CN107410033B - The rapid propagation method of national spice berry snippings - Google Patents
The rapid propagation method of national spice berry snippings Download PDFInfo
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- CN107410033B CN107410033B CN201710903541.9A CN201710903541A CN107410033B CN 107410033 B CN107410033 B CN 107410033B CN 201710903541 A CN201710903541 A CN 201710903541A CN 107410033 B CN107410033 B CN 107410033B
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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
Abstract
The present invention provides a kind of rapid propagation method of national spice berry snippings (P.magen B.Q.Cheng ex C.L.long&J.Yang).This method comprises: including choosing quick propagation material, Optimal Medium and establishing sterile clone culture, promote clump bud to be formed, culture of rootage step; strong operability of the present invention; can rapid expansion seedling radix, for increasingly rare snippings (P.magen B.Q.Cheng ex C.L.long&J.Yang) solve the problems, such as it breed it is slow, provide technical guarantee to meet the Dai nationality compatriot to the hobby of the fragrance and protection application and to protect the species in the future, introducing a fine variety recurrence.
Description
Technical field
The invention belongs to field of biotechnology, and in particular, to national spice berry snippings ((P.magen B.Q.Cheng
Ex C.L.long&J.Yang)) rapid propagation method
Technical background:
Piper (Piper) is the maximum category of Piperaceae, about 1000-2000 kinds.The category is mainly distributed on tropical ground
Area, about 60 kinds (Cheng et al.1999) of China, wherein have more than 30 kinds of pepper platymisciums that there is good medical value,
There is the effect of expelling wind and clearing away cold, the promoting flow of qi and blood circulation, removing blood stasis and acesodyne, analgesia in folk tradition application.
Snippings (P.magen B.Q.Cheng ex C.L.long&J.Yang) climbs up by holding on to liana to be wooden, is mainly grown in cloud
On the lime rock wall of South-South portion In Xishuangbanna height above sea level about 1500m.Snippings has special fragrance, in local a small number of people
Community, race is used as the special flavorant of one kind and eats, and the Dai nationality that version is received crosses the Dai Nationality and nearly all to go to buy several two snippings over the years.By
Stock number in field is few, the price of this kind of fragrance is also rising all the way 200-500 member/kg in locality, due to pecuniary benefit and
Local community eats driving for custom, and stock number of the snippings in field sharply declines, degree aggravation in imminent danger.Current appearance out of office
The main propagation method of the snippings observed is bred using seed, however, snippings belongs to dioecian plant, is seen in field
To seedling be nearly all young plant, approximately time of 5-10 from the young to adult plants, civil main feeding at
Year plant, adult plants destruction are particularly acute, and almost can not see the flower of the plant of adult and the seed of maturation in field, with kind
Son breeding is almost without possible.The species in August, 2017 is just used as novel species formally to deliver, and breeds currently without any about snippings
Relevant report.Therefore the breeding problem that snippings is solved using artificial breeding method is to solve snippings protection and sustainable use
Important technology.
Summary of the invention:
It is an object of that present invention to provide a kind of national spice berry snippings (P.magen B.Q.Cheng ex C.L.long&
J.Yang rapid propagation method and in-vitro conservation method)).
In order to realize above-mentioned purpose of the invention, the present invention provides the following technical solutions:
The rapid propagation method of national spice berry snippings, this method include choosing quick propagation material, Optimal Medium
With establish sterile clone culture, promote clump bud is formationed, culture of rootage, test tube seedling transplant step, take germinate after grow three months
Snippings belt segment budling aseptic water washing 2 times, use 0.1%HgCl with 75% ethanol disinfection 10s2Water-soluble liquid disinfectant 6min is used
Aseptic water washing 5-6 times, each 1min cut off wound part 0.2cm, cut later with the sterile worry paper suck dry moisture after disinfection
At belt segment 0.5cm segment, the ready culture medium for inducing stem section to grow axillary bud 1. 1/4MS+BA0.02mg/L+ is accessed
NAA0.05mg/L；Culture is transferred to the culture medium for inducing stem section to grow clump bud 2. 1/4MS+BA0.2mg/L+IAA0.1mg/ after 35 days
L and 3. 1/4MS+BA0.5mg/L；Culture is transferred to the culture medium of root induction as 4. 1/4MS+IAA1mg/L+PP333 after 60 days
0.1mg/L。
According to the rapid propagation method of the national spice berry snippings, wherein the quick propagation material step of the selection
Suddenly it is the snippings used on the lime rock wall for picking up from south of Yunnan In Xishuangbanna height above sea level 1400m-1600m, takes back indoor cultivation
Kind is to (sedendary soil equipped with snippings growing environment: red soil: the basin of the soil of fertile soil 1:1:1) in basin, after ten months
Sprouting is issued, sprouting is used as the explant quickly bred after growing 3 months.
