CN108849523B - A kind of Calotropis gigantea tissue culture and rapid propagation method - Google Patents
A kind of Calotropis gigantea tissue culture and rapid propagation method Download PDFInfo
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- CN108849523B CN108849523B CN201810909980.5A CN201810909980A CN108849523B CN 108849523 B CN108849523 B CN 108849523B CN 201810909980 A CN201810909980 A CN 201810909980A CN 108849523 B CN108849523 B CN 108849523B
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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 present invention provides a kind of Calotropis gigantea tissue culture and rapid propagation methods, belong to plant tissue culture technical field.The method of the invention is inoculated in primary inducing culture after disinfection the following steps are included: 1) using Calotropis gigantea tender tissue as explant and carries out Fiber differentiation, obtains sprout primary；2) sprout primary for obtaining step 1) is cut into the stem section comprising 1~2 axillary bud, is placed in subculture medium and carries out squamous subculture, obtains tufted seedling；3) tufted seedling for obtaining step 2), which is placed on root media, carries out culture of rootage, obtains Calotropis gigantea tissue-culturing rapid propagation seedling.Make 4.83~5.93/25d of Calotropis gigantea growth coefficient using method of the invention；The glass rate of tissue-cultured seedling is reduced to 4.44~8.89%, and continuous multi-generation culture, glass rate maintains always 8% hereinafter, difference is not significant between each generation.
Description
Technical field
The invention belongs to plant tissue culture technical fields, and in particular to a kind of Calotropis gigantea tissue culture and rapid propagation method.
Background technique
Calotropis gigantea (Calotropis giganteaL.) is Asclepiadaceae Calotropis shrub, is distributed mainly on cloud in China
South, Sichuan, Guangxi and Guangdong etc. are regional, be grown on more low altitude area endroit, wilderness and seashore, in dry-hot valley, salt
Alkali etc. adaptability is stronger under habitats, can play the role of improving the ecological environment.In recent years, as domestic and foreign scholars are to ox horn
The further investigation of melon, it was found that a variety of application and development values of Calotropis gigantea.
Firstly, akund density is 0.9g/cm3, it is the most light fiber of nature quality, the structure class of Calotropis gigantea suede
It is similar to bombax cotton and there is more excellent mechanical performance, the mixed yarn of Calotropis gigantea suede and cotton can satisfy wanting substantially for dress ornament
It asks, is known as after the another new type natural plant fiber with wide application prospect of cotton fiber.
It is toxic but there is medical value secondly, Calotropis gigantea complete stool is rich in white milk.The ox extracted from Calotropis gigantea milk
The substances such as angle melon glycosides have cardiac effect；The extract of Calotropis gigantea root and flower have the effects that it is anti-inflammatory, ease pain, bring down a fever；In addition, ox
The different extracts of angle melon also have protect liver, promote a variety of effects such as wound healing, anti-oxidant.Early in Kunming the 1960s
The virtuous equal calotropin just extracted to the Calotropis gigantea root skin in Yunnan of medical college scholar Deng is studied, and uses the amphibian living body heart respectively
Dirty, mammal isolated heart etc. is experimental subjects, and discovery calotropin has cardiac effect, and it is lethal most to calculate birds
It is low dose of.Foreign scholar has conducted extensive research the chemical component and its medical mechanism of Calotropis gigantea, about the anti-swollen of cardiac glycoside
Tumor effect is patented in the U.S..
In addition, Calotropis gigantea is referred to as " petroleum plant ", a large amount of hydrocarbons and some hydrocarbon substance are contained in juice,
It is approximate with natural oil ingredient, be containing fat content in the Calotropis gigantea plant produced there are many chemical component, India in juice
4.7%, calorific value and raw coal are close, are considered can be used to develop liquid fuel and useful chemicals.Due to the Calotropis gigantea speed of growth
Fastly, weekly can long 30cm, can more batches of harvests, biomass is big.Therefore, it is of great significance as energy-source plant exploitation.
Confirm that Calotropis gigantea is in cotton spinning field, field of medicaments and biomass energy by the research of domestic and international researcher
Source development field etc. all has important application value, but Calotropis gigantea has a very wide distribution and more dispersed, up to the present, and
A set of effective introduction and acclimatization and intensive culture technique are not formed, the serious popularization and application for hindering Calotropis gigantea.
Although Calotropis gigantea tissue culture technology is currently reported, research is relatively fewer, and there are two influence Industrial Efficiency
Main problem does not solve simultaneously: first is that Calotropis gigantea tissue-cultured seedling growth coefficient is lower, only 3.9/25d (Tang Junrong etc., 2016)；Second is that
Easily vitrifying during Calotropis gigantea tissue-culturing rapid propagation, and increasing with subculture number, vitrifying aggravate, and eventually lead to negative increasing
It grows.
