CN108812310A - A kind of efficient method for inducing magnificent Paris polyphylla sapling multiplication - Google Patents
A kind of efficient method for inducing magnificent Paris polyphylla sapling multiplication Download PDFInfo
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- CN108812310A CN108812310A CN201810563864.2A CN201810563864A CN108812310A CN 108812310 A CN108812310 A CN 108812310A CN 201810563864 A CN201810563864 A CN 201810563864A CN 108812310 A CN108812310 A CN 108812310A
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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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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 discloses a kind of methods for efficiently inducing magnificent Paris polyphylla sapling multiplication, this method is using the magnificent Paris polyphylla seed of differentiated radicle out as explant, by callus induction and Multiplying culture, after reaching higher breeding potential, using differentiation culture and stem tuber expands and culture of rootage, transplanting, with mass propagation China Paris polyphylla seedling and can shorten magnificent Paris polyphylla medicinal material growth period.The problem of it is low that the present invention solves magnificent Paris polyphylla sapling multiplication rate existing in the prior art, slow growth, is not able to satisfy existing market Paris polyphylla seedling demand to China.Using the method for the present invention, 2 years breeding potentials are more many fastly than conventional seminal propagation up to 60000 times or more.Not only breeding potential is high by the present invention, also shortens the field growing phase of magnificent Paris polyphylla medicinal material, and the planting survival rates of tissue culture stem tuber are high, the large-scale production suitable for magnificent Paris polyphylla.
Description
Technical field
The invention belongs to breeding plant technical fields, and in particular to a method of efficiently induce magnificent Paris polyphylla sapling multiplication.
Background technique
Magnificent Paris polyphylla also known as paris polyphylla are precious Chinese herbal medicines.Magnificent Paris polyphylla seed is low because having natural propagation rate, embryo hair
It educates not exclusively, endosperm is hard and plumular axis after-ripening etc. has the characteristic of obvious " dual suspend mode ", therefore its seed after planting usually needs
Undergo two winters that could sprout, seedling growth time is long.And conventional rhizome dissection nursery, due to the soil moisture and humidity
Be unable to control, wound healing is undesirable, easy infection germ class and cause monolith rhizome to fester, germplasm materials loss is high；Furthermore it dives
Lie prostrate that bud sprout time is long, and germination rate is low, polygerm sprouts less simultaneously, and so as to cause total breeding coefficient reduction, breeding is produced
Seedling quality it is poor.
It is quickly bred using the method for tissue cultures, has big breeding coefficient, virus removal, rapid more new varieties etc. excellent
Point, there are many successfully reports on lily.And the tissue culture of magnificent Paris polyphylla is there is not yet system research and application.
The tissue cultures of China's Paris polyphylla are concentrated mainly on the axenic germination, callus induction and culture aspect of seed at present,
Research in terms of research and test tube rhizome about magnificent Paris polyphylla tissue culture seedling proliferation and in terms of taking root are proliferated and expand is less.
Chinese patent CN201310529238.9 discloses a kind of rapid propagation method of Paris polyphylla plant, which uses
Be that the rhizome of magnificent Paris polyphylla makees explant, have the following disadvantages：Since rhizome is long in the soil, lead to Initial culture pollution very
Seriously, additionally due to rhizome itself is medicinal material, and generally only the sprout of front one, two can induce out callus, therefore should
Method can waste a large amount of raw material.
Summary of the invention
That the purpose of the present invention is to provide a kind of reproduction speeds is fast, the efficient side for inducing magnificent Paris polyphylla sapling multiplication more than quantity
Method, to meet the market demand.
