CN105684898B - Method with plant regeneration occurs for a kind of efficiently induction hybrid santal somatic embryo - Google Patents
Method with plant regeneration occurs for a kind of efficiently induction hybrid santal somatic embryo Download PDFInfo
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- CN105684898B CN105684898B CN201610038247.1A CN201610038247A CN105684898B CN 105684898 B CN105684898 B CN 105684898B CN 201610038247 A CN201610038247 A CN 201610038247A CN 105684898 B CN105684898 B CN 105684898B
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- somatic embryo
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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
-
- 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
Abstract
The method with plant regeneration occurs the invention discloses a kind of efficiently induction hybrid santal somatic embryo.For the present invention from excellent cultivation hybrid santal children tender stem as explant, the surface sterilization of explant is carried out using gradation disinfectant program can obtain higher success rate.Sterilize successful aseptic explant and contain 2, cultivated on 4 D embryo callus culture medium, induce fibrous embryo callus, it is then transferred to somatic embryo inducement, maturation, sprouting, cultivates on plant regeneration culture medium, regeneration plant can be obtained after 3 months, breeding cycle is short, quantity is more, the high feature of stability, it is not high to solve reproductive efficiency present in hybrid santal tissue culture propagating seedling, seedling-raising technique will be provided to carry out the quick breeding of hybrid santal seedling, or efficient genetic transformation provides Regeneration Ways.The present invention has the advantages that strong operability, reproductive efficiency and high application value.
Description
Technical field：
The invention belongs to field of plant reproduction, and in particular to a kind of efficiently induction hybrid santal somatic embryo occurs and planted
Strain regeneration method.
Background technology：
Santal (Santalum album L.) is that Santalaceae (Santalaceae) santal category (Santalum) semiparasite is normal
Green arbor, it is rare, rare plant, is distributed mainly on India, Indonesia, some Australian and Pacific archipelagos.
Its existing thousands of years history of human use, it is especially famous because its timber can be used for carving and refine sandalwood oil, it is known as " green
The title of gold ".The Chinese primary distribution without santal, its finished product or semi-finished product are in the past always from external import.Chinese section in 1962
Institute South China Botanical Garden introduces santal seed growing success from Indonesia first in continent, and it is high-quality to introduce " Laoshan is fragrant " from India thereafter
Introduces a collection also succeeds.By the development test of decades, nowadays santal can set seeds breeding and can Edgeworthia chrysantha in South China Botanical Garden.
In the late two decades, our seminar also promote to domestic other areas plants experimentally santal.
Research data shows：In Fiji, the sexual hybridization kind F1 generation of santal and Fijian santal (hybrid santal S.album ×
S.yasi obvious hybrid vigour) is shown, its growth potential is higher by 300% than maternal Fijian santal, then higher by 32% than male parent santal；
In terms of Edgeworthia chrysantha, santal needs 10 years, and hybrid santal then needs 6-7；In terms of heartwood growth, general every plant of santal needs to plant
Plant 25-30 heartwoods and reach 40-80 kilograms, and hybrid santal F1 need to only plant 15-20.In recent years we strengthen with both at home and abroad
On the basis of exchange, imperial Takeshima santal study base researcher is wanted from overseas introduction hybrid santal to imperial bamboo with Zhaoqing Guangdong height
Island santal study base, well-grown；Although hybrid santal can be solid on a small quantity, characters of progenies seriously separates, it is necessary to seeks
Pierre technology is to maintain hybrid vigour.
At present, have no that hybrid santal somatic embryo occurs and plant regeneration report and patent application both at home and abroad.
The content of the invention：
It is an object of the invention to provide a kind of strong operability, reproductive efficiency and application value are high, can be the big rule of hybrid santal
Mould quickly breeds the efficient induction hybrid santal somatic embryo generation for providing seedling and the method for plant regeneration
The present invention uses hybrid santal tender stem as explant, carries out somatic embryo induction, sprouting, maturation, regeneration plant,
The features such as quantity is more, and breeding is fast, and stability is high can be obtained by embryo callus subculture somatic embryos.Can be successfully to carry out hybrid
Santal seedling, which is quickly bred, to be provided seedling-raising technique and provides a kind of effective path for transformation for transgenic experiments, it is achieved thereby that this
The purpose of invention.
