CN108056023A - No germplasm genotype limits high-frequency somatic embryo regeneration culture method - Google Patents
No germplasm genotype limits high-frequency somatic embryo regeneration culture method Download PDFInfo
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- CN108056023A CN108056023A CN201810116768.3A CN201810116768A CN108056023A CN 108056023 A CN108056023 A CN 108056023A CN 201810116768 A CN201810116768 A CN 201810116768A CN 108056023 A CN108056023 A CN 108056023A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Abstract
The invention discloses no germplasm genotype to limit high-frequency somatic embryo regeneration culture method, comprises the following steps：1）Selection and the growth of sterile sprout domestication culture；2）Leaf bud former base somatic embryo inducement is cultivated；3）Embryoid is proliferated and grown cultures；4）The seedling-growing rapid breeding culture of Multiple Buds；5）Strengthening seedling and rooting culture；6）It Transplantation of Regenerated Plantlets and survives.The invention also discloses a kind of high-frequency somatic embryo regeneration culture cultural methods of Korea lawn grass.The present invention is by selecting suitable explant and adjusting culture medium prescription and hormonal components concentration and corresponding condition of culture etc. in time according to the explant crucial stage of development, these factors is made to reach good mutually synergistic effect, are effectively overcomed not of the same race in Korea lawn grass culture（Kind）And genotype restricted problem and high-frequency somatic embryo regeneration problem, increase substantially somatic embryo occur frequency and regeneration frequencies.
Description
Technical field
The present invention relates to field of plant breeding, and in particular to and no germplasm genotype limits high-frequency somatic embryo regeneration culture method,
The high-frequency somatic embryo regeneration culture method of more particularly to a kind of gramineous grass more particularly to a kind of Korea lawn grass limit without germplasm genotype
High-frequency somatic embryo regeneration culture method.
Background technology
Zoysia (Zoysia genus) plant is distributed mainly in east Asia broad area, and north and south is across about 20
Latitude, thing account for 30 longitudes；In coastal strip of the main distributed areas in China from northeast Liaoning to southern Guangxi and other places.
Since Korea lawn grass ecological environment complexity is various, the stringent cross-pollination characteristic of typical case results in Korea lawn grass interspecific hybridization and day
Right hybrid largely exists, and intraspecific variablity is very big, but also maintaining abundant natural variation, gene between wild population at individual
Type difference and genetic diversity.
Korea lawn grass with its high wear tolerance, strong adaptability and high resiliency to compacted soil, the barren-resistant property of strong drought resistant and
Saline alkali tolerance and the first choice and hair for becoming high quality football pitch and sports turf suitable for characteristics such as the extensive property management of low-maintenance
Open up the Steppe Plants of the excellent ecology, soil and slope protection and the water and soil conservation that have a high potential；Also universally acknowledged fine turfgrass species are become；
China's Korea lawn grass germ plasm resource ranks the first in the world and uniquely produces in the world wild zoysia seed and with foreign exchange earning
The country of ability.In view of the good characteristic of Korea lawn grass, the conventional choosing that the developed countries such as the U.S., Japan take the lead in having carried out new varieties is trained
Educate, but use of the Korea lawn grass in production at present be mostly or based on wild resource, improved variety (except orchid draws 3 extras) it is wide
General application is also seldom seen.
The choosing that Korea lawn grass new varieties are carried out using biotechnology combination conventional breeding methods is cultivated, and is both to improve Korea lawn grass to educate
The effective way of kind efficiency, Ye Shu worlds breeding forward position.Kind or the improvement of germplasm genotype and germplasm are carried out using biotechnology
Fast numerous most effective approach is exactly to establish high frequency somatic embryo regeneration techniques system；Because somatic embryo is unicellular origin,
Somatic embryo Regeneration Ways have not only recurred the morphogenetic process of zygotic embryo, realize again seed bearing source and germplasm
Preservation, seedling detoxifying fast breeding, inducing somatic become the preferable receptor of exclusive or genetic transformation；Somatic embryo is fast numerous excellent except can guarantee
The genetic identity and stability of population at individual genotype, moreover it is possible to keep the specificity of new germ plasm.So somatic embryo regenerates skill
Art is also the research direction of most attraction in plant tissue culture clone.
