CN106613970B - The quick breeding by group culture method of sealwort leaf elegant jessamine - Google Patents
The quick breeding by group culture method of sealwort leaf elegant jessamine Download PDFInfo
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- CN106613970B CN106613970B CN201611117758.9A CN201611117758A CN106613970B CN 106613970 B CN106613970 B CN 106613970B CN 201611117758 A CN201611117758 A CN 201611117758A CN 106613970 B CN106613970 B CN 106613970B
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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
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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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a kind of quick breeding by group culture methods of sealwort leaf elegant jessamine, include the following steps：1), select the adventitious bud of the root-like stock of sealwort leaf elegant jessamine as explant；2), the disinfection of adventitious bud；3), the Primary culture of adventitious bud：It will be inoculated into primary culture medium after the blade of adventitious bud stripping periphery after disinfection, carry out adventitious bud growth and the induction of Multiple Buds；4), the rapid amplifying of adventitious bud：It is divided into after the bud clump containing 1 sprouting switching in being cultivated on proliferated culture medium the Multiple Buds obtained by step 3)；5), the elongation of adventitious bud and culture of rootage：Multiple Buds obtained by step 4) are cut into be transferred to for the one clump of adventitious bud clump containing 2~3 plants take root/elongation medium on cultivate；6), the plant of having taken root obtained by step 5) is taken out, obtain can bottle outlet plantation seedling.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of quick breeding by group culture method of sealwort leaf elegant jessamine.
Background technology
Plant is the natural treasure-house of drug, and human use's medicinal plant is come existing thousands of years history of preventing and curing diseases.Plant is closed
It is widely used in the industries such as drug, foods and cosmetics at the secondary metabolites with accumulation.China is medicinal plant in the world
Most abundant one of the country of resource, with the fast development of Chinese Medicine Industry, demand of the home and abroad to natural resources of Chinese medicinal materials is increasing rapidly
Add, China also there are a large amount of extracts from medicinal plant to export to all over the world every year, these all accelerate China's Chinese medicine
The consumption of resource, to resources of medicinal plant in imminent danger bring it is destructive endanger (Xiao Chen Shilin Baconic, 2006；High text far Xiao ＆
Baconic, 2008).Industrialization is also constantly increasing the pressure of natural resources plus urbanization.Due to ecological environment destruction and plunder
The excessive harvesting for taking formula by force causes many natural resources of Chinese medicinal materials amounts of containing to decline, or even exhausts, some types it is endangered (Huang Luqi,
2001).Flourishing for biotechnology provides a brand-new method fundamentally to change the production of traditional medicine.Fast
The horizontal to meet the needs of drug and reduce harvesting natural resources in situ of useful secondary metabolites is protected and enhances in speed breeding
Aspect, plant tissue culture technique possess huge potentiality (Bapat etc., 2008).
Sealwort leaf Gelsemium (Croomia) plant is Stemonaceae (Stemonaceae) herbaceos perennial, 4 base of flower portion
Number, monocot genus plant netted veins monoid.Only 6 kinds of the category, 4 kinds (C.heterosepala, C.hyugaensis,
C.saitoana and C.kinoshitae) be Japan it is peculiar (Kato＆Ebihara, 2011；Kadota, 2010,2012), a kind
(C.japonica) it is China and Japan's distribution, a kind (C.pauciflora) is that the U.S. is peculiar.The overall distribution of the platymiscium is in
Ancient East Asia and North America Disjunct distribution is rendered as the Distribution Pattern on China's Mainland-Japan island again in Asia.The platymiscium
Chromosome research shows 3 kinds of (C.heterosepala, C.japonica and C.pauciflora) chromosome numbers of the category
All be 2n=24 (Duyfjes, 1991；Oginuma etc., 2001), but also there is research to point out sealwort leaf elegant jessamine (C.japonica)
Chromosome number be 2n=26 (Li Linchu, 1986).Li Enxiang's (2006) research shows that the breeding system of the platymiscium is
The mixing mating system of self-fertility.Chloroplast DNA (mono- F of trnL) research shows that：Two kinds of East Asia are sister's monoids, point
The discrimination time is in Pleistocene middle and advanced stage；East Asia kind and the bifurcation solution of U.S.'s kind are in late Pliocene to Late Plestocene-Holocene (Li
Deng 2008).Fang Ming (2012) is based on SSR molecular marker and single-copy nuclear gene Agtl sequences research shows that sealwort leaf elegant jessamine is
Ancestors' kind, and by it derived Japan C.heterosepala and North America C.pauciflora.The Tian Mu Shan Mountain of China can
Can be distribution and the centre of origin of the platymiscium.