Summary of the invention
In view of this, Calotropis gigantea group can be inhibited the purpose of the present invention is to provide a kind of Calotropis gigantea tissue culture and rapid propagation method
The vitrifying of seedling is trained, and improves growth coefficient.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of Calotropis gigantea tissue culture and rapid propagation methods, comprising the following steps:
1) it using Calotropis gigantea tender tissue as explant, is inoculated in after disinfection in primary inducing culture and carries out Fiber differentiation,
Obtain sprout primary；The primary inducing culture is basic culture medium with MS culture medium, comprising: 25~35gL of sucrose-1And fine jade
3.4~4gL of rouge-1；
2) sprout primary for obtaining step 1) is cut into the stem section comprising 1~2 axillary bud, is placed in subculture medium
Squamous subculture is carried out, tufted seedling is obtained；The subculture medium is basic culture medium with MS culture medium, comprising: 6-BA 0.5~
2.2mg·L-1, 0.02~0.15mgL of NAA-1、AgNO30.5~2.5mgL-1, 25~35gL of sucrose-1With agar 3.4
~4gL-1；
3) tufted seedling for obtaining step 2), which is placed on root media, carries out culture of rootage, and it is fast to obtain Calotropis gigantea tissue culture
Numerous seedling；The root media is basic culture medium with 1/2MS culture medium, comprising: 0.5~1.5mgL of NAA-1, sucrose 15~
25g·L-1With 3.4~4gL of agar-1。
Preferably, step 1) the Calotropis gigantea tender tissue includes the stem section with axillary bud.
Preferably, the step 1) disinfection includes: that explant is placed in the dish washing liquid aqueous solution that mass concentration is 1~2%
Middle immersion 5min, is rinsed well after taking-up, then is placed in the ethanol water that volumetric concentration is 70~75% and is impregnated 50~70s,
The HgCl that mass concentration is 0.1~0.2% is placed in after taking-up again25~10min is impregnated in aqueous solution, is taken out, sterile water wash 3
~5 times.
Preferably, the step 1) Fiber differentiation is illumination cultivation, and the light application time of the illumination cultivation is 10~15h/
D, intensity of illumination are 35~45 μm of olm-2·s-1, the temperature of the Fiber differentiation is 22~32 DEG C, the Fiber differentiation when
Between be 20~28d.
Preferably, the step 2) squamous subculture be illumination cultivation, 10~15h/d of light application time of the illumination cultivation,
Intensity of illumination is 35~45 μm of olm-2·s-1, the temperature of the squamous subculture is 22~32 DEG C, the time of the squamous subculture
For 21~30d.
Preferably, the step 3) culture of rootage is illumination cultivation, and the light application time of the illumination cultivation is 10~15h/
D, intensity of illumination are 35~45 μm of olm-2·s-1, the temperature of the culture of rootage is 22~32 DEG C, the culture of rootage when
Between be 10~20d.
It preferably, further include hardening and transplanting after the step 3) culture of rootage.
Preferably, the hardening is that Calotropis gigantea tissue-culturing rapid propagation seedling is set hardening 5~7 days in greenhouse, the shading of the greenhouse
Degree is 60~80%.
Preferably, to enter the tissue-culturing rapid propagation transplantation of seedlings after hardening in matrix, the matrix includes sandy loam for the transplanting
And fertile soil.
Preferably, the sandy loam and volume ratio humous are (1~4): 1.
The present invention provides a kind of Calotropis gigantea tissue culture and rapid propagation methods, carry out tissue culture by explant of Calotropis gigantea tender tissue,
Using the successively culture of primary inducing culture, subculture medium and root media, make Calotropis gigantea growth coefficient 4.83
~5.93/25d, hence it is evident that be higher than prior art growth coefficient achieved；And the glass rate of tissue-cultured seedling be reduced to 4.44~
8.89%, continuous multi-generation culture, glass rate maintains always 8% hereinafter, difference is not significant between each generation.Using the present invention
Method solve Calotropis gigantea tissue-cultured seedling easily it is vitrified simultaneously, moreover it is possible to maintain the higher growth coefficient of Calotropis gigantea Multiplying culture,
Advantage is created for the seedling tissue-culturing rapid propagation of Calotropis gigantea high efficiency, low cost.
Detailed description of the invention
Fig. 1 is Multiplying culture result figure in the embodiment of the present invention 1；
Fig. 2 is 2 Multiplying culture result figure of the embodiment of the present invention；
Fig. 3 is 3 Multiplying culture result figure of the embodiment of the present invention；
Fig. 4 is 4 Multiplying culture result figure of the embodiment of the present invention；
Fig. 5 is 5 Multiplying culture result figure of the embodiment of the present invention；
Fig. 6 is comparative example 3 of the present invention proliferation passage result figure.
