CN107047298A - A kind of method of David's-harp tissue culture and rapid proliferation - Google Patents
A kind of method of David's-harp tissue culture and rapid proliferation Download PDFInfo
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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
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Abstract
The invention belongs to technical field of plant culture, and in particular to the method for David's-harp tissue culture and rapid proliferation.The invention discloses a kind of method of David's-harp tissue culture and rapid proliferation, comprise the following steps successively：（1）The sterilization and inoculation of David's-harp stem section：The new stem of the David's-harp extracted out then is taken, the moderate lignifying stem section with axillary bud is cut into and is inoculated with；（2）The induction of Multiple Buds；（3）The propagation of adventitious bud；（4）The culture of rootage of regeneration plant；（5）Acclimatization and transplantses.The culture medium of heterogeneity and proportioning is employed in above-mentioned steps.Using method of the invention, it is possible to rapidly obtain David's-harp plant, beneficial to industrialization production.Using the inventive method, the adventitious bud induction frequencies of David's-harp Multiple Buds is up to 91.7%, adventitious bud number 9.5, and adventitious bud proliferation multiple is up to 8.6, and plantlet of transplant survival rate is up to more than 95%.
Description
Technical field
The invention belongs to technical field of plant culture, and in particular to the method for David's-harp tissue culture and rapid proliferation.
Background technology
David's-harp (Polygonatum cyrtonema Hua), is Liliaceae Polygonatum herbaceos perennial, distribution
In Jiangsu, Anhui, Zhejiang, Jiangxi, Hunan, Fujian, Guangdong (the north), the dark and damp rich soil of sylvan life, shrubbery or careless slope is born in
In, because of its root-like stock in locality so that sealwort is used as medicine and largely plants, while also edible.David's-harp in production is numerous at present
Grow using seed and root-like stock, mainly based on root-like stock, have the shortcomings that breeding coefficient is low, the breeding cycle is long, therefore build
The tissue culture rapid propagation system of vertical David's-harp, is the another effective way for realizing its industrialization production.
At present David's-harp and sealwort, the explant of use are focused primarily upon for the tissue cultures that Liliaceae Polygonatum is carried out
Body is mainly root-like stock or the bud produced by root-like stock.Because root-like stock is grown on underground, surface carries many miscellaneous bacterias, and also deposits
In endophyte, therefore the control relative difficult of its pollution rate, take long enough just induce adventitious bud after sterilizing in addition.Substantially
The optimal minimal medium that the report of upper all HUANGJING ZANYU CAPSULE tissue cultures is used is the MS culture mediums of various concentrations, wherein
Process before root induction mainly uses MS culture mediums, also there is the report that adventitious bud inducing is carried out using 1/2MS；During root induction
It is main to use 1/2MS culture mediums, also there are using MS culture mediums and 1/3MS culture mediums.The induction of Polygonatum adventitious bud is even more
Basic element of cell division 6-BA has often been used in the induction of injured tissue, and the optium concentration that many document report 6-BA are used is 4.0mg/L；
In culture of rootage be used alone auxin or its combination, or even have 2,4-D can hestening rooting report.Current David's-harp is with flower
Valve is that the tissue cultures of explant and quick breeding have no report.
The content of the invention
The technical problems to be solved by the invention are：In view of the deficienciess of the prior art, providing a kind of survival rate height, profit
In the method for the David's-harp tissue culture and rapid proliferation for realizing industrialization production.
To realize the purpose of the present invention, it is achieved using following technical scheme：A kind of David's-harp tissue cultures with it is fast
The method of speed breeding, comprises the following steps successively：
(1) sterilization of David's-harp bud
The bud of the David's-harp not yet bloomed is taken May then, 4 DEG C of deepfreeze 0-48h of refrigerator are put into, added after taking-up
Enter appropriate liquid detergent solution immersion 2min, rinsed with flowing water after 0.5-1.0h, stem section is transferred to 70% wine in super-clean bench
1min is soaked in smart solution, with aseptic water washing 3 times, immersion dropwise addition there are 5 drop Tween-80s, and mass fraction is 0.1% HgCl2
Soaking disinfection 9-10min in solution, is then fully embathed 3 times with sterilized water, the explant material being sterilized.