Sealwort leaf elegant jessamine platymiscium is that a kind of population is smaller and the kind of compartmentalization distribution, the breeding of population and spread mainly according to
Rely the vegetative propagation of rhizome.Due to rhizome activity it is very limited, make the platymiscium become easily be disturbed, plant easily in imminent danger
(Tomlinson and Ayensu, 1968).Therefore, which is all listed in rare extinction plants name in China, Japan and the U.S.
Record (Fourier state, 1992；Estill&Cruzan,2001；Chafin, 2007；Kato&Ebihara,2011).
Sealwort leaf elegant jessamine also known as Buddha's warrior attendant are big, are distributed in China Zhejiang, Anhui, Jiangxi, Fujian and Japan.The root of the plant
Shape stem is civil common drug, has wind-dispelling removing toxic substances, controls bruise damage, venomous snake bite and other effects (zhejiang plants will, 1994).Woods
The chemical composition of essay etc. (1993) root big to Buddha's warrior attendant and cauline leaf research shows that its main component be croomine
(Croomine) and dehydrocroomine ((didehydrocroomine), in addition there be Pachysamine A root
(pachysamine A) and two steroid alkaloids of croomionidine (Croomionidine) and cupreol..
Genetic variation and genetic differentiation is high between sealwort leaf elegant jessamine population, and the genetic diversity in population is low (li etc., 2008), simultaneously because
Population is smaller, serious by the effect of human activity, therefore urgently protects.Since the setting percentage of this kind of plant is relatively low, pass through
The modes of reproduction of germination is limited, and the breeding by rhizome is the major way of its field breeding, but this method is also very limited.
Therefore, develop and imperative using the method for rapid propagation in vitro.In order to protect wild sealwort leaf elegant jessamine resource, while can expire again
The application of sufficient medical industry, it is therefore necessary to establish the vitro Regeneration System of sealwort leaf elegant jessamine, this is to enriching Plant Tissue Breeding
Theory and Applied Biotechnology protection germ plasm resource and to meet clinical application etc. significant.
So far, the research in terms of sealwort leaf elegant jessamine tissue cultures both at home and abroad or limited and preliminary, only
Only be using the stem section of sealwort leaf elegant jessamine induce callus and prevent callus browning research (Li Wen equalitys, 2012；
Chen Beibei etc., 2012), have no that callus successfully differentiates the report of test tube seedling.
The bibliography being related to is specific as follows：
1、Bapat V A,Yadav SR,Dixit GB.Rescue of endangered plants through
biotechnological applications[J].National Academy Science Letters,2008,31(7&
8):(Bapat V A, Yadav SR, Dixit GB. save endangered plants [J] nationals academy of sciences to 201-211 by biotechnology
Science communicates, 2008,31 (7＆8):201-211)；
2、Chafin L G.Field guide to the rare plants of Georgia[M].University
Of Georgia Press, 2007. (publish the Georgia States the Chafin L G. Universities of Georgia rare plant field guide [M]
Society, 2007.)；
3、Duyfjes B.E.E.Stemonaceae and Pentastemonaceae；with miscellaneous
notes on members of both families[J].Blumea,1991,36:239-252. (Stemonaceae and the squama tuber of stemona
Section：Diversified annotation [J] .Blumea of the two sections member, 1991,36:239-252.)；
4, Estill JC, Cruzan MB.Phytogeography of rare plant species endemic to
the Southeastern United States[J].Castanea,2001,66(1-2):3-23. (Estill JC,
Cruzan MB. southeastern USs are peculiar, plant geography research [J] .Castanea, 2001,66 (1-2) of rare plant:3-
23.)；
5、Kato M&Ebihara A.Endemic plants of Japan[M].Tokai University Press,