Specific embodiment
The present invention provides a kind of Calotropis gigantea tissue culture and rapid propagation methods, comprising the following steps:
1) it using Calotropis gigantea tender tissue as explant, is inoculated in after disinfection in primary inducing culture and carries out Fiber differentiation,
Obtain sprout primary；The primary inducing culture is basic culture medium with MS culture medium, comprising: 25~35gL of sucrose-1And fine jade
3.4~4gL of rouge-1；
2) sprout primary for obtaining step 1) is cut into the stem section comprising 1~2 axillary bud, is placed in subculture medium
Squamous subculture is carried out, tufted seedling is obtained；The subculture medium is basic culture medium with MS culture medium, comprising: 6-BA 0.5~
2.2mg·L-1, 0.02~0.15mgL of NAA-1、AgNO30.5~2.5mgL-1, 25~35gL of sucrose-1With agar 3.4
~4gL-1；
3) tufted seedling for obtaining step 2), which is placed on root media, carries out culture of rootage, and it is fast to obtain Calotropis gigantea tissue culture
Numerous seedling；The root media is basic culture medium with 1/2MS culture medium, comprising: 0.5~1.5mgL of NAA-1, sucrose 15~
25g·L-1With 3.4~4gL of agar-1。
In Calotropis gigantea tissue culture and rapid propagation method of the present invention, using Calotropis gigantea tender tissue as explant, it is inoculated with after disinfection
Fiber differentiation is carried out in primary inducing culture, obtains sprout primary；The primary inducing culture is based on MS culture medium
Culture medium, comprising: 25~35gL of sucrose-1With 3.4~4gL of agar-1.Calotropis gigantea tender tissue of the present invention preferably wraps
Include the stem section with axillary bud.The step of disinfection of the present invention preferably includes: it is 1~2% to wash that explant, which is placed in mass concentration,
5min is impregnated in clean essence aqueous solution, is rinsed well after taking-up, then is placed in the ethanol water that volumetric concentration is 70~75% and soaks
50~70s is steeped, is placed in the HgCl that mass concentration is 0.1~0.2% after taking-up again25~10min is impregnated in aqueous solution, is taken out, nothing
Bacterium water cleans 3~5 times.Primary inducing culture of the present invention includes sucrose, and the sucrose is in the primary inducing culture
In concentration be preferably 26~33gL-1, more preferably 28~32gL-1, most preferably 30gL-1.It is of the present invention primary
Induced medium includes agar, and concentration of the agar in the primary inducing culture is preferably 3.45~3.8gL-1,
More preferably 3.55~3.7gL-1, most preferably 3.6gL-1.The present invention tries each raw material of the primary inducing culture
There is no particular determinations in agent source, utilize the conventional reagent of this field.System of the present invention to the primary inducing culture
There is no particular determinations for Preparation Method, after preferably obtaining the primary inducing culture, adjust the primary inducing culture pH
It is 5.8.Fiber differentiation of the present invention is preferably illumination cultivation, and the light application time of the illumination cultivation is preferably 10~15h/d,
More preferably 11~13h/d, most preferably 12h/d.The intensity of illumination of illumination cultivation of the present invention is preferably 35~45 μ
mol·m-2·s-1, more preferably 38~42 μm of olm-2·s-1, most preferably 40 μm of olm-2·s-1.Light of the present invention
Temperature according to culture is preferably 22~32 DEG C, more preferably 25~31 DEG C, most preferably 26~30 DEG C.Illumination training of the present invention
The feeding time is preferably 20~28d, more preferably 22~26d, most preferably 25d.Using induced medium of the invention into
Row Fiber differentiation can promote the axillary bud development on explant to grow, and form the seedling of 3~4cm high, inductivity up to 53.3~
83.3%.