(2) induction of Multiple Buds
Explant material obtained in the previous step is put on aseptic filter paper, cuts end to end, stamen and gynoecium is removed, by petal
It is cut into 2cm2In size, the SH minimal mediums for being connected to addition TDZ 0-2.0mg/L, NAA 0-1.0mg/L, AC 0-2.0g/L,
Sucrose addition in the culture medium is 30g/L, and agar addition is 8.0g/L, and pH is 5.8-6.0, and culture medium is once in 121 DEG C
Lower sterilizing 20min, similarly hereinafter.The culture medium for connecting petal is put prior to cultivating 0-7d in dark, and cultivation temperature is 28 ± 2 DEG C；Then
Illumination cultivation is transferred to, condition of culture is：Cultivation temperature is (28 ± 2) DEG C, and light application time is 14h light/10h dark, intensity of illumination 30-
40μmol/(m2·s)；
(3) propagation of adventitious bud
Induce the adventitious bud obtained clump to be cut into the sprout tuber of 2-3 adventitious bud previous step, be inoculated into added with TDZ 0-
Cultivated in 2.0mg/L, 6-BA0-5.0mg/L, CH 0-1.0mg/L, AC 0-2.0g/L SH minimal mediums, cultivate bar
Part is：Cultivation temperature is (28 ± 2) DEG C, and light application time is 14h light/10h dark, 30-40 μm of ol/ (m of intensity of illumination2·s)；
(4) culture of rootage of regeneration plant
Cut that blade is light green, grow vigorous and 3-4 pieces of blade adventitious bud, insertion is added with 6-BA 0-4.0, NAA 0-
Taken root in the SH minimal mediums of 2.0mg/L, banana puree 0-150g/L 12.5%-100% concentration, the sugarcane added in culture medium
Sugar is 20-30g/L, and agar addition is 8.0g/L, pH 5.8-6.0, and once in the 20min that sterilized at 121 DEG C；
(5) acclimatization and transplantses
The height of seedling length of the tissue-cultured seedling of under growth root is to more than 5cm, when root length is more than 5cm, the bottle cap of tissue culture bottle is unclamped, into bottle
A small amount of sterilized water is injected to prevent culture medium dry and cracked, bottle cap is moved away in 6h is later half, and adds sterilized water, after 18h, removes bottle
Lid, allows 30-40 μm of ol/ (m2S) during which the light of light intensity add sterilized water several times directly according to regenerated plant culture 2d.Then it is small
Heart smashs culture medium to pieces, takes out regeneration plant, carefully rinses the agar of residual well with flowing water, plant is colonized in and once disappeared
The perlite that poison is crossed, vegetable garden soil weight ratio is 1：In the mixed-matrix of (50-500), pour permeable and with transparent vinyon
Film covering is controlled between 24-28 DEG C with heat and moisture preserving, indoor temperature.Sealed polyethylene plastic corner, next day are opened after 3d
Sealed polyethylene plastic is all opened, after after young leaves expansion can balled transplanting to crop field.
Preferably：The material of inoculation is the bud not yet bloomed in the step (1), and in 4 DEG C of low temperature of refrigerator
Refrigerate 12-24h.
Preferably：SH culture mediums in the step (2) are added with TDZ 0.5mg/L, NAA 0.2mg/L,
AC0.5-1.0mg/L, be inoculated with petal after in dark cultivate 3-5d after be transferred to illumination cultivation again.
Preferably：SH culture mediums are added with TDZ 0.1mg/L, 6-BA 2.0mg/L, CH in the step (3)
0.2-0.5g/L, AC 0.5-1.0mg/L.