To achieve the above object, the present invention uses following technical scheme：
A kind of efficient method for inducing magnificent Paris polyphylla sapling multiplication, the acquisition including aseptic explant are trained by the following steps
It supports：
1）Disinfection treatment：Select it is differentiated go out radicle magnificent Paris polyphylla seed, first rinsed well with flowing water, then with detergent immersion,
It is gently brushed with brush, is then rinsed well with tap water, then cut off radicle completely, remaining part is as explant, super
Explant is first impregnated with alcohol on net workbench, then is sterilized with mercuric chloride, is finally used aseptic water washing 3~4 times；
2）Fiber differentiation：On the induction medium by the explant inoculation after disinfection treatment, in 20 ± 2 DEG C of temperature, dark condition
Lower culture 50-60 d obtains callus；
The induced medium is MS culture medium, and each ingredient and its concentration are as follows in induced medium：
6- BA 2.0-3.0 mg·L-1
NAA 0.3-0.5 mg·L-1
2,4-D 0.1-0.3 mgL-1
Sucrose 28000-30000 mgL-1
Agar 7200-7500 mgL-1
3）Multiplying culture：Aseptically, by step 2）The callus of acquisition cuts into 1-3 cm3Proliferation is transferred to after size
In culture medium, at 24 ± 2 DEG C of temperature, 30-50 d is cultivated under dark condition, callus is largely proliferated；
The proliferated culture medium is MS culture medium, and each ingredient and its concentration are as follows in proliferated culture medium：
6- BA 1.0-2.0 mg·L-1
NAA 0.2 mg·L-1
2,4-D 0.1 mgL-1
Sucrose 28000-30000 mgL-1
Agar 7200-7500 mgL-1
4）Differentiation culture：By step 3）The callus of acquisition cuts into 1-3 cm3It is transferred in differential medium after size, in temperature
24 ± 2 DEG C of degree, cultivates 50-70 d under dark condition, callus can differentiate protrusion（That is tubercle）；
The differential medium is MS culture medium, and each ingredient and its concentration are as follows in differential medium：
6- BA 1.0 mg·L-1
NAA 0.2 mg·L-1
Sucrose 28000-30000 mgL-1
Agar 7200-7500 mgL-1
5）It expands and culture of rootage：By step 4）The tubercle of acquisition cut into it is single after be transferred to expand in root media,
At 24 ± 2 DEG C of temperature, 70-90 d is cultivated under dark condition, tubercle is expanded and taken root rapidly；
Described expand with root media is MS culture medium, is expanded as follows with ingredient each in root media and its concentration：
6- BA 1.0 mg·L-1
NAA 0.1 mg·L-1
IAA 0.1 mg·L-1
Sucrose 58000-60000 mgL-1
Agar 7200-7500 mgL-1
6）Transplanting：When step 5）When the root long of the stem tuber of culture is 0.5-2.0 cm, stem tuber is taken out, after the culture medium for washing away root
It is transplanted in matrix and grows, survival rate is up to 93% or more.
Step 1）In, the alcohol concentration of the immersion is 70-75%, and soaking time is 25-35 s.
Step 1）In, the mercuric chloride concentration of the disinfection is 0.1-0.15%, and disinfecting time is 8-10 min.
Step 2）Step 5）In, the pH of culture medium is 5.6-6.0, and preferably 5.8.
Step 6）In, mixed-matrix that the matrix is made of turf and perlite.
Further, the volume ratio of base turf and perlite is 1 in the mixed-matrix:1.
The invention adopts the above technical scheme, using the magnificent Paris polyphylla seed of differentiated radicle out as explant, by induction
Callus and Multiplying culture, after reaching higher breeding potential, using differentiation culture and stem tuber expands and culture of rootage, transplanting
Plant is formed, can realize the quick breeding of magnificent Paris polyphylla in a short time, and shorten the field growing phase.
The present invention has the following advantages that：（1）Using the magnificent Paris polyphylla seed of differentiated radicle out as explant, seed amount
More, disinfection is simple, and Initial culture pollution is few.（2）Breeding coefficient is high, and 2 years breeding potentials are up to 60000 times or more, than conventional seed
Breeding is fast very much.（3）The stem tuber neat and consistent produced using the method for the present invention can be unified to plant, convenient for management.（4）Due to adopting
It is transplanted with Tuberous root, without cauline leaf, washes seedling quickly and easily, convenient transportation, and degeneration-resistant border ability is strong, plants high survival rate, fits
Large-scale production for magnificent Paris polyphylla.
Specific embodiment：
The embodiment of the present invention is given below in substantive content in order to better illustrate the present invention, but the contents of the present invention are not
It is only limitted to these.