2011. (endemic plant [M] the Tokai Universities publishing houses of Kato M＆Ebihara A. Japan, 2011.)；
6、Kadota Y.Two new species of Croomia(Stemonaceae)from miyazaki
prefecture,kyushu southern Japan[J].The Journal of Japanese Botany,2010,85
(5):277-288. (learn miscellaneous by sealwort leaf Gelsemium novel species [J] Japanese plants of 2, Kadota Y. EMUs for Kyushu of Japan south county of Miyazaki
Will, 2010,85 (5):277-288.)；
7、Kadota Y.The new species of Croomia(Stemonaceae)from shikoku,
western Japan[J].The Journal of Japanese Botany,2012,87:79-84. (Kadota Y. Japan
Western part-four countries, one novel species [J] Japanese plants magazine of sealwort leaf Gelsemium, 2012,87:79-84.)；
8、Li EX,Sun Y,Qiu,YX,et al.Phylogeography of two East Asian species
in Croomia(Stemonaceae)inferred from chloroplast DNA and ISSR fingerprinting
variation[J].Molecular Phylogenetics and Evolution,2008,49:702-714.(Li EX,Sun
Y, Qiu, YX, et al. disclose Stemonaceae sealwort leaf 2 East Asia species of Gelsemium based on chloroplast DNA and the variation of ISSR fingerprints
Phylogeography [J] molecular evolutions and systematic growth, 2008,49:702-714.)；
9、Oginuma,K.,Horiuchi K,Fukuhara T.Karyomorphology of two genera in
Stemonaceae[J].Acta phytotaxonomica et geobotanica,2001,52(1):57-63. (Stemonaceae two
STUDY ON THE KARYOTYPE [J] Acta Phytotaxonomica Sinicas of a category, 2001,52 (1):57-63.)；
10, Tomlinson PB, Ayensu ES.Anatomy of Croomia Pauciflora [J] .Journal of
the Arnold Arboretum,1968,49:260-277. (Tomlinson PB, Ayensu ES.Croomia
Dissection [J] the Arnolds botanical garden magazine of Pauciflora, 1968,49:260-277.)；
11, the Beijing Chen Shilin, Xiao Peigen natural resources of Chinese medicinal materials sustainable use introduction [M]:Chinese medicine science publishing house,
2006；
The discussion Zhejiang Agriculture science of He mushroom in the sealwort leaf elegant jessamine tissue cultures such as 12, Chen Beibei, Wang Anle, Jiang Ming,
2012,12(4):490-492；
13, the bright .Agt1 in side is large based on the sealwort leaf Gelsemium Phylogeography of sequence and nSSR research [D] University Of Nanchang
Bachelorship paper, 2012；
14, one Beijing rare extinction plants [M] of Fourier state .1992. Chinese Plants Red Data Book:Science Press；
15, Gao Wenyuan, Xiao Peigen biotechnologies protect [J] Chinese herbal medicines, 2008,39 (7) with resources of medicinal plant:
961-964；
16, the people's republicanism of Chinese Pharmacopoeia Commission China is total to the Beijing pharmacopeia [M]:China Medical Science Press, 2010；
17, Discussions of The Related Issues [J] World Sciences of Huang Luqi, Li Hui, Chen Jing litchi rare and endangered TMRs protection
Technology-the modernization of Chinese medicine, 2001,3 (6):46-49；
18, the Zhejiang the phyletic evolution of the Phylogeography of Lee's grace perfume (or spice) sealworts leaf Gelsemium and its Relatives research [D]
University Ph.D. dissertation, 2006；
19, chromosome observation [J] guangxi plant of some domestic plants of the first of Li Lin, 1986.6 (l-2):99-105；
The sealwort leaf elegant jessamine callus inductions such as 20, Li Wenping, Chen Beibei, Jiang Ming study [J] Jiangsu's agriculture science,
2012,40(6):43-44,79；
21, Lin Wenhan, Cai Mengshen, Ying Baiping, wait research [J] Acta Pharmaceutica Sinicas of the big chemical composition of Buddha's warrior attendants, and 1993,28
(3):202-206；
22, the Zhejiang zhejiang plants will editorial board zhejiang plants will [M] science tech publishing house, 1993,7:373.
Abbreviation letter mentioned in text and see following breviary vocabulary：
Invention content
The technical problem to be solved in the present invention is to provide a kind of quick breeding by group culture methods of sealwort leaf elegant jessamine, using this hair
Bright method can go out a large amount of test tube seedlings with quickly breeding in the short time.