In general, plant cell cultured in vitro, induces dedifferentiation and atomization can both be controlled by genotype again,
With culture environment it is closely related in addition grasp condition of culture it is more important；Many researchs show：Select suitable explant, explant
Body different development stage selects the concentration of the plant growth regulator added in suitable culture medium and culture medium, species and matches somebody with somebody
Than and condition of culture (temperature, illumination, light intensity etc.) cultivation results can all be had an impact；But how what is more important makes this
A little factors are used cooperatively in due course, by the mutual synergistic effect between them, farthest play its complementary effect so as to reach
Should be its key technology to expected effect.
Theoretically, the body cell of various plants all has totipotency, passes through somatic embryo fetal hair in cultured in vitro
Raw approach can form regeneration plant, however in fact some plants are easy, some plants are difficult, mainly people up to now
The regeneration condition of culture of these plants is also understood there are no complete with grasping.Turfgrass is also like the same quilt of many monocotyledons
It is considered to be difficult with somatic embryo development ways regeneration induction plant, is particularly some warm season turf relevant reports very
It is few.Usually most of turfgrass is on the solid medium of the basic element of cell division of the auxin and low concentration of higher concentration with regard to energy
Callus is formed, but whether can induce out whether the cells,primordial in embryo callus and embryo callus can pass through
Somatic embryo development pathway formed embryoid then with the age of explant, physiological status, the composition of culture medium, hormone species
It is related with concentration, condition of culture etc..Wherein, the composition of culture medium, the species of hormone and concentration are the key factors of condition of culture,
Suitable condition of culture is exactly to add suitable hormone and suitable concentration in due course on suitable culture medium, so as to reach plant
In the stage accordingly broken up to the needs of growth and development.Hormone is mutually promoted to the physiological effect of variety classes plant or phase
The effect mutually resisted, influence are related to the various aspects such as synthesis, transport, metabolism and the effect of hormone.Plant cell growth and point
The adjusting of change be often a variety of hormone comprehensive functions as a result, and interaction between them it is extremely complex.
It is reported that Korea lawn grass is the species of more difficult culture in grass family, the material for largely studying selection is with 1~3
Kind or germplasm genotype based on, the germplasm genotype negligible amounts that involve；Tissue culture regeneration is mostly with the adventitious organogenesis of many cells
Based on；And becoming embryo callus from callus and regeneration plant is all more difficult, tissue culture regeneration rate is low, and regenerative process is long.
The yield and quality that Korea lawn grass somatic embryo is mentioned in some researchs is depended on to the excellent of the conditions such as culture medium composition and plant hormone
Change, but also do not see the report that Korea lawn grass high-frequency somatic embryo regeneration system is established by somatic embryo development ways so far,
The report for the high frequency somatic embryo regeneration techniques for establishing Korea lawn grass difference germplasm genotype is not seen.So the studies above is neither
It can solve the problems, such as that Korea lawn grass germplasm genotype limits, also not involve Korea lawn grass difference germplasm genotype high-frequency somatic embryo regeneration
The problem of, these problems also become Korea lawn grass biotechnology breeding and the fast numerous bottleneck of elite germplasm.Therefore, establish Korea lawn grass without
The high-frequency somatic embryo regeneration culture technique of germplasm genotype limitation realizes that Korea lawn grass Genetic improvement is efficiently utilized with elite germplasm
Key factor has important practical significance.
The content of the invention
Goal of the invention：For overcome the deficiencies in the prior art, there is provided no kind for the technical problems to be solved by the invention
Matter genotype limits high-frequency somatic embryo regeneration culture method.
Also there is provided a kind of high-frequency somatic embryo regeneration culture methods of gramineous grass for technical problems to be solved by the present invention.
There is provided a kind of high-frequency somatic embryo regeneration culture cultural methods of Korea lawn grass for the present invention also technical problems to be solved.