Method with plant regeneration occurs for the efficient induction hybrid santal somatic embryo of the present invention, it is characterised in that bag
Include following steps：
A, the induction of embryo callus and propagation：Using the young tender stem of hybrid santal as explant, carry out disinfection, will disappear
Explant after poison is seeded on embryonic callus induction culture medium and cultivated, and embryo callus is obtained, by embryo callus subculture group
Knit and be transferred to progress shoot proliferation culture in new embryonic callus induction culture medium, obtain cells,primordial, every 2 week switching
1 time, the power of regeneration of embryo callus subculture can be maintained, every liter described of embryonic callus induction culture medium contains 2,4-D (2,4-
Dichlorphenoxyacetic acid) 0.5-1.0mg, sucrose 20-30g, agar 6-7g, surplus is MS culture mediums, and described hybrid santal is father
This santal (Santalum album) and the sexual hybridization kind F1 generation of maternal Fijian santal (Santalum yasi)；
B, the induction of somatic embryo and maturation：It is thin that cells,primordial is transferred to inductor on somatic embryo inducing culture
The generation of blastula, somatic embryo is obtained, select somatic embryo and be transferred in somatic embryo maturation culture medium and cultivate, obtain the son of maturation
Leaf embryo, every liter described of somatic embryo inducing culture contain 6-BA (6- benzyls aminoadenine) 0.2-0.5mg, sucrose 20-
30g, agar 6-7g, surplus are MS culture mediums, and (6- benzyl amino glands are fast containing 6-BA for every liter described of somatic embryo maturation culture medium
Purine) 0.05-0.1mg, sucrose 20-30g, agar 6-7g, surplus is MS culture mediums；
C, somatic embryo sprouting and plant regeneration：The ripe cotyledonary embryos of picking, which are transferred in somatic embryo germination medium, to be trained
Support, obtain the somatic embryo of sprouting, the somatic embryo of sprouting is transferred on plant regeneration culture medium and cultivated, obtain completely again
Raw plant, every liter described of somatic embryo germination medium contain 6-BA (6- benzyls aminoadenine) 0.2-0.5mg, GA3It is (red mould
Element) 0.5-1mg, sucrose 20-30g, agar 6-7g, surplus is MS culture mediums, and every liter described of plant regeneration culture medium contains 6-
BA (6- benzyls aminoadenine) 0.2-1mg, IBA (indolebutyric acid) 0.2-1mg, sucrose 20-30g, agar 6-7g, surplus 1/
2MS culture mediums.
The described young tender stem using hybrid santal carries out disinfection as explant, explant is first preferably used into volume integral
The alcohol water blend of number 50% wipes explant 2-3 times, then first water-soluble with the alcohol of volume fraction 75% on superclean bench
Liquid is soaked 30 seconds, and 5~6min of immersion in mass fraction 0.1%HgC1 solution is put into after aseptic water washing, rocks 1 at intervals of two minutes
It is secondary, aseptic water washing 2-3 times, then with volume fraction 6-12% hydrogenperoxide steam generators sterilize 6-8min, aseptic water washing 3-5 times,
Explant after being sterilized.
It is preferred that being seeded to the explant after sterilization on embryonic callus induction culture medium for described step a is cultivated,
Embryo callus is obtained, embryo callus is transferred in new embryonic callus induction culture medium and carries out shoot proliferation
Culture；
The described step b hair that cells,primordial is transferred to somatic embryos on somatic embryo inducing culture
It is raw, somatic embryo is obtained, somatic embryo is selected and is transferred in somatic embryo maturation culture medium and cultivate；
The ripe cotyledonary embryos of described step c picking, which are transferred in somatic embryo germination medium, to be cultivated, and obtains sprouting
Somatic embryo, the somatic embryo of sprouting is transferred on plant regeneration culture medium and cultivated；
Its condition of culture is all intensity of illumination 1000-2000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature.
MS culture mediums are international culture medium, and its composition and collocation method are shown in Murashige T, Skoog F
(1962)(A revised medium for rapid growth and bioassay with tobacco tissue
cultures.Physiol Plant15:473–497).1/2MS culture mediums refer to that by a great number of elements dosage in MS culture mediums be original
1/2 come, remaining composition is constant.
The present invention, as explant, explant is carried out using gradation disinfectant program from excellent cultivation hybrid santal children tender stem
Surface sterilization can obtain higher success rate.Successful aseptic explant is sterilized in the embryo callus training for containing 2,4-D
Support and cultivated on base, induce fibrous embryo callus, be then transferred to somatic embryo inducement, maturation, sprouting, plant again
Cultivated on raw culture medium, can obtain regeneration plant after 3 months, the breeding cycle is short, quantity is more, the high feature of stability, solves miscellaneous
Reproductive efficiency present in kind santal tissue culture propagating seedling is not high, quickly will breed provide for development hybrid santal seedling and educate
Seedling technology, or efficient genetic transformation provide Regeneration Ways.The present invention has strong operability, reproductive efficiency and application value
The advantages that high.
