CN107155881A - A kind of method suitable for polytype micropropagation of plants - Google Patents
A kind of method suitable for polytype micropropagation of plants Download PDFInfo
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- CN107155881A CN107155881A CN201710315912.1A CN201710315912A CN107155881A CN 107155881 A CN107155881 A CN 107155881A CN 201710315912 A CN201710315912 A CN 201710315912A CN 107155881 A CN107155881 A CN 107155881A
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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 a kind of method suitable for polytype micropropagation of plants, comprise the following steps：(1) selection of explant：Honeysuckle stem segments are selected, cordate houttuynia root-like stock, Japanese cryptotaenia stem and leaf terminal bud, four count kamuning seeds for explant, cultivated after sterilization；(2) Initial culture：Stem section, root-like stock, terminal bud or seed after step (1) is sterilized are seeded on ER+6 BA0.6~2.5mg/L+KT0.2~1.5mg/L, and the solid medium added with sucrose and agar and cultivated 20~30 days, induction budding；(3) proliferation and subculture：The aseptic seedling or stem section that step (2) is obtained are seeded in ER+KT0.01~3.0mg/L+ 0.2~3.0mg/L of paclobutrazol, and are cultivated in the solid medium added with sucrose and agar, after cultivating 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 field, it is related to a kind of core technology suitable for polytype plant fast propagation.
Background technology
Honeysuckle (Lonicerajaponica Thund.) also known as honeysuckle, honeysuckle, honeysuckle flower, two precious flowers, are Caprifoliaceae honeysuckles
Belong to perennial half evergreen liana, be clinical conventional Chinese herbal medicine, be the distinctive rare traditional Chinese medicine of China.Honeysuckle has heat-clearing solution
Poison, anti-inflammatory detumescence, effect of anti-aging, can effectively prevent the viruses such as " SARS ", bird flu and Influenza A H1N1, also have
The effect of anticancer, anti-AIDS.In addition, honeysuckle also contain a variety of oxidation-resistant active ingredients, can reducing blood lipid, for prophylactic treatment
Cardiovascular and cerebrovascular disease.With the development of the green revolution, the mankind's gradually back to nature, natural drug is because with effect is steady, poison is secondary
The features such as acting on small, more and more concerns and attention by modern, its purposes is also extended to diet, health care, cosmetics, green
The various aspects of the human lives such as color agricultural chemicals, using honeysuckle as raw material, various health drinks and food are developed using high-tech technology
Product, for preventing and treating common disease, frequently-occurring disease, ensure that people are healthy, will give play to positive role, empty with huge market
Between, development prospect is wide.Honeysuckle is bred in production based on cutting propagation, but takes root slow and rooting rate is not high, its is numerous
Grow coefficient and reproduction speed is all limited, it is difficult to meet in the market to honeysuckle improved seeds number and the demand of 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, category dicotyledon Saururaceae heartleaf houttuynia category, because of it
Root, stem, leaf have a fish like smell, therefore named cordate houttuynia.The tender rhizome edible of cauline leaf, fertilizer, its delicious flavour, with clearing heat and detoxicating, profit
The effects such as urinating detumescence, pleasant helping digestion, analgesia, cough-relieving, dispelling-wind invigorating stomach.Meanwhile, cordate houttuynia and energy flu-prevention, pneumonia, eczema etc.
A variety of diseases, are the health-care vegetables of dietotherapeutic.Formally it is defined as by health ministry " being both medicine, be food again "
One of resource.In traditional mode of production, cordate houttuynia typically uses plant division, the seedling raising mannerses 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 in reproductive process that more pest and disease damage, current fish can be produced
Measure or medicine that white Silk diseases, the zonate spot that raw meat grass occurs also are produced without 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 disease such as renal shutdown and pyogenic infections is effective in cure.In recent years, Japanese cryptotaenia stem and leaf goes on people's as staple vegetable
On dining table.As the improvement of people's living standards, the pursuit to quality of life is increasing, as delicious, the battalion of the class of Japanese cryptotaenia stem and leaf one
Foster edible wild herbs, also increasing development prospect is very wide for its market demand.Japanese cryptotaenia stem and leaf relies primarily on seminal propagation, but its step
Cumbersome, longer cycle of emerging, low reproduction rate and reproductive-cost are high, it is impossible to which providing a large amount of high quality seedlings in a short time is used to give birth to
Production.