After sprout primary, the sprout primary is cut into the stem section comprising 1~2 axillary bud by the present invention, is placed in after being commissioned to train
It supports in base and carries out squamous subculture, obtain tufted seedling；The subculture medium is basic culture medium with MS culture medium, comprising: 6-BA
0.5~2.2mgL-1, 0.02~0.15mgL of NAA-1、AgNO30.5~2.5mgL-1, 25~35gL of sucrose-1And fine jade
3.4~4gL of rouge-1.It include 6-BA, concentration of the 6-BA in the subculture medium in subculture medium of the present invention
Preferably 0.8~2mgL-1, more preferably 1.2~1.8mgL-1, most preferably 1.5mgL-1.It is of the present invention after being commissioned to train
Supporting in base includes NAA, and concentration of the NAA in the subculture medium is preferably 0.03~0.1mgL-1, more preferably
0.04~0.06mgL-1, most preferably 0.05mgL-1.It include AgNO in subculture medium of the present invention3, the AgNO3
Concentration in the subculture medium is preferably 0.6~2mgL-1, more preferably 0.8~1.5mgL-1, most preferably
1mg·L-1.Subculture medium of the present invention includes sucrose, and concentration of the sucrose in the subculture medium is preferably 26
~33gL-1, more preferably 28~32gL-1, most preferably 30gL-1.Subculture medium of the present invention includes agar,
Concentration of the agar in the subculture medium is preferably 3.45~3.8gL-1, more preferably 3.55~3.7gL-1,
Most preferably 3.6gL-1.Especially AgNO in induced medium of the present invention3, can inhibit the vitrifying of tufted seedling, make glass
Glass rate is reduced to 4.44~8.89%, and continuous multi-generation culture, glass rate maintains always 8% hereinafter, poor between each generation
It is different not significant.There is no particular determinations in each source chemicals source of the present invention to the subculture medium, utilize the normal of this field
Advise reagent.There is no particular determinations for preparation method of the present invention to the subculture medium, preferably obtain the subculture
After culture medium, adjusting the subculture medium pH is 5.8.Squamous subculture of the present invention is preferably illumination cultivation, the illumination
The light application time of culture is preferably 10~15h/d, more preferably 11~13h/d, most preferably 12h/d.Illumination of the present invention
The intensity of illumination of culture is preferably 35~45 μm of olm-2·s-1, more preferably 38~42 μm of olm-2·s-1, most preferably 40
μmol·m-2·s-1.The temperature of illumination cultivation of the present invention is preferably 22~32 DEG C, and more preferably 25~31 DEG C, most preferably
It is 26~30 DEG C.The time of illumination cultivation of the present invention is preferably 21~30d, more preferably 23~28d, most preferably 25d.
Subculture is carried out using subculture medium of the present invention, glass rate can be reduced, and improve growth coefficient, make glass
Glass rate is reduced to 4.44~8.89%, and growth coefficient is increased to 4.83~5.93/25d.
After obtaining tufted seedling, the tufted seedling is placed on root media and carries out culture of rootage by the present invention, obtains Calotropis gigantea group
Train fast propagating seedling；The root media is basic culture medium with 1/2MS culture medium, comprising: 0.5~1.5mgL of NAA-1, sucrose
15~25gL-1With 3.4~4gL of agar-1.Root media of the present invention includes NAA, and the NAA is in the training of taking root
The concentration supported in base is preferably 0.6~1.3mgL-1, more preferably 0.8~1.1mgL-1, most preferably 1.0mgL-1.This
Inventing the root media includes sucrose, and concentration of the sucrose in the root media is preferably 26~33gL-1,
More preferably 28~32gL-1, most preferably 30gL-1.Root media of the present invention includes agar, and the agar exists
Concentration in the root media is preferably 3.45~3.8gL-1, more preferably 3.55~3.7gL-1, most preferably
3.6g·L-1.There is no particular determinations in each source chemicals source of the present invention to the root media, utilize the normal of this field
Advise reagent.There is no particular determinations for preparation method of the present invention to the root media, preferably obtain described take root
After culture medium, adjusting the root media pH is 5.8.Culture of rootage of the present invention is preferably illumination cultivation, the illumination
The light application time of culture is preferably 10~15h/d, more preferably 11~13h/d, most preferably 12h/d.Illumination of the present invention
The intensity of illumination of culture is preferably 35~45 μm of olm-2·s-1, more preferably 38~42 μm of olm-2·s-1, most preferably 40
μmol·m-2·s-1.The temperature of illumination cultivation of the present invention is preferably 22~32 DEG C, and more preferably 25~31 DEG C, most preferably
It is 26~30 DEG C.The time of illumination cultivation of the present invention is preferably 10~20d, more preferably 12~16d, most preferably 14d.
Culture of rootage is carried out using root media of the present invention, seedling rooting of growing thickly can be promoted, root system is healthy and strong, and rooting rate reaches
100%.
The present invention is after the culture of rootage, and it is also preferable to include hardenings and transplanting.Hardening of the present invention is preferably by ox
Angle melon tissue-culturing rapid propagation seedling sets hardening 5~7 days in greenhouse, and the shade density of the greenhouse is preferably 60~80%, more preferably 65~
78%, most preferably 70%.In the present invention, root system is still during succulent, tissue-culturing rapid propagation seedling hardening for Calotropis
Undeveloped, the ability that draws water is weaker, and its stalk is more very thin weak, easily lodges.Shade greenhouse can to avoid it by direct sunlight,
Its transpiration is reduced, while controlling canopy temperature between 25~32 DEG C, it is avoided to fall because of high temperature and humidity occurrence of large-area
Volt.The present invention transplants the Calotropis gigantea tissue-culturing rapid propagation seedling after hardening, and the transplanting is preferably by the tissue-culturing rapid propagation after hardening
Transplantation of seedlings enters in matrix, and the matrix includes sandy loam and fertile soil.Sandy loam of the present invention and volume ratio humous are excellent
It is selected as (1~4): 1, more preferably (1.5~3:1), most preferably 2:1.The sandy loam can increase matrix in the present invention
Water penetration, fertile soil can guarantee that matrix sufficient nutrient supplies, can prevent ox during hardening by combination
Because of matrix ponding stem rot occurs for angle melon tissue-cultured seedling, increases hardening survival rate.