Preferably：Prescription of rooting medium is 50%SH, 6-BA 0.5, NAA 0.5mg/ in the step (4)
L, banana puree 80-100g/L.
Compared with prior art, the beneficial effects of the invention are as follows：Using the present invention method, compared with it has been reported that text
Offer, it is low to reduce shortening pre-treatment time, explant pollution rate, shortens adventitious bud time of origin, so as to faster be spent more
Sealwort regeneration plant, beneficial to industrialization production.Using the inventive method, the adventitious bud induction frequency of David's-harp Multiple Buds is up to
91.7%, adventitious bud number 9.5, adventitious bud proliferation multiple is up to 8.6, and plantlet of transplant survival rate is up to more than 95%.
The initialism of the present invention：
AC activated carbons
6-BA 6- benzyl aminoadenines
CH caseinhydrolysates
NAA methyl α-naphthyl acetates
TDZ Thidiazurons (Thidiazuron).
Embodiment
Embodiment 1
A kind of method of David's-harp tissue culture and rapid proliferation, comprises the following steps successively：
(1) sterilization of David's-harp bud
The bud of the David's-harp not yet bloomed is taken May then, 4 DEG C of deepfreeze 12-24h of refrigerator are put into, after taking-up
Appropriate liquid detergent solution immersion 2min is added, is rinsed with flowing water after 0.5-1.0h, stem section is transferred to 70% in super-clean bench
1min is soaked in alcoholic solution, with aseptic water washing 3 times, immersion dropwise addition there are 5 drop Tween-80s, and mass fraction is 0.1%
HgCl2Soaking disinfection 9-10min in solution, is then fully embathed 3 times with sterilized water, and the explant material being sterilized disappears
Malicious success rate is up to more than 90%.
(2) induction of Multiple Buds
Explant material obtained in the previous step is put on aseptic filter paper, cuts end to end, stamen and gynoecium is removed, by petal
It is cut into 2cm2, should in size, the SH minimal mediums for being connected to addition TDZ 0.5mg/L, NAA 0.2mg/L, AC 0.5-1.0g/L
Sucrose addition in culture medium is 30g/L, and agar addition is 8.0g/L, and pH is 5.8-6.0, and culture medium is once at 121 DEG C
Sterilize 20min, similarly hereinafter.The culture medium for connecting petal is put prior to cultivating 3-5d in dark, and cultivation temperature is 28 ± 2 DEG C；Then turn
Enter illumination cultivation, condition of culture is：Cultivation temperature is (28 ± 2) DEG C, and light application time is 14h light/10h dark, intensity of illumination 30-40
μmol/(m2·s)；Starting to occur petal section during culture 15d has protuberance, and adventitious bud clump can have been found during 20d, indefinite during 30d
Bud can grow to 3cm or so, and the inductivity of adventitious bud is up to 91.7%, and the adventitious bud number of the adventitious bud clump of induction has 9.5.
(3) propagation of adventitious bud
Induce the adventitious bud obtained clump to be cut into the sprout tuber of 2-3 adventitious bud previous step, be inoculated into added with TDZ
Cultivated, cultivated in 0.1mg/L, 6-BA2.0mg/L, CH 0.2-0.5mg/L, AC 0.5-1.0g/L SH minimal mediums
Condition is：Cultivation temperature is (28 ± 2) DEG C, and light application time is 14h light/10h dark, 30-40 μm of ol/ (m of intensity of illumination2·s)；Training
Support and counted after 30d, proliferation times 8.6, adventitious bud grows fine.
(4) culture of rootage of regeneration plant
Cut that blade is light green, grow vigorous and 3-4 pieces of blade adventitious bud, insertion is added with 6-BA 0.5, NAA
Taken root in the SH minimal mediums of 0.5mg/L, banana puree 80-100g/L 50% concentration, the sucrose added in culture medium is 20-
30g/L, agar addition is 8.0g/L, pH 5.8-6.0, and once in the 20min that sterilized at 121 DEG C；Cultivate 30d and find rooting rate
Up to more than 95%, spherosome is formed, root is generated thereon, root is about 6cm, sturdy, with many roots hair.
