CN106171975B - The in-vitro culture method of Decaisnea fargesii - Google Patents
The in-vitro culture method of Decaisnea fargesii Download PDFInfo
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- CN106171975B CN106171975B CN201610529979.0A CN201610529979A CN106171975B CN 106171975 B CN106171975 B CN 106171975B CN 201610529979 A CN201610529979 A CN 201610529979A CN 106171975 B CN106171975 B CN 106171975B
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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
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- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of embryos using immature fruit as explant, successively obtains Decaisnea fargesii regeneration plant by the induction of callus, adventitious buds differentiation induction, adventitious bud proliferation culture and rooting induction culture.Before the induction for carrying out callus, further include: the sterilizing of explant: take Decaisnea fargesii children okra fruit real, the brushing surface after immersion 20-30min first in the detergent liquid of every 100ml drop containing 6-10, it is rinsed with water 1-2h later, then in gnotobasis first with 75% alcohol sterilization treatment children okra fruit reality 30-60s, later with the 0.1%HgCl of every 100ml drop containing 3-4 soil temperature 402Solution sterilizes to it 15min, then with sterile water wash 4-6 times, each 1-3min, finally takes out the tender seed of children, squeezes out the tender embryo of children, using obtaining explant after the tender embryo of rinsed with sterile water children.The method of the present invention can induce to high-frequency a large amount of Decaisnea fargesii and grow thickly adventitious bud, and transplanting survival rate is high.The present invention is the breeding coefficient of raising Decaisnea fargesii, is that the preserving seed of the species and its popularization and application are had laid a good foundation.
Description
Technical field
The present invention relates to a kind of in-vitro culture methods of Decaisnea fargesii.
Background technique
Decaisnea fargesii (Decaisnea insignis (Griff.) Hook.f.et Thoms.) is Lardizabalaceae
(Lardizabalaceae) Decaisnea fargesii category (Decaisnea) upright machaka, is distributed widely in Southwestern China portion to middle part
Area, be grown on 900-3600 meters of height above sea level hillside shrubbery or cheuch shaw under dark and damp place (answer pretty raw etc., 2001).Decaisnea fargesii is again
Name cat dung melon is bluish violet strip after its fruit maturation, slightly bent, likeness in form cat dung (Hou Yun and wait, 2011).
The fruit of Decaisnea fargesii is berry, and juice rich in is rich in sugar, vitamin and mineral nutrition etc., Ke Yizuo
It is edible for fruit, it can be used for production fruit juice, wine and sugaring；Contain a large amount of rubber constituent in pericarp, ratio is reachable
10%-20% can be used for the production (Hou Yun and wait, 2010) of rubber product, in the increasingly depleted situation of petroleum resources today
Under, Decaisnea fargesii can be considered a kind of wild rubber plant being of great rarity.Grease abundant is rich in Decaisnea fargesii seed, ratio is about
For 18%-22%, oil yield is close with cottonseed, and lubricant component in containing a large amount of unsaturated fatty acid (Liu Jirui etc.,
2011), meet the theory that current people pursue natural health products, have in terms of exploitation medicinal plant oil and health care product very big
Potentiality (Sun Xiangyu etc. 2012) are a kind of wild oil trees with very high economic benefit.The root of Decaisnea fargesii and fruit can
It is sweet in flavor, pungent to be used as medicine, it is mild-natured, have a clearing lung and relieving cough, dispelling wind and eliminating dampness, the function of moisturizing, be mainly used for treating pulmonary tuberculosis, cough,
The diseases such as pruritus vulvae and hernia (Hu Zhenghai etc., 1991), show the multiple compounds contained in Decaisnea fargesii stem by mouse experiment in vivo
With antineoplastic action (Kong Jie, 1996), it is seen that Decaisnea fargesii has very high medical value.The florescence of Decaisnea fargesii tree and fruiting period
It is up to 4 months, blooms in 4-7 month, the 7-10 month is fruiting period, and flower is light green, and fruit is blue or bluish violet, in gardens
With very high appreciating (class's litchi etc., 2014).In addition, the blade and fruit of Decaisnea fargesii are also the main of Shennongjia Rhinopithecus roxellana
One of food source plant (iron army etc., 2011).
The adaptability of Decaisnea fargesii itself is extremely strong, and the ability of disease and insect resistance is also very prominent, in its growth course, root
Prosperity is mutually wound, and the branches and leaves and Stem nematode of ground are luxuriant, and area coverage is big, conducive to preventing erosion and soil sandification
(Hou Yun and wait, 2011).Decaisnea fargesii is currently the wild tree species that China is not yet exploited, and can be used as edible fruit tree, medicinal
Fruit tree uses tree species with ornamental, and potential value is huge, therefore the introducing and planting of Decaisnea fargesii and the meaning of utilization and extention are extremely great.
Decaisnea fargesii mainly passes through the modes vegetative propagation such as cuttage, press strip and plant division, distribution of the elite stand plant by wild resource
It is difficult to breed on a large scale with quantity limitation；Decaisnea fargesii seed kind skin is rich in grease, germinates slow and irregular, and germination percentage is low, uses
Sexual reproduction method the defects of there are the seedling growth period is long, and emergence rate is unstable, it is unable to meet production the demand with market.
