CN107027622B - A kind of Herba Andrographitis Polyploid Induction Methods - Google Patents
A kind of Herba Andrographitis Polyploid Induction Methods Download PDFInfo
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Classifications
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
-
- 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
-
- 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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- Engineering & Computer Science (AREA)
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Abstract
The present invention relates to a kind of Herba Andrographitis Polyploid Induction Methods, this method is using Herba Andrographitis Multiple Buds as induced material, through containing 100~2000mgL‑1Colchicine and the solution of 1% dimethyl sulfoxide pre-processed, after sterile water wash clean, it is transferred to corresponding culture medium and continues to cultivate, culture medium is constantly replaced according to upgrowth situation, polyploid Multiple Buds are obtained, then taken root, practice the plant that seedling culture obtains normal growth.The present invention in conjunction with used method for inducing and cultivating, significantly improves the inductivity of Herba Andrographitis polyploid, up to 62.86%, survival rate 79.05%, and reduce the incidence of chimera for the first time using the regenerated Multiple Buds of Herba Andrographitis axillary bud tissue as induced material.The Herba Andrographitis tetraploid leaf area of acquisition, stem etc. are all huge compared with diploid, are conducive to the yield for improving andrographolide and Dehydro and drographolide, meet the needs of people are to Herba Andrographitis medicinal material and its Related product.
Description
Technical field
The present invention relates to plant tissue culture technical fields, and in particular to a kind of Herba Andrographitis Polyploid Induction Methods.
Background technique
Herba Andrographitis is the dry ground of acanthaceous plant Herba Andrographitis (Andrographispaniculata (Burm.F.) Nees)
Upper part, is clinically used for cold, fever, abscess of throat, aphthae etc., and chemical component is mainly andrographolide class and Huang
Ketone compounds.Andrographolide class compound has the effects that antibacterial, anti-inflammatory, antiviral, anti-infective, anticancer, antitumor,
It is the raw medicinal material of a variety of famous-brand and high-quality Chinese patent drug products (chuanxinlian tablet, XIAOYAN LIDAN PIAN etc.).Commodity herb main product Guangdong, Fujian,
It is on sale throughout the country.
Herba Andrographitis plant is mainly harvest object hyoscine with the nutrition organs of plant, is particularly suitable for thus using polyploid
The method of induction improves yield and quality.Because plant chromosome after doubling, shows nutrition organs " huge property "
Feature, can also meet well with vegetable nutritorium's (root, stem, leaf) is the production of crude drugs requirement for harvesting object, improves medicinal material
Yield.In addition, after being doubled due to plant cell chromosome, gene regulation active chemical content also more normal two on chromosome
Times body obviously increases, so carrying out multiploid induction to Herba Andrographitis medicinal plant extremely has the yield for improving effective component
Benefit.
Polyploid plant morphological feature, physio-biochemical characteristics and yield, in terms of be all varied, wherein having one
A little good characteristics are that diploid is unexistent, thus is widely used in plant breeding work.Polyploid Induction Methods early stage
Research is mainly using revulsion in situ, i.e., the seed of test plant, the seedling being growing, stem apex etc. under the conditions of Non in vitro
It is handled with the mutagens of various concentration, so that induced chromosome doubles.The method of processing have infusion method, injection method, semar technique and
Investment etc..Although these methods can obtain polyploid, often there is the high phenomenon of chimera probability.Recently as life
The development of object technology, in vitro culture double method start prevailing, and from the point of view of having been reported, the main path of this method has two: one
It is directly to handle culture materials with colchicine solution in tissue culture procedures；Second is that with the solid containing colchicine
Material culture for a period of time, then is transferred to differential medium, then differentiate intact plant by mutant by culture medium.It doubles in vitro
Cultivation is since experimental condition is easily controllable, and back mutation phenomenon caused by variation plant is affected by environment is reduced, test result
It is repeated strong, it is generallyd use in multiploid induction.
