CN106665361A - Method for collecting syzygium buxifolium tissue culture initial bud - Google Patents
Method for collecting syzygium buxifolium tissue culture initial bud Download PDFInfo
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- CN106665361A CN106665361A CN201710077546.0A CN201710077546A CN106665361A CN 106665361 A CN106665361 A CN 106665361A CN 201710077546 A CN201710077546 A CN 201710077546A CN 106665361 A CN106665361 A CN 106665361A
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- explant
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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
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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 method for collecting a syzygium buxifolium tissue culture initial bud. The method comprises the procedures of explant preparation, explant disinfection treatment and initial bud induction, and comprises the following specific steps: collecting a syzygium buxifolium annual shoot to serve as an explant; disinfecting the explant; inoculating the obtained sterile explant into an initial bud induction culture medium; culturing in a proper environment for 20 to 30 days to obtain a syzygium buxifolium initial bud which grows quickly and has high vitality. The annual syzygium buxifolium shoot is selected as a tissue culture propagation material, a disinfection treatment way of using a plurality of agents with certain concentration and disinfection time is adopted, so that the sterile explant collection rate is over 90 percent; an optimized induction culture medium and an optimized culture way are adopted, so that browning of the explant is reduced, the initial bud generation rate is high, and the bud grows quickly and has high vitality; the method has good economical benefit and social benefit.
Description
Technical field
The invention belongs to field of plant tissue culture technique, more particularly, to a kind of initial bud acquisition side of Radix Syzygii Buxifolii tissue culture
Method.
Background technology
Radix Syzygii Buxifolii (Syzygium buxifolium Hook. et Arn.), category Myrtaceae Syzygium category, is shrub or little Qiao
Wood, twig has rib, pitchy after doing.Blade leathery, oblong to ellipse, wealthy obovate sometimes, tip circle or blunt, sometimes
There is blunt point head, base portion wealthy wedge shape or blunt, crineous after doing above, tarnish is slightly light below, there is gland point, and lateral vein is more close,
Above not substantially, slightly projection below；Style is equal with stamen.Fruit is spherical, diameter 5-7 millimeters.The month at florescence 6-8.Produce China
The provinces and regions such as Anhui, Zhejiang, Taiwan, Fujian, Jiangxi, Hunan, Guangdong, Guangxi, Guizhou.It is born in low mountain sparse woods or shrubbery.Radix Syzygii Buxifolii is liked
Light, also shade tolerant, moisture-proof, the climatic environment of suitable warm and moist.High temperature resistant, not cold-resistant, short-term can restrain oneself -13 DEG C extremely low
Temperature, but slightly prolonged less than 0 DEG C low temperature can make it be subject to serious cold damage.Radix Syzygii Buxifolii penjing art value is high, unusual ancient red
It is difficult to estimate that nanmu miniature gardening is even more true valency.Its root and bark can be used as medicine, and the crust for having the medical value of resolving sputum of relievining asthma, fruit can be eaten
With being the relatively common wild fruit in rural area.
Radix Syzygii Buxifolii tissue culture difficulty is primarily due to explant sterilization difficulty, also during initial bud inducement cultivation,
Easily the bottleneck problem such as browning, initial bud incidence rate low, sprout does not extend, poor activity, seriously limits Radix Syzygii Buxifolii superior clone
Promote and application.
The content of the invention
The present invention is for explant sterilization difficulty, browning, initial bud incidence rate in existing Radix Syzygii Buxifolii tissue culture be low, sprout work
Property difference etc. technical problem, there is provided a kind of Radix Syzygii Buxifolii aseptic explant prepare and initial bud induction method.