Therefore, the effective way for exploring quickly breeding Decaisnea fargesii becomes urgent problem to be solved.Tissue cultures have need quantity of material it is few,
The features such as breeding is fast, not by seasonal effect is quick breeding one important method of xylophyta.In recent years, for Decaisnea fargesii
Study on tissue culture only has Aseptic seedling, axillary bud does explant evoking adventive bud and (Yang Jingtian etc. 2009 occurs；Guo Bangli
2013) type of culture medium, the combination of plant growth regulator, are analyzed and is influenced with the induction for comparing axillary bud and adventitious bud,
But breeding coefficient is lower.Based on the above issues, there is an urgent need to pass through explant, plant growth regulator, minimal medium and training
The screening for the condition of supporting establishes and stablizes Decaisnea fargesii occurs for high frequency adventitious shoot regeneration and rapid propagation system, improves its breeding coefficient, is cat
Germ-plasma resources protection, biological control, utilization and extention and the biotechnology breeding of youngster's dung provide good experiment porch.
Summary of the invention
It is excellent it is an object of the invention to solve at least the above problems and/or defect, and provide at least to will be described later
Point.
A further object of the present invention is to provide a kind of in-vitro culture method of Decaisnea fargesii.
For this purpose, technical solution provided by the invention are as follows:
A kind of in-vitro culture method of Decaisnea fargesii, comprising: using the embryo of immature fruit as explant, successively by being cured
Induction, adventitious buds differentiation induction, adventitious bud proliferation culture and the rooting induction culture of injured tissue obtain Decaisnea fargesii regeneration plant.
Preferably, it in the in-vitro culture method of the Decaisnea fargesii, before the induction for carrying out callus, also wraps
It includes:
The sterilizing of explant: it takes Decaisnea fargesii children okra fruit real, is impregnated in the detergent liquid of every 100ml drop containing 6-10 first
Brushing surface after 20-30min is rinsed with water 1-2h later, then first young with 75% alcohol sterilization treatment in gnotobasis
Okra fruit reality 30-60s, later with the 0.1%HgCl of every 100ml drop containing 3-4 soil temperature 402Solution sterilizes to it 15min, then with sterile
Water cleans 4-6 times, each 1-3min, finally takes out the tender seed of children, squeezes out the tender embryo of children, uses tender kind of children described in rinsed with sterile water
Explant is obtained after embryo.
It preferably,, will be described in the induction of the callus tissue in the in-vitro culture method of the Decaisnea fargesii
Explant is placed in callus inducing medium and carries out inducing 32 days to obtain callus, the callus inducing medium packet
Contain: the 6-nonylaminopurine of MS, 0.1-2.0mg/L and 2, the 4- dichlorphenoxyacetic acid of 0.5-4.0mg/L.
Preferably, in the in-vitro culture method of the Decaisnea fargesii, in the adventitious buds differentiation induction, by callus
The callus that Fiber differentiation obtains, which is placed on differential medium, induces 30 days generation adventitious buds, and the differential medium includes:
The α-naphthylacetic acid of 6- benzyl purine (6-BA) and 0.01-0.5mg/L of MS, 0.1-2.0mg/L.
Preferably, in the in-vitro culture method of the Decaisnea fargesii, in the adventitious bud proliferation culture, by adventitious bud point
Change adventitious bud obtained in induction and is placed in induction generation adventitious bud proliferation 15-30 days, the proliferated culture medium packet in proliferated culture medium
Contain: the 6- benzyl purine of 1/2MS, 2.0-3.0mg/L and the α-naphthylacetic acid of 0.2-1.0mg/L.
Preferably, in the in-vitro culture method of the Decaisnea fargesii, in the rooting induction culture, by adventitious bud proliferation
The adventitious bud generated in culture is placed in root induction on rooting induction culture medium, obtains Decaisnea fargesii regeneration plant, and described take root lures
Leading culture medium includes: 1/2MS and 0.3-0.9mg/L is grinned the α-naphthylacetic acid of diindyl butyric acid or 1/2MS and 0.3-0.9mg/L.
Preferably, in the in-vitro culture method of the Decaisnea fargesii, the induction of the callus, adventitious buds differentiation are lured
It leads, in adventitious bud proliferation culture and rooting induction culture, condition of culture are as follows: 25 ± 2 DEG C of temperature, light application time 16h/ days, illumination
Intensity 2000lux.
Preferably, in the in-vitro culture method of the Decaisnea fargesii, Decaisnea fargesii is obtained again in the rooting induction culture
After raw plant further include:
Hardening: by the long Decaisnea fargesii regeneration plant to 5-8cm high in outdoor 20-25 DEG C of lower refining seedling 5-7 days.
Preferably, in the in-vitro culture method of the Decaisnea fargesii, after the hardening further include: transplanting: will refine
Decaisnea fargesii Transplantation of Regenerated Plantlets after seedling enters to be cultivated in the mixed-matrix of river sand and detritus soil, river sand and detritus soil it is mixed
Composition and division in a proportion example is 0.5-1:1.
Preferably, in the in-vitro culture method of the Decaisnea fargesii, the fruit in the crude fruit actually 90 days fruit ages.