Technical solution：In order to solve the above technical problem, the present invention provides limit high-frequency somatic embryo again without germplasm genotype
Raw cultural method, comprises the following steps：
1) selection and the growth of sterile sprout domestication culture；
2) leaf bud former base somatic embryo inducement is cultivated：It chooses aseptic seedling eustipes part and starts the leaf bud former base sprouted work
For explant, cut leaf bud former base and be transferred to the induction that somatic embryo is carried out on somatic embryo inducement culture medium, light culture 28 ±
2d, the somatic embryo inducement culture medium, including modified MS medium, 2~4mg/L 2,4-D, 0.1~0.2mg/L NAA,
0.05~0.1mg/L 6-BA, 40~50g/L sucrose, 0.6~0.7% agar, pH value is between 5.8~6.0；
3) embryoid multiplication and grown cultures；
4) the seedling-growing rapid breeding culture of Multiple Buds；
5) strengthening seedling and rooting culture；
6) Transplantation of Regenerated Plantlets and survive.
Wherein, the step 1) selection and the growth of sterile sprout domestication incubation step are as follows：Take half tender stem in growth period
The section of band 2~4 crawls stem section as sterilization treatment material, is cleaned through flowing water, disinfection, sterile material is seeded in sterile sprout
The growth domestication culture of sprouting of sterile bud seedling is carried out on culture medium, wherein, the sterilizing stem with bud of inoculation includes 2~3 sections；Training
Foster 28 ± 2 DEG C of temperature daytime, 20 ± 2 DEG C of night；
Wherein, step 3) the embryoid multiplication and grown cultures step are as follows：The leaf bud protocorm that step 2) is cultivated
Blast be transferred to embryoid multiplication and growth medium on, be first put into 6 ± 2 DEG C of low temperature artificial climate incubator light cultures 6~
8d, then be transferred to intensity of illumination to cultivate 26~28d under 600~800lx low-light intensity, 28 ± 2 DEG C of cultivation temperature daytime, night
20 ± 2 DEG C, count embryoid formation rate.
Wherein, step 3) the embryoid multiplication and growth medium are MS+0.5~1mg/L NAA+0.05~0.1mg/
+ 0.7% agar of L 6-BA+0.1~0.2mg/L 2,4-D+30g/L sucrose.
Wherein, the seedling-growing rapid breeding incubation step of the step 4) Multiple Buds is as follows：The embryoid that step 3) is obtained is grown thickly
The explant that bud cuts into 0.5cm sizes is transferred on differentiation seedling-growing rapid breeding culture medium, and 2000~2500lx illumination is strong
Degree lower culture 28~30d statistics differentiation rates.
Wherein, the seedling-growing rapid breeding culture medium of the step 4) Multiple Buds is MS+1~2mg/L 6-BA+0.02~0.05mg/
+ 0.7% agar of L NAA+30g/L sucrose.
Wherein, step 5) the strengthening seedling and rooting incubation step is as follows：The Multiple Buds switching that step 4) is obtained is given birth in strong sprout
The rooting rate of 26~30d statistics regeneration plants is cultivated on root culture medium, under 2000~2500lx intensities of illumination.
Wherein, the strengthening seedling and rooting culture medium is+0.7% agar of 1/2MS+20g/L white sugar.
Wherein, the step 6) Transplantation of Regenerated Plantlets and to survive step as follows：The regeneration taken root that step 5) is obtained is planted
Strain is transplanted in greenhouse small flower, and basin local soil type point and weight percent are：Vermiculite：Peat soil：Garden mould=2：4：4 mixing；It will be small
Flowerpot is put into shelter from the sun, and watering 1~2 time daily of transplanting initial stage gradually decreases irrigation times after seedling survives, unites after 15~20d
Count survival rate.
Wherein, the kind of the germplasm genotype is gramineous grass, is preferably the knot thread of different population that is wild or being bred as
Grass.