(5) acclimatization and transplantses
The height of seedling length of the tissue-cultured seedling of under growth root is to more than 5cm, when root length is more than 5cm, the bottle cap of tissue culture bottle is unclamped, into bottle
A small amount of sterilized water is injected to prevent culture medium dry and cracked, bottle cap is moved away in 6h is later half, and adds sterilized water, after 18h, removes bottle
Lid, allows 30-40 μm of ol/ (m2S) during which the light of light intensity add sterilized water several times directly according to regenerated plant culture 2d.Then it is small
Heart smashs culture medium to pieces, takes out regeneration plant, carefully rinses the agar of residual well with flowing water, plant is colonized in and once disappeared
The perlite that poison is crossed, vegetable garden soil weight ratio is 1：In 100-200 mixed-matrix, pour permeable and thin with transparent vinyon
Film covering is controlled between 24-28 DEG C with heat and moisture preserving, indoor temperature.Sealed polyethylene plastic corner is opened after 3d, next day will
Sealed polyethylene plastic is all opened, after after young leaves expansion can balled transplanting to crop field.Practice shoot survival percent and transplanting survival rate
Up to more than 98%.
Embodiment 2
The present embodiment and the difference of embodiment 1 are step (1)：The sterilization of David's-harp bud：Taken not yet May then
The bud of the David's-harp bloomed, is put into 4 DEG C of deepfreeze 0-48h of refrigerator, and appropriate liquid detergent solution immersion is added after taking-up
2min, is rinsed after 0.5-1.0h with flowing water, stem section is transferred in 70% alcoholic solution in super-clean bench and soaks 1min, with sterile
Water is rinsed 3 times, and immersion dropwise addition has 5 drop Tween-80s, and mass fraction is 0.1% HgCl2Soaking disinfection 9-10min in solution, so
Fully embathed with sterilized water 3 times afterwards, the explant material being sterilized.Find out in inoculation 30d statistical results, bud is used afterwards
Obtained pollution rate is carried out disinfection as explant will be well below root-like stock be used as explant, while bud is as outer
The time of implant evoking adventive bud also significantly faster than will use root-like stock for explant, and also be significantly larger than root on adventitious bud number
Shape stem is explant, and in the comparison of Cold pretreatment, be all petal as explant, pollution rate is basically unchanged, but is entered
The inductivity of Multiple Buds has significantly raised after row Cold pretreatment, and adventitious bud number has a small amount of increase.It follows that using right
(bud is used as explant, Cold pretreatment 24h) is handled in claim optimal performance.
The influence of table 1 different explants and Cold pretreatment to pollution rate and inducing clumping bud
Embodiment 3
The present embodiment and the difference of embodiment 1 are step (2)：The induction of Multiple Buds：By explant obtained in the previous step
Material is put on aseptic filter paper, is cut end to end, is removed stamen and gynoecium, petal is cut into 2cm2Size, is connected to addition TDZ 0-
In 2.0mg/L, NAA 0-1.0mg/L, AC 0-2.0g/L SH minimal mediums, the sucrose addition in the culture medium is
30g/L, agar addition is 8.0g/L, and pH is 5.8-6.0, and culture medium is once in the 20min that sterilized at 121 DEG C, similarly hereinafter.Connect petal
Culture medium put prior to cultivating 0-7d in dark, cultivation temperature is 28 ± 2 DEG C；Illumination cultivation is then continued at, condition of culture is：Training
It is (28 ± 2) DEG C to support temperature, and light application time is 14h light/10h dark, 30-40 μm of ol/ (m of intensity of illumination2·s)；Hair is counted after 30d
Existing, addition TDZ 0.5mg/L, NAA 0.2mg/L, AC 0.5g/L SH culture mediums have highest adventitious bud induction frequency and not
Normal bud number, and the growth of adventitious bud is best, shows as that leaf color is dark green, and stem is high and sturdy；The different disposal of TDZ concentration is shown, low
The TDZ (0.1mg/L) of concentration has than the low adventitious bud induction frequency of TDZ 0.5mg/L processing and adventitious bud number, the growth of adventitious bud
Also it is weaker, and the TDZ (2.0mg/L) of high concentration processing handles slightly higher adventitious bud inducing and slightly lower adventitious bud compared with TDZ 0.5mg/L
Number, the growth of adventitious bud shows vitrification phenomenon；Compared with SH culture mediums, be incubated at identical hormone and concentration of activated carbon group
The adventitious bud induction frequency and adventitious bud number of the MS culture mediums of conjunction are weaker, and the growth of adventitious bud is similar.