In order to solve the above technical problem, the present invention provides a kind of quick breeding by group culture methods of sealwort leaf elegant jessamine, including
Following steps：
1) it, draws materials：
Select the adventitious bud of the root-like stock of sealwort leaf elegant jessamine as explant；
2), the disinfection of adventitious bud：
Adventitious bud on root-like stock obtained by step 1) is fully washed and (micro liquid detergent first is added with tap water to carry out
Washing is then placed in flowing water in plastics mesh bag and rinses 10~15min), it is then operated on super-clean bench, first with 75% (volume %)
Alcohol 20~30sec of immersion treatment after aseptic water washing, is sterilized 8~10min with containing 0.1% (quality %) mercuric chloride, sterile water punching
It washes 3~5 times；
3), the Primary culture of adventitious bud：
Obtained by the adventitious bud (step 2) after disinfection) it is inoculated into after the blade (being carried out in super-clean bench) of stripping periphery and opens
On dynamic culture medium, adventitious bud growth and the induction of Multiple Buds are carried out；
The primary culture medium be MS+BA0.6~3mg/L+NAA0~0.2mg/L+ 20~30g/l+ of sucrose agar 5~
8g/l, pH are 5.6~5.8；
Inducing culturing condition is：Illumination in 16 hours, 30~45 μm of olm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；8 hours
Light culture, temperature are 21 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
When at least three Multiple Buds (that is, adventitious bud protrusion) occurs in the base portion of the adventitious bud in primary culture medium, terminate to open
Dynamic culture (incubation time about 2~2.5 months)；
The production method of above-mentioned primary culture medium is specific as follows：Based on MS minimal mediums, be separately added into BA, NAA,
Sucrose, agar uniformly mix, and it is 5.6~5.8 to adjust pH using the HCl of the KOH or 1mol/L of 1mol/L；MS per 1L is basic
The NAA of the BA of 0.6~3mg, 0~0.2mg, 20~30g sucrose, 5~8g agar is added in culture medium；
4), the rapid amplifying of adventitious bud：
It transfers after Multiple Buds obtained by step 3) are divided into the bud clump containing 1 sprouting (base portion of sprouting has 2~3 protrusions)
In being cultivated on proliferated culture medium, proliferated culture medium be 0.6~3mg/L+NAA of MS+BA, 0.2~0.5mg/L+KT 0~
1.0mg/L+2,4-D 0~0.5mg/L+0.8g/L PVP+ sucrose 20~30g/L+ agar 5~8g/L, pH are 5.6~5.8；
Condition of culture is：Illumination in 16 hours, 30~45 μm of olm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；Dark training in 8 hours
It supports, temperature is 21 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
It (is about cultivated 35~42 days) when the sprouting on proliferated culture medium at least grows 3 Multiple Buds, terminates this step
Culture；
The production method of above-mentioned proliferated culture medium is specific as follows：Based on MS minimal mediums, be separately added into BA, NAA,
KT, 2,4-D, PVP, sucrose, agar；Uniformly mixing, it is 5.6~5.8 to adjust pH using the HCl of the KOH or 1mol/L of 1mol/L；
The MS minimal mediums addition BA of 0.6~3mg of every 1L, the 2 of the NAA of 0.2~0.5mg, the KT of 0~1.0mg, 0~0.5mg,
The PVP of 4-D, 0.8g, 20~30g sucrose, 5~8g agar.
5), the elongation of adventitious bud and culture of rootage：
Multiple Buds obtained by step 4) are cut into and are transferred to culture of taking root/extend containing 2~3 plants for one clump of adventitious bud clump
Cultivated on base (that is, to realize can elongation growth and meanwhile but also take root), take root/elongation medium be MS+
BA0.6mg/L+NAA0.5mg/L+0.8g/L PVP+ sucrose 20~30g/L+ agar 5~8g/L, pH are 5.6~5.8；
Condition of culture is：Illumination in 16 hours, 30~45 μm of oLm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；Dark training in 8 hours
It supports, temperature is 20 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
When adventitious bud clump grows to 4.0~6.0cm high and base portion there are at least 5 root (5~10 roots), terminate this step
Culture；
This takes root/and the production method of elongation medium is specific as follows：Based on MS minimal mediums, be separately added into BA,
NAA, PVP, sucrose, agar uniformly mix, and it is 5.6~5.8 to adjust pH using the HCl of the KOH or 1mol/L of 1mol/L；Per 1L
MS minimal mediums be added 0.6mg BA, 0.5mg NAA, 0.8g PVP, 20~30g sucrose, 5~8g agar.
6), the plant of having taken root obtained by step 5) is taken out, obtain can bottle outlet plantation seedling.
The seedling is cultivated in river sand：Peat soil is 1:On 1 mixed-matrix.
The improvement of the quick breeding by group culture method of sealwort leaf elegant jessamine as the present invention：
With the Multiple Buds alternative steps 3 obtained by step 4)) obtained by Multiple Buds, repeat the fast of the adventitious bud of step 4)
Speed amplification；When the sprouting on proliferated culture medium grows at least three Multiple Buds, it is split.
Remarks explanation：General 5~9 of the number of new Multiple Buds differs.
The quick breeding by group culture further improvements in methods of sealwort leaf elegant jessamine as the present invention：
The step 5) is：
Adventitious bud clump is transferred to take root/elongation medium on, by culture in 28~35 days, adventitious bud can extend life
It is long, while root is generated in the base portion of plant, it can induce within 7~14 days and form root, form flourishing root system after being further cultured for.