Advantageous effect：Referring now to the prior art, the present invention has the following advantages：
1) the Growth of Somatic Cells stage, by adding suitable hormone and suitable concentration in due course, inducing somatic into
Enter the needs that somatic embryo development pathway and embryoid are differentiated to form, the present invention is by the basis of modified MS medium
Plant growth regulator 2 is added, the components such as 4-D, NAA, 6-BA, sucrose, agar effectively overcome Korea lawn grass high-frequency somatic embryo
Different cultivars and genotype barrier problem in regeneration culture, increase substantially somatic embryo occur frequency and regeneration frequencies；
2) based on grand nutrition element formula of the present invention is formulated with MS, ferrous sulfate and ethylenediamine tetrem during MS is formulated
The molysite that acid disodium is prepared changes iron edta sodium salt into, not only saves the link of mother liquid of iron salt preparation, also avoids molysite
Prepare improper the problem of being also easy to produce deposition invalidation, reducing utilization rate；
3) based on modified MS medium Meso- and micro-nutrients formula of the invention is formulated with SH, and the concentration of copper and cobalt compared with
Other formulas significantly improve, and are conducive to the induction of somatic embryo, and experiment effect proves to be more easy to obtain somatic embryo really；
4) present invention is by selecting suitable explant and adjusting culture medium prescription in time according to the explant crucial stage of development
With hormonal components concentration and corresponding condition of culture etc., these factors is made to reach good mutually synergistic effect, efficiently against finishing
(kind) and genotype restricted problem and high-frequency somatic embryo regeneration problem not of the same race in thread grass culture, increase substantially embryogensis
Raw frequency and regeneration frequencies.Body embryo is fast numerous can not only realize regeneration seed, it is ensured that the uniformity of good variety population character
And stability and the specificity of new germ plasm；It is the most effective approach of preserving seed, seedling detoxifying fast breeding, Genetic improvement.Body embryo
Regeneration is high except reproductive efficiency, and same period seed floor space or vegetative estabilshment are also substantially better than in terms of growth potential, Phenotypic traits.So
Korea lawn grass the Development of Somatic Embryogenesis and sprouting and rooting system are established, not only opens the new way of Korea lawn grass industrialization development
Footpath, and to the Korea lawn grass of quick breeding seed source difficulty, there is important theory significance and application value.
Description of the drawings
The generation of Fig. 1 somatic embryos and embryoid growth and development process；1 is Korea lawn grass of the present invention and Qingdao knot thread
Some somatic cell transformations are into the schematic diagram of cells,primordial in careless callus；2 be Korea lawn grass somatic embryo of the present invention
The schematic diagram of globular embryo and torpedo embryo in growth course；3 draw No. III Korea lawn grass and mascarene grass for orchid of the present invention
Globular embryo and scultellum embryo and the blue signal for drawing a tool root hair on No. III Korea lawn grass callus in somatic embryo growth course
Figure；4 for globular embryo, torpedo embryo and scultellum embryo in manilagrass somatic embryo growth course of the present invention signal
Figure；5 be the globular embryo and embryoid in Zoysia matrella cv.Huanan Korea lawn grass somatic embryo growth course of the present invention
The schematic diagram of bud clump；6 are divided into Multiple Buds for Korea lawn grass somatic embryo different development stage of the present invention and embryoid
Schematic diagram；
Fig. 2 embryoids Multiple Buds (left figure) and rapid propagation cultivation (right figure)；
Fig. 3 Multiple Buds growth and development processes；1 and 2 be respectively that orchid of the present invention draws No. III Korea lawn grass and ditch leaf knot thread
The schematic diagram of careless embryoid Multiple Buds explant embryo callus differentiation and development；3 are cured for Korea lawn grass embryo of the present invention
Injured tissue differentiates the schematic diagram of young shoot bud clump；4 draw No. III Korea lawn grass embryo callus for orchid of the present invention divides completely
Turn to the schematic diagram of Multiple Buds；
The Multiple Buds seedling-growing rapid breeding culture of the different germplasm genotype of Fig. 48；Fig. 1~8 are successively：Zg、Zd、Zq、Z1、
The schematic diagram of the Multiple Buds of the different germplasm genotype of Z3, Zh, Zx, Zz 8；
Fig. 5 strengthening seedling and rooting cultures；Left figure develops the schematic diagram of young root for Korea lawn grass differentiation of the present invention；
Right figure draws No. III Korea lawn grass differentiation and provides showing for root, stem and leaf intact plant for mascarene grass of the present invention and orchid
It is intended to；
Fig. 6 Transplantation of Regenerated Plantlets and different germplasm genotype survive plant.Left figure regenerates for Korea lawn grass of the present invention
The schematic diagram of plantlet of transplant hardening；Right figure is 8 of the present invention different germplasm genotype Korea lawn grass and a cuttage control
Korea lawn grass Transplantation of Regenerated Plantlets survives and well-grown schematic diagram.