However, there is also some urgent problems to be solved in multiploid induction: firstly, although colchicine is to use at present
At most, maximally efficient chemical inducer, but its price is more expensive and has severe toxicity, and the artificial increased environment side of body is said it is to plant
The factor of compeling, can make plant cell internal environment change, promote the ROS generated into the cell normal under certain condition
It eliminates and makes apoptosis.Secondly, the chromosome number of not all meristematic cell all similarly doubles after mutagenic treatment, have
It may be that certain cells double, and some cells do not double and lead to occur chimera, chimera phenomenon is to polyploid breeding
Difficulty is brought, so that the really polyploid underfrequency of same ploidy level.
In multiploid induction, the selection of induced material mainly has: callus, adventitious bud, Multiple Buds, stem apex, seed, son
Leaf and blade, root etc..Wherein Multiple Buds are the group's crowd shoots come out by plant organ and healing tissue development, are mostly
Many cells are formed, bipolarity, structural integrity, can be formed directly in plantlet, it is the preferable material of plant polyploid nursery that planting percent is high
Material.The country is not with the relevant report of Herba Andrographitis inducing clumping bud polyploid nursery at present.For this purpose, using Herba Andrographitis Multiple Buds as material
Material reduces the induced concentration of colchicine by improvement inductive condition, mitigates its toxic action to cell, improves survival rate,
Chimera incidence is reduced simultaneously, provides practical advice to improve the genetic breeding work of Herba Andrographitis.
Summary of the invention
To solve the above-mentioned problems, the present invention is established by choosing specific induced material, improving induction and cultural method
A kind of abductive approach for the Herba Andrographitis polyploid that inductivity is high, chimera incidence is low.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
The present invention provides a kind of Herba Andrographitis Polyploid Induction Methods comprising following steps:
(1) 0.1% mercuric chloride of common Herba Andrographitis diploid seed the culture of aseptic seedling: is sterilized into primary, immersion 3min；Disappear
Seed after poison is rinsed 4~5 times with sterile distilled water；The seed rinsed impregnates spare for 24 hours in sterile water；Seed is inoculated with
It is cultivated 20~30 days in initial culture base, obtains aseptic seedling；
(2) induction of Multiple Buds: aseptically, the band axillary bud stem of aseptic seedling is transferred in adventitious shoots culture base and is trained
It supports 30~45 days, obtains Multiple Buds；
(3) processing of colchicine and dimethyl sulfoxide: aseptically, the Multiple Buds stem apex of 0.5~1cm long of clip
It is soaked in containing 100~2000mgL-1Colchicine and 1% dimethyl sulfoxide solution in, under dark condition vibrate 12~
72h；
(4) renewal cultivation: aseptically, by the Multiple Buds stem apex nothing after colchicine and dmso treatment
Bacterium washing is clean, is inoculated in after cultivating 12~14 days in the MS culture medium for do not add any hormone, is inoculated in adventitious shoots culture
It is cultivated 20~30 days in base, obtains polyploid Multiple Buds；
(5) Multiplying culture: polyploid Multiple Buds are inoculated into proliferated culture medium and are carried out Multiplying culture 20~25 days；
(6) polyploid is identified: the stem apex progress Chromosome Number Observation of Multiple Buds after Multiplying culture is taken, on a tip of a root
Observe that 5 or more split coil method chromosome numbers double simultaneously, can confirm the variation strain is polyploid；
(7) culture of rootage: aseptically, the strain for being accredited as polyploid is cut from base portion, is transferred to training of taking root
It supports and is cultivated 15~25 days in base, obtain rooted seedling；
(8) hardening: taking out rooted seedling, be transferred in the basin equipped with vermiculite, sand and soil, and hardening culture indoors 28~
40 days；
(9) it transplants seedlings: after hardening, basin seedling being moved on into outdoor, there is the negative canopy of 70~85% sunshade rates in screening, cultivate 8~12 days
Afterwards, negative canopy is removed, cultivation management is carried out.
Preferably, the initial culture base of above-mentioned steps (1) are as follows:+30~32gL of MS minimal medium-1Sucrose+2.0~
3.0g·L-1Agar.
Preferably, the adventitious shoots culture base of above-mentioned steps (2) are as follows:+0.5~1.0mgL of MS basal medium-16-BA+30
~32gL-1Sucrose+6.0~7.0gL-1Agar.