According to the rapid propagation method of the national spice berry snippings, wherein the Optimal Medium and establishing nothing
Bacterium clone incubation step is using condition of culture are as follows: minimal medium 1/4MS, the culture medium that induction stem section grows axillary bud are
①1/4MS+BA0.02mg/L+NAA0.05mg/L；The culture medium for inducing young stem section clump bud proliferation is 2. 1/4MS+BA0.2mg/L+
IAA0.1mg/L and 3. 1/4MS+BA0.5mg/L；The culture medium of root induction is 4. 1/4MS+IAA1mg/L+PP333 0.1mg/
L；1. 2. 3. sugared concentration is 3% to culture medium, and 4. sugared concentration is 2% to culture medium；Condition of culture is equal are as follows: all culture medium PH 5.8,
26 ± 2 DEG C of cultivation temperature, light application time 12h.d-1, 20-40 μm of ol.m of intensity of illumination-2.S-1。
According to the rapid propagation method of the national spice berry snippings, wherein the promotion clump bud forming step is
Snippings children stem through 75% alcoholic solution impregnate 10s, aseptic water washing 2 times, with 0.1% mercuric chloride solution handle 6min after, sterile water
It rinses 4 times, removes blade with sterilizing operating scissors, be trimmed to the dissection for being about 1 section of 0.5cm band, be placed on culture medium 1. upper culture, warp
After 35d culture, each stem section section grows axillary bud 1-2, and inductivity 98%, the average of the bud of every block of material is 1.2, takes
2. the bud issued in lower stem section above cultivates 60d, subculture cycle 60d in culture medium, build up snippings and quickly breed clone, increases
Rate is grown up to 1:4, then 2. the upper culture by 60d, growth rate reach 1:4, reach wanting for snippings the factorial production in culture medium
It asks.
According to the rapid propagation method of the national spice berry snippings, wherein the culture of rootage step is to train
The 3. upper culture of feeding base, after 3-5 clumps/bottle bud is inoculated with 35d, every bottle obtains height up to 3-4cm or more budling 4-6, while also having increasing
It grows, moon growth rate is 1:4, chooses budling height in 3cm or more, cuts and get off to be placed on culture medium 4. 1/4MS+IAA1mg/L
It being cultivated on+PP333 0.1mg/L, 10d-25d starts to take root, as incubation time extends rooting rate up to 99%, every plant of 2-5 item
Root, root long 0.8-1.2cm, plant strain growth are healthy and strong.
According to the rapid propagation method of the national spice berry snippings, wherein the test tube seedling transplant step is bud
After item accesses root media 30 days, when root long 1.4cm or more, is transplanted on cultivation matrix, the cultivation matrix are as follows: cultivation soil:
Fertile soil: red soil: perlite 3:1:0.5, adding 0.1% Bravo to stir evenly, three days dress basins on heap are spare, and bottle seedling of taking root is first
It puts at normal temperature, unclamps bottle cap 1 day, fully open within second day bottle cap, dew is two days later, the 4th day light the culture medium being attached on root
It rinses completely, after drying 30min, plants in ready basin, sprinkle profoundly water, be put into 70% shading net of outer cover, average temperature later
It is 99% that survival rate is counted in the greenhouse of 21 DEG C ± 2 DEG C of degree, after 20 days.
According to the rapid propagation method of the national spice berry snippings, this method further progress Plantlet in vitro, institute
The Plantlet in vitro step stated is to carry out Plantlet in vitro with the culture type of vitro rooting in test tube seedling, is taken short in slowly growth preservation
The mode of phase Plantlet in vitro, with growth inhibitor CCC1mg/L, 5mg/L, 10mg/L；PP333 0.5mg/L,1mg/L,5mg/L
With ABA0.5mg/L processing；Or using the 4 DEG C of progress Plantlet in vitro of method dark culture for reducing cultivation temperature, extend subculture cycle
To 1 year and a half.