Specific embodiment：
Below by embodiment, the present invention is further explained.
Embodiment 1：Somatic embryo inducement culture medium
Somatic embryo inducement culture medium：Improve MS+2mg/L 2,4-D+0.2mg/L NAA+0.1mg/L 6-BA+40g/L
+ 0.7% agar of sucrose, pH value 5.8.
The modified MS medium includes grand nutrition element, micronutrient element and organic reagent：
The component of grand nutrition element concentration corresponding with its is as follows：
The component of micronutrient element concentration corresponding with its is as follows：
The component of organic reagent concentration corresponding with its is as follows：
2 somatic embryo inducement culture medium of embodiment
Substantially the same manner as Example 1, institute's difference is, the somatic embryo inducement culture medium, to improve MS+4mg/L 2,4-
+ 0.7% agar of D+0.2mg/L NAA+0.1mg/L 6-BA+40g/L sucrose, pH value 5.8.
3 somatic embryo inducement culture medium of embodiment
Substantially the same manner as Example 1, institute's difference is, the somatic embryo inducement culture medium, to improve MS+2mg/L 2,4-
+ 0.6% agar of D+0.1mg/L NAA+0.05mg/L 6-BA+50g/L sucrose, pH value 6.0.
4 embryoid of embodiment multiplication growth combination culture medium
Culture medium occurs for leaf bud former base somatic embryo inducement：It is identical with the nutrient media components of embodiment 1；
Embryoid is proliferated and growth medium：MS+1mg/L NAA+0.05mg/L 6-BA+0.2mg/L 2,4-D+30g/L
+ 0.7% agar of sucrose；
Embodiment 5---- embryoids multiplication growth combination culture medium
Culture medium occurs for leaf bud former base somatic embryo inducement：Substantially the same manner as Example 1, institute's difference is, the embryoid
Multiplication and growth medium：+ 0.7% agar of MS+1mg/L NAA+0.1mg/L 6-BA+0.2mg/L 2,4-D+30g/L sucrose；
Embodiment 6-- embryoids multiplication growth combination culture medium
Culture medium occurs for leaf bud former base somatic embryo inducement：It is identical with the nutrient media components of embodiment 2；
Embryoid is proliferated and growth medium：MS+1mg/L NAA+0.05mg/L 6-BA+0.2mg/L 2,4-D+30g/L
+ 0.7% agar of sucrose；
7 embryoid of embodiment multiplication growth combination culture medium
Culture medium occurs for leaf bud former base somatic embryo inducement：Substantially the same manner as Example 3, institute's difference is, the embryoid
Multiplication and growth medium：+ 0.7% agar of MS+1mg/L NAA+0.1mg/L 6-BA+0.2mg/L 2,4-D+30g/L sucrose；
Embodiment 8 obtains intact plant combination culture medium -- and no germplasm genotype limits high-frequency somatic embryo regeneration culture medium
Culture medium occurs for leaf bud former base somatic embryo inducement：It is identical with the nutrient media components of embodiment 4；
Embryoid is proliferated and growth medium：It is identical with the nutrient media components of embodiment 4；
The seedling-growing rapid breeding culture medium of Multiple Buds：+ 0.7% fine jade of MS+1mg/L 6-BA+0.05mg/L NAA+30g/L sucrose
Fat；
Strengthening seedling and rooting culture medium：+ 0.7% agar of 1/2MS+20g/L white sugar；
PH is 5.8 or so after above-mentioned all culture medium high pressure sterilizations.