Preferably, the proliferated culture medium of above-mentioned steps (5) are as follows:+0.5~1.0mgL of MS basal medium-1KT+0.1~
0.3mg·L-1NAA+30~32gL-1Sucrose+6.0~7.0gL-1Agar.
Preferably, the root media of above-mentioned steps (7) are as follows:+0.8~1.0mgL of 1/2MS basal medium-1IBA+30
~32gL-1Sucrose+6.0~7.0gL-1Agar.
Preferably, the condition of culture of above-mentioned steps (1) aseptic seedling are as follows: 26~30 DEG C of temperature, intensity of illumination 1000~
1200Lx, illumination/14 8~10h of periodicity of illumination~16h are dark.
Step (2) adventitious shoots culture condition, step (5) Multiplying culture condition, step (7) culture of rootage condition, step (8)
Hardening condition of culture are as follows: 25~28 DEG C of temperature, 1000~1200Lx of intensity of illumination, illumination/12 10~12h of periodicity of illumination~14h
It is dark.
Preferably, the volume ratio of vermiculite, sand and soil is 1:(1~1.5 in above-mentioned steps (8)): 1.
The present invention uses colchicine and dimethyl sulfoxide for inducer, and the dimethyl sulfoxide is by improving mitogenetic group
The penetration power knitted promotes colchicine to rapidly soak into plant tissue, improves inductivity, alleviate because be impregnated with unevenly generate it is embedding
Fit phenomenon, and the induced concentration of colchicine can be reduced, mitigate its toxic action to cell, improves survival rate.
The present invention investigates the induction that different inducing treatments generates polyploid with different Herba Andrographitis induced materials
The influence that rate, survival rate, chimera occur.Specially using cotton ball stem apex cladding process respectively to Herba Andrographitis seedling stem apex, at
Ripe plant stem apex carries out multiploid induction, using regeneration breeding after infusion process to Herba Andrographitis callus and Herba Andrographitis Multiple Buds into
Row multiploid induction, as a result, it has been found that, the inductivity that mature plant stem apex generates polyploid is minimum, and only 6.45%, callus
The survival rate for generating polyploid plant is minimum, and only 10.26%, it is secondly that the inductivity of seedling stem apex generation polyploid is
11.11%, the survival rate of plant is 36.59%, and using Multiple Buds as induced material, using restoring after infusion process and regenerate numerous
Grow carry out multiploid induction, the survival rate highest of inductivity and plant, respectively 62.86% and 79.05%, and chimera is sent out
Raw rate is minimum, is 2 plants 11 plants of chimera/detection.
Compared with prior art, present invention has an advantage that
(1) present invention utilizes colchicine and two for the first time using the regenerated Multiple Buds of Herba Andrographitis axillary bud tissue as induced material
The stem apex of Multiple Buds is carried out multiploid induction by methyl sulfoxide infusion process, then the stem apex after induction is broken up to obtain polyploid plant again
Strain.Induced material of the invention combines used method for inducing and cultivating, significantly improves the inductivity of Herba Andrographitis polyploid, high
Up to 62.86%, survival rate 79.05%, and reduce the incidence of chimera.
(2) Herba Andrographitis tetraploid leaf area, stem that the present invention obtains etc. is all huge compared with diploid, is conducive to improve Herba Andrographitis
The yield of lactone and Dehydro and drographolide promotes the improvement of andrographolide and Dehydro and drographolide quality, realizes punching
The purpose of lotus good quality and high output meets the needs of people are to Herba Andrographitis medicinal material and its Related product.
Detailed description of the invention
Fig. 1 is Herba Andrographitis Diploid and Tetraploid growth figure, A: Seedling Stage Herba Andrographitis diploid (left side) and four times of Herba Andrographitis
Body (right side), B: maturity period Herba Andrographitis tetraploid (left side) and Herba Andrographitis diploid (right side).
Fig. 2 is Herba Andrographitis Diploid and Tetraploid plant porosity chart, A: Herba Andrographitis liploid plant porosity chart, B: Herba Andrographitis
Tetraploid plant porosity chart.