The quick breeding by group culture further improvements in methods of sealwort leaf elegant jessamine as the present invention：
The culture vessel equipped with rooted plantlet obtained by step 6) is placed in the environment of natural temperature, illumination and tempers 5~7
It, then opens bottle cap, after placing 1~2 day, by taking root for plant base portion/and elongation medium (this is refered in particular to take root/extend culture
Agar in base) it cleans, by transplantation of seedlings to river sand：Peat soil is 1：The mixed-matrix culture of 1 (weight ratio) 20~35 days starts
10~14 days temperature be 23~25 DEG C, relative humidity be 70~80%.
The quick breeding by group culture further improvements in methods of sealwort leaf elegant jessamine as the present invention：
As preferred version：
Primary culture medium is：MS+BA 2mg/L+NAA0.2mg/L+ sucrose 20~30g/L+ agar 5~8g/L, pH 5.6
~5.8；
Proliferated culture medium be it is following any one：
MS+BA0.6mg/L+NAA 0.5mg/L+0.8g/L PVP+ sucrose 20~30g/L+ agar 5~8g/L, pH 5.6
~5.8；
MS+BA3mg/L+NAA0.2mg/L+0.8g/L PVP+ sucrose 20~30g/L+ agar 5~8g/L, pH be 5.6~
5.8；
MS+BA2mg/L+NAA 0.2mg/L+0.8g/L PVP+ sucrose 20~30g/L+ agar 5~8g/L, pH be 5.6~
5.8；
20~30g/ of MS+BA2mg/L+KT 1mg/L+NAA 0.2mg/L+2,4-D 0.5mg/L+0.8g/L PVP+ sucrose
L+ agar 5~8g/L, pH are 5.6~5.8.
In the present invention, full spectrum T5 energy-saving lamps can be used in headlamp.
Using the method for the present invention, the adventitious bud disinfection of sealwort leaf elegant jessamine root-like stock, inoculated and cultured can be lured for 60~75 days
Lead to form Multiple Buds, on this basis squamous subculture, Multiple Buds can mass propagation, average proliferation primary every about 42 days subcultures
Multiple is 6.4.Multiple Buds after subculture are transferred to elongation provided by the invention, can largely be sent out within 14~28 days in root media
Root, for rooting rate 93% or so, being cultivated for 7-14 days can bottle outlet hardening.
The present invention has following technical advantage：
1, the present invention is using/elongation medium of specifically taking root, to realize the elongation growth of adventitious bud and training of taking root
A step is supported to complete.
And the culture of conventional Stemonaceae Stemona platymisciums, elongation growth and taking root are completed in two steps, culture of rootage need
Individually to add the hormones such as NAA, IAA or IBA.
2, the plant hormone that proliferated culture medium of the present invention uses is the combination of the BA and NAA of low concentration；It can realize adventitious bud
Amplification.
3, incubation time needed for step 3) of the present invention is 2~2.5 months, and the incubation time needed for step 4) is about 42 days,
Incubation time needed for step 5) is about 42 days.
Advantages of the present invention：(1) production of sealwort leaf elegant jessamine can carry out under artificial control condition, not by season, gas
The restriction of the factors such as time condition and soil.2) growth rate is fast, with short production cycle, and equipment is simple, and floor space is few, can save people
Power, material resources etc., convenient for being factory produced.(3) technical method solves sealwort leaf elegant jessamine quickly breeding and the stable weight taken root
Sport technique segment is wanted, reaches consistent, the high requirement of breeding coefficient can be applied to the purpose of industrialized production.(4) this hair is used
Sealwort leaf elegant jessamine seedling that the group culturation rapid propagating technology of bright offer obtains carries out artificial cultivation, and it is small to obtain individual difference, quality
Uniform sealwort leaf elegant jessamine can provide afforestation or provide homogeneity good root-like stock finished product, can be clinical application
The raw material of stay in grade are provided.(5) germ plasm resource of sealwort leaf elegant jessamine can be preserved, while being conducive to protect wild resource,
Reduce the destruction to natural resources.
In conclusion the present invention carried out as explant using the adventitious bud on the root-like stock of sealwort leaf elegant jessamine it is fast numerous.
Disinfection including adventitious bud, the startup of adventitious bud growth, the induction of Multiple Buds and fast breeding, the elongation of Multiple Buds and training of taking root
Foster and test tube seedling transplanting.The present invention adds plant hormone based on MS culture mediums：6-benzyladenine, kinetin,
Methyl α-naphthyl acetate, 2,4- dichlorphenoxyacetic acids etc., for sealwort leaf elegant jessamine adventitious bud induction, the groups of critical stages such as be proliferated and take root
Knit culture.The present invention is successfully realized the tissue-culturing rapid propagation of sealwort leaf elegant jessamine, can go out a large amount of test tube seedlings with quickly breeding in the short time,
A kind of practicable exploration project is provided for the plant of Chinese medicine GAP kinds.
