of 10 hours/day Jade-green callus；Well-grown callus is transferred in proliferated culture medium, carries out Multiplying culture culture Environmental condition is：25~27 DEG C of temperature, 1500~2000lx of illumination, hour/day of light application time 10.Growth coefficient reaches within 45 days 10.8, callus color is dark green.See accompanying drawing 3 and 4.
Evoked callus culture composition：
B5：Improve MS+6-BA0.5mg/L+2,4-D0.2mg/L+VB21.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B6：Improve MS+6-BA0.5mg/L+2,4-D0.2mg/L+VB26.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B7：Improve MS+6-BA0.5mg/L+2,4-D0.5mg/L+VB21.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B8：Improve MS+6-BA0.5mg/L+2,4-D0.5mg/L+VB26.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B9：Improve MS+6-BA0.5mg/L+2,4-D0.2mg/L+VB23.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
Evoked callus increment medium component：
B10：Improve MS+6-BA0.3mg/L+NAA0.05mg/L+VB22.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B11：Improve MS+6-BA0.3mg/L+NAA0.1mg/L+VB23.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B12：Improve MS+6-BA0.5mg/L+NAA0.3mg/L+VB21.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B13：Improve MS+6-BA0.5mg/L+NAA0.4mg/L+VB22.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
(3) differentiation of Pterospermi Heterophylli callus.Callus is cut into 1.0cm2The block of size is transferred to differentiation culture In base, under conditions of temperature is 24~28 DEG C, intensity of illumination is 2000~3000lx, light application time is 10 hours/day, culture 45~60 days, callus grew root before this, started long budding part in root long to 2.0cm or so, bud is high about after 45 days 1.5cm, true leaf 2-4 pieces；See accompanying drawing 5 and 6.
B14：Improve MS+6-BA0.5mg/L+NAA0.1mg/L+VB22.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B15：Improve MS+6-BA0.5mg/L+NAA0.3mg/L+VB23.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B16：Improve MS+6-BA0.8mg/L+NAA0.1mg/L+VB24.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B17：Improve MS+6-BA0.8mg/L+NAA0.3mg/L+VB25.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
(4) Pterospermi Heterophylli inducing clumping bud.After seed is sprouted 30 days, 2~4 true leaves have typically been grown, you can by its belt segment Stem cut and be connected in the culture medium of induction adventitious buds proliferation, be placed in intensity of illumination to cultivate under 1500~2000lx, daily Illumination cultivation 12 hours；The high about 3.0~5.0cm of plant after cultivating 30-40 days, growth coefficient 2.8.See accompanying drawing 7 and Fig. 8.
Inducing clumping bud medium component：
B18：Improve MS+6-BA0.05mg/L+NAA0.1mg/L+VB22.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B19：Improve MS+6-BA0.05mg/L+NAA0.3mg/L+VB225mg/L+ white sugar 30g/L+ agar 3.6g/L；
B20：Improve MS+6-BA0.08mg/L+NAA0.1mg/L+VB21.0mg/L+ white sugar 30g/L+ agar 3.6g/L；
B21：Improve MS+6-BA0.08mg/L+NAA0.3mg/L+VB23.5mg/L+ white sugar 30g/L+ agar 3.6g/L；
(5) Pterospermi Heterophylli strong seedling culture：Multiple Buds through differentiation are cut off into base portion, are inoculated in following strong seedling culture base：And Intensity of illumination is placed in be cultivated under 2000~3000lx, daily illumination cultivation 12 hours；Plant is high by about 3.0 after cultivating 30-40 days ~4.0cm, radical 4~6,2~3cm of root long.See accompanying drawing 9, Figure 10, Figure 11.