Brief description of the drawings：
Fig. 1 is hybrid santal somatic embryo occur and plant regeneration, wherein A, embryo callus；B, the body cell of induction
Embryo；C, each stage of somatic embryo development：Globular embryo, heart-shape embryo, torpedo-shape embryo, cotyledon shape embryo, ripe cotyledonary embryos；
D regeneration plants.A-C scale=2 millimeter, D scale=1 centimetre.
Embodiment：
Following examples are to further explanation of the invention, rather than limitation of the present invention.
Embodiment 1：Efficiently the method with plant regeneration occurs for induction hybrid santal somatic embryo, comprises the following steps：
(1) explant and its processing
Pick up from Guangdong height and take imperial Takeshima Sandalwood cultivation base introducing and planting elite plant strain hybrid santal (the male parent santal of 6 years
(Santalum album) and the sexual hybridization kind F1 generation of maternal Fijian santal (Santalum yasi)) stem segments are as explant
Body, first wipe explant 2-3 times with the alcohol water blend of volume fraction 50%, volume fraction is then first used on superclean bench
75% alcohol water blend soaks 30 seconds, is put into after aseptic water washing in mass fraction 0.1%HgC1 solution and soaks 5-6min or so,
Rock at intervals of two minutes 1 time, aseptic water washing 2-3 times, then about 6-8min is sterilized with the hydrogenperoxide steam generator of volume fraction 6%, it is sterile
Water rinses 3-5 times, obtains the tender stem after surface sterilization processing.
(2) induction of embryo callus and propagation
Tender stem after surface sterilization is handled with sharp sterile scalpel is cut into 1-1.5cm band knot stem section, sterile tweezer
Son places stem section to embryonic callus induction culture medium.Condition of culture, intensity of illumination 1000lx, light application time 10h/d, training
Support 26 ± 2 DEG C of temperature.Stem section starts expansion after 7-10d and incision starts to sprout, the vigorous embryo of visible division after culture 20d
Callus (Fig. 1-A), callus induction rate is up to more than 80%.Then embryo callus is transferred into new embryo to be cured
Shoot proliferation culture is carried out in injured tissue inducing culture, obtains cells,primordial.Every 2 week transfers 1 time, can maintain embryo callus subculture
Power of regeneration.Every liter described of embryonic callus induction culture medium contain 2,4-D (2,4- dichlorphenoxyacetic acid) 1.0mg,
Sucrose 30g, agar 7g, surplus are MS culture mediums, and pH5.8, its compound method is after composition is well mixed, and adjusts pH value, sterilizing
It is standby.
(3) induction of somatic embryo and maturation
Take the cells,primordial of 6d after squamous subculture, the hair of somatic embryos on somatic embryo inducing culture
It is raw.Condition of culture, intensity of illumination 1000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature.The visible body of naked eyes is thin after 15d
The appearance of blastula, during to 30d, it is seen that substantial amounts of somatic embryo (Fig. 1-B).Select somatic embryo and be transferred to somatic embryo maturation training
Support base, condition of culture, intensity of illumination 1000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature.Obtain ripe cotyledonary embryos
(its growth course is as shown in Fig. 1-C).
Every liter of described somatic embryo inducing culture containing 6-BA (6- benzyls aminoadenine) 0.2mg, sucrose 30g,
Agar 7g, surplus are MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust pH value, is sterilized standby.
Every liter described of somatic embryo maturation culture medium contains 6-BA (6- benzyls aminoadenine) 0.1mg, sucrose 30g, fine jade
Fat 6g, surplus are MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust pH value, is sterilized standby.
(4) somatic embryo sprouting and plant regeneration
The ripe cotyledonary embryos for selecting 1-2cm or so are transferred on somatic embryo germination medium and cultivated, intensity of illumination
1000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature, 2 weeks or so two panels cotyledons of culture start to expand, and start to grow children
Tender root, the somatic embryo after being sprouted.Somatic embryo after sprouting is transferred on plant regeneration culture medium, intensity of illumination
1000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature, cultivate 2 weeks, further bud into complete regeneration plant (figure
1-D)。
Every liter described of somatic embryo germination medium contains 6-BA (6- benzyls aminoadenine) 0.5mg, GA3(gibberellin)
1mg, sucrose 30g, agar 7g, surplus are MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust pH value,
Sterilize standby.