The present invention is include at least the following beneficial effects:
The present invention provides a kind of in-vitro culture methods of resource plant Decaisnea fargesii.This method is using the tender embryo of children as outside
Implant obtains regeneration plant by adventitious bud inducing approach.Exist using the tender embryo of children as the advantage that explant carries out Regeneration in Vitro
Strong in induction differentiation capability, long term subculture process is not easy to degenerate.The method of the invention can induce to high-frequency largely
Decaisnea fargesii grow thickly adventitious bud, the Transplantation of Regenerated Plantlets high survival rate after taking root, up to 98%.The present invention is to improve Decaisnea fargesii
Breeding coefficient, the preserving seed for the species and its popularization and application have laid a good foundation, have a extensive future.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is the photo of the initial callus of the young tender kind of embryonal induction of Decaisnea fargesii of the present invention；
Fig. 2 is the photo of the callus of the shoot proliferation of Decaisnea fargesii of the present invention；
Fig. 3 is the photo for the Multiple Buds that the callus of Decaisnea fargesii of the present invention breaks up；
Fig. 4 is that the adventitious bud proliferation of Decaisnea fargesii of the present invention obtains the photo of larger seedling；
Fig. 5 is the photo of the healthy and strong root system of the larger seedling of Decaisnea fargesii of the present invention；
Fig. 6 is the photo of the Decaisnea fargesii seedling of transplant survival of the present invention.
Specific embodiment
The present invention will be further described in detail below with reference to the embodiments, to enable those skilled in the art referring to specification
Text can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
The present invention provides a kind of in-vitro culture method of Decaisnea fargesii, comprising: using the embryo of immature fruit as explant,
Successively Decaisnea fargesii is obtained by the induction of callus, adventitious buds differentiation induction, adventitious bud proliferation culture and rooting induction culture
Regeneration plant.
It is of the invention in one embodiment, preferably, carry out callus induction before, further includes:
The sterilizing of explant: it takes Decaisnea fargesii children okra fruit real, is impregnated in the detergent liquid of every 100ml drop containing 6-10 first
Brushing surface after 20-30min is rinsed with water 1-2h later, then first young with 75% alcohol sterilization treatment in gnotobasis
Okra fruit reality 30-60s, later with the 0.1%HgCl of every 100ml drop containing 3-4 soil temperature 402Solution sterilizes to it 15min, then with sterile
Water cleans 4-6 times, each 1-3min, finally takes out the tender seed of children, squeezes out the tender embryo of children, uses tender kind of children described in rinsed with sterile water
Explant is obtained after embryo.Because the pericarp of Decaisnea fargesii is rich in rubber and gland point is more, surface is easy the excessive dust of bonding, and not
It is easy to clean.So micro detergent, which is added, when cleaning can effectively remove the dust of pericarp bonding, it is ensured that children is tender
Fruit is cleaner, strips for the tender embryo of children and provides basis with sterile culture.The detergent be it is commercially available it is common it is without phosphorus wash it is clean
It is net.
It is of the invention in one embodiment, will be described outer preferably, in the induction of the callus tissue
Implant is placed in callus inducing medium and carries out inducing 32 days to obtain callus, the callus inducing medium packet
Contain: the 6-nonylaminopurine of MS, 0.1-2.0mg/L and 2, the 4- dichlorphenoxyacetic acid of 0.5-4.0mg/L.
It is of the invention in one embodiment, preferably, the adventitious buds differentiation induction in, callus is lured
It leads the obtained callus of culture and is placed on differential medium and induce 30 days generation adventitious buds, the differential medium includes: MS,
The α-naphthylacetic acid of 6- benzyl purine (6-BA) and 0.01-0.5mg/L of 0.1-2.0mg/L.
It is of the invention in one embodiment, preferably, in the adventitious bud proliferation culture, by adventitious buds differentiation
Adventitious bud obtained in induction is placed in induction in proliferated culture medium and generates adventitious bud proliferation 15-30 days, which includes:
The 6- benzyl purine of 1/2MS, 2.0-3.0mg/L and the α-naphthylacetic acid of 0.2-1.0mg/L.
It is of the invention in one embodiment, preferably, adventitious bud proliferation is trained in the rooting induction culture
The adventitious bud generated in supporting is placed in root induction on rooting induction culture medium, obtains Decaisnea fargesii regeneration plant, the rooting induction
Culture medium includes: 1/2MS and 0.3-0.9mg/L is grinned the α-naphthylacetic acid of diindyl butyric acid or 1/2MS and 0.3-0.9mg/L.
It is of the invention in one embodiment, preferably, the induction of the callus, adventitious buds differentiation lure
It leads, in adventitious bud proliferation culture and rooting induction culture, condition of culture are as follows: 25 ± 2 DEG C of temperature, light application time 16h/ days, illumination
Intensity 2000lux.
It is of the invention in one embodiment, preferably, the rooting induction culture obtain Decaisnea fargesii regeneration
After plant further include:
Hardening: by the long Decaisnea fargesii regeneration plant to 5-8cm high in outdoor 20-25 DEG C of lower refining seedling 5-7 days.