Strong seedling culture base：
B22：Improve MS+NAA1.0mg/L+6-BA0.5mg/L+VB22.0mg/L+GA31.0mg/L+ sucrose 30g/L+ agar 3.6g/L；
B23：Improve MS+NAA1.5mg/L+6-BA0.1mg/L+VB22.0mg/L+GA31.0mg/L+ sucrose 30g/L+ agar 3.6g/L；
B24：Improve MS+NAA2.0mg/L+6-BA0.1mg/L+VB22.0mg/L+GA31.0mg/L+ sucrose 30g/L+ agar 3.6g/L；
B25：Improve MS+NAA2.0mg/L+6-BA1.0mg/L+VB22.0mg/L+GA31.0mg/L+ sucrose 30g/L+ agar 3.6g/L；
(6) Pterospermi Heterophylli culture of rootage：Plant after strong sprout is cut off into base portion to be inoculated in root media：It is placed in light It is to be cultivated under 2000~3000lx according to intensity, daily illumination cultivation 12 hours；Culture 30~40 days after plant it is high by about 3.0~ 4.0cm, 80% goes out root, radical 4~6,2~3cm of root long.See accompanying drawing 12, Figure 13, Figure 14, Figure 15.
Culture of rootage based component：
B26：1/2 improvement MS+NAA1.0mg/L+IBA1.0mg/L+GA32.0mg/L+ sucrose 30g/L+ agar 4g/L；
B27：1/2 improvement MS+NAA2.0mg/L+IBA1.0mg/L+GA32.0mg/L+ sucrose 30g/L+ agar 4g/L；
B28：1/2 improvement MS+NAA1.0mg/L+IBA2.0mg/L+GA32.0mg/L+ sucrose 30g/L+ agar 4g/L；
B29：1/2 improvement MS+NAA2.0mg/L+IBA2.0mg/L+GA32.5mg/L+ sucrose 30g/L+ agar 4g/L；
Table 1, table 2 and table 3 show improvement MS, 1/2 improvement MS, MS component.
Table 1 improves MS
Table 2 1/2 improves MS
Table 3MS
Above-described is only presently preferred embodiments of the present invention, all timess made in the range of the spirit and principles in the present invention What modifications, equivalent substitutions and improvements etc., should be included in the scope of the protection.

Claims (1)

1. a kind of Pterospermi Heterophylli seed sprouts the tissue culture and rapid propagation method of approach, it is characterised in that：Comprise the following steps：
The foundation of step 1, sterilizable material：Pterospermi Heterophylli capsule is taken to take seed to carry out aseptic process after drying；
Step 2, seed sprout culture：It is inoculated in culture medium and cultivates after seed disinfection after step 1 is handled, is sprouted Seed；Described culture medium includes following components：Modified MS medium, GA30.2~0.8mg/L, VB2It is 1.0~5.0mg/L, white Sugared 30g/L, agar 3.6g/L；Cultivation temperature is 25~27 DEG C, and intensity of illumination is 1500~2000lx, light application time is 10 small When/day；
Step 3, evoked callus：The hypocotyl of the seed of sprouting after step 2 is handled cuts into stem as induced material Duan Hou, it is inoculated in culture medium, culture obtains callus；Described culture medium includes following components：Modified MS medium, 6- BA0.5mg/L, 2,4-D0.2~0.5mg/L, VB21.0~6.0mg/L, white sugar 30g/L, agar 3.6g/L；Cultivation temperature is 25 ~27 DEG C, intensity of illumination is 1500~2000lx, light application time is 10 hours/day；
Step 4, callus proliferation：The callus that step 3 is obtained is transferred in culture medium, carries out Multiplying culture；It is described Culture medium include following components：Modified MS medium, 6-BA0.3~0.5mg/L, NAA0.05~0.4mg/L, VB21.0~ 3.0mg/L, white sugar 30g/L, agar 3.6g/L；Cultivation temperature is 25~27 DEG C, when intensity of illumination is 1500~2000lx, illumination Between be 10 hours/day；