Every liter described of plant regeneration culture medium contains 6-BA (6- benzyls aminoadenine) 1mg, IBA (indolebutyric acid)
0.2mg, sucrose 30g, agar 7g, surplus are 1/2MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust
PH value, sterilizing are standby.
Embodiment 2：Efficiently the method with plant regeneration occurs for induction hybrid santal somatic embryo, comprises the following steps：
(1) explant and its processing
Pick up from Guangdong height and take imperial Takeshima Sandalwood cultivation base introducing and planting elite plant strain hybrid santal (the male parent santal of 6 years
(Santalum album) and the sexual hybridization kind F1 generation of maternal Fijian santal (Santalum yasi)) stem segments are as explant
Body, first wipe explant 2-3 times with the alcohol water blend of volume fraction 50%, volume fraction is then first used on superclean bench
75% alcohol water blend soaks 30 seconds, is put into after aseptic water washing in mass fraction 0.1%HgC1 solution and soaks 5-6min or so,
Rock at intervals of two minutes 1 time, aseptic water washing 2-3 times, then about 6-8min, nothing are sterilized with the hydrogenperoxide steam generator of volume fraction 12%
Bacterium water rinses 3-5 times, obtains the tender stem after surface sterilization processing.
(2) induction of embryo callus and propagation
Tender stem after surface sterilization is handled with sharp sterile scalpel is cut into 1-1.5cm band knot stem section, sterile tweezer
Son places stem section to embryonic callus induction culture medium.Condition of culture, intensity of illumination 2000lx, light application time 10h/d, training
Support 26 ± 2 DEG C of temperature.Stem section starts expansion after 7-10d and incision starts to sprout, the vigorous embryo of visible division after culture 20d
Callus, callus induction rate is up to more than 80%.Then embryo callus is transferred to new embryo callus
Shoot proliferation culture is carried out in inducing culture, obtains cells,primordial.Every 2 week transfers 1 time, can maintain the regeneration of embryo callus subculture
Ability.Every liter described of embryonic callus induction culture medium contains 2,4-D (2,4- dichlorphenoxyacetic acid) 0.5mg, sucrose
20g, agar 6g, surplus are MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust pH value, is sterilized standby.
(3) induction of somatic embryo and maturation
Take the cells,primordial of 6d after squamous subculture, the hair of somatic embryos on somatic embryo inducing culture
It is raw.Condition of culture, intensity of illumination 2000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature.The visible body of naked eyes is thin after 15d
The appearance of blastula, during to 30d, it is seen that substantial amounts of somatic embryo.Select somatic embryo and be transferred to somatic embryo maturation culture medium, train
The condition of supporting, intensity of illumination 2000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature.Obtain ripe cotyledonary embryos.
Every liter of described somatic embryo inducing culture containing 6-BA (6- benzyls aminoadenine) 0.5mg, sucrose 20g,
Agar 6g, surplus are MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust pH value, is sterilized standby.
Every liter described of somatic embryo maturation culture medium contains 6-BA (6- benzyls aminoadenine) 0.05mg, sucrose 20g, fine jade
Fat 7g, surplus are MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust pH value, is sterilized standby.
(4) somatic embryo sprouting and plant regeneration
The ripe cotyledonary embryos for selecting 1-2cm or so are transferred on somatic embryo germination medium and cultivated, intensity of illumination
2000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature, 2 weeks or so two panels cotyledons of culture start to expand, and start to grow children
Tender root, the somatic embryo after being sprouted.Somatic embryo after sprouting is transferred on plant regeneration culture medium, intensity of illumination
2000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature, cultivate 2 weeks, further bud into complete regeneration plant.
Every liter described of somatic embryo germination medium contains 6-BA (6- benzyls aminoadenine) 0.2mg, GA3(gibberellin)
0.5mg, sucrose 20g, agar 6g, surplus are MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust pH
Value, sterilizing are standby.
Every liter described of plant regeneration culture medium contains 6-BA (6- benzyls aminoadenine) 0.2mg, IBA (indolebutyric acid)
1mg, sucrose 20g, agar 6g, surplus are 1/2MS culture mediums, and pH5.8, its compound method is after composition is well mixed, to adjust pH
Value, sterilizing are standby.
