CN107155881B - One kind being suitable for polytype plant rapid propagation method - Google Patents
One kind being suitable for polytype plant rapid propagation method Download PDFInfo
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- CN107155881B CN107155881B CN201710315912.1A CN201710315912A CN107155881B CN 107155881 B CN107155881 B CN 107155881B CN 201710315912 A CN201710315912 A CN 201710315912A CN 107155881 B CN107155881 B CN 107155881B
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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
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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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Abstract
The invention discloses one kind to be suitable for polytype plant rapid propagation method, the following steps are included: the selection of (1) explant: selection honeysuckle stem segments, cordate houttuynia rhizomes, Japanese cryptotaenia stem and leaf terminal bud, four number kamuning seeds are explant, are cultivated after disinfection；(2) stem section, rhizomes, terminal bud or the seed after step (1) disinfection Initial culture: are seeded in ER+6-BA0.6~2.5mg/L+KT0.2~1.5mg/L, and it is cultivated 20~30 days on the solid medium added with sucrose and agar, induction budding；(3) aseptic seedling or stem section that step (2) obtain proliferation and subculture: are seeded in 0.2~3.0mg/L of ER+KT0.01~3.0mg/L+ paclobutrazol, and it is cultivated in the solid medium added with sucrose and agar, after culture 30 days, synchronization gain bud and root.Polytype plant high-volume high quality seedling is provided in time for large-scale development.
Description
Technical field
The invention belongs to plant tissue culture technical fields, are related to a kind of suitable for the fast numerous core technology of polytype plant.
Background technique
Honeysuckle (Lonicerajaponica Thund.) also known as honeysuckle, honeysuckle, honeysuckle flower, two precious flowers, are Caprifoliaceae honeysuckles
Belong to perennial half evergreen liana, is the distinctive rare traditional Chinese medicine in China for clinical common Chinese herbal medicine.Honeysuckle has heat-clearing solution
Poison, anti-inflammatory detumescence, the effect of delaying senescence, it is viral effectively to prevent " SARS ", bird flu and Influenza A H1N1 etc., also has
The effect of anticancer, anti-AIDS.In addition, honeysuckle also contains there are many oxidation-resistant active ingredient, can reducing blood lipid, be used for prophylactic treatment
Cardiovascular and cerebrovascular disease.With the development of the green revolution, gradually back to nature, natural drug are steady, malicious secondary because having effect by the mankind
Act on the features such as small, more and more concerns and attention by modern, purposes is also extended to diet, health care, cosmetics, green
The various aspects of the human lives such as color pesticide develop various health drinks and food using high-tech technology using honeysuckle as raw material
Product ensure people's health, will play positive effect, have huge market empty for preventing and treating common disease, frequently-occurring disease
Between, development prospect is wide.Honeysuckle is bred in production based on cutting propagation, but slow and rooting rate of taking root is not high, it is numerous
It grows coefficient and reproduction speed is all limited, it is difficult to meet in the market the needs of to honeysuckle excellent variety number and amount, be unfavorable for it and push away
Extensive farming is planted.
Cordate houttuynia (Herba houttuyniae) also known as the folding basal part of the ear, belong to dicotyledon Saururaceae heartleaf houttuynia category, because of it
Root, stem, leaf have a fish like smell, therefore named cordate houttuynia.The tender rhizome of cauline leaf, fertilizer is edible, and delicious flavour has clearing heat and detoxicating, sharp
Urine detumescence, helping digestion is pleasant, analgesia, cough-relieving, dispelling-wind invigorating stomach and other effects.Meanwhile cordate houttuynia and energy flu-prevention, pneumonia, eczema etc.
A variety of diseases are the health-care vegetables of dietotherapeutic.Formally it is determined as " being both drug and food " by health ministry
One of resource.In traditional mode of production, cordate houttuynia generally uses plant division, the seedling raising manners transplanted with Propagation of Rhizomes, but carries out all the year round
Vegetative propagation easily causes the degeneration of cordate houttuynia seedling stem, is primarily due to that more pest and disease damage can be generated in reproductive process, at present fish
The measure or drug that southern blight that raw meat grass occurs, zonate spot are generated there are no good controlling disease, are the industrialization of cordate houttuynia
The bottleneck of production.