It is of the invention in one embodiment, preferably, after the hardening further include: transplanting: by hardening
Decaisnea fargesii Transplantation of Regenerated Plantlets later enters to be cultivated in the mixed-matrix of river sand and detritus soil, the mixing of river sand and detritus soil
Ratio is 0.5-1:1.
It is of the invention in one embodiment, preferably, the fruit in the crude fruit actually 90 days fruit ages.
It is of the invention in one embodiment, the in-vitro culture method of Decaisnea fargesii of the present invention specifically includes following
Step:
(1) explant sterilizes:
It is real to choose Decaisnea fargesii children okra fruit, impregnates 20- in the detergent liquid that concentration is, for example, every 100ml drop containing 6-10
Then it is spare to rush 1-2h with tap water for brushing surface after 30min.It is first gone out in fact with 75% alcohol in superclean bench to young okra fruit
Bacterium handles 30-60s, with the 0.1%HgCl of every 100ml drop containing 3-4 soil temperature 402Solution sterilization 15min, with sterile water wash 4-6
It is secondary, each 1-3min.It wears sterile gloves and strips the tender seed of children in fruit, squeeze out the tender embryo of children, be used as explant after rinsed with sterile water
Body inoculation.The detergent is commercially available common without phosphorus detergent.
(2) the induction of callus: placed it in more using the tender embryo of children in immature fruit after sterilization treatment as explant
Evoked callus in injured tissue induced medium；At 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination 2000Lux's
Under the conditions of cultivate 32 days.The callus inducing medium is to be added to 6-nonylaminopurine on the basis of MS culture medium
(KT) and 2,4 dichlorophenoxyacetic acid (2,4-D)；Wherein the concentration of 6-nonylaminopurine (KT) is 0.1-2.0mg/L, preferably
For 0.1mg/L, the concentration of 2,4- dichlorphenoxyacetic acids (2,4-D) is 0.5-4.0mg/L, preferably 2.0mg/L.
(3) the callus that the callus Fiber differentiation obtains adventitious bud differentiation and culture: is inoculated into adventitious bud differentiation and culture
On base, culture 30 days is carried out under conditions of 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination 2000lux.It is described not
Normal bud differential medium are as follows: 6-benzyladenine (6-BA) and α-naphthylacetic acid (NAA) are added on the basis of MS culture medium.Institute
The concentration for stating 6-benzyladenine in differential medium is 0.1-2.0mg/L, and the concentration of preferably 2.0mg/L, α-naphthylacetic acid are
0.01-0.5mg/L, preferably 0.5mg/L；
(4) the adventitious bud seedling that the differentiation incubation step obtains adventitious bud proliferation culture: is cut into the small stem of simple bud point
Section, is seeded in adventitious bud proliferation culture medium, at 25 ± 2 DEG C of temperature, light application time 16h/ days, the item of intensity of illumination 2000lux
Culture 15-30 days is carried out under part.The adventitious bud proliferation culture medium are as follows: be added to 6- on the basis of 1/2MS minimal medium
Benzyladenine (6-BA) and α-naphthylacetic acid (NAA).The concentration of 6-benzyladenine is 2.0- in the proliferated culture medium
3.0mg/L, preferably 3.0mg/L, the concentration of α-naphthylacetic acid are 0..2-1.0mg/L, preferably 0.5mg/L；
(5) culture of rootage and plant regeneration: isolating single plant for healthy and strong adventitious bud and be placed in root induction on root media,
Culture 15-30 days is carried out under conditions of 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination 2000lux.It is described to take root
Culture medium is grin diindyl butyric acid (IBA) or the α-naphthylacetic acid (NAA) for being added to concentration and being 0.3-0.9mg/L, preferably 0.6mg/L's
α-naphthylacetic acid；
(6) hardening and transplanting: by step (5) in the regeneration plant that is obtained after culture of rootage carry out method for transplanting are as follows: to again
When raw plant is long intensively healthy and strong to 5-8cm high, root system, it is gradually opened sealed membrane after removing culturing room, makes regeneration plant and nature
Air contact, can transplant for lower refining seedling 5-7 days at 20-25 DEG C.By the Transplantation of Regenerated Plantlets Jing Guo hardening to river sand and detritus soil
Mixed-matrix in transplant, in mixed-matrix the ratio of sandy soil and detritus soil be 0.5-1:1.Greenhouse production management is moved to, is transplanted
Survival rate is up to 98%.
Embodiment 1, the in vitro culture of Decaisnea fargesii
MS minimal medium: it can refer to document (Murashige T, Skoog F.A revised medium for
rapid grouth and bioassays with tobacco tissue cultures.Physiol.Plant,1962,
15:473-497) prepare.
The in-vitro culture method of Decaisnea fargesii of the present invention, comprising the following steps:
(1) sterilizing of young okra fruit reality explant
It takes Decaisnea fargesii children okra fruit real, scrubs table after impregnating 30min in the detergent liquid that concentration is every 100ml drop containing 6-10
Then face rushes 2h with tap water, be placed in superclean bench and first impregnate 60s with 75% alcohol in fact to young okra fruit, then with every
0.1%HgCl of the 100ml containing 4 drop soil temperatures 402Solution sterilization 15min, with sterile water wash 5 times.It wears sterile gloves and strips fruit
The interior tender seed of children squeezes out the tender embryo of children, is inoculated with after rinsed with sterile water as explant.