Embodiment 1
A kind of efficient method for inducing magnificent Paris polyphylla sapling multiplication, the acquisition including aseptic explant：It is trained by the following steps
It supports：
1）Disinfection treatment：The magnificent Paris polyphylla seed for selecting differentiated radicle out, is first rinsed well with flowing water, with detergent immersion, is used
Brush is gently brushed, and is then rinsed well with tap water, is then cut off radicle completely, remaining part is as explant, ultra-clean
75% alcohol of explant is impregnated into 30 s on workbench, then sterilizes 10 min with 0.1% mercuric chloride, finally uses aseptic water washing
4 times；
2）Fiber differentiation：On the induction medium by the explant inoculation after disinfection treatment, under 20 DEG C of temperature, dark condition
It cultivates 55d and obtains callus；
The induced medium is the MS culture medium of pH=5.8, and each ingredient and its concentration are as follows in induced medium：
6- BA 2.5mg·L-1
NAA 0.4 mg·L-1
2,4-D 0.2 mgL-1
30000 mgL of sucrose-1
7500 mgL of agar-1
3）Multiplying culture：Aseptically, by step 2）The callus of acquisition cuts into 2cm3Proliferation training is transferred to after size
It supports in base, cultivates 40 d under 24 DEG C of temperature, dark condition, callus is largely proliferated；
The increment culture medium is the MS culture medium of pH=5.8, and each ingredient and its concentration are as follows in the culture medium that rises in value：
6- BA 1.5 mg·L-1
NAA 0.2 mg·L-1
2,4-D 0.1 mgL-1
30000 mgL of sucrose-1
7500 mgL of agar-1
4）Differentiation culture：By step 3）The callus of acquisition cuts into 2 cm3It is transferred in differential medium after size, in temperature
24 DEG C, 60 d are cultivated under dark condition, callus can differentiate protrusion（That is tubercle）；
The differential medium is the MS culture medium of pH=5.8, and each ingredient and its concentration are as follows in differential medium：
6- BA 1.0 mg·L-1
NAA 0.2 mg·L-1
30000 mgL of sucrose-1
7500 mgL of agar-1
5）It expands and culture of rootage：By step 4）The tubercle of acquisition cut into it is single after be transferred to expand in root media,
80 d are cultivated under 24 DEG C of temperature, dark condition, tubercle is expanded and taken root rapidly；
It is described expand be with root media pH=5.8 MS culture medium, expand and each ingredient and its dense in culture of rootage basis
It spends as follows：
6- BA 1.0 mg·L-1
NAA 0.1 mg·L-1
IAA 0.1 mg·L-1
60000 mgL of sucrose-1
7500 mgL of agar-1
6）Transplanting：When step 5）When the root long of the stem tuber of culture is 1.5 cm, stem tuber is taken out, is transplanted after washing away the culture medium of root
It is 1 to turf and perlite volume ratio:It is grown in 1 mixed-matrix.
Embodiment 2
A kind of efficient method for inducing magnificent Paris polyphylla sapling multiplication, the acquisition including aseptic explant：It is trained by the following steps
It supports：
1）Disinfection treatment：The magnificent Paris polyphylla seed for selecting differentiated radicle out, is first rinsed well with flowing water, with detergent immersion, is used
Brush is gently brushed, and is then rinsed well with tap water, is then cut off radicle completely, remaining part is as explant, ultra-clean
70% alcohol of explant is impregnated into 35 s on workbench, then sterilizes 8 min with 0.15% mercuric chloride, finally uses aseptic water washing
3 times；
2）Fiber differentiation：On the induction medium by the explant inoculation after disinfection treatment, under 18 DEG C of temperature, dark condition
It cultivates 60 d and obtains callus；
The induced medium is the MS culture medium of pH=5.6, and each ingredient and its concentration are as follows in induced medium：
6- BA 2.0 mg·L-1
NAA 0.3 mg·L-1
2,4-D 0.1 mgL-1
28000 mgL of sucrose-1
7200 mgL of agar-1
3）Multiplying culture：Aseptically, by step 2）The callus of acquisition cuts into 1cm3Proliferation training is transferred to after size
It supports in base, cultivates 50 d under 22 DEG C of temperature, dark condition, callus is largely proliferated；
The proliferated culture medium is the MS culture medium of pH=5.6, and each ingredient and its concentration are as follows in proliferated culture medium：
6- BA 1.0 mg·L-1
NAA 0.2 mg·L-1
2,4-D 0.1 mgL-1
28000 mgL of sucrose-1
7200 mgL of agar-1
4）Differentiation culture：By step 3）The callus of acquisition cuts into 1cm3It is transferred in differential medium after size, in temperature
22 DEG C, 70 d are cultivated under dark condition, callus can differentiate protrusion（That is tubercle）；
The differential medium is the MS culture medium of pH=5.6, and each ingredient and its concentration are as follows in differential medium：
6- BA 1.0 mg·L-1
NAA 0.2 mg·L-1
28000 mgL of sucrose-1
7200 mgL of agar-1
5）It expands and culture of rootage：By step 4）The tubercle of acquisition cut into it is single after be transferred to expand in root media,
90 d are cultivated under 22 DEG C of temperature, dark condition, tubercle is expanded and taken root rapidly；
It is described expand be with root media pH=5.8 MS culture medium, expand and each ingredient and its dense in culture of rootage basis
It spends as follows：
6- BA 1.0 mg·L-1
NAA 0.1 mg·L-1
IAA 0.1 mg·L-1
58000 mgL of sucrose-1
7200 mgL of agar-1
6）Transplanting：When step 5）When the root long of the stem tuber of culture is 0.5cm, stem tuber is taken out, is transplanted after washing away the culture medium of root
It is 1 to turf and perlite volume ratio:It is grown in 1 mixed-matrix.