Calotropis gigantea tissue culture and rapid propagation method provided by the invention is described in detail below with reference to embodiment, but cannot
They are interpreted as limiting the scope of the present invention.
Embodiment 1
1) it using Calotropis gigantea stem section as explant, is placed in the dish washing liquid solution that mass concentration is 1% and impregnates 5min, take out
It rinses well under flowing tap water afterwards, then is placed on superclean bench and impregnates 60s with the ethyl alcohol that volumetric concentration is 70%, take
The HgCl that mass concentration is 0.1% is placed in after out again2Middle immersion 7min takes out, with sterile water wash 5 times.It will be outer after disinfection
Implant is inoculated in primary inducing culture, and illumination cultivation 20 days, the growth of explant axillary bud development formed the children of 3~4cm high
Seedling, inductivity 83.3%；Primary inducing culture formula used are as follows: MS+ sucrose 30gL-1+ agar 3.6gL-1, pH
5.8.Condition of culture are as follows: temperature is 28 ± 2 DEG C, light application time 12h/d, and intensity of illumination is 40 μm of olm-2·s-1。
2) it is the stem section with 1 axillary bud by resulting aseptic seedlings sub-cut, gained stem section is transferred to shoot proliferation culture
It is cultivated in base, illumination cultivation 25 days, obtains the tufted seedling that height is 5~7cm, Multiplying culture is as shown in Figure 1, growth coefficient
5.1/25d glass rate 8.89%；Subculture multiplication medium formula used are as follows: MS+6-BA 1.5mgL-1+NAA
0.05mg·L-1+AgNO30.5g·L-1+ sucrose 30gL-1+ agar 3.6gL-1, pH 5.8.Condition of culture are as follows: temperature 28
± 2 DEG C, light application time 12h/d, intensity of illumination is 40 μm of olm-2·s-1。
3) tufted seedling plant division is transferred in root media, is transferred to 10~12 plants in each culture bottle, illumination cultivation 14 days,
Calotropis gigantea tissue-culturing rapid propagation seedling is obtained, rooting rate 100% is counted；Prescription of rooting medium used are as follows: 1/2MS+NAA 1.0mgL-1
+ sucrose 20gL-1+ agar 3.6gL-1, pH 5.8.Condition of culture are as follows: temperature is 28 ± 2 DEG C, light application time 12h/d, light
It is 40 μm of olm according to intensity-2·s-1。
4) Calotropis gigantea rooted seedling is placed in the greenhouse of 70% shade density after hardening 5~7 days, takes out and cleans root culture
Base is transplanted in the nutrition cup (12cm × 12cm) equipped with sterilized transplanting medium (sandy loam: fertile soil=2:1), in temperature
After room greenhouse culture 30 days, height of seedling is up to 20cm or so, survival rate 95%.
Embodiment 2
Except the chemical formulation change with step 2) subculture medium in embodiment 1 are as follows: MS+6-BA 1.5mgL-1+NAA
0.05mg·L-1+AgNO31.0g·L-1+ sucrose 30gL-1+ agar 3.6gL-1, outside pH 5.8, remaining condition is all the same, increases
It grows and cultivates the growth coefficient 5.8/25d as shown in Fig. 2, gained tufted seedling, glass rate 6.11%.
It is the stem section with 1 or 2 axillary bud by sprout primary sub-cut obtained in the present embodiment, stem section is transferred to after being commissioned to train
It supports and be proliferated fast numerous in base, every 25 days are a generation, continuous to cultivate 5 generations, the first generation to second generation growth coefficient 5.8/25d, glass
Glass rate 6.11%；The second generation is to third generation growth coefficient 5.93/25d, glass rate 5.56%；The third generation is proliferated to forth generation
Coefficient 5.6/25d, glass rate 6.67%；Forth generation is to the 5th generation growth coefficient 5.78/25d, glass rate 6.11%, each generation
Growth coefficient and glass rate are all without significant difference between generation.
Embodiment 3
Except the chemical formulation change with step 2) subculture medium in embodiment 1 are as follows: MS+6-BA 1.5mgL-1+NAA
0.05mg·L-1+AgNO31.5g·L-1+ sucrose 30gL-1+ agar 3.6gL-1, outside pH 5.8, remaining condition is all the same, increases
It grows and cultivates the growth coefficient 5.93/25d as shown in figure 3, gained tufted seedling, glass rate 5.56%.