Influence of the different disposal of table 2 to inducing clumping bud
Embodiment 4
The present embodiment and the difference of embodiment 1 are step (3)：The propagation of adventitious bud：Step (2) induction is obtained not
Normal bud clump is cut into the sprout tuber of 2-3 adventitious bud, is inoculated into added with TDZ 0-2.0mg/L, 6-BA 0-5.0mg/L, CH 0-
Cultivated in 1.0mg/L, AC 0-2.0g/L SH minimal mediums, condition of culture is：Cultivation temperature is (28 ± 2) DEG C, light
It is 14h light/10h dark, 30-40 μm of ol/ (m of intensity of illumination according to the time2·s)；Count and find after 30d, adventitious bud in each culture medium
Proliferation times between 5.7-9.8, but proliferation times difference substantially, with the rise of TDZ concentration, although improve propagation
Multiple, but bring serious vitrifying, the leaf deformity of growth, stem also water stainization, the adventitious bud of acquisition is in culture of rootage
It can not take root completely；But compared with (proliferation times 5.7) without TDZ, TDZ presence promotes propagation (propagation really
Multiple 8.6)；Research also found that CH's is added with the propagation for being beneficial to adventitious bud, but finite concentration above effect is not further added by.
6-BA concentration also has an impact for the propagation of adventitious bud, and the effect of low concentration is also weaker than debita spissitudo, high concentration but
Bring the vitrifying of adventitious bud.Table 3 in general, with the addition of TDZ 0.1mg/L, 6-BA 2.0mg/L, CH 0.5g/L, AC
0.5g/L SH minimal mediums are best suitable for the propagation of adventitious bud.
Influence of the different disposal of table 3 to adventitious bud proliferation
Embodiment 5
The present embodiment and the difference of embodiment 1 are step (4)：The culture of rootage of regeneration plant：Cut blade light green, raw
Long vigorous and 3-4 pieces of blade adventitious bud, insertion is added with 6-BA 0-4.0, NAA 0-2.0mg/L, banana puree 0-150g/L's
Taken root in the SH minimal mediums of 12.5%-100% concentration, the sucrose added in culture medium is 20-30g/L, agar addition
For 8.0g/L, pH 5.8-6.0, and once in the 20min that sterilized at 121 DEG C.During 30d count find, rooting rate in 12.5-97.5%,
Earliest rootage duration 12-22d.It is extremely low without the SH minimal medium rooting rates of the low concentration (12.5%) of any hormone, and
The growth of tissue-cultured seedling is not good, and 100%SH is also unfavorable for taking root, and 50%SH is suitable；6-BA addition, although for rooting rate
It is several without influence, but it is favourable for the growth of tissue-cultured seedling overground part, promotes the growth of overground part；NAA is served for taking root
Crucial effect, the NAA of low concentration can cause thin and delicate root system, with increasing for NAA concentration, and root is thicker to shorten, therefore also uncomfortable
Work as high concentration；Addition AC and banana puree considerably increase rooting rate, and AC can provide the dark surrounds needed for root growth, and banana
Mud may also function as the effect for promoting tissue-cultured seedling growth, it may be possible to because it is internal with growth promoting substance.Consolidated statement 4, optimal
50%SH, 6-BA 0.5mg/L, NAA0.5mg/L are combined as, banana puree 80g/L, sucrose concentration is 20-30g/L, its rooting rate
Reachable 97.5, radical 4.8, root is slightly moderate, suitable for acclimatization and transplantses.