Embodiment 3
A kind of efficient method for inducing magnificent Paris polyphylla sapling multiplication, the acquisition including aseptic explant：It is trained by the following steps
It supports：
1）Disinfection treatment：The magnificent Paris polyphylla seed for selecting differentiated radicle out, is first rinsed well with flowing water, with detergent immersion, is used
Brush is gently brushed, and is then rinsed well with tap water, is then cut off radicle completely, remaining part is as explant, ultra-clean
75% alcohol of explant is impregnated into 25s on workbench, then sterilizes 10 min with 0.1% mercuric chloride, finally uses aseptic water washing 4
It is secondary；
2）Fiber differentiation：On the induction medium by the explant inoculation after disinfection treatment, under 22 DEG C of temperature, dark condition
It cultivates 50d and obtains callus；
The induced medium is the MS culture medium of pH=6.0, and each ingredient and its concentration are as follows in induced medium：
6- BA 3.0 mg·L-1
NAA 0.5 mg·L-1
2,4-D 0.3 mgL-1
30000 mgL of sucrose-1
7500 mgL of agar-1
3）Multiplying culture：Aseptically, by step 2）The callus of acquisition cuts into 3cm3Proliferation training is transferred to after size
It supports in base, cultivates 30 d under 26 DEG C of temperature, dark condition, callus is largely proliferated；
The increment culture medium is the MS culture medium of pH=6.0, and each ingredient and its concentration are as follows in the culture medium that rises in value：
6- BA 2.0 mg·L-1
NAA 0.2 mg·L-1
2,4-D 0.1 mgL-1
30000 mgL of sucrose-1
7500 mgL of agar-1
4）Differentiation culture：By step 3）The callus of acquisition cuts into 3cm3It is transferred in differential medium after size, in temperature
26 DEG C, 50 d are cultivated under dark condition, callus can differentiate protrusion（That is tubercle）；
The differential medium is the MS culture medium of pH=6.0, and each ingredient and its concentration are as follows in differential medium：
6- BA 1.0 mg·L-1
NAA 0.2 mg·L-1
30000 mgL of sucrose-1
7500 mgL of agar-1
5）It expands and culture of rootage：By step 4）The tubercle of acquisition cut into it is single after be transferred to expand in root media,
70 d are cultivated under 26 DEG C of temperature, dark condition, tubercle is expanded and taken root rapidly；
It is described expand be with root media pH=6.0 MS culture medium, expand and each ingredient and its dense in culture of rootage basis
It spends as follows：
6- BA 1.0 mg·L-1
NAA 0.1 mg·L-1
IAA 0.1 mg·L-1
60000 mgL of sucrose-1
7500 mgL of agar-1
6）Transplanting：When step 5）When the root long of the stem tuber of culture is 2.0 cm, stem tuber is taken out, is transplanted after washing away the culture medium of root
It is 1 to turf and perlite volume ratio:It is grown in 1 mixed-matrix.