(2) induction of callus
Preferred callus inducing medium be added on the basis of MS minimal medium 0.1mg/L KT and
The 2,4-D of 2.0mg/L.
Using step, (1) young tender embryo is inoculated on above-mentioned callus inducing medium as explant, such as each
It is inoculated with 15 in culture dish, each combination 10 culture dishes of inoculation, at 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination
Evoked callus under 2000Lux.
As shown in Figure 1, radicle and cotyledon start callus after explant is inoculated with 7 days, bulk pale green is formed after culture 30 days
Color callus, inductivity is up to 92%.In shoot proliferation incubation, a small number of callus have fragmentary adventitious buds differentiation to produce
Raw (Fig. 2).
(3) adventitious buds differentiation Fiber differentiation
Preferred differential medium is the 6-BA and 0.5mg/L that 2.0mg/L is added on the basis of MS minimal medium
NAA.
By step (2) in 30 days callus of induction transfer and carry out adventitious buds differentiation Fiber differentiation on above-mentioned culture medium,
25 ± 2 DEG C of temperature, light application time 16h/ days, after cultivating 30 days under intensity of illumination 2000lux, callus Surface Differentiation was largely not
Normal bud (Fig. 3).
(4) adventitious bud proliferation culture
Preferred adventitious bud proliferation culture medium is the 6-BA that 3.0mg/L is added on the basis of 1/2MS minimal medium
With the NAA of 0.5mg/L.
By step, (3) the middle larger seedling for obtaining 4-7cm high cuts into the small stem section of simple bud point (band 1-2 piece leaf), each seedling
3 sections can be averagely cut into, inserts in proliferated culture medium and cultivates, at 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination
After cultivating 30 days under 2000lux, small stem section base portion proliferation generates adventitious bud of growing thickly, and each stem section can averagely be proliferated generation 5 not
Normal bud, then each seedling average proliferation generates 15 adventitious buds, i.e., breeding coefficient is up to 15 (Fig. 4).
(5) culture of rootage and plant regeneration
Preferred root media is that (a great number of elements for including is the 1/ of MS minimal medium full dose in 1/2MS culture medium
2) NAA of 0.6mg/L is added on the basis of.
(4) adventitious bud of growing thickly that step is obtained is isolated single plant and is transferred on root media at 25 ± 2 DEG C of temperature, light
According to the time 16h/ days, root induction under intensity of illumination 2000Lux.Adventitious bud base portion gradually grows white within culture of rootage 7 days or so
Tender, 15d or so adventitious root length reaches 1cm or so, and by the culture of 30d or so, adventitious root is 4-6cm, is formed strong
It strengthens intensive root system (Fig. 5), obtains complete regenerated plant, rooting rate is up to 95%.
(6) hardening and transplanting
By step (5) in the regeneration plant that is obtained after culture of rootage carry out method for transplanting are as follows: plant to be regenerated is long to 5-
When 8cm high, root system are intensively healthy and strong, it is gradually opened sealed membrane after removing culturing room, contacts regeneration plant with natural air,
20-25 DEG C can transplant for lower refining seedling 5-7 days.By the river sand and detritus of the Transplantation of Regenerated Plantlets Jing Guo hardening to 0.5-1:1 ratio
In native mixed-matrix, greenhouse production management is moved to, survival rate is up to 98% (Fig. 6).
Embodiment 2, the in vitro culture of Decaisnea fargesii
MS minimal medium: it can refer to document (Murashige T, Skoog F.Arevised medium for rapid
grouth and bioassays with tobacco tissue cultures.Physiol.Plant,1962,15:473-
497) it prepares.
The in-vitro culture method of Decaisnea fargesii of the present invention, comprising the following steps:
(1) the sterilizing of young okra fruit reality explant
It takes Decaisnea fargesii children okra fruit real, scrubs table after impregnating 20min in the detergent liquid that concentration is every 100ml drop containing 4-8
Then face rushes 1h with tap water, be placed in superclean bench and first impregnate 40s with 70% alcohol in fact to young okra fruit, then with every
0.1%HgCl of the 100ml containing 3 drop soil temperatures 402Solution sterilization 15min, with sterile water wash 4 times.It wears sterile gloves and strips fruit
The interior tender seed of children squeezes out the tender embryo of children, is inoculated with after rinsed with sterile water as explant.
(2) the induction of callus
Callus inducing medium is the KT and 1.0mg/L that 1.0mg/L is added on the basis of MS minimal medium
2,4-D.
Using step, (1) young tender embryo is inoculated on above-mentioned callus inducing medium as explant, such as each
It is inoculated with 15 in culture dish, each combination 10 culture dishes of inoculation, at 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination
Evoked callus under 2000Lux.
After explant is inoculated with 7 days, radicle and cotyledon start callus (figure), form bulk light green callus after culture 30 days
Tissue, inductivity is up to 86%.In shoot proliferation incubation, a small number of callus have fragmentary adventitious buds differentiation to generate (figure
2)。
(3) adventitious buds differentiation Fiber differentiation
Differential medium is the NAA that the 6-BA and 0.1mg/L of 2.0mg/L are added on the basis of MS minimal medium.