Fig. 3 be Herba Andrographitis Diploid and Tetraploid plant chromosome map, A: Herba Andrographitis liploid plant chromosome map (2n=
2x=48), B: Herba Andrographitis tetraploid plant chromosome map (2n=4x=96).
Specific embodiment
The present invention is further described below by way of specific embodiment, but the present invention is not limited only to following embodiment.
The induction of 1 Herba Andrographitis polyploid of embodiment
(1) 0.1% mercuric chloride of common Herba Andrographitis diploid seed the culture of aseptic seedling: is sterilized into primary, immersion 3min；Disappear
Seed after poison is rinsed 4 times with sterile distilled water；The seed rinsed impregnates spare for 24 hours in sterile water；Seed is inoculated in
MS+30g·L-1Sucrose+2.5gL-1In the initial culture base of agar, in 28 DEG C of temperature, intensity of illumination 1200Lx, periodicity of illumination
It is cultivated 25 days under conditions of 10h illumination/14h is dark, obtains aseptic seedling；
(2) induction of Multiple Buds: aseptically, the band axillary bud stem of aseptic seedling is transferred to MS+0.8mgL-16-BA
+30g·L-1Sucrose+6.5gL-1In the adventitious shoots culture base of agar, in 28 DEG C of temperature, intensity of illumination 1200Lx, periodicity of illumination
It is cultivated 40 days under conditions of 12h illumination/12h is dark, obtains Multiple Buds；
(3) processing of colchicine and dimethyl sulfoxide: aseptically, the Multiple Buds stem apex of clip 0.8cm long soaks
It steeps in containing 2000mgL-1Colchicine and 1% dimethyl sulfoxide solution in, vibrate 72h under dark condition；
(4) renewal cultivation: aseptically, by the Multiple Buds stem apex nothing after colchicine and dmso treatment
Bacterium washing is clean, is inoculated in after cultivating 14 days in the MS culture medium for do not add any hormone, is inoculated in adventitious shoots culture base
Culture 25 days obtains polyploid Multiple Buds；
(5) polyploid Multiple Buds Multiplying culture: are inoculated into MS+0.8mgL-1KT+0.2mg·L-1NAA+30g·L-1
Sucrose+6.5gL-1In the proliferated culture medium of agar, in 28 DEG C of temperature, intensity of illumination 1200Lx, periodicity of illumination 12h illumination/12h
It is carried out Multiplying culture 25 days under conditions of dark；
(6) polyploid is identified: the stem apex progress Chromosome Number Observation of Multiple Buds after Multiplying culture is taken, on a tip of a root
Observe that 5 or more split coil method chromosome numbers double simultaneously, can confirm the variation strain is polyploid；
(7) culture of rootage: aseptically, the strain for being accredited as polyploid is cut from base portion, is transferred to 1/2MS+
0.8mg·L-1IBA+30g·L-1Sucrose+6.5gL-1In the root media of agar, in 28 DEG C of temperature, intensity of illumination
It is cultivated 20 days under 1200Lx, periodicity of illumination 12h illumination/12h dark condition, obtains rooted seedling；
(8) hardening: taking out rooted seedling, is transferred in the basin equipped with vermiculite, sand and soil that volume ratio is 1:1.5:1,
Hardening culture 35 days under conditions of 28 DEG C of temperature, intensity of illumination 1200Lx, periodicity of illumination 12h illumination/12h are dark indoors；
(9) it transplants seedlings: after hardening, basin seedling being moved on into outdoor, there is the negative canopy of 85% sunshade rate to remove in screening after culture 10 days
Negative canopy carries out cultivation management.
The multiploid induction effect of the different Herba Andrographitis induced materials of embodiment 2 compares
Multiploid induction is carried out to Herba Andrographitis seedling stem apex and mature plant stem apex with cotton ball stem apex cladding process respectively, with
Regeneration breeding is compared Herba Andrographitis callus and Herba Andrographitis Multiple Buds progress multiploid induction after infusion process, from polyploid
Inductivity, the survival rate of polyploid plant, evaluate different methods for inducing in terms of chimera incidence three and induce different Herba Andrographitis
The effect of material generation polyploid.