Influence of the different disposal of table 4 to inducing clumping bud
Claims (5)
1. a kind of method of David's-harp tissue culture and rapid proliferation, it is characterised in that comprise the following steps successively：
The sterilization of David's-harp bud
The bud of the David's-harp not yet bloomed is taken May then, 4 DEG C of deepfreeze 0-48 h of refrigerator are put into, added after taking-up
Appropriate liquid detergent solution soaks 2 min, is rinsed with flowing water after 0.5-1.0 h, and stem section is transferred to 70% alcohol in super-clean bench
1 min is soaked in solution, with aseptic water washing 3 times, immersion dropwise addition there are 5 drop Tween-80s, and mass fraction is 0.1% HgCl2It is molten
Soaking disinfection 9-10 min in liquid, are then fully embathed 3 times with sterilized water, the explant material being sterilized；
（2）The induction of Multiple Buds
Explant material obtained in the previous step is put on aseptic filter paper, is cut end to end, is removed stamen and gynoecium, petal is cut into
2 cm2In size, the SH minimal mediums for being connected to addition TDZ 0-2.0 mg/L, NAA 0-1.0 mg/L, AC 0-2.0 g/L,
Sucrose addition in the culture medium is 30 g/L, and agar addition is 8.0 g/L, and pH is 5.8-6.0, and culture medium is once in 121
Sterilize 20 min at DEG C, similarly hereinafter；The culture medium for connecting petal is put prior to cultivating 0-7 d in dark, and cultivation temperature is 28 ± 2 DEG C；
Illumination cultivation is then continued at, condition of culture is：Cultivation temperature is (28 ± 2) DEG C, and light application time is that 14 h light/10 h are dark, illumination
30-40 μm of ol/ (m of intensity2·s)；
（3）The propagation of adventitious bud
Induce the adventitious bud obtained clump to be cut into the sprout tuber of 2-3 adventitious bud previous step, be inoculated into added with TDZ 0-2.0 mg/
Cultivated in L, 6-BA 0-5.0 mg/L, CH 0-1.0 mg/L, AC 0-2.0 g/L SH minimal mediums, condition of culture
For：Cultivation temperature is (28 ± 2) DEG C, and light application time is that 14 h light/10 h are dark, 30-40 μm of ol/ (m of intensity of illumination2·s)；
（4）The culture of rootage of regeneration plant
Cut that blade is light green, grow vigorous and 3-4 pieces of blade adventitious bud, insertion is added with 6-BA 0-4.0, NAA 0-2.0
Taken root in the SH minimal mediums of mg/L, banana puree 0-150 g/L 12.5%-100% concentration, the sucrose added in culture medium is
20-30 g/L, agar addition is 8.0 g/L, pH 5.8-6.0, and once in 20 min that sterilized at 121 DEG C；
（5）Acclimatization and transplantses
The height of seedling length of the tissue-cultured seedling of under growth root is to 5 more than cm, when root length is more than 5 cm, unclamps the bottle cap of tissue culture bottle, is noted into bottle
Enter a small amount of sterilized water to prevent culture medium dry and cracked, move bottle cap away in 6 h are later half, and add sterilized water, after 18 h, remove bottle
Lid, allows 30-40 μm of ol/ (m2S) during which the light of light intensity add sterilized water several times directly according to the d of regenerated plant culture 2；Then
Carefully culture medium is smashed to pieces, regeneration plant is taken out, carefully the agar of residual is rinsed well with flowing water, plant is colonized in once
The perlite sterilized, vegetable garden soil weight ratio is 1：In 100-200 mixed-matrix, pour permeable and moulded with transparent polyethylene
Film covering is expected with heat and moisture preserving, and indoor temperature is controlled between 24-28 DEG C；Sealed polyethylene plastic corner is opened after 3 d, it is secondary
Day sealed polyethylene plastic is all opened, after after young leaves expansion can balled transplanting to crop field.