By step (2) in 30 days callus of induction transfer and carry out adventitious buds differentiation Fiber differentiation on above-mentioned culture medium,
25 ± 2 DEG C of temperature, light application time 16h/ days, after cultivating 30 days under intensity of illumination 2000lux, callus Surface Differentiation was largely not
Normal bud (Fig. 3).
(4) adventitious bud proliferation culture
Adventitious bud proliferation culture medium be added on the basis of 1/2MS minimal medium 3.0mg/L 6-BA and
The NAA of 1.0mg/L.
By step, (3) the middle larger seedling for obtaining 4-7cm high cuts into the small stem section of simple bud point (band 1-2 piece leaf), each seedling
3 sections can be cut into, is placed in proliferated culture medium and cultivates, under 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination 2000lux
After culture 30 days, small stem section base portion proliferation generates adventitious bud of growing thickly, and each stem section, which can be averagely proliferated, generates 4 adventitious buds, then often
A seedling average proliferation generates 12 adventitious buds, i.e., breeding coefficient is up to 12 (Fig. 4).
(5) culture of rootage and plant regeneration
Root media is the base in 1/2MS culture medium (a great number of elements for including is the 1/2 of MS minimal medium full dose)
The IBA of 0.6mg/L is added on plinth.
The adventitious bud of growing thickly that step (4) obtain is isolated single plant to be transferred on root media at 25 ± 2 DEG C of temperature, light
According to the time 16h/ days, root induction under intensity of illumination 2000Lux.Culture of rootage 15d or so adventitious root length reaches 1cm or so,
By the culture of 30d or so, adventitious root is 4-6cm, forms healthy and strong intensive root system (Fig. 5), obtains complete regenerated plant, raw
Root rate is up to 90%.
(6) hardening and transplanting
The regeneration plant obtained after culture of rootage in step (5) is subjected to method for transplanting are as follows: plant to be regenerated is long to 6-
When 8cm high, root system are intensively healthy and strong, it is gradually opened sealed membrane after removing culturing room, contacts regeneration plant with natural air,
20-25 DEG C can transplant for lower refining seedling 5-7 days.By the river sand and detritus of the Transplantation of Regenerated Plantlets Jing Guo hardening to 0.5-1:1 ratio
In native mixed-matrix, greenhouse production management is moved to, survival rate is up to 95% (Fig. 6).
Embodiment 3, the in vitro culture of Decaisnea fargesii
MS minimal medium: it can refer to document (Murashige T, Skoog F.Arevised medium for rapid
grouth and bioassays with tobacco tissue cultures.Physiol.Plant,1962,15:473-
497) it prepares.
The in-vitro culture method of Decaisnea fargesii of the present invention, comprising the following steps:
(1) sterilizing of young okra fruit reality explant
It takes Decaisnea fargesii children okra fruit real, scrubs table after impregnating 30min in the detergent liquid that concentration is every 100ml drop containing 6-10
Then face rushes 2h with tap water, be placed in superclean bench and first impregnate 60s with 75% alcohol in fact to young okra fruit, then with every
0.1%HgCl of the 100ml containing 5 drop soil temperatures 402Solution sterilization 15min, with sterile water wash 5 times.It wears sterile gloves and strips fruit
The interior tender seed of children squeezes out the tender embryo of children, is inoculated with after rinsed with sterile water as explant.
(2) induction of callus
Preferred callus inducing medium be added on the basis of MS minimal medium 1.0mg/L KT and
The 2,4-D of 3.0mg/L.
Using step, (1) young tender embryo is inoculated on above-mentioned callus inducing medium as explant, such as each
It is inoculated with 15 in culture dish, each combination 10 culture dishes of inoculation, at 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination
Evoked callus under 2000Lux.
As shown in Figure 1, radicle and cotyledon start callus after explant is inoculated with 7 days, bulk pale green is formed after culture 30 days
Color callus, inductivity is up to 92%.In shoot proliferation incubation, a small number of callus have fragmentary adventitious buds differentiation to produce
Raw (Fig. 2).
(3) adventitious buds differentiation Fiber differentiation
Preferred differential medium is the 6-BA and 0.01mg/L that 1.0mg/L is added on the basis of MS minimal medium
NAA.
By step (2) in 30 days callus of induction transfer and carry out adventitious buds differentiation Fiber differentiation on above-mentioned culture medium,
25 ± 2 DEG C of temperature, light application time 16h/ days, after cultivating 30 days under intensity of illumination 2000lux, callus Surface Differentiation was largely not
Normal bud (Fig. 3).
(4) adventitious bud proliferation culture
Preferred adventitious bud proliferation culture medium is the 6-BA that 2.0mg/L is added on the basis of 1/2MS minimal medium
With the NAA of 0.2mg/L.
By step, (3) the middle larger seedling for obtaining 4-7cm high cuts into the small stem section of simple bud point (band 1-2 piece leaf), each seedling
3 sections can be averagely cut into, inserts in proliferated culture medium and cultivates, at 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination
After cultivating 30 days under 2000lux, small stem section base portion proliferation generates adventitious bud of growing thickly, and each stem section can averagely be proliferated generation 5 not
Normal bud, then each seedling average proliferation generates 15 adventitious buds, i.e., breeding coefficient is up to 15 (Fig. 4).