Embodiment 4
Except the chemical formulation change with step 2) subculture medium in embodiment 1 are as follows: MS+6-BA 1.5mgL-1+NAA
0.05mg·L-1+AgNO32.0g·L-1+ sucrose 30gL-1+ agar 3.6gL-1, outside pH 5.8, remaining condition is all the same, increases
It grows and cultivates the growth coefficient 5.27/25d as shown in figure 4, gained tufted seedling, glass rate 5.0%.
Embodiment 5
Except the chemical formulation change with step 2) subculture medium in embodiment 1 are as follows: MS+6-BA 1.5mgL-1+NAA
0.05mg·L-1+AgNO32.5g·L-1+ sucrose 30gL-1+ agar 3.6gL-1, outside pH 5.8, remaining condition is all the same, increases
It grows and cultivates the growth coefficient 4.83/25d as shown in figure 5, gained tufted seedling, glass rate 4.44%.
Comparative example 1
1) it using Calotropis gigantea stem section as explant, is placed in the dish washing liquid solution that mass concentration is 0.5% and impregnates 5min, take
It is rinsed well under flowing tap water after out, then is placed on superclean bench and impregnates 40s with the ethyl alcohol that volumetric concentration is 65%,
The HgCl that mass concentration is 0.05% is placed in after taking-up again2Middle immersion 4min takes out, with sterile water wash 2 times.After disinfection
Explant is inoculated in initial culture base MS+ sucrose 20gL-1+ agar 3.3gL-1, in pH 5.8, illumination cultivation 20 days, explant
The growth of body axillary bud development, forms the seedling of 2-3cm high, inductivity 11.1%；Condition of culture are as follows: temperature is 28 ± 2 DEG C, when illumination
Between be 8h/d, intensity of illumination be 40 μm of olm-2·s-1。
3) it is the stem section with 1 or 2 axillary bud by resulting aseptic seedlings sub-cut, gained stem section is transferred to shoot proliferation training
Support base MS+6-BA 1.5mgL-1+NAA 0.05mg·L-1+AgNO30.2mg·L-1+ sucrose 20gL-1+ agar 3.3gL-1, cultivated in pH 5.8, illumination cultivation 25 days, growth coefficient 4.4/25d, glass rate 21.11%, illumination cultivation condition
Are as follows: temperature is 28 ± 2 DEG C, light application time 8h/d, and intensity of illumination is 40 μm of olm-2·s-1。
4) tufted seedling of 4cm or more is chosen, plant division is transferred to root media 1/2MS+NAA 0.3mgL-+ sucrose 12g
L-1+ agar 3.3gL-1In (pH 5.8), it is transferred to 10-12 plants in each culture bottle, illumination cultivation 14 days, obtains Calotropis gigantea group
Fast propagating seedling is trained, through counting rooting rate 78%；Illumination cultivation condition are as follows: temperature is 28 ± 2 DEG C, light application time 8h/d, and illumination is strong
Degree is 40 μm of olm-2·s-1。
5) Calotropis gigantea rooted seedling is placed in the greenhouse of 50% shade density after hardening 2 days, takes out and clean root culture medium,
It transplants in the nutrition cup (12cm × 12cm) equipped with sterilized transplanting medium (sandy loam: fertile soil=2:1), it is big in greenhouse
After canopy culture 25 days, height of seedling is 15cm or so, survival rate 66%.
Comparative example 2
1) it using Calotropis gigantea stem section as explant, is placed in the dish washing liquid solution that mass concentration is 2.5% and impregnates 5min, take
It is rinsed well under flowing tap water after out, then is placed on superclean bench and impregnates 80s with the ethyl alcohol that volumetric concentration is 80%,
The HgCl that mass concentration is 0.3% is placed in after taking-up again2Middle immersion 11min takes out, with sterile water wash 6 times.After disinfection
Explant is inoculated in initial culture base MS+ sucrose 40gL-1+ agar 4.2gL-1In (pH 5.8), illumination cultivation 20 days, outside
The growth of implant axillary bud development, forms the seedling of 1~2cm high, and gained seedling stalk is very thin, yellow leaf, survival rate 9.44%；Training
The condition of supporting are as follows: temperature is 28 ± 2 DEG C, light application time 16h/d, and intensity of illumination is 40 μm of olm-2·s-1。
3) it is the stem section with 1 or 2 axillary bud by resulting aseptic seedlings sub-cut, gained stem section is transferred to shoot proliferation training
Support base MS+6-BA 1.5mgL-1+NAA 0.05mg·L-1+AgNO33.0mg·L-1+ sucrose 40gL-1+ agar 4.2gL-1, cultivated in pH 5.8, illumination cultivation 25 days, gained seedling plants are downgraded, yellow leaf, growth coefficient 4.2/25d, glass
Glass rate 3.33%, illumination cultivation condition are as follows: temperature is 28 ± 2 DEG C, light application time 16h/d, and intensity of illumination is 40 μm of ol
m-2·s-1。
4) tufted seedling of 4cm or more is chosen, plant division is transferred to root media 1/2MS+NAA 2.0mgL-1+ sucrose 30g
L-1+ agar 4.2gL-1, in pH 5.8, it is transferred to 10~12 plants in each culture bottle, illumination cultivation 14 days, it is raw to obtain Calotropis gigantea
The root system of offspring, gained rooted seedling is more very thin, through counting rooting rate 86%；Illumination cultivation condition are as follows: temperature is 28 ± 2 DEG C,
Light application time is 16h/d, and intensity of illumination is 40 μm of olm-2·s-1。
5) Calotropis gigantea rooted seedling is placed in the greenhouse of 90% shade density after hardening 8 days, takes out and clean root culture medium,
It transplants in the nutrition cup (12cm × 12cm) equipped with sterilized transplanting medium (sandy loam: fertile soil=2:1), it is big in greenhouse
After canopy culture 60 days, height of seedling is 25cm or so, survival rate 72%.