Japanese cryptotaenia stem and leaf (Umbelliferae) also known as three leaves, wild hollyhock, umbellate form flower section.Traditional Chinese medicine thinks that Japanese cryptotaenia stem and leaf complete stool can
It is used as medicine, in poor health, the diseases such as renal shutdown and pyogenic infections are effective in cure.In recent years, Japanese cryptotaenia stem and leaf has been used as main vegetables to go on people's
On dining table.As the improvement of people's living standards, the pursuit to quality of life is increasing, deliciousness, battalion as Japanese cryptotaenia stem and leaf one kind
Feeding edible wild herbs, also increasing development prospect is very wide for the market demand.Japanese cryptotaenia stem and leaf relies primarily on seminal propagation, but its step
Cumbersome, longer period of emerging, low reproduction rate and reproductive-cost height, can not provide a large amount of high quality seedlings for giving birth in a short time
It produces.
Four numbers kamuning (Murraya tetramera) are Rutaceae Murraya plant, and defoliation small arbor, leaf has strong
Fragrance.Be used as medicine with root, leaf, pungent, slight bitter, slightly warm in nature, can using fresh herb or dry in the shade it is spare, tool expel pathogenic wind from the body surface, promoting qi circulation and relieving pain, Huoxue San "
The effect of becoming silted up, be anti-inflammatory, easing pain, is antipyretic, for treating cold, fever, bronchitis, asthma, stomachache, rheumatic arthralgia, bruise silt
The diseases such as swollen, skin itching, venomous snake bite, eczema, malaria.Its habitat causes to provide by artificial or naturally destruction in recent years
Source atrophy, species are in imminent danger, or even are in extinction trend.
For these reasons, it is necessary to carry out honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four systems for counting kamuning tissue cultures
Research.Not only reproduction speed is fast for method for tissue culture, breeding coefficient is high, can also keep the fine quality of maternal plant well；And
Tissue-culturing rapid propagation is carried out to peculiar rare medicinal plant, not only can reach the purpose of breeding preservation, and can ensure that the supply of medicine source,
Have very great significance to the further investigation tool of the fast development and genetic improvement etc. that promote its industry.
Tissue-culturing rapid propagation is to apply to obtain a most commonly used emerging practical technique during sapling multiplication in recent years.Mesh
Before, have thousands of kinds of plants in the world and is succeeded by tissue cultures.Each group training research center, research object all will not be
Single variety, usually two or more master grind product, but the suitable explant of every kind of plant, tissue culture propagation method are all
It is not identical, the tissue culture propagation method for exploring the efficient stable of every kind of plant need to be expended considerable time and effort, so significantly
The man power and material for increasing investment directly affects the factorial production cost.
Summary of the invention
The purpose of the present invention is in view of the shortcomings of the prior art, providing one kind to be suitable for polytype plant includes: honeysuckle, fish
Raw meat grass, Japanese cryptotaenia stem and leaf, four number kamuning etc. the rapid propagation methods of tissue cultures be to reach indoor large-scale breeding seedling
Large-scale development provides various plants high-volume high quality seedling in time.The purpose of the present invention can be realized by the following technologies:
One kind being suitable for polytype plant rapid propagation method, comprising the following steps:
(1) selection of explant: selection honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four tissues for counting kamuning are explant, are disappeared
It is cultivated after poison；
(2) Initial culture: by step (1) disinfection after stem section, rhizomes, terminal bud or seed be seeded in ER+6-BA0.6~
It cultivates 20~30 days, induces on 2.5mg/L+KT0.2~1.5mg/L, and the solid medium added with sucrose and agar
Bud；Emergence rate is up to 92% or more, and most of explant Bud Differentiation, only the four number a small amount of seeds of kamuning do not send out sprouting；Especially fish
Raw meat grass culture 10d or so, sprouts sprouting, and 30d or so gradually differentiates Multiple Buds；
(3) aseptic seedling or stem section that step (2) obtain proliferation and subculture: are seeded in ER+KT0.01~3.0mg/L+ multiple-effect
It is cultivated in 0.2~3.0mg/L of azoles, and the solid medium added with sucrose and agar, after culture 30 days, 100% synchronizes and obtains
Obtain bud and root.