(5) culture of rootage and plant regeneration
Preferred root media is that (a great number of elements for including is the 1/ of MS minimal medium full dose in 1/2MS culture medium
2) NAA of 0.3mg/L is added on the basis of.
(4) adventitious bud of growing thickly that step is obtained is isolated single plant and is transferred on root media at 25 ± 2 DEG C of temperature, light
According to the time 16h/ days, root induction under intensity of illumination 2000Lux.Adventitious bud base portion gradually grows white within culture of rootage 7 days or so
Tender, 15d or so adventitious root length reaches 1cm or so, and by the culture of 30d or so, adventitious root is 4-6cm, is formed strong
It strengthens intensive root system (Fig. 5), obtains complete regenerated plant, rooting rate is up to 95%.
(6) hardening and transplanting
By step (5) in the regeneration plant that is obtained after culture of rootage carry out method for transplanting are as follows: plant to be regenerated is long to 5-
When 8cm high, root system are intensively healthy and strong, it is gradually opened sealed membrane after removing culturing room, contacts regeneration plant with natural air,
20-25 DEG C can transplant for lower refining seedling 5-7 days.By the river sand and detritus of the Transplantation of Regenerated Plantlets Jing Guo hardening to 0.5-1:1 ratio
In native mixed-matrix, greenhouse production management is moved to, survival rate is up to 98% (Fig. 6).
Embodiment 4, the in vitro culture of Decaisnea fargesii
MS minimal medium: it can refer to document (Murashige T, Skoog F.Arevised medium for rapid
grouth and bioassays with tobacco tissue cultures.Physiol.Plant,1962,15:473-
497) it prepares.
The in-vitro culture method of Decaisnea fargesii of the present invention, comprising the following steps:
(1) the sterilizing of young okra fruit reality explant
It takes Decaisnea fargesii children okra fruit real, scrubs table after impregnating 20min in the detergent liquid that concentration is every 100ml drop containing 4-8
Then face rushes 1h with tap water, be placed in superclean bench and first impregnate 40s with 70% alcohol in fact to young okra fruit, then with every
0.1%HgCl of the 100ml containing 3 drop soil temperatures 402Solution sterilization 15min, with sterile water wash 4 times.It wears sterile gloves and strips fruit
The interior tender seed of children squeezes out the tender embryo of children, is inoculated with after rinsed with sterile water as explant.
(2) the induction of callus
Callus inducing medium is the KT and 4.0mg/L that 1.0mg/L is added on the basis of MS minimal medium
2,4-D.
Using step, (1) young tender embryo is inoculated on above-mentioned callus inducing medium as explant, such as each
It is inoculated with 15 in culture dish, each combination 10 culture dishes of inoculation, at 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination
Evoked callus under 2000Lux.
After explant is inoculated with 7 days, radicle and cotyledon start callus (figure), form bulk light green callus after culture 30 days
Tissue, inductivity is up to 86%.In shoot proliferation incubation, a small number of callus have fragmentary adventitious buds differentiation to generate (figure
2)。
(3) adventitious buds differentiation Fiber differentiation
Differential medium is the NAA that the 6-BA and 0.1mg/L of 2.0mg/L are added on the basis of MS minimal medium.
By step (2) in 30 days callus of induction transfer and carry out adventitious buds differentiation Fiber differentiation on above-mentioned culture medium,
25 ± 2 DEG C of temperature, light application time 16h/ days, after cultivating 30 days under intensity of illumination 2000lux, callus Surface Differentiation was largely not
Normal bud (Fig. 3).
(4) adventitious bud proliferation culture
Adventitious bud proliferation culture medium be added on the basis of 1/2MS minimal medium 3.0mg/L 6-BA and
The NAA of 1.0mg/L.
By step, (3) the middle larger seedling for obtaining 4-7cm high cuts into the small stem section of simple bud point (band 1-2 piece leaf), each seedling
3 sections can be cut into, is placed in proliferated culture medium and cultivates, under 25 ± 2 DEG C of temperature, light application time 16h/ days, intensity of illumination 2000lux
After culture 30 days, small stem section base portion proliferation generates adventitious bud of growing thickly, and each stem section, which can be averagely proliferated, generates 4 adventitious buds, then often
A seedling average proliferation generates 12 adventitious buds, i.e., breeding coefficient is up to 12 (Fig. 4).
(5) culture of rootage and plant regeneration
Root media is the base in 1/2MS culture medium (a great number of elements for including is the 1/2 of MS minimal medium full dose)
The IBA of 0.9mg/L is added on plinth.
The adventitious bud of growing thickly that step (4) obtain is isolated single plant to be transferred on root media at 25 ± 2 DEG C of temperature, light
According to the time 16h/ days, root induction under intensity of illumination 2000Lux.Culture of rootage 15d or so adventitious root length reaches 1cm or so,
By the culture of 30d or so, adventitious root is 4-6cm, forms healthy and strong intensive root system (Fig. 5), obtains complete regenerated plant, raw
Root rate is up to 90%.