To achieve these goals, technical scheme is as follows
A kind of initial bud acquisition methods of Radix Syzygii Buxifolii tissue culture, including explant prepares, explant is disinfected and initial bud induction
Operation, collection Radix Syzygii Buxifolii gives birth to then twig as explant, and after disinfection, the aseptic explant of acquisition is inoculated in initial bud
In inducing culture, after cultivating 20-30 days in control environment, the initial bud of Radix Syzygii Buxifolii that growth is fast, flush is obtained, its operation
Step is as follows：
（1）Explant prepares：
In at least sunny for three days on end weather, the twig raw then of robust growth of Radix Syzygii Buxifolii is gathered as explant；
（2）Explant is disinfected：
By explant with aseptic water washing it is clean after, be placed in volumetric concentration be 0.1-0.3 % wide-spectrum bactericide in soak 20-30
Min, takes out explant and is placed on volumetric concentration to soak 1-2 min in 70 % ethanol, then moves into volumetric concentration for 5-8 %'s
10-20 min are soaked in liquor natrii hypochloritises, taking-up is put in the mixing of the mercuric chloride that volumetric concentration is 10-25 % and 2-5 drop tweens
In liquid after stirring immersion 20-30 min, finally with aseptic washing 3-4 time, 3-5 min are rinsed every time；Explant is cut into into band extremely
Few 1 axillary bud, the stem section of 1.5-3cm length is standby depending in stem section degree of lignification classified placing container；
By step（2）The explant for obtaining is inoculated in inducing culture；Described inducing culture is：Improvement 1/2MS cultures
Base+Thidiazuron（TDZ）0.5-2.0 mg·L-1 + MT 1.0-3.0 mg·L-1 + NAA 1.0-3.0 mg·L-1 + Portugal
Grape 30 gL of sugar-1 The gL of+carrageenan 3.5-1 + 0.1-0.3 % wide-spectrum bactericides；The initial bud inducement cultivation cycle is：20-
30 days, it is placed in temperature：20 ± 3 DEG C, the illumination cultivation time is：16 h, intensity of illumination is：Carry out in the environment of≤1000 lx just
Beginning bud inducement cultivation, the initial bud of Radix Syzygii Buxifolii for obtain robust growth, flushing.Initial bud sprouts rate more than 90%.
Above-described modified MS medium is consisted of：：KNO3 1800 mg·L-1；NH4NO3 1200 mg·L-1；
CaCl2·2H2O 130 mg·L-1；MgSO4·7H2O 380 mg·L-1；Ca(NO3)2·4H2O 280 mg·L-1；KH2PO4
310 mg·L-1；MnSO4·H2O 21.6 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025
mg·L-1；H3BO3 6.2 mg·L-1；Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O
0.025 mg·L-1；Vitamin B1 1.5 mg·L-1；Vitamin B6 0.6 mg·L-1；The mgL of nicotinic acid 0.5-1；Glycine
2.0 mg·L-1；The mgL of inositol 200-1。
Preferably, above suitable condition is：Temperature：20 ± 3 DEG C, the illumination cultivation time is：16 h, intensity of illumination is：≤
1000 lx。
In contrast to prior art, advantages of the present invention and good effect are as follows：
1st, it is tissue culture propagation material that the present invention chooses the twig of Radix Syzygii Buxifolii life then, and the rudiment bar juvenile form degree of explant is high, greatly
Improve the effectiveness of explant, so as to be successfully established a kind of Radix Syzygii Buxifolii stem section tissue culture method, this method is to accelerating Radix Syzygii Buxifolii high-quality
The production of strong sprout, promotes country's material fast-developing, is significant.
2nd, the present invention successively using the wide-spectrum bactericide of certain concentration and disinfecting time, ethanol, sodium hypochlorite, mercuric chloride and
The mode disinfected that tween is engaged, aseptic explant acquisition rate is high, reaches more than 90%.
3rd, the present invention is using optimization inducing culture and training method, and Brown is few, and initial bud incidence rate is high, sprout
Elongation is fast, flush, with preferable economic benefit and social benefit.
Specific embodiment
With reference to specific embodiment, the present invention is further described.
Embodiment 1：
In at least sunny for three days on end weather, gather Radix Syzygii Buxifolii robust growth gives birth to then twig as explant.By explant
With aseptic water washing it is clean after, be placed in the wide-spectrum bactericide that volumetric concentration is 0.1 % and soak 30 min, take out explant by its
Volumetric concentration is placed in soak 1 min in 70 % ethanol, then is moved in the liquor natrii hypochloritises that volumetric concentration is 5 % and is soaked 20
Min, taking-up is put in the mixed liquor of the mercuric chloride that volumetric concentration is 10 % and 2 drop tweens after stirring 30 min of immersion, finally with nothing
Bacterium is washed 3 times, and 5 min are rinsed every time；Explant is cut into into band at least one axillary bud, the stem section of 1.5-2.0cm length, depending on stem section wood
It is standby in matter degree classified placing container；The explant obtained after disinfecting is inoculated in inducing culture；Described
Inducing culture is：Improvement 1/2MS culture medium+Thidiazuron（TDZ）0.5 mg·L-1 + MT 1.0 mg·L-1 + NAA
1.0mg·L-1 The gL of+glucose 30-1 The gL of+carrageenan 3.5-1 + 0.1 % wide-spectrum bactericides；Initial bud inducement cultivation
Cycle is：20-35 days, it is placed in temperature：20 ± 3 DEG C, the illumination cultivation time is：16 h, intensity of illumination is：The ring of≤1000 lx
Carry out initial bud inducement cultivation in border, the initial bud of Radix Syzygii Buxifolii for obtain robust growth, flushing, initial bud is sprouted rate 96%.