Compared with prior art, the present invention has following excellent benefit:
The present invention snippings be the newfound plant novel species of the present inventor, rapid propagation method operability of the invention
By force, energy rapid expansion seedling radix, for increasingly rare snippings (P.magen B.Q.Cheng ex C.L.long&J.Yang)
Effective propagation method is provided, solve the problems, such as it breed it is slow, to meet numerous people, especially the Dai nationality compatriot is to this
The hobby of fragrance and the protection application of snippings provide technical guarantee to protect the species future, introducing a fine variety recurrence.
The invention 2. 1/4MS+BA0.2mg/L+IAA0.1mg/L and 3. 1/4MS+BA0.5mg/L culture medium, not only solve
Snippings lateral bud of having determined forms difficult problem, and lateral bud robust growth is also allowed to be conducive to take root and subsequent transplant survival, is rapid expansion
Seedling radix provides technical guarantee.
Specific embodiment:
Essentiality content of the invention is further illustrated with the embodiment of the present invention below, but this is not limited with this
Invention.
Embodiment 1:
Plant snippings (P.magen B.Q.Cheng ex C.L.long&J.Yang)) quick breeding:
Include: sterile clonal foundation, medium optimization selection, screen the formation of promotion clump bud and culture of rootage,
In: induction stem section grows the culture medium of axillary bud as 1. 1/4MS+BA0.02mg/L+NAA0.05mg/L；Young stem section clump bud is induced to increase
The culture medium grown is 2. 1/4MS+BA0.2mg/L+IAA0.1mg/L and 3. 1/4MS+BA0.5mg/L；The culture medium of root induction
For 4. 1/4MS+IAA1mg/L+PP3330.1mg/L
Propagation method:
One, sterile clonal foundation:
Material source: plant snippings (P.magen B.Q.Cheng ex C.L.long&J.Yang) picks up from south of Yunnan west
Double versions receive regional height above sea level about 1500m lime rock wall on.
The foundation of aseptic strain is the disinfection and sterilizing of explant first, and the sterilizing of explant material is the important ring in tissue culture
Section, initially sterilizes more difficult in tissue culture.Both the microorganism for thoroughly killing external-talent surface had been required, has injured external-talent's group less as far as possible again
It knits and superficial cell, ideal effect has been reached by the research to sterilisation program, specific practice is as follows:
Process from field to greenhouse:
Culture materials plant snippings (P.magen B.Q.Cheng ex C.L.long&J.Yang)) from field take back plantation
In to basin (sedendary soil equipped with snippings growing environment: red soil: the basin of the soil of fertile soil 1:1:1), sent out after ten months
Sprouting out, sprouting are used as quick propagation material after growing 3 months
Two, condition of culture: minimal medium is 1/4MS (MS a great number of elements a quarter, remaining ingredient and MS are same), induction
Stem section grows the culture medium of axillary bud as 1. 1/4MS+BA0.02mg/L+NAA0.05mg/L；Induce the culture of young stem section clump bud proliferation
Base is 2. 1/4MS+BA0.2mg/L+IAA0.1mg/L and 3. 1/4MS+BA0.5mg/L；The culture medium of root induction is 4. 1/4MS
+IAA1mg/L+PP333 0.1mg/L；1. 2. 3. sugared concentration is 3% to culture medium, and 4. sugared concentration is 2% to culture medium；Condition of culture
Are as follows: all culture medium PH 5.8,26 ± 2 DEG C of cultivation temperature, light application time 12h.d-1, 20-40 μm of ol.m of intensity of illumination-2.S-1。
Three, quickly breeding
Snippings children stem impregnates 10s through 75% alcoholic solution, aseptic water washing 2 times, handles 6min with 0.1% mercuric chloride solution
Afterwards, aseptic water washing 4 times remove blade with sterilizing operating scissors, are trimmed to the dissection for being about 1 section of 0.5cm band, are placed on culture medium 1.
Upper culture, about after 35d is cultivated, each stem section section grows axillary bud 1-2, and inductivity 98%, the bud of every block of material is averaged
Number is 1.2.It removes the bud issued in stem section and 2. above cultivates 60d (subculture cycle 60d) in culture medium, just built up snippings
Quickly breeding clone, multiplication rate is up to 1:4, then in the culture medium 2. upper culture by 60d, and growth rate is up to 1:4.It reaches
To the requirement of snippings the factorial production.There are two types of clump bud regenerations: one is stem section axils to grow, and takes sterile skewer after cutting
Slotting mode can be continuously proliferated down；Another way is to form Multiple Buds in stem section base portion incision, generates position still in leaf
At armpit, because contact culture medium nutrition supply is sufficient herein, bud occurs and grows quickly, so be the form appearance of Multiple Buds,
The material cultivated on this culture medium, no callus generate, and illustrate the examination that the bud that this Clonal regeneration mode obtains generates
Pipe seedling heredity is stable.