(4) transplanting and management: the healthy and strong rooted seedling that step (3) are obtained opens bottle cap hardening at least 3d, takes out cleaning root
Culture medium survives then by young seedling direct transplantation to crop field and reaches 90% or more.
Step (1) selects honeysuckle stem section, cordate houttuynia rhizomes, Japanese cryptotaenia stem and leaf terminal bud, four number kamuning kinds in the above method
Son is explant.
It is that explant is cultivated that step (1), which cuts the stem section of honeysuckle annotinous branch, in the above method, and stem section is cut into
1.0~2.0cm is spare；After cordate houttuynia rhizomes first cleans up, places and cultivate 10d or so in clear water, then rhizomes is cut into
It is spare after 1.0~2.0cm；Japanese cryptotaenia stem and leaf selection removes root, petiole and leaf compared with plantlet, retains base portion 2cm or so height, strips outer
It encloses spare after several layers of bracts；The crust for stripping four number kamuning seeds is spare；75% alcohol of ready explant is impregnated
30~60s, then 12~15min is sterilized with 0.1% mercuric chloride, it is spare after then using sterile water wash 5~6 times；
In the above method step (2) by after disinfection honeysuckle stem section and cordate houttuynia rhizomes be directly inoculated into Initial culture
On base (ER+6-BA0.6~2.5mg/L+KT0.2~1.5mg/L)；The explant of Japanese cryptotaenia stem and leaf after disinfection is stripped into outer layer bud again
Piece takes out terminal bud and is inoculated on initial culture base (ibid)；Four number kamuning seeds after disinfection are cut in half and are inoculated into just
For on culture medium (ibid).3~7 explants of every bottle of culture medium inoculated are cultivated.
In the above method step (3) by aseptic seedling base portion or stem section that step (2) obtains be seeded in ER+KT0.01~
0.2~3.0mg/L of 3.0mg/L+ paclobutrazol.5~20 explants of every bottle of culture medium inoculated are cultivated.
Initial culture and proliferation and subculture condition of culture in the above method: inoculation moves back culturing room and cultivates, and cultivates room temperature
Be 24~26 DEG C, culturing room's relative humidity be 30~50%, daily illumination 10~12 hours, intensity of illumination be 1000~
2000lx.3~5% sucrose and 0.75% agar are added in culture medium used in Initial culture and proliferation and subculture culture.
Compared with prior art, the present invention the beneficial effects of the present invention are:
1, method of the invention is applicable not only to herbaceous plant cordate houttuynia and Japanese cryptotaenia stem and leaf and liana honeysuckle carries out
Quickly breeding applies also for the number kamuning of dungarunga plant four and is quickly bred.
2, method choice difference plant difference explant of the invention, identical tissue culture propagation method, 4 kinds of plants can obtain
The cultivating system of efficient stable greatly reduces the man power and material of investment in this way, directly reduction the factorial production cost.
3, select ER for minimal medium, suitable for draft, liana and the plant regeneration of magaphanerophytes；Using the present invention into
Row various plants difference explant, induces sprouting, and inductivity is up to 92% or more.
4, it can be synchronized on explant subculture medium and generate bud and root, especially honeysuckle and cordate houttuynia, synchronize to form clump
It sprouts；It is step completion that the present invention, which will be proliferated with culture of rootage program simplification,.