Four number kamuning (Murraya tetramera) are Rutaceae Murraya plant, and defoliation small arbor, leaf has strong
Fragrance.It is used as medicine with root, leaf, pungent, slight bitter, slightly warm in nature using fresh herb or can dry in the shade standby, tool expels pathogenic wind from the body surface, promoting qi circulation and relieving pain, Huoxue San "
Silt, anti-inflammatory, analgesia, it is antipyretic the effect of, for treat cold, fever, bronchitis, asthma, stomachache, arthralgia pain due to rheumatism, bruise become silted up
The diseases such as swollen, skin itching, venomous snake bite, eczema, malaria.Its habitat causes money by artificial or naturally destruction in recent years
Source atrophy, species are in imminent danger, or even in extinction trend.
For these reasons, it is necessary to carry out honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, the system of four number kamuning tissue cultures
Research.Not only reproduction speed is fast for method for tissue culture, breeding coefficient is high, and the fine quality of maternal plant can be also kept well；And
Tissue-culturing rapid propagation is carried out to peculiar rare medicinal plant, the purpose of breeding preservation is not only can reach, and can ensure that the supply of medicine source,
The further investigation tool in terms of fast-developing and genetic improvement to promoting its industry has very great significance.
Tissue-culturing rapid propagation is to apply to obtain a most commonly used emerging practical technique during sapling multiplication in the last few years.Mesh
Before, existing thousands of kinds of plants are succeeded by tissue cultures in the world.Each group training research center, research object is all without being
Single variety, is usually that two or more master grinds product, but the suitable explant of every kind of plant, tissue culture propagation method are all
Differ, the tissue culture propagation method for the efficient stable for exploring every kind of plant need to be expended considerable time and effort, so significantly
The man power and material of input is added, factorial praluction cost is directly affected.
The content of the invention
The purpose of the present invention is included in view of the shortcomings of the prior art, providing one kind and being suitable to polytype plant：Honeysuckle, fish
The rapid propagation method of the tissue cultures of raw meat grass, Japanese cryptotaenia stem and leaf, four number kamuning etc., to reach indoor large-scale breeding seedling, be
Large-scale development provides various plants high-volume high quality seedling in time.The purpose of the present invention can be realized by following technology：
A kind of method suitable for polytype micropropagation of plants, comprises the following steps：
(1) selection of explant：Honeysuckle is selected, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning are organized as explant, disappeared
Cultivated after poison；
(2) Initial culture：By step (1) sterilize after stem section, root-like stock, terminal bud or seed be seeded in ER+6-BA0.6~
Cultivate 20~30 days, induce on 2.5mg/L+KT0.2~1.5mg/L, and the solid medium added with sucrose and agar
Bud；Emergence rate is up to more than 92%, most of explant Bud Differentiation, and the only 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, 30d or so progressively differentiates Multiple Buds；
(3) proliferation and subculture：The aseptic seedling or stem section that step (2) is obtained are seeded in ER+KT0.01~3.0mg/L+ multiple-effect
Cultivated in 0.2~3.0mg/L of azoles, and the solid medium added with sucrose and agar, after cultivating 30 days, 100% synchronously obtains
Obtain bud and root.
(4) transplanting and management：The healthy and strong rooted seedling that step (3) is obtained, opens bottle cap hardening at least 3d, takes out cleaning root
Culture medium, young seedling direct transplantation then survived to crop field and reach more than 90%.
Step (1) selects honeysuckle stem section, cordate houttuynia root-like stock, Japanese cryptotaenia stem and leaf terminal bud, four number kamuning kinds in the above method
Son is explant.
Step (1) cuts the stem section of honeysuckle annotinous branch and cultivated for explant in the above method, and stem section is cut into
1.0~2.0cm is standby；After cordate houttuynia root-like stock is first cleaned up, culture 10d or so in clear water is placed, then root-like stock is cut into
It is standby after 1.0~2.0cm；Japanese cryptotaenia stem and leaf is selected compared with plantlet, removes root, petiole and leaf, retains base portion 2cm or so height, is stripped outer
Enclose standby after several layers of bracts；The crust for stripping four number kamuning seeds is standby；Ready explant is alcohol-pickled with 75%
30~60s, then 12~15min is sterilized with 0.1% mercuric chloride, then with standby after sterile water wash 5~6 times；
The honeysuckle stem section and cordate houttuynia root-like stock after sterilization are directly inoculated into Initial culture by step (2) in the above method
On base (ER+6-BA0.6~2.5mg/L+KT0.2~1.5mg/L)；The explant of Japanese cryptotaenia stem and leaf after sterilization is stripped into outer layer bud again
Piece, takes out terminal bud and is inoculated into Initial culture base (ibid)；Four number kamuning seeds after sterilization 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.