Four, culture of rootage
In culture medium 3. upper culture, after 3-5 clumps/bottle bud is inoculated with 35d, every bottle can be obtained height up to 3-4cm or more budling 5
Left and right, while also having proliferation, moon growth rate is 1:4.Budling height is chosen in 3cm or more, cuts and gets off to be placed on culture medium
4. cultivating on 1/4MS+IAA1mg/L+PP333 0.1mg/L, 10d-25d just starts to take root as incubation time extension almost reaches
99% rate, every plant of 2-5 item root, root long about 1cm, plant strain growth are healthy and strong.
Five, test tube seedling (hardening) is transplanted
After access root media 30 days, when root long about 1.5cm, can be transplanted.Concrete operation method 1, cultivation soil: fertile soil,
Red soil, perlite (3:1:0.5) add 0.1% Bravo to stir evenly three days dress basins on heap spare.2, bottle seedling of taking root is firstly placed on
It under room temperature, unclamps bottle cap 1 day, fully opens within second day bottle cap, dew two days later, is gently washed the culture medium being attached on root on the 4th day
Completely, (30min) is dried slightly to plant in ready basin, is sprinkled profoundly water.It is put into 70% shading net of outer cover, mean temperature later
In 21 DEG C ± 2 DEG C of greenhouse, count after 20 days: survival rate is 99%.
Six, result
The young stem section clump bud of induction, which is filtered out, by optimum choice is proliferated best culture medium as 2. 1/4MS+BA0.2mg/L+
IAA0.1mg/L and 3. 1/4MS+BA0.5mg/L, the bud which derives is more healthy and stronger, moves to improve rooted seedling below
It plants survival rate and quality material is provided；The culture medium of root induction is 4. 1/4MS+IAA1mg/L+PP333 0.1mg/L, this is taken root
It is well developed root system that culture medium derives, neat, therefore transplanting is easy to survive, and provides technical guarantee for industrialization production from now on
Seven, Plantlet in vitro
Culture type in Plantlet in vitro is selected first, at room temperature, snippings Multiple Buds and test tube seedling are carried out
Whether determining subculture, as a result (with culture yellowing leaf, the withered of budling is standard more than 20%, for the comparison of subculture cycle
Display: Multiple Buds subculture cycle is 70d, and vitro rooting in test tube seedling subculture cycle is 8 months.Herein with the culture of vitro rooting in test tube seedling
Type carries out Plantlet in vitro.The mode for mainly taking the middle or short term Plantlet in vitro that slowly growth saves, is testing growth inhibition
Agent CCC1mg/L (unit is similarly hereinafter), 5,10；After 0.5,1,5 and ABA0.5 of PP333, and use the method for reducing cultivation temperature (dark
4 DEG C of culture) carry out Plantlet in vitro.Subculture cycle can be extended to 1 year and a half.During the cultivation process, snippings culture under former condition of culture
Object 60d-70d must subculture.
Claims (7)
1. the rapid propagation method of national spice berry snippings, it is characterised in that this method includes choosing quick propagation material, excellent
Change culture medium and establish sterile clone culture, promote clump bud formation, culture of rootage, test tube seedling transplant step, takes life after germination
Long trimestral snippings belt segment budling aseptic water washing 2 times, uses 0.1%HgCl with 75% ethanol disinfection 10s2Water-soluble liquid disinfectant
6min, later with the sterile worry paper suck dry moisture after disinfection, cuts off wound part with aseptic water washing 5-6 times, each 1min
0.2cm is cut into belt segment 0.5cm segment, accesses the ready culture medium for inducing stem section to grow axillary bud 1. 1/4MS+BA0.02mg/
L+NAA0.05mg/L；Culture is transferred to the culture medium for inducing stem section to grow clump bud 2. 1/4MS+BA0.2mg/L+ after 35 days
IAA0.1mg/L and 3. 1/4MS+BA0.5mg/L；Culture is transferred to the culture medium of root induction as 4. 1/4MS+IAA1mg/ after 60 days
L+PP333 0.1mg/L。
2. the rapid propagation method of nationality's spice berry snippings according to claim 1, it is characterised in that the selection
Quick propagation material step is with the fiber crops on the lime rock wall for picking up from south of Yunnan In Xishuangbanna height above sea level 1400m-1600m
Root takes back interior and is planted to the sedendary soil equipped with snippings growing environment: red soil: in the basin of fertile soil 1:1:1, after ten months
Sprouting is issued, sprouting is used as the explant quickly bred after growing 3 months.