Detailed description of the invention
Fig. 1 be honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four synchronous with the stem section induced buds of number kamuning squamous subculture aseptic seedlings or
The bud growing state of Multiple Buds and root；
Fig. 2 be honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four synchronous with the stem section induced buds of number kamuning squamous subculture aseptic seedlings or
The root growth situation of Multiple Buds and root.
Specific embodiment
It is intended to further illustrate the present invention with reference to embodiments, is not intended to limit the present invention.
Embodiment 1, tissue-culturing quick-propagation honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning
One, culture medium is prepared and sterilizes
Initial culture base :+0.75% agar of ER+6-BA1.2mg/L+KT0.5mg/L+3% sucrose.
Proliferated culture medium :+0.75% agar of ER+KT2.0mg/L+ paclobutrazol 1.0mg/L+3% sucrose.The above culture medium
At 121 DEG C, sterilize 20 minutes.
Two, Initial culture
It is that explant is cultivated that step (1), which selects the stem section of honeysuckle annotinous branch, in the above method, and stem section is cut into
1.0~2.0cm is spare；After cordate houttuynia rhizomes first cleans up, places and cultivate 10d or so in clear water, then rhizomes is cut into
It is spare after 1.0~2.0cm；Japanese cryptotaenia stem and leaf selection removes root, petiole and leaf compared with plantlet, retains base portion 2cm or so height, strips outer
It encloses spare after several layers of bracts；The crust for stripping four number kamuning seeds is spare；75% alcohol of ready explant is impregnated
30~60s, then 12~15min is sterilized with 0.1% mercuric chloride, it is spare after then using sterile water wash 5~6 times；Then after sterilizing
Honeysuckle stem section and cordate houttuynia rhizomes be directly inoculated into initial culture base；The explant of Japanese cryptotaenia stem and leaf after disinfection is shelled again
Outer layer bract is taken, terminal bud is taken out and is inoculated into initial culture base；Four number kamuning seeds after disinfection are cut in half and are inoculated into
In initial culture base.3~7 explants of every bottle of culture medium inoculated are cultivated；Cultivating room temperature is 24~26 DEG C, culturing room's phase
It is 30~50% to humidity, daily illumination 10~12 hours, intensity of illumination is 1000~2000lx.Culture 20~30 days, emergence
Rate is up to 92% or more, and most of explant Bud Differentiation, only the four number a small amount of seeds of kamuning do not send out sprouting；Especially cordate houttuynia is cultivated
10d or so, sprouts sprouting, and 30d or so gradually differentiates Multiple Buds；127 strains are obtained altogether.
Three, squamous subculture
Above-mentioned 127 strains are taken to carry out successive propagation, the specific method is as follows: by step 2 honeysuckle, cordate houttuynia, four nine periods
In the fragrant aseptic seedling stem with bud obtained be cut into 1.0~2.0cm, be transferred on proliferated culture medium, cultivate 30 days or so, 100% is same
Step obtains bud and root；The aseptic seedling that step 2 Japanese cryptotaenia stem and leaf obtains is removed into root, petiole and leaf, retains base portion 2cm or so height, is transferred to
It on proliferated culture medium, cultivates 30 days or so, 100% synchronization gain bud and root；It is primary every 25~30d subculture, every bottle of culture medium
5~20 explants of inoculation are cultivated.Synchronous induced bud or Multiple Buds and root growth situation are as shown in Figs. 1-2；Condition of culture
Same step 2.
Four, transplanting and management
Select honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning 3-6cm high rooted seedling, open bottle cap hardening 3d, take
Cleaning root culture medium survives then by young seedling direct transplantation to crop field and reaches 90% or more out.
Honeysuckle provided by the present invention, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning tissue culture propagations, according to not
With the characteristic of plant, select stem section, rhizomes, terminal bud and seed for explant, materials are easy, and quantity is big, and genetic stability is high.
During Initial culture, different plants difference explant induces sprouting in same medium.It in the squamous subculture stage, adopts
With the method for bud or Multiple Buds and root, the method for createing forming seedling through one step culture not only simplifies health seedling production routine, Er Qiesheng
It produces at low cost, improves production efficiency.