2. a kind of method of David's-harp tissue culture and rapid proliferation according to claim 1, it is characterised in that：It is described
Step（1）The material of middle inoculation is the bud not yet bloomed, and in 4 DEG C of deepfreeze 12-24 h of refrigerator.
3. a kind of method of David's-harp tissue culture and rapid proliferation according to claim 1, it is characterised in that：It is described
Step（2）In SH culture mediums be added with TDZ 0.5 mg/L, NAA 0.2 mg/L, AC 0.5-1.0 mg/L, be inoculated with flower
Valve is transferred to illumination cultivation again after 3-5 d are cultivated in dark.
4. a kind of method of David's-harp tissue culture and rapid proliferation according to claim 1, it is characterised in that：It is described
Step（3）Middle SH culture mediums are added with TDZ 0.1 mg/L, 6-BA 2.0 mg/L, CH 0.2-0.5 g/L, AC 0.5-1.0
mg/L。
5. a kind of method of David's-harp tissue culture and rapid proliferation according to claim 1, it is characterised in that：It is described
Step（4）In root media be 50%SH, 6-BA 0.5 mg/L, NAA 0.5 mg/L, banana puree 80-100 g/L.
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| CN107996333A (en) * | 2017-11-29 | 2018-05-08 | 福建三明林业学校 | A kind of David's-harp tissue culture method for transplanting |
| CN108849508A (en) * | 2018-07-03 | 2018-11-23 | 福建农林大学 | A kind of method of Sanming City wild foundation of rhizoma polygonati sterile system and bud induction |
| CN110089429A (en) * | 2019-04-25 | 2019-08-06 | 浙江省农业科学院 | A method of quickly breeding bletilla seedling using method for tissue culture |
| CN111264391A (en) * | 2020-03-30 | 2020-06-12 | 贵州大学 | Method for inducing cluster buds of polygonatum sibiricum and forming test-tube plantlets |
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| CN107996333A (en) * | 2017-11-29 | 2018-05-08 | 福建三明林业学校 | A kind of David's-harp tissue culture method for transplanting |
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| CN108849508A (en) * | 2018-07-03 | 2018-11-23 | 福建农林大学 | A kind of method of Sanming City wild foundation of rhizoma polygonati sterile system and bud induction |
| CN110089429A (en) * | 2019-04-25 | 2019-08-06 | 浙江省农业科学院 | A method of quickly breeding bletilla seedling using method for tissue culture |
| CN111264391A (en) * | 2020-03-30 | 2020-06-12 | 贵州大学 | Method for inducing cluster buds of polygonatum sibiricum and forming test-tube plantlets |
| CN111937747A (en) * | 2020-08-24 | 2020-11-17 | 三明市农业科学研究院 | Breeding method for forming strong and healthy rootstocks of polygonatum cyrtonema with multiple buds |
| CN111937747B (en) * | 2020-08-24 | 2021-10-01 | 三明市农业科学研究院 | Breeding method for forming strong and healthy rootstocks of polygonatum cyrtonema with multiple buds |
| CN115581202A (en) * | 2022-09-29 | 2023-01-10 | 中国科学院合肥物质科学研究院 | Method for regenerating and in-vitro seedling of new variety of polygonatum cyrtonema |
| CN115581202B (en) * | 2022-09-29 | 2023-11-14 | 中国科学院合肥物质科学研究院 | Method for regenerating new variety of Polygonatum cyrtonema Fabricius and in vitro seedling |
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