Claims (3)
1. the method with plant regeneration occurs for a kind of efficiently induction hybrid santal somatic embryo, it is characterised in that including following
Step：
A, the induction of embryo callus and propagation：Using the young tender stem of hybrid santal as explant, carry out disinfection, after sterilization
Explant be seeded on embryonic callus induction culture medium and cultivate, obtain embryo callus, embryo callus turned
Progress shoot proliferation culture in new embryonic callus induction culture medium is connected to, obtains cells,primordial, described embryo callus subculture
Every liter of inducing culture of tissue contains 2,4-D 0.5-1.0mg, sucrose 20-30g, agar 6-7g, and surplus is MS culture mediums, described
Hybrid santal be male parent santal (Santalum album) and maternal Fijian santal (Santalum yasi) sexual hybridization kind
F1 generation；
B, the induction of somatic embryo and maturation：Cells,primordial is transferred to somatic embryos on somatic embryo inducing culture
Generation, obtain somatic embryo, select somatic embryo and be transferred in somatic embryo maturation culture medium and cultivate, obtain the cotyledon of maturation
Embryo, every liter described of somatic embryo inducing culture contain 6-BA 0.2-0.5mg, sucrose 20-30g, agar 6-7g, surplus
For MS culture mediums, every liter described of somatic embryo maturation culture medium contains 6-BA 0.05-0.1mg, sucrose 20-30g, agar 6-
7g, surplus are MS culture mediums；
C, somatic embryo sprouting and plant regeneration：The ripe cotyledonary embryos of picking, which are transferred in somatic embryo germination medium, to be cultivated, and is obtained
The somatic embryo that must be sprouted, the somatic embryo of sprouting is transferred on plant regeneration culture medium and cultivated, obtained complete regeneration and plant
Strain, every liter described of somatic embryo germination medium contain 6-BA 0.2-0.5mg, GA30.5-1mg, sucrose 20-30g, agar
6-7g, surplus are MS culture mediums, and every liter described of plant regeneration culture medium contains 6-BA 0.2-1mg, IBA 0.2-1mg, sucrose
20-30g, agar 6-7g, surplus are 1/2MS culture mediums.
2. the method with plant regeneration occurs for efficiently induction hybrid santal somatic embryo according to claim 1, it is special
Sign is that the young tender stem using hybrid santal carries out disinfection as explant, is that explant is first used into volume fraction 50%
Alcohol water blend wipe explant 2-3 time, then first with the immersion of the alcohol water blend of volume fraction 75% on superclean bench
30 seconds, mass fraction 0.1%HgCl is put into after aseptic water washing25~6min is soaked in solution, rocks 1 time at intervals of two minutes, nothing
Bacterium water rinses 2-3 times, then sterilizes 6-8min with volume fraction 6-12% hydrogenperoxide steam generators, aseptic water washing 3-5 times, is disappeared
Explant after poison.
3. the method with plant regeneration occurs for efficiently induction hybrid santal somatic embryo according to claim 1, it is special
Sign is,
Being seeded to the explant after sterilization on embryonic callus induction culture medium for described step a is cultivated, and obtains embryo
Callus, embryo callus is transferred to progress shoot proliferation culture in new embryonic callus induction culture medium；
The described step b generation that cells,primordial is transferred to somatic embryos on somatic embryo inducing culture, is obtained
Somatic embryo, select somatic embryo and be transferred in somatic embryo maturation culture medium and cultivate；
The ripe cotyledonary embryos of described step c picking, which are transferred in somatic embryo germination medium, to be cultivated, and the body for obtaining sprouting is thin
Blastula, the somatic embryo of sprouting is transferred on plant regeneration culture medium and cultivated；
Its condition of culture is all intensity of illumination 1000-2000lx, light application time 10h/d, 26 ± 2 DEG C of cultivation temperature.
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| CN108207385B (en) * | 2018-02-09 | 2021-01-15 | 中国林业科学研究院热带林业研究所 | Chemical regulation and control method for improving sandalwood seedling quality and resistance |
| CN112586349A (en) * | 2020-11-11 | 2021-04-02 | 江西省中国科学院庐山植物园 | Method for rapidly propagating clematis chinensis seedlings through somatic embryogenesis |
| CN115250913B (en) * | 2022-07-25 | 2023-06-20 | 三峡大学 | Rhus chinensis somatic embryogenesis and plant regeneration method |
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