Step 5, callus differentiation, the callus after step 4 is bred are cut into block and are transferred to medium culture, had There is the tissue of true leaf；Described culture medium includes following components：Modified MS medium, 6-BA0.5 or 0.8mg/L, NAA0.05~ 0.3mg/L, native white sugar 30g/L, agar 3.6g/L；Cultivation temperature is 24~28 DEG C, intensity of illumination is 2000~3000lx, illumination Time is 10 hours/day；
Step 6, inducing clumping bud, it is inoculated into after the true leaf in step 5 is cut in culture medium, the Multiple Buds broken up；Institute The culture medium stated includes following components：Modified MS medium, 6-BA0.5~0.8mg/L, NAA0.1~0.3mg/L, VB21.0~ 5.0mg/L, white sugar 30g/L, agar 3.6g/L；Intensity of illumination is 1500~2000lx, daily illumination cultivation 12 hours；
After step 7, the Multiple Buds for the differentiation for obtaining step 6 cut off base portion, it is inoculated in culture medium, obtains strong sprout；Described Culture medium includes following components：Modified MS medium, NAA1.0~2.0mg/L, 6-BA0.1~1.0mg/L, VB22.0mg/L、 GA31.0mg/L, sucrose 30g/L, agar 3.6g/L；Intensity of illumination is 2000~3000lx, daily illumination cultivation 12 hours
Step 8：After the strong sprout that step 7 is obtained cuts off base portion, it is inoculated in culture medium, carries out culture of rootage；Described culture Base includes following components：1/2 modified MS medium, NAA1.0mg/L, IBA1.0mg/L, GA32.0mg/L, sucrose 30g/L, fine jade Fat 3.6g/L；Intensity of illumination is 2000~3000lx, illumination cultivation 12 hours；
Included in described modified MS medium：NH4NO3340mg/L；KNO31318mg/L；MgSO4·7H2O370mg/L； KH2PO4170mg/L；CaCl2·2H2O440mg/L；MnSO4·4H2O22.3mg/L；ZnSO4·7H2O8.6mg/L； H3BO36.2mg/L；KI0.83mg/L；Na2MoO4·2H2O0.25mg/L；CuSO4·5H2O0.025mg/L；CoCl2· 6H2O0.025mg/L；FeSO4·7H2O27.8mg/L；Na2-EDTA·2H2O37.3mg/L；Glycine 2.0mg/L；Thiamine hydrochloride Plain 0.1mg/L；Puridoxine hydrochloride 0.5mg/L；IVB nicotinic acid 0.5mg/L；Inositol 100mg/L；
Included in 1/2 described modified MS medium：NH4NO3170mg/L；KNO3659mg/L；MgSO4·7H2O185mg/L； KH2PO485mg/L；CaCl2·2H2O220mg/L；MnSO4·4H2O22.3mg/L；ZnSO4·7H2O8.6mg/L； H3BO36.2mg/L；KI0.83mg/L；Na2MoO4·2H2O0.25mg/L；CuSO4·5H2O0.025mg/L；CoCl2· 6H2O0.025mg/L；FeSO4·7H2O27.8mg/L；Na2-EDTA·2H2O37.3mg/L；Glycine 2.0mg/L；Thiamine hydrochloride Plain 0.1mg/L；Puridoxine hydrochloride 0.5mg/L；IVB nicotinic acid 0.5mg/L；Inositol 100mg/L.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102301958A (en) * 2011-07-27 2012-01-04 地缘（厦门）生物科技有限公司 Method for culturing in vitro tissues of Semiliquidambar cathayensis
CN105325185A (en) * 2015-12-04 2016-02-17 湖南省森林植物园 Grafting propagation method for semiliquidambar cathayensis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102301958A (en) * 2011-07-27 2012-01-04 地缘（厦门）生物科技有限公司 Method for culturing in vitro tissues of Semiliquidambar cathayensis
CN105325185A (en) * 2015-12-04 2016-02-17 湖南省森林植物园 Grafting propagation method for semiliquidambar cathayensis

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Molecular evidence for natural intergeneric hybridization between Liquidambar and Altingia;Wei Wu,et al.;《J Plant Res》;20091126;第123卷;第231-239页 *
半枫荷实生播种育苗技术;唐邦权等;《林业科技》;20121130;第37卷(第6期);第33-34页 *
濒危植物半枫荷Semiliquidambar cathayensis 组织培养快繁技术研究;胡刚等;《种子》;20121231;第31卷(第12期);第116-120页 *

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