(6) hardening and transplanting
The regeneration plant obtained after culture of rootage in step (5) is subjected to method for transplanting are as follows: plant to be regenerated is long to 6-
When 8cm high, root system are intensively healthy and strong, it is gradually opened sealed membrane after removing culturing room, contacts regeneration plant with natural air,
20-25 DEG C can transplant for lower refining seedling 5-7 days.By the river sand and detritus of the Transplantation of Regenerated Plantlets Jing Guo hardening to 0.5-1:1 ratio
In native mixed-matrix, greenhouse production management is moved to, survival rate is up to 95% (Fig. 6).
Module number and treatment scale described herein are for simplifying explanation of the invention.To Decaisnea fargesii of the invention
The application of in-vitro culture method, modifications and variations will be readily apparent to persons skilled in the art.
As described above, according to the present invention, due to carrying out Regeneration in Vitro using the tender embryo of children as explant, having long-term
Squamous subculture and process are not easy the effect degenerated.The present invention be improve Decaisnea fargesii breeding coefficient, be the species preserving seed
And its promote and apply and have laid a good foundation, it has a extensive future.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and embodiment shown and described herein.
Claims (5)
1. a kind of in-vitro culture method of Decaisnea fargesii characterized by comprising using the embryo of immature fruit as explant,
Successively Decaisnea fargesii is obtained by the induction of callus, adventitious buds differentiation induction, adventitious bud proliferation culture and rooting induction culture
Regeneration plant；
Before the induction for carrying out callus, further includes:
The sterilizing of explant: it takes Decaisnea fargesii children okra fruit real, impregnates 20- in the detergent liquid of every 100ml drop containing 6-10 first
Brushing surface after 30min is rinsed with water 1-2 h later, then in gnotobasis first with 75% alcohol sterilization treatment children's okra fruit
Real 30-60s, later with 0.1% HgCl of every 100ml drop containing 3-4 polysorbate402Solution sterilizes to it 15min, then uses sterile water
The tender seed of children is finally taken out in cleaning 4-6 times, each 1-3min, squeezes out the tender embryo of children, uses the tender embryo of children described in rinsed with sterile water
After obtain explant；
In the induction of the callus, the explant is placed in after callus inducing medium carries out induction 30 days and is obtained
Callus, the callus inducing medium addition: MS, the 6-nonylaminopurine of 0.1-2.0 mg/L and 0.5-4.0
2, the 4- dichlorphenoxyacetic acid of mg/L；
In the adventitious buds differentiation induction, the callus that induction of callus is obtained is placed on differential medium and induces
Adventitious bud, the differential medium addition: the 6- benzyl purine (6-BA) and 0.01- of MS, 0.1-2.0 mg/L are generated after 30 days
The α-naphthylacetic acid of 0.5 mg/L；
In the adventitious bud proliferation culture, by adventitious buds differentiation induce obtained in adventitious bud be placed in proliferated culture medium and induce not
Bud multiplication 15-30 days, proliferated culture medium addition: the 6- benzyl purine and 0.2-1.0 mg/L of 1/2MS, 2.0-3.0 mg/L
α-naphthylacetic acid；
In the rooting induction culture, the adventitious bud generated in adventitious bud proliferation culture is placed on rooting induction culture medium and is induced
Take root, obtain Decaisnea fargesii regeneration plant, the rooting induction culture medium addition: 1/2MS and 0.3-0.9 mg/L indolebutyric acid or
The α-naphthylacetic acid of 1/2MS and 0.3-0.9 mg/L.
2. the in-vitro culture method of Decaisnea fargesii as described in claim 1, which is characterized in that the induction of the callus, no
In normal bud induction, adventitious bud proliferation culture and rooting induction culture, condition of culture are as follows: 25 ± 2 DEG C of temperature, light application time
16h/ days, 2000 lux of intensity of illumination.
3. the in-vitro culture method of Decaisnea fargesii as described in claim 1, which is characterized in that obtained in the rooting induction culture
After Decaisnea fargesii regeneration plant further include:
Hardening: by the long Decaisnea fargesii regeneration plant to 5-8 cm high in outdoor 20-25 DEG C of lower refining seedling 5-7 days.
4. the in-vitro culture method of Decaisnea fargesii as claimed in claim 3, which is characterized in that after the hardening further include:
Transplanting: the Decaisnea fargesii Transplantation of Regenerated Plantlets after hardening is entered and is cultivated in the mixed-matrix of river sand and detritus soil, river sand and
The mixed proportion of detritus soil is 0.5-1:1.
5. the in-vitro culture method of Decaisnea fargesii as described in claim 1, which is characterized in that the crude fruit actually 90 days fruits
The fruit in age.
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| 猫儿屎的愈伤组织诱导和植株再生;魏文桃等;《分子植物育种》;20171231;第15卷(第4期);第1473-1477页 |
| 猫屎瓜的组织培养与快速繁殖初探;郭邦利;《陕西农业科学》;20131231(第1期);第69-70页 |
| 矮杞树的组织培养和快速繁殖;杨敬天等;《植物生理学通讯》;20090831;第45卷(第8期);第803页 |
| 超声对三叶木通叶片愈伤组织生长及代谢影响的研究;刘香;《中国优秀硕士学位论文全文数据库 农业科技辑》;20100615(第6期);第D047-214页 |
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