Wherein, cotton ball stem apex cladding process specifically: the phase has been successfully established Herba Andrographitis regenerating system prior to the study
On the basis of obtain aseptic seedling.It will be transplanted in culture plate after the uniform aseptic seedling hardening of growing way, be divided into 5 groups, every group 3.With
The colchicine that mass concentration containing 1%DMSO is 0.1% is handled.Various concentration medical fluid, which is dipped in, with absorbent cotton is placed in seedling top
End keeps cotton wet, handles 36h.Then remove absorbent cotton, clear water rinses top 3 times.It is placed under light and cultivates, identified after 30d.
Above-mentioned step is referred to the method that cotton ball stem apex cladding process induction Herba Andrographitis maturation plant stem apex generates polyploid
Suddenly.
Breeding method is regenerated after infusion process specifically: Herba Andrographitis tissue cultures material Multiple Buds are placed in various concentration colchicum
12~72h is impregnated in element, it is clean with aseptic water washing after material is soaking, it is transferred to corresponding culture medium and continues to cultivate, according to life
Long situation constantly replaces culture medium, observes statistics after 30d.It is transferred to 1/2MS+1.0mgL-1IBA+30g·L-1Sucrose+
6.5g·L-1In the root media of agar, when length to 10cm or so, its aberration rate is counted after hardening, transplanting 30d.
Above-mentioned step is referred to the method that breeding method induction Herba Andrographitis callus generates polyploid is regenerated after infusion process.
As a result it see the table below shown.
The multiploid induction effect of the different Herba Andrographitis induced materials of table 1 compares
The results show that the inductivity that mature plant stem apex generates polyploid is minimum, and only 6.45%, callus generates more
The survival rate of times body plant is minimum, and only 10.26%, it secondly be the inductivity that seedling stem apex generates polyploid is 11.11%, plant
The survival rate of strain is 36.59%, and using Multiple Buds as induced material, more times are carried out using recovery after infusion process and regeneration breeding
Body induction, the survival rate highest of inductivity and plant, respectively 62.86% and 79.05%, and chimera incidence is minimum,
It is 2 plants 11 plants of chimera/detection.Show to carry out polyploid, tool to Multiple Buds using Herba Andrographitis abductive approach provided by the invention
There is preferable effect, inductivity is best, polyploid plant survival rate highest, and chimera incidence is minimum.
The identification of 3 Herba Andrographitis stomata characteristics of embodiment
It takes common plant 5 of growth 6 months to randomly select 4 representational leaves with every group of every plant of processing variating seedling to tear
Epidermis is removed, Guard Cell Length, width, chloroplast number are measured.Every leaf measures 20 stomatas, calculates average value, counts gas
As a result Kong Mi is shown in Fig. 2 and table 2.
2 Herba Andrographitis diploid of table is compared with tetraploid stomatal character
Note: Indexes Comparison corresponding with Herba Andrographitis diploid,*P < 0.05,**P<0.01。
By table 2 and Fig. 2 it is found that compared with diploid, the gentle hole width of stomata length of tetraploid is noticeably greater than two times
Body (P < 0.01 or P < 0.05)；The stomatal frequency of tetraploid shows provided by the invention more significantly less than diploid (P < 0.05)
Times body abductive approach improves the kind of Herba Andrographitis.
4 stem tip chromosome identification method of embodiment
Herba Andrographitis stem apex is pre-processed into 2h with 0.10% colchicine, Ka Nuoshi liquid is fixed for 24 hours, and 60 DEG C of 1.0mol/L hydrochloric acid
Lower dissociation 8min, hypotonic 15min after distilled water, carbolfuchsin dye 25min, gained Herba Andrographitis stem tip chromosome production effect compared with
It is good, the chromosome medium cell more visible compared with dispersion, kinetochore is observed in field of microscope, sees Fig. 3.