The present invention has grasped stretching for the startup of adventitious bud in sealwort leaf elegant jessamine tissue cultures, the proliferation of Multiple Buds and adventitious bud
The technology of the critical stages such as growing, take root makes this rare medicinal, ornamental plant of sealwort leaf elegant jessamine realize fast seedling growing, batch production
Cultivation is possibly realized.This rare wild plant resource is can simultaneously be effectively protected using the present invention, reduces private digging coyoting, shape
At the mutually coordinated benign cycle of sustainable use and reasonable development.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is the tissue-culturing rapid propagation figure of sealwort leaf elegant jessamine；
A, the wounded in the battle plant of the sealwort leaf elegant jessamine in the Tian Mu Shan Mountain is picked up from；
B, the adventitious bud on root-like stock；
C, growth and adventitious bud week of the adventitious bud on the Fiber differentiation that principal component is MS+BA 2mg/L+NAA0.2mg/L
Cross the Multiple Buds of existing green white；
D, Multiple Buds are cut into the bud clump of 2~3 buds in the culture medium that principal component is MS+BA 2mg/L+NAA0.2mg/L
On proliferation；
E, Multiple Buds are elongation and culture of rootage on MS+BA0.6mg/L+NAA 0.5mg/L culture mediums in principal component；
F, one month tissue-cultured seedling is grown after transplanting.
Specific implementation mode
Embodiment 1, a kind of quick breeding by group culture method of sealwort leaf elegant jessamine, follow the steps below successively：
1) it, draws materials：
Select the adventitious bud of the root-like stock of sealwort leaf elegant jessamine as explant；Specially：Select pick up from the Tian Mu Shan Mountain April
Adventitious bud on sealwort leaf elegant jessamine root-like stock is as explant.
2), the disinfection of adventitious bud：
Adventitious bud on root-like stock obtained by step 1) is fully washed：First with per 2~3 drop liquid detergents of 1L tap water addition
It is washed, is then placed in flowing water in mesh bag and rinses 10~15min；Then adventitious bud is transferred on superclean bench, is first used
75% (volume %) alcohol 20~30sec of immersion treatment, after aseptic water washing, with containing 0.1% (quality %) mercuric chloride (per 100mL
0.1% mercuric chloride adds 2~3 drop polysorbas20s) 8~10min of sterilizing, after aseptic water washing 3~5 times, it is used for the inoculation of subsequent step.
3), the Primary culture of adventitious bud：
Obtained by the adventitious bud (step 2) after disinfection) it is inoculated into after the blade (being carried out in super-clean bench) of stripping periphery and opens
On dynamic culture medium, adventitious bud growth and the induction of Multiple Buds are carried out；
The primary culture medium is MS+BA 2mg/L+NAA0.2mg/L+ sucrose 30g/l+ agar 8g/l, pH is 5.6~
5.8；
Inducing culturing condition is：Illumination in 16 hours, 35 μm of olm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；Dark training in 8 hours
It supports, temperature is 21 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
When Multiple Buds (that is, adventitious bud protrusion) of at least three occurs in the base portion of the adventitious bud in primary culture medium, terminate
Primary culture (incubation time is about 2~2.5 months)；
4), the rapid amplifying of adventitious bud：
Multiple Buds obtained by step 3) are cut after being divided into the bud clump containing 1 sprouting (base portion of sprouting there are 2~3 protrusions)
It transfers in being cultivated on proliferated culture medium, proliferated culture medium is MS+BA2mg/L+NAA0.2mg/L+0.8g/L PVP+ sucrose
30g/L+ agar 8g/L, pH are 5.6~5.8；
Condition of culture is：Illumination in 16 hours, 30~45 μm of olm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；Dark training in 8 hours
It supports, temperature is 21 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
When the sprouting on proliferated culture medium grows at least three Multiple Buds, it is divided into corresponding at least 3 with scalpel
A sprouting (sprouting base portion has 2-3 protrusion).In general, after 35~42 days, each sprouting can obtain 5~9 and grow thickly again
Bud.
Remarks explanation：In order to obtain more Multiple Buds, the higher relatively strong sprouting of gained can be repeated step 4)
The rapid amplifying of adventitious bud；When the sprouting on proliferated culture medium grows at least three Multiple Buds, then cut accordingly.
After 6 weeks, sprouting in each culture vessel it is renewable go out 5~9 Multiple Buds.Average proliferation multiple is about 6.8.