Claims (7)
1. a kind of method for efficiently inducing magnificent Paris polyphylla sapling multiplication, the acquisition including aseptic explant, it is characterised in that：Under
Column step is cultivated：
1）Disinfection treatment：Radicle is cut off clean, remaining part work by the magnificent Paris polyphylla seed for selecting differentiated radicle out after cleaning
For explant, explant is first impregnated with alcohol on superclean bench, then is sterilized with mercuric chloride, aseptic water washing 3~4 is finally used
It is secondary；
2）Fiber differentiation：On the induction medium by the explant inoculation after disinfection treatment, in 20 ± 2 DEG C of temperature, dark condition
Lower culture 50-60 d obtains callus；
The induced medium is MS culture medium, and each ingredient and its concentration are as follows in induced medium：
6- BA 2.0-3.0 mg·L-1
NAA 0.3-0.5 mg·L-1
2,4-D 0.1-0.3 mgL-1
Sucrose 28000-30000 mgL-1
Agar 7200-7500 mgL-1
3）Multiplying culture：Aseptically, by step 2）The callus of acquisition cuts into 1-3 cm3Proliferation is transferred to after size
In culture medium, at 24 ± 2 DEG C of temperature, 30-50 d is cultivated under dark condition, callus is largely proliferated；
The proliferated culture medium is MS culture medium, and each ingredient and its concentration are as follows in proliferated culture medium：
6- BA 1.0-2.0 mg·L-1
NAA 0.2 mg·L-1
2,4-D 0.1 mgL-1
Sucrose 28000-30000 mgL-1
Agar 7200-7500 mgL-1
4）Differentiation culture：By step 3）The callus of acquisition cuts into 1-3 cm3It is transferred in differential medium after size, in temperature
24 ± 2 DEG C of degree, cultivates 50-70 d under dark condition, callus can differentiate tubercle；
The differential medium is MS culture medium, and each ingredient and its concentration are as follows in differential medium：
6- BA 1.0 mg·L-1
NAA 0.2 mg·L-1
Sucrose 28000-30000 mgL-1
Agar 7200-7500 mgL-1
5）It expands and culture of rootage：By step 4）The tubercle of acquisition cut into it is single after be transferred to expand in root media,
At 24 ± 2 DEG C of temperature, 70-90 d is cultivated under dark condition, tubercle is expanded and taken root rapidly；
Described expand with root media is MS culture medium, is expanded as follows with ingredient each in root media and its concentration：
6- BA 1.0 mg·L-1
NAA 0.1 mg·L-1
IAA 0.1 mg·L-1
Sucrose 58000-60000 mgL-1
Agar 7200-7500 mgL-1
6）Transplanting：When step 5）When the root long of the stem tuber of culture is 0.5-2.0 cm, stem tuber is taken out, after the culture medium for washing away root
It is transplanted in matrix and grows.
2. a kind of method for efficiently inducing magnificent Paris polyphylla sapling multiplication according to claim 1, it is characterised in that：Step 1）
In, the alcohol concentration of the immersion is 70-75%, and soaking time is 25-35 s.
3. a kind of method for efficiently inducing magnificent Paris polyphylla sapling multiplication according to claim 1, it is characterised in that：Step 1）
In, the mercuric chloride concentration of the disinfection is 0.1-0.15%, and disinfecting time is 8-10 min.
4. a kind of method for efficiently inducing magnificent Paris polyphylla sapling multiplication according to claim 1, it is characterised in that：Step 2）-
Step 5）In, the pH of culture medium is 5.6-6.0.
5. a kind of method for efficiently inducing magnificent Paris polyphylla sapling multiplication according to claim 4, it is characterised in that：Step 2）-
Step 5）In, the pH of culture medium is 5.8.
6. a kind of method for efficiently inducing magnificent Paris polyphylla sapling multiplication according to claim 1, it is characterised in that：Step 6）
In, mixed-matrix that the matrix is made of turf and perlite.
7. a kind of method for efficiently inducing magnificent Paris polyphylla sapling multiplication according to claim 6, it is characterised in that：Step 6）
In, the volume ratio of base turf and perlite is 1 in the mixed-matrix:1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109526745A (en) * | 2018-12-29 | 2019-03-29 | 广西壮族自治区农业科学院生物技术研究所 | A method of seedling is bred with paris polyphylla blade |
| CN113951140A (en) * | 2021-11-11 | 2022-01-21 | 广西壮族自治区中国科学院广西植物研究所 | Method for promoting rapid propagation of seedlings of paris polyphylla young plants |
-
2018
- 2018-06-04 CN CN201810563864.2A patent/CN108812310A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 刘银花等: "华重楼植株的快速繁殖研究", 《中草药》 * |
| 熊海浪等: "影响濒危药用植物滇重楼愈伤组织发生的因素", 《时珍国医国药》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109526745A (en) * | 2018-12-29 | 2019-03-29 | 广西壮族自治区农业科学院生物技术研究所 | A method of seedling is bred with paris polyphylla blade |
| CN113951140A (en) * | 2021-11-11 | 2022-01-21 | 广西壮族自治区中国科学院广西植物研究所 | Method for promoting rapid propagation of seedlings of paris polyphylla young plants |
| CN113951140B (en) * | 2021-11-11 | 2022-07-08 | 广西壮族自治区中国科学院广西植物研究所 | Method for promoting rapid propagation of seedlings of paris polyphylla young plants |
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