From the figure 3, it may be seen that Herba Andrographitis liploid plant chromosome number is 2n=2x=48, the tetraploid after inducing and doubling
Plant chromosome number is 2 times of original liploid plant, i.e. 2n=4x=96.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (7)
1. a kind of Herba Andrographitis Polyploid Induction Methods, which comprises the following steps:
(1) 0.1% mercuric chloride of common Herba Andrographitis diploid seed the culture of aseptic seedling: is sterilized into primary, immersion 3min；After disinfection
Seed with sterile distilled water rinse 4~5 times；The seed rinsed impregnates spare for 24 hours in sterile water；Seed is inoculated in just
It is cultivated 20~30 days in culture medium, obtains aseptic seedling；
(2) induction of Multiple Buds: aseptically, the band axillary bud stem of aseptic seedling is transferred in adventitious shoots culture base and cultivates 30
~45 days, obtain Multiple Buds；
(3) processing of colchicine and dimethyl sulfoxide: aseptically, the Multiple Buds stem apex of 0.5~1cm long of clip impregnates
In contain 100~2000mgL-1Colchicine and 1% dimethyl sulfoxide solution in, under dark condition vibrate 12~72h；
(4) renewal cultivation: aseptically, by the Multiple Buds stem apex sterile water after colchicine and dmso treatment
Wash clean is inoculated in after cultivating 12~14 days in the MS culture medium for do not add any hormone, is inoculated in adventitious shoots culture base
Culture 20~30 days obtains polyploid Multiple Buds；
(5) Multiplying culture: polyploid Multiple Buds being inoculated into proliferated culture medium and are carried out Multiplying culture 20~25 days, Multiplying culture
Base are as follows:+0.5~1.0mgL of MS basal medium-1KT+0.1~0.3mgL-1NAA+30~32gL-1Sucrose+6.0~
7.0g·L-1Agar；
(6) polyploid is identified: taking the stem apex progress Chromosome Number Observation of Multiple Buds after Multiplying culture, on a tip of a root simultaneously
Observe that 5 or more split coil method chromosome numbers double, can confirm the variation strain is polyploid；
(7) culture of rootage: aseptically, the strain for being accredited as polyploid is cut from base portion, is transferred to root media
Middle culture 15~25 days, obtains rooted seedling；
(8) hardening: rooted seedling is taken out, is transferred in the basin equipped with vermiculite, sand and soil, hardening culture indoors 28~40
It；
(9) it transplants seedlings: after hardening, basin seedling being moved on into outdoor, there is the negative canopy of 70~85% sunshade rates to move in screening after culture 8~12 days
Negative canopy is removed, cultivation management is carried out.
2. Herba Andrographitis Polyploid Induction Methods according to claim 1, which is characterized in that first being commissioned to train of the step (1)
Support base are as follows:+30~32gL of MS minimal medium-1Sucrose+2.0~3.0gL-1Agar.
3. Herba Andrographitis Polyploid Induction Methods according to claim 1, which is characterized in that the Multiple Buds of the step (2)
Culture medium are as follows:+0.5~1.0mgL of MS basal medium-16-BA+30~32gL-1Sucrose+6.0~7.0gL-1Agar.
4. Herba Andrographitis Polyploid Induction Methods according to claim 1, which is characterized in that the training of taking root of the step (7)
Support base are as follows:+0.8~1.0mgL of 1/2MS basal medium-1IBA+30~32gL-1Sucrose+6.0~7.0gL-1Agar.
5. Herba Andrographitis Polyploid Induction Methods according to claim 1, which is characterized in that step (1) aseptic seedling
Condition of culture are as follows: 26~30 DEG C of temperature, 1000~1200Lx of intensity of illumination, illumination/14 8~10h of periodicity of illumination~16h are dark.
6. Herba Andrographitis Polyploid Induction Methods according to claim 1, which is characterized in that step (2) the Multiple Buds training
Support condition, step (5) Multiplying culture condition, step (7) culture of rootage condition, step (8) hardening condition of culture are as follows: temperature 25~
28 DEG C, 1000~1200Lx of intensity of illumination, illumination/12 10~12h of periodicity of illumination~14h dark.
7. Herba Andrographitis Polyploid Induction Methods according to claim 1, which is characterized in that vermiculite, sand in the step (8)
The volume ratio of son and soil is 1:(1~1.5): 1.
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