5), the elongation of adventitious bud and culture of rootage：
Multiple Buds obtained by step 4) are cut into and are transferred to culture of taking root/extend containing 2-3 plants for one clump of adventitious bud clump
Cultivated on base (that is, to realize can elongation growth and meanwhile but also take root), take root/elongation medium be MS+
BA0.6mg/L+NAA0.5mg/L+0.8g/L PVP+ sucrose 30g/L+ agar 8g/L, pH are 5.6~5.8；
Condition of culture is：Illumination in 16 hours, 30~45 μm of oLm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；Dark training in 8 hours
It supports, temperature is 20 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
When adventitious bud grows to 4.0~6.0cm high and base portion has at least 5 root (proximal process are grown to 3~5 centimetres), knot
The culture of this step of beam.
In general：Adventitious bud is transferred to take root/elongation medium on, by culture in 28~35 days, adventitious bud was both
Energy elongation growth, while can generate root in the base portion of plant again.It, 7~14 days can be with that is, 10~14 days adventitious buds can extend rapidly
Seeing has root restriction to be formed, and completion in 21~28 days is taken root；It can be achieved with adventitious bud within 35~42 days and grow to 4.0~6.0cm high and base
There are at least 5 roots in portion.
6), the plant of having taken root obtained by step 5) is taken out, obtain can bottle outlet plantation seedling, which cultivates in river
It is husky：Peat soil is 1:On 1 mixed-matrix.
Specially：Culture vessel equipped with rooted plantlet is placed in the environment of natural temperature, illumination and tempers 5~7 days, so
After open bottle cap, pour one layer of water in media surface, after placing 1~2 day, the taking root of plant base portion/elongation medium (refered in particular to
This takes root/elongation medium in agar) clean, by transplantation of seedlings to river sand：Peat soil is 1：1 mixed-matrix culture 20~35
It, 10~14 days temperature of beginning are 23~25 DEG C, and relative humidity is 70~80%.Natural ring can be then gradually transitions
Border condition.Hardening is transplanted to crop field after one month, average viability reaches 92%.
Embodiment 2,
Proliferated culture medium in step 4) is changed to：MS+BA2mg/L+KT1mg/L+NAA0.2mg/L+2,4-D 0.5mg/
L+0.8%PVP+ sucrose 30g/L+ agar 8g/L, pH are 5.6~5.8；Remaining is equal to embodiment 1.
In step 4), after 6 weeks, bud clump in each culture vessel it is renewable go out 5~7 Multiple Buds；Proliferation times are about
5.9。
Final survival rate is 90%.
Embodiment 3,
Proliferated culture medium in step 4) is changed to MS+BA3mg/L+NAA0.2mg/L+0.8g/L PVP+ sucrose 30g/L+ fine jades
Fat 8g/L, pH are 5.6~5.8；Remaining is equal to embodiment 1.
In step 4), after 6 weeks, bud clump in each culture vessel it is renewable go out 5~8 Multiple Buds.Proliferation times are about
6.6。
Final survival rate is 90%.
Comparative example 1-1,
The concentration of BA in 1 step 4) proliferated culture medium of embodiment makes into " 4.5mg/L (belongs to common dense by " 2mg/L "
Degree) ", remaining is equal to embodiment 1.
In step 4), after 6 weeks, bud clump in each culture vessel it is renewable go out about 6~8 Multiple Buds；Proliferation times are about
7.2。
The method is used, although proliferation times increase, since BA concentration is too high, plant strain growth is downgraded, and seedling generates one
Fixed vitrifying, final survival rate are only 68%.
Comparative example 1-2,
" BA of 2mg/L " in 1 step 4) proliferated culture medium of embodiment is made into " KT of 7mg/L ", remaining is equal to implementation
Example 1.
In step 4), after 6 weeks, bud clump in each culture vessel it is renewable go out about 4~6 Multiple Buds；Proliferation times are about
4.7。
The Multiple Buds are thin and delicate, and growth is not healthy and strong, and final survival rate is only 52%.