3. the rapid propagation method of nationality's spice berry snippings according to claim 1, it is characterised in that the optimization
Culture medium is using condition of culture with sterile clone incubation step is established are as follows: minimal medium 1/4MS, induction stem section are grown
The culture medium of axillary bud is 1. 1/4MS+BA0.02mg/L+NAA0.05mg/L；The culture medium for inducing young stem section clump bud proliferation is 2. 1/
4MS+BA0.2mg/L+IAA0.1mg/L and 3. 1/4MS+BA0.5mg/L；The culture medium of root induction is 4. 1/4MS+IAA1mg/
L+PP333 0.1mg/L；1. 2. 3. sugared concentration is 3% to culture medium, and 4. sugared concentration is 2% to culture medium；Condition of culture is equal are as follows: all
Culture medium PH 5.8,26 ± 2 DEG C of cultivation temperature, light application time 12h.d-1, 20-40 μm of ol.m of intensity of illumination-2.S-1。
4. the rapid propagation method of nationality's spice berry snippings according to claim 1, it is characterised in that the promotion
Clump bud forming step is snippings children stem through 75% alcoholic solution immersion 10s, aseptic water washing 2 times, is handled with 0.1% mercuric chloride solution
After 6min, aseptic water washing 4 times, removes blade with sterilizing operating scissors, be trimmed to the dissection for being about 1 section of 0.5cm band, be placed on culture
Base 1. upper culture, after 35d is cultivated, each stem section section grows axillary bud 1-2, and inductivity 98%, the bud of every block of material is put down
Mean is 1.2, removes the bud issued in stem section and 2. above cultivates 60d in culture medium, it is fast to build up snippings by subculture cycle 60d
Speed breeding clone, multiplication rate reaches 1:4, then 2. the upper culture by 60d, growth rate reach 1:4, reach snippings in culture medium
The requirement of the factorial production.
5. the rapid propagation method of nationality's spice berry snippings according to claim 1, it is characterised in that described takes root
Incubation step is in culture medium 3. upper culture, and after 3-5 clumps/bottle bud is inoculated with 35d, every bottle obtains height up to 3-4cm or more budling 4-6
It is a, while also having proliferation, moon growth rate is 1:4, chooses budling height in 3cm or more, cuts and get off to be placed on culture medium 4.
It is cultivated on 1/4MS+IAA1mg/L+PP333 0.1mg/L, 10d-25d starts to take root, and reaches as incubation time extends rooting rate
99%, every plant of 2-5 item root, root long 0.8-1.2cm, plant strain growth stalwartness.
6. the rapid propagation method of nationality's spice berry snippings according to claim 1, it is characterised in that the test tube
Transplantation of seedlings step is after budling is accessed root media 30 days, and when root long 1.4cm or more is transplanted on cultivation matrix, the cultivation
Matrix are as follows: cultivation soil: fertile soil: red soil: perlite 3:1:0.5 adds 0.1% Bravo to stir evenly three days dress basins on heap standby
With bottle seedling of taking root is firstly placed under room temperature, is unclamped bottle cap 1 day, is fully opened within second day bottle cap, and two days later, the 4th day being attached to for dew
Culture medium on root gently wash clean, after drying 30min, plants in ready basin, sprinkles profoundly water, be put into outer cover 70% later
It is 99% that survival rate is counted in shading net, 21 DEG C ± 2 DEG C of mean temperature of greenhouse, after 20 days.
7. the rapid propagation method of nationality's spice berry snippings according to claim 1, it is characterised in that this method is into one
Step carries out Plantlet in vitro, and the Plantlet in vitro step is to carry out Plantlet in vitro with the culture type of vitro rooting in test tube seedling, takes
The mode for the middle or short term Plantlet in vitro that slowly growth saves, with growth inhibitor CCC1mg/L, 5mg/L, 10mg/L；PP333
0.5mg/L, 1mg/L, 5mg/L and ABA0.5mg/L processing；Or using reduce 4 DEG C of method dark culture of cultivation temperature carry out from
Body saves, and extends subculture cycle to 1 year and a half.
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