Embodiment 2, tissue-culturing quick-propagation honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning
One, culture medium is prepared and sterilizes
Initial culture base :+0.75% agar of ER+6-BA1.5mg/L+KT0.5mg/L+3% sucrose.
Proliferated culture medium :+0.75% agar of ER+KT2.5mg/L+ paclobutrazol 1.5mg/L+3% sucrose.The above culture medium
At 121 DEG C, sterilize 20 minutes.
Take tissue-culturing quick-propagation honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning different explants, it is basic to grasp
It is consistent with embodiment 1 to make method；Effect is not much different with embodiment 1, and only four number kamuning basal part of stem break up more brown
Substance, but its growth is not influenced.
Claims (4)
1. one kind is suitable for polytype plant rapid propagation method, which comprises the following steps:
(1) selection of explant: selection honeysuckle stem section, cordate houttuynia rhizomes, Japanese cryptotaenia stem and leaf terminal bud, four number kamuning seeds are outer
Implant is cultivated after disinfection；
(2) Initial culture: honeysuckle stem section and cordate houttuynia rhizomes after step (1) is sterilized directly are inoculated into initial culture base
On；The explant of Japanese cryptotaenia stem and leaf after disinfection is stripped into outer layer bract again, terminal bud is taken out and is inoculated into initial culture base；After sterilizing
Four number kamuning seeds cut in half and be inoculated into initial culture base；Initial culture 20~30 days, induction budding, described was first
It is 0.2~1.5mg/L of ER+6-BA0.6~2.5mg/L+KT, and the solid culture added with sucrose and agar for culture medium
Base；
(3) aseptic seedling or stem section that step (2) obtain proliferation and subculture: are seeded in ER+KT0.01~3.0 mg/L+paclobutrazol
Cultivated in 0.2~3.0 mg/L, and the solid medium added with sucrose and agar, culture 30 days after, synchronization gain bud and
Root；
(4) transplanting and management: the healthy and strong rooted seedling that step (3) are obtained opens bottle cap hardening at least 3 d, takes out the training of cleaning root
Base is supported, then by young seedling direct transplantation to crop field；
Step (2) Initial culture and step (3) proliferation and subculture condition of culture: cultivation temperature is 24~26 DEG C, relative humidity 30
~50%, daily illumination 10~12 hours, intensity of illumination is 1000~2000lx；
3~5% sucrose and 0.75% fine jade are added in culture medium used in step (2) Initial culture and step (3) proliferation and subculture culture
Rouge.
2. according to claim 1 be suitable for polytype plant rapid propagation method, which is characterized in that
The stem section that step (1) cuts honeysuckle annotinous branch is that explant is cultivated, and it is standby that stem section is cut into 1.0~2.0 cm
With；After cordate houttuynia rhizomes first cleans up, places in clear water and cultivate at least 10 d, then rhizomes is cut into 1.0~2.0 cm
It is spare afterwards；Japanese cryptotaenia stem and leaf selection removes root, petiole and leaf compared with plantlet, retains base portion 2cm high, strips the several layers of bract standby in periphery
With；The crust for stripping four number kamuning seeds is spare；75% alcohol of ready explant is impregnated into 30~60 s, then is used
0.1% mercuric chloride sterilizes 12~15 min, spare after then using sterile water wash 5~6 times.
3. according to claim 1 be suitable for polytype plant rapid propagation method, which is characterized in that in step (2)
3~7 explants of every bottle of culture medium inoculated are cultivated.
4. according to claim 1 be suitable for polytype plant rapid propagation method, which is characterized in that in step (3)
5~20 explants of every bottle of culture medium inoculated are cultivated.
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| CN104642108A (en) * | 2015-02-03 | 2015-05-27 | 中国科学院亚热带农业生态研究所 | Method suitable for tissue culture mass production of multiple plants |
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