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
Bright to be not limited to above example, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (5)
1. the quick breeding by group culture method of sealwort leaf elegant jessamine, it is characterized in that including the following steps：
1) it, draws materials：
Select the adventitious bud of the root-like stock of sealwort leaf elegant jessamine as explant；
2), the disinfection of adventitious bud：
Adventitious bud on root-like stock obtained by step 1) is fully washed, is then operated on super-clean bench, is first soaked with 75% alcohol
It steeps and handles 20~30sec, after aseptic water washing, with containing 0.1% mercuric chloride, 8~10min of sterilizing, aseptic water washing 3~5 times；
3), the Primary culture of adventitious bud：
It will be inoculated into primary culture medium after the blade of adventitious bud stripping periphery after disinfection, carry out adventitious bud growth and Multiple Buds
Induction；
The primary culture medium is that MS+BA0.6~3mg/L+NAA0.2mg/L+ sucrose 20~30g/l+ agar 5~8g/l, pH are
5.6~5.8；
Inducing culturing condition is：Illumination in 16 hours, 30~45 μm of olm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；Dark training in 8 hours
It supports, temperature is 21 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
When at least three Multiple Buds occurs in the base portion of the adventitious bud in primary culture medium, terminate Primary culture；
4), the rapid amplifying of adventitious bud：
Switching is proliferated in being cultivated on proliferated culture medium after Multiple Buds obtained by step 3) are divided into the bud clump containing 1 sprouting
Culture medium is 0~0.5mg/L+ of MS+BA 0.6~3mg/L+NAA, 0.2~0.5mg/L+KT, 0~1.0mg/L+2,4-D
0.8g/L PVP+ sucrose 20~30g/L+ agar 5~8g/L, pH are 5.6~5.8；
Condition of culture is：Illumination in 16 hours, 30~45 μm of olm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；8 hours light cultures, temperature
Degree is 21 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
When the sprouting on proliferated culture medium at least grows 3 Multiple Buds, terminate the culture of this step；
5), the elongation of adventitious bud and culture of rootage：
Multiple Buds obtained by step 4) are cut into be transferred to for the one clump of adventitious bud clump containing 2~3 plants take root/elongation medium on
Cultivated, take root/elongation medium be MS+BA0.6mg/L+NAA0.5mg/L+0.8g/L PVP+ sucrose 20~30g/L+ fine jades
Fat 5~8g/L, pH are 5.6~5.8；
Condition of culture is：Illumination in 16 hours, 30~45 μm of oLm of intensity of illumination-2s-1, temperature is 25 ± 1 DEG C；8 hours light cultures, temperature
Degree is 20 ± 1 DEG C；Above-mentioned illumination and light culture are alternately；
When adventitious bud clump grows to 4.0~6.0cm high and base portion there are at least 5 roots, terminate the culture of this step；
6), the plant of having taken root obtained by step 5) is taken out, obtain can bottle outlet plantation seedling.
2. the quick breeding by group culture method of sealwort leaf elegant jessamine according to claim 1, it is characterized in that：
With the Multiple Buds alternative steps 3 obtained by step 4)) obtained by Multiple Buds, repeat the quick expansion of the adventitious bud of step 4)
Increase；When the sprouting on proliferated culture medium grows at least three Multiple Buds, it is split.
3. the quick breeding by group culture method of sealwort leaf elegant jessamine according to claim 1 or 2, it is characterized in that：
The step 5) is：
Adventitious bud clump is transferred to take root/elongation medium on, by culture in 28~35 days, adventitious bud can elongation growth,
Root is generated in the base portion of plant simultaneously, can induce within 7~14 days and form root, form flourishing root system after being further cultured for.
4. the quick breeding by group culture method of sealwort leaf elegant jessamine according to claim 1 or 2, it is characterized in that：
The culture vessel equipped with rooted plantlet obtained by step 6) is placed in the environment of natural temperature, illumination and is taken exercise 5~7 days,
Then it opens bottle cap, after placing 1~2 day, the taking root of plant base portion/elongation medium is cleaned, by transplantation of seedlings to river sand：Peat
Soil is 1：1 mixed-matrix culture 20~35 days, 10~14 days temperature of beginning are 23~25 DEG C, relative humidity is 70~
80%.
5. the quick breeding by group culture method of sealwort leaf elegant jessamine according to claim 1 or 2, it is characterized in that：
Primary culture medium is：MS+BA2mg/L+NAA0.2mg/L+ sucrose 20~30g/L+ agar 5~8g/L, pH be 5.6~
5.8；
Proliferated culture medium be it is following any one：
MS+BA3mg/L+NAA0.2mg/L+0.8g/L PVP+ sucrose 20~30g/L+ agar 5~8g/L, pH are 5.6~5.8；
MS+BA2mg/L+NAA 0.2mg/L+0.8g/L PVP+ sucrose 20~30g/L+ agar 5~8g/L, pH are 5.6~5.8；
MS+BA2mg/L+KT 1mg/L+NAA0.2mg/L+2,4-D 0.5mg/L+0.8g/L PVP+ sucrose 20~30g/L+ fine jades
Fat 5~8g/L, pH are 5.6~5.8.
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