Described modified MS medium is consisted of：：KNO3 1800 mg·L-1；NH4NO3 1200 mg·L-1；
CaCl2·2H2O 130 mg·L-1；MgSO4·7H2O 380 mg·L-1；Ca(NO3)2·4H2O 280 mg·L-1；KH2PO4
310 mg·L-1；MnSO4·H2O 21.6 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025
mg·L-1；H3BO3 6.2 mg·L-1；Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O
0.025 mg·L-1；Vitamin B1 1.5 mg·L-1；Vitamin B6 0.6 mg·L-1；The mgL of nicotinic acid 0.5-1；Glycine
2.0 mg·L-1；The mgL of inositol 200-1。
Embodiment 2：
In at least sunny for three days on end weather, gather Radix Syzygii Buxifolii robust growth gives birth to then twig as explant.By explant
With aseptic water washing it is clean after, be placed in the wide-spectrum bactericide that volumetric concentration is 0.2 % and soak 25 min, take out explant by its
Volumetric concentration is placed in soak 2 min in 70 % ethanol, then is moved in the liquor natrii hypochloritises that volumetric concentration is 6 % and is soaked 15
Min, taking-up is put in the mixed liquor of the mercuric chloride that volumetric concentration is 15 % and 4 drop tweens after stirring 25 min of immersion, finally with nothing
Bacterium is washed 3 times, and 5 min are rinsed every time；Explant is cut into into band at least one axillary bud, the stem section of 2.0-2.5 cm length, depending on stem section wood
It is standby in matter degree classified placing container；The explant obtained after disinfecting is inoculated in inducing culture；Described
Inducing culture is：Improvement 1/2MS culture medium+Thidiazuron（TDZ）1.5 mg·L-1 + MT 2.0 mg·L-1 + NAA
2.0 mg·L-1 The gL of+glucose 30-1 The gL of+carrageenan 3.5-1 + 0.1-0.3 % wide-spectrum bactericides；Initial bud is lured
Leading cultivation cycle is：20-25 days, it is placed in temperature：20 ± 3 DEG C, the illumination cultivation time is：16 h, intensity of illumination is：≤1000
Carry out initial bud inducement cultivation in the environment of lx, the initial bud of Radix Syzygii Buxifolii for obtain robust growth, flushing, initial bud is sprouted rate
95%。
Described modified MS medium is consisted of：：KNO3 1800 mg·L-1；NH4NO3 1200 mg·L-1；
CaCl2·2H2O 130 mg·L-1；MgSO4·7H2O 380 mg·L-1；Ca(NO3)2·4H2O 280 mg·L-1；KH2PO4
310 mg·L-1；MnSO4·H2O 21.6 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025
mg·L-1；H3BO3 6.2 mg·L-1；Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O
0.025 mg·L-1；Vitamin B1 1.5 mg·L-1；Vitamin B6 0.6 mg·L-1；The mgL of nicotinic acid 0.5-1；Glycine
2.0 mg·L-1；The mgL of inositol 200-1。
Embodiment 3：
In at least sunny for three days on end weather, gather Radix Syzygii Buxifolii robust growth gives birth to then twig as explant.By explant
With aseptic water washing it is clean after, be placed in the wide-spectrum bactericide that volumetric concentration is 0.3 % and soak 20 min, take out explant by its
Volumetric concentration is placed in soak 2 min in 70 % ethanol, then is moved in the liquor natrii hypochloritises that volumetric concentration is 8 % and is soaked 10
Min, taking-up is put in the mixed liquor of the mercuric chloride that volumetric concentration is 25 % and 5 drop tweens after stirring 20 min of immersion, finally with nothing
Bacterium is washed 4 times, and 3 min are rinsed every time；Explant is cut into into band at least one axillary bud, the stem section of 2.5-3cm length is wooden depending on stem section
It is standby in change degree classified placing container；The explant obtained after disinfecting is inoculated in inducing culture；Described lures
Leading culture medium is：Improvement 1/2MS culture medium+Thidiazuron（TDZ）2.0 mg·L-1 + MT 3.0 mg·L-1 + NAA 3.0
mg·L-1 The gL of+glucose 30-1 The gL of+carrageenan 3.5-1 + 0.3 % wide-spectrum bactericides；Initial bud inducement cultivation week
Phase is：25-30 days, it is placed in temperature：20 ± 3 DEG C, the illumination cultivation time is：16 h, intensity of illumination is：The environment of≤1000 lx
In carry out initial bud inducement cultivation, the initial bud of Radix Syzygii Buxifolii for obtain robust growth, flushing, initial bud is sprouted rate 92%.