The aseptic seedling base portion or stem section that step (3) obtains step (2) in the above method be seeded in ER+KT0.01~
0.2~3.0mg/L of 3.0mg/L+ paclobutrazols.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：Culturing room's culture is moved on to after inoculation, room temperature is cultivated
For 24~26 DEG C, culturing room's relative humidity is 30~50%, daily illumination 10~12 hours, intensity of illumination is 1000~
2000lx.3~5% sucrose and 0.75% agar are added in culture medium used in Initial culture and proliferation and subculture culture.
The present invention compared with prior art, the beneficial effects of the present invention are：
1st, method of the invention is applicable not only to herbaceous plant cordate houttuynia and Japanese cryptotaenia stem and leaf, and liana honeysuckle is carried out
Quick breeding, applies also for the number kamuning of dungarunga plant four and is quickly bred.
2nd, the different explants of the different plants of method choice of the invention, identical tissue culture propagation method, 4 kinds of plants can obtain
The cultivating system of efficient stable, so greatly reduces the man power and material of input, directly reduces factorial praluction cost.
4th, selection ER is minimal medium, it is adaptable to draft, liana and the plant regeneration of magaphanerophytes；Entered using the present invention
Row various plants difference explant, induces sprouting, and inductivity is up to more than 92%.
6th, bud and root, especially honeysuckle and cordate houttuynia can be synchronously produced on explant subculture medium, it is synchronous to form clump
Sprout；The present invention completes propagation with culture of rootage program simplification for a step.
Brief description of the drawings
Fig. 1 be honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning squamous subculture aseptic seedlings and the synchronous induced bud of stem section or
The bud growing state of Multiple Buds and root；
Fig. 2 be honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning squamous subculture aseptic seedlings and the synchronous induced bud of stem section or
The root growth situation of Multiple Buds and root.
Embodiment
The present invention is intended to further illustrate 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
First, culture medium is prepared and sterilized
Initial culture base：The agar of ER+6-BA1.2mg/L+KT0.5mg/L+3% sucrose+0.75%.
Proliferated culture medium：The agar of ER+KT2.0mg/L+ paclobutrazol 1.0mg/L+3% sucrose+0.75%.Above culture medium
At 121 DEG C, sterilize 20 minutes.
2nd, Initial culture
Step (1) is cultivated from the stem section of honeysuckle annotinous branch for explant in the above method, and stem section is cut into
1.0~2.0cm is standby；After cordate houttuynia root-like stock is first cleaned up, culture 10d or so in clear water is placed, then root-like stock is cut into
It is standby after 1.0~2.0cm；Japanese cryptotaenia stem and leaf is selected compared with plantlet, removes root, petiole and leaf, retains base portion 2cm or so height, is stripped outer
Enclose standby after several layers of bracts；The crust for stripping four number kamuning seeds is standby；Ready explant is alcohol-pickled with 75%
30~60s, then 12~15min is sterilized with 0.1% mercuric chloride, then with standby after sterile water wash 5~6 times；Then after sterilizing
Honeysuckle stem section and cordate houttuynia root-like stock be directly inoculated into Initial culture base；The explant of Japanese cryptotaenia stem and leaf after sterilization 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 sterilization are cut in half and are inoculated into
In Initial culture base.3~7 explants of every bottle of culture medium inoculated are cultivated；It is 24~26 DEG C, culturing room's phase to cultivate room temperature
It is 30~50% to humidity, daily illumination 10~12 hours, intensity of illumination is 1000~2000lx.Culture 20~30 days, emerges
Rate is up to more than 92%, most of explant Bud Differentiation, and the only 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 progressively differentiates Multiple Buds；127 strains are obtained altogether.
3rd, squamous subculture
Above-mentioned 127 strains are taken to carry out successive propagation, 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 is obtained removes root, petiole and leaf, retains base portion 2cm or so height, is transferred to
On proliferated culture medium, cultivate 30 days or so, 100% synchronization gain bud and root；Every 25~30d subcultures once, 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 Figure 1-2；Condition of culture
Same step 2.
4th, transplanting and management
The rooted seedling for selecting honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning 3-6cm high, opens bottle cap hardening 3d, takes
Go out to clean root culture medium, then young seedling direct transplantation is survived to crop field and reaches more than 90%.
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, selection stem section, root-like stock, terminal bud and seed are explant, and materials are easy, and quantity is big, and genetic stability is high.
During Initial culture, different plants difference explant induces sprouting in same medium.In the squamous subculture stage, adopt
With the method for bud or Multiple Buds and root, the method for createing forming seedling through one step culture not only simplify health seedling production routine, Er Qiesheng
Produce cost low, improve production efficiency.
Embodiment 2, tissue-culturing quick-propagation honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning
First, culture medium is prepared and sterilized
Initial culture base：The agar of ER+6-BA1.5mg/L+KT0.5mg/L+3% sucrose+0.75%.