Comparative example 3
1) callus induction, culture medium prescription MS+2,4-D 2.0mg are carried out with Calotropis gigantea aseptic bladeL-1, lure
Conductance is 86%.
2) callus differential medium are as follows: MS+6-BA 0.5mgL-1+NAA 0.1mg·L-1；Each explant is flat
It sprouts number 17.6, the period is two months or so, and is conducive to the differentiation and proliferation of bud.
3) root media is 1/2MS+NAA 0.5mgL-1, rooting rate 100% after 25d, transplanting survival rate is high.But
During the cultivation process as shown in fig. 6, a large amount of vitrifyings of Calotropis gigantea tissue-cultured seedling, and as generation change vitrifying degree aggravates, arrive
Forth generation almost all glass.
Comparative example 4
1) explant sterilizes: cutting off Calotropis gigantea terminal bud blade, rinses 10-20min with tap water, wash off the ash on explant
Dirt is cut into the length of 3cm or so；After 75% alcohol dampening, explant is put into superclean bench, it is molten with 0.1% mercuric chloride
Liquid carries out disinfection, and handles 10min, then uses aseptic water washing 3-4 times, aseptic filter paper suck dry moisture.Tender shoots base portion is cut
It removes, terminal bud length is retained in 2cm or so, is inoculated into MS+6-BA 0.3mg/L+NAA 0.1mg/L on Primary culture base；Each place
15 bottles of reason inoculation, every bottle of 1 tender shoots is repeated 3 times, and survival rate is 82.2% after 20d.
2) Multiplying culture: proliferated culture medium is that the proliferation times after MS+6-BA 1.0mg/L+NAA 0.1mg/L, 25d are
3.9。
3) culture of rootage: culture medium is 1/2MS+IBA 0.1mg/L, and rooting rate is up to 100%.
4) even transplantation of seedlings: by rooted seedling be transplanted to mixed-matrix (red heart soil: fertile soil: perlite=2: in 2: 1), 45d
Transplanting survival rate afterwards is 90% or more.
But a large amount of vitrifyings of Calotropis gigantea tissue-cultured seedling during the cultivation process, and as generation change vitrifying degree aggravates,
Forth generation almost all glass is arrived.
Comparative example 5
1) it using Calotropis gigantea stem section as explant, is placed in the dish washing liquid solution that mass concentration is 1% and impregnates 5min, take out
It rinses well under flowing tap water afterwards, then is placed on superclean bench and impregnates 60s with the ethyl alcohol that volumetric concentration is 70%, take
The HgCl that mass concentration is 0.1% is placed in after out again2Middle immersion 7min takes out, with sterile water wash 5 times.It will be outer after disinfection
Implant is inoculated in initial culture base, and illumination cultivation 20 days, the growth of explant axillary bud development formed the seedling of 3~4cm high, institute
It is bud green to obtain seedling leaves, robust plant, inductivity 83.3%；Initial culture based formulas used are as follows: MS+ sucrose 30gL-1+ fine jade
Rouge 3.6gL-1, pH 5.8.Condition of culture are as follows: temperature is 28 ± 2 DEG C, light application time 12h/d, and intensity of illumination is 40 μm of ol
m-2·s-1。
2) it is the stem section with 1 or 2 axillary bud by resulting aseptic seedlings sub-cut, gained stem section is transferred to shoot proliferation training
It supports and is cultivated in base, illumination cultivation 25 days, obtain the tufted seedling that height is 5~7cm, growth coefficient 4.6/25d, glass rate
33.9%；Subculture multiplication medium formula used are as follows: MS+6-BA 1.5mgL-1+NAA 0.05mg·L-1+ sucrose 30gL-1
+ agar 3.6gL-1, pH 5.8.Condition of culture are as follows: temperature is 28 ± 2 DEG C, light application time 12h/d, and intensity of illumination is 40 μ
mol·m-2·s-1。
3) tufted seedling of 4cm or more is chosen, plant division is transferred in root media, 10~12 plants are transferred in each culture bottle,
Illumination cultivation 14 days, Calotropis gigantea rooted seedling is obtained, through counting rooting rate 100%；Prescription of rooting medium used are as follows: 1/2MS+
NAA 1.0mg·L-1+ sucrose 20gL-1+ agar 3.6gL-1, pH 5.8.Condition of culture are as follows: temperature is 28 ± 2 DEG C, illumination
Time is 12h/d, and intensity of illumination is 40 μm of olm-2·s-1。
4) Calotropis gigantea rooted seedling is placed in the greenhouse of 70% shade density after hardening 5~7 days, takes out and cleans root culture
Base is transplanted in the nutrition cup (12cm × 12cm) equipped with sterilized transplanting medium (sandy loam: fertile soil=2:1), in temperature
After room greenhouse culture 30 days, height of seedling is up to 20cm or so, survival rate 95%.