Described modified MS medium is consisted of：：KNO3 1800 mg·L-1；NH4NO3 1200 mg·L-1；
CaCl2·2H2O 130 mg·L-1；MgSO4·7H2O 380 mg·L-1；Ca(NO3)2·4H2O 280 mg·L-1；KH2PO4
310 mg·L-1；MnSO4·H2O 21.6 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025
mg·L-1；H3BO3 6.2 mg·L-1；Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O
0.025 mg·L-1；Vitamin B1 1.5 mg·L-1；Vitamin B6 0.6 mg·L-1；The mgL of nicotinic acid 0.5-1；Glycine
2.0 mg·L-1；The mgL of inositol 200-1。
Claims (3)
1. initial bud acquisition methods of a kind of Radix Syzygii Buxifolii tissue culture, it is characterised in that：Including explant prepare, explant disinfect with
And initial bud induction operation, collection Radix Syzygii Buxifolii gives birth to then twig as explant, after disinfection, the aseptic explant of acquisition
In being inoculated in initial bud inducement cultivation base, after cultivating 20-30 days in control environment, the Radix Syzygii Buxifolii that growth is fast, flush is obtained
Initial bud, its operating procedure is as follows：
（1）Explant prepares：
In at least sunny for three days on end weather, the twig raw then of robust growth of Radix Syzygii Buxifolii is gathered as explant；
Explant is disinfected：
By explant with aseptic water washing it is clean after, be placed in volumetric concentration be 0.1-0.3 % wide-spectrum bactericide in soak 20-30
Min, takes out explant and is placed on volumetric concentration to soak 1-2 min in 70 % ethanol, then moves into volumetric concentration for 5-8 %'s
10-20 min are soaked in liquor natrii hypochloritises, taking-up is put in the mixing of the mercuric chloride that volumetric concentration is 10-25 % and 2-5 drop tweens
In liquid after stirring immersion 20-30 min, finally with aseptic washing 3-4 time, 3-5 min are rinsed every time；Explant is cut into into band extremely
Few 1 axillary bud, the stem section of 1.5-3cm length is standby depending in stem section degree of lignification classified placing container；
（3）Initial bud induction：By step（2）The explant for obtaining is inoculated in inducing culture；Described inducing culture is：
Improvement 1/2MS culture medium+Thidiazuron（TDZ）0.5-2.0 mg·L-1 + MT 1.0-3.0 mg·L-1 + NAA 1.0-3.0
mg·L-1 The gL of+glucose 30-1 The gL of+carrageenan 3.5-1 + 0.1-0.3 % wide-spectrum bactericides；Initial bud induction training
The foster cycle is：20-30 days, being placed under suitable condition carried out initial bud inducement cultivation, the Radix Syzygii Buxifolii for obtain robust growth, flushing
Initial bud.
2. initial bud acquisition methods of Radix Syzygii Buxifolii tissue culture according to claim 1, it is characterised in that：Described improvement MS cultures
Base is consisted of：KNO3 1800 mg·L-1；NH4NO3 1200 mg·L-1；CaCl2·2H2O 130 mg·L-1；MgSO4·
7H2O 380 mg·L-1；Ca(NO3)2·4H2O 280 mg·L-1；KH2PO4 310 mg·L-1；MnSO4·H2O 21.6
mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025 mg·L-1；H3BO3 6.2 mg·L-1；
Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O 0.025 mg·L-1；Vitamin B1
1.5 mg·L-1；Vitamin B6 0.6 mg·L-1；The mgL of nicotinic acid 0.5-1；The mgL of glycine 2.0-1；Inositol 200
mg·L-1。
3. Radix Syzygii Buxifolii individual plant explant according to claim 1 is prepared and initial bud abductive approach, it is characterised in that：Step
（3）Described suitable condition is：Temperature：20 ± 3 DEG C, the illumination cultivation time is：16 h, intensity of illumination is：≤1000 lx.
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2017
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| CN103477988A (en) * | 2013-09-22 | 2014-01-01 | 丽水市林业科学研究院 | Culture in vitro and rapid propagation method for syzygium grijsii |
| CN104255448A (en) * | 2014-08-22 | 2015-01-07 | 广西壮族自治区林业科学研究院 | A bud induction method for primary culture of baeckea frutescens |
| CN104756869A (en) * | 2015-04-09 | 2015-07-08 | 广西壮族自治区林业科学研究院 | Method for preparation of pinus massoniana lamb superior individual sterile explant and induction of initial bud |
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Application publication date: 20170517 |