Proliferated culture medium：The agar of ER+KT2.5mg/L+ paclobutrazol 1.5mg/L+3% sucrose+0.75%.Above culture medium
At 121 DEG C, sterilize 20 minutes.
Take tissue-culturing quick-propagation honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, the different explants of four number kamuning, basic behaviour
Make method be the same as Example 1 consistent；Effect is more or less the same with embodiment 1, and simply four number kamuning basal part of stem break up more brown
Material, but do not influence it to grow.
Claims (9)
1. a kind of method suitable for polytype micropropagation of plants, it is characterised in that comprise the following steps：
(1) selection of explant：Selection honeysuckle, cordate houttuynia, Japanese cryptotaenia stem and leaf, four number kamuning are organized as explant, after sterilization
Cultivated；
(2) Initial culture：By step (1) sterilize after stem section, root-like stock, terminal bud or seed be seeded in ER+6-BA0.6~
Cultivate 20~30 days, induce on 2.5mg/L+KT0.2~1.5mg/L, and the solid medium added with sucrose and agar
Bud；
(3) proliferation and subculture：The aseptic seedling or stem section that step (2) is obtained are seeded in ER+KT0.01~3.0mg/L+ paclobutrazols 0.2
Cultivated in~3.0mg/L, and the solid medium added with sucrose and agar, after cultivating 30 days, synchronization gain bud and root；
(4) transplanting and management：The healthy and strong rooted seedling that step (3) is obtained, opens bottle cap hardening at least 3d, takes out the culture of cleaning root
Base, then by young seedling direct transplantation to crop field.
2. the method according to claim 1 suitable for polytype micropropagation of plants, it is characterised in that
Step (1) selects honeysuckle stem section, and cordate houttuynia root-like stock, Japanese cryptotaenia stem and leaf terminal bud, four count kamuning seeds for explant.
3. the method according to claim 2 suitable for polytype micropropagation of plants, it is characterised in that
Step (1) cuts the stem section of honeysuckle annotinous branch and cultivated for explant, and it is standby that stem section is cut into 1.0~2.0cm；
After cordate houttuynia root-like stock is first cleaned up, place in clear water and cultivate at least 10d, then root-like stock is cut into 1.0~2.0cm standby
With；Japanese cryptotaenia stem and leaf is selected compared with plantlet, removes root, petiole and leaf, and reservation base portion 2cm is high, strips standby after peripheral several layers of bracts；Stripping
Take the crust of four number kamuning seeds standby；By ready explant with 75% alcohol-pickled 30~60s, then with 0.1% liter
Mercury sterilizes 12~15min, then with standby after sterile water wash 5~6 times.
4. the method suitable for polytype micropropagation of plants according to claim 1 or 2 or 3, it is characterised in that
Honeysuckle stem section and cordate houttuynia root-like stock after sterilization is directly inoculated into Initial culture base by step (2)；After sterilizing
The explant of Japanese cryptotaenia stem and leaf strip outer layer bract again, take out terminal bud and be inoculated into Initial culture base；By in four nine periods after sterilization
Fragrant seed, which cuts in half, to be inoculated into Initial culture base.
5. the method according to claim 1 suitable for polytype micropropagation of plants, it is characterised in that in step (1)
3~7 explants of every bottle of culture medium inoculated are cultivated.
6. the method according to claim 1 suitable for polytype micropropagation of plants, it is characterised in that in step (3)
The aseptic seedling base portion or stem section that step (2) is obtained are seeded on proliferation and subculture culture medium.
7. the method suitable for polytype micropropagation of plants according to claim 1 or 6, it is characterised in that step (3)
In 5~20 explants of every bottle of culture medium inoculated are cultivated.
8. the method according to claim 1 suitable for polytype micropropagation of plants, it is characterised in that step (2) is just
It is commissioned to train and supports and step (3) proliferation and subculture condition of culture：Cultivation temperature is 24~26 DEG C, and relative humidity is 30~50%, per the daylight
According to 10~12 hours, intensity of illumination was 1000~2000lx.
9. the method according to claim 1 suitable for polytype micropropagation of plants, it is characterised in that step (2) is just
It is commissioned to train and supports and 3~5% sucrose of addition and 0.75% agar in the culture medium used in step (3) proliferation and subculture culture.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109928504A (en) * | 2019-03-12 | 2019-06-25 | 中国科学院华南植物园 | A method of utilizing saururus chinensis purifying eutrophication water body |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109928504A (en) * | 2019-03-12 | 2019-06-25 | 中国科学院华南植物园 | A method of utilizing saururus chinensis purifying eutrophication water body |
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