It, not only can be with by above-described embodiment and comparative example it is found that the present invention provides a kind of Calotropis gigantea tissue culture and rapid propagation methods
The growth coefficient for improving Calotropis gigantea tissue-cultured seedling, reaches 4.83~5.93/25d, hence it is evident that is higher than comparative example growth coefficient achieved；
And the glass rate of tissue-cultured seedling is reduced to 4.44~8.89%, continuous multi-generation culture, glass rate maintain always 8% hereinafter,
Difference is not significant between each generation.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of method of Calotropis gigantea tissue-culturing rapid propagation, comprising the following steps:
1) it using Calotropis gigantea tender tissue as explant, is inoculated in after disinfection in primary inducing culture and carries out Fiber differentiation, obtained just
For sprout；The primary inducing culture are as follows: 25~35gL of sucrose-1With 3.4~4gL of agar-1+ MS culture medium；It is described to lure
Leading culture is illumination cultivation, and the light application time of the illumination cultivation is 10~15h/d, and intensity of illumination is 35~45 μm of olm-2·
s-1, the temperature of the Fiber differentiation is 22~32 DEG C, and the time of the Fiber differentiation is 20~28d；Described tender group of Calotropis gigantea children
It is woven to the stem section with axillary bud；
2) sprout primary for obtaining step 1) is cut into the stem section comprising 1~2 axillary bud, is placed in subculture medium and carries out
Squamous subculture obtains tufted seedling；The subculture medium are as follows: 0.5~2.2mgL of 6-BA-1, 0.02~0.15mgL of NAA-1、
AgNO30.5~2.5mgL-1, 25~35gL of sucrose-1With 3.4~4gL of agar-1+ MS culture medium；The squamous subculture
For illumination cultivation, 10~15h/d of light application time of the illumination cultivation, intensity of illumination is 35~45 μm of olm-2·s-1, described
The temperature of squamous subculture is 22~32 DEG C, and the time of the squamous subculture is 21~30d；
3) tufted seedling for obtaining step 2), which is placed on root media, carries out culture of rootage, obtains Calotropis gigantea tissue-culturing rapid propagation
Seedling；The root media are as follows: 0.5~1.5mgL of NAA-1, 15~25gL of sucrose-1With 3.4~4gL of agar-1+1/
2MS culture medium；The culture of rootage is illumination cultivation, and the light application time of the illumination cultivation is 10~15h/d, and intensity of illumination is
35~45 μm of olm-2·s-1, the temperature of the culture of rootage is 22~32 DEG C, and the time of the culture of rootage is 10~20d.
2. the method according to claim 1, wherein the step 1) disinfection includes: that explant is placed in quality
5min is impregnated in the dish washing liquid aqueous solution that concentration is 1~2%, is rinsed well after taking-up, then being placed in volumetric concentration is 70~75%
Ethanol water in impregnate 50~70s, be placed in again after taking-up mass concentration be 0.1~0.2% HgCl2It is impregnated in aqueous solution
5~10min takes out, sterile water wash 3~5 times.
3. the method according to claim 1, wherein further including hardening and shifting after the step 3) culture of rootage
It plants.
4. according to the method described in claim 3, it is characterized in that, the hardening is to set Calotropis gigantea tissue-culturing rapid propagation seedling in greenhouse
Hardening 5~7 days, the shade density of the greenhouse was 60~80%.
5. according to the method described in claim 3, it is characterized in that, the transplanting is to enter the tissue-culturing rapid propagation transplantation of seedlings after hardening
In matrix, the matrix includes sandy loam and fertile soil.
6. according to the method described in claim 5, it is characterized in that, the sandy loam and volume ratio humous are (1~4):
1。
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