The acquisition intact plant of embodiment 9 combination culture medium-without germplasm genotype limitation high-frequency somatic embryo regeneration culture medium
Institute's difference is that culture medium occurs for leaf bud former base somatic embryo inducement：It is identical with the nutrient media components of embodiment 5；
Embryoid is proliferated and growth medium：It is identical with the nutrient media components of embodiment 5；It is other substantially the same manner as Example 8；
The acquisition intact plant of embodiment 10 combination culture medium-without germplasm genotype limitation high-frequency somatic embryo regeneration culture medium
Institute's difference is that culture medium occurs for leaf bud former base somatic embryo inducement：It is identical with the nutrient media components of embodiment 6；
Embryoid is proliferated and growth medium：It is identical with the nutrient media components of embodiment 6；It is other substantially the same manner as Example 8；
The acquisition intact plant of embodiment 11 combination culture medium-without germplasm genotype limitation high-frequency somatic embryo regeneration culture medium
Culture medium occurs for leaf bud former base somatic embryo inducement：It is identical with the nutrient media components of embodiment 7；
Embryoid is proliferated and growth medium：It is identical with the nutrient media components of embodiment 7；It is other with 8 basic phase of embodiment
Together；
The acquisition intact plant of embodiment 12 combination culture medium-without germplasm genotype limitation high-frequency somatic embryo regeneration culture medium
Substantially the same manner as Example 8, institute's difference is, the seedling-growing rapid breeding culture medium of the Multiple Buds：MS+2mg/L 6-BA+
+ 0.7% agar of 0.05mg/L NAA+30g/L sucrose；
The acquisition intact plant of embodiment 13 combination culture medium-without germplasm genotype limitation high-frequency somatic embryo regeneration culture medium
Substantially the same manner as Example 9, institute's difference is, the seedling-growing rapid breeding culture medium of the Multiple Buds：MS+2mg/L 6-BA+
+ 0.7% agar of 0.05mg/L NAA+30g/L sucrose；
Experimental example：
1st, selection and sterilization treatment：
Selection：To be respectively derived from Liaoning, the Qingdao peninsula；Jiangsu and Yangtze River Delta surrounding area；And Guangdong and surrounding area
The wild or germplasm genotype of 5 kinds of 8 populations of Korea lawn grass that is bred as experimental study material, including Korea lawn grass (Zoysia
Japonica Steud. (Z1)), Zoysia sinica (Z.sinica Hance (Zz)), manilagrass (Z.matrella (L.)
Merr (Zg)), Zoysia macrostachya (Z.macrostachya Franch.et Saw. (Zd)), mascarene grass
(Z.tenuifolia Willd.ex Trin. (Zx)) Qingdao Korea lawn grass (Zoysia japonica cv.Qingdao (Zq)),
Orchid draws No. III Korea lawn grass (Zoysia japonica cv.lanyin3 (Z3)), Zoysia matrella cv.Huanan (Z.matrella
Cv.Huanan (Zh)) etc., it is represented respectively with symbols Z 1, Zz, Zg, Zd, Zx, Zq, Z3, Zh.
Sterilization treatment：The half tender stem band 2~4 of Korea lawn grass for cutting growth period saves stem section of crawling, and first uses plus liquid detergent water rinses
10 minutes or so, then with 3% sodium hypochlorite liquid disinfectant 15 minutes, in desinfection chamber 75% alcohol disinfecting 20~30 seconds, sterile water
It rinses 3~5 times, 0.1% mercuric chloride sterilizes 6~8 minutes, aseptic water washing 5~8 times, and sterilize blotting paper suck dry moisture, and clip contains 2
~3 section explants are inoculated into the growth domestication of sprouting of progress sterile bud seedling on sterile sprout culture medium and cultivate, 1000~
16h/d under 1500lx illumination cultivates 26~28d.Cultivation temperature is 2 DEG C of 28 scholar of room temperature on daytime, 20 ± 2 DEG C of (cultivation temperatures of night
If it is the same to be not particularly illustrated later step), it is similar below.
The sterile sprout culture medium：1/4MS+GA3+ 0.7% agar of 0.1mg/L+6-BA0.2mg/L+20g/L white sugar.
2nd, leaf bud protocorm embryonal induction is cultivated：
It chooses sterile sprout eustipes part and starts the leaf bud former base sprouted as explant, cut the sterile leaf of 0.5cm sizes
Bud former base is transferred to somatic embryo inducement culture medium i.e. 28 ± 2d of upper light culture of Examples 1 to 3 preparation.More than 3 kinds of embodiments
Take embodiment 1 as optimal, 8 different germplasm genotype Korea lawn grass somatic embryo inducement rates minimum 49.9%, highest 85.3%,
Averagely reach 70.3%；And embodiment 1 minimum 35.2%, highest 61.7%, average out to 51.5%；Take second place compared with embodiment 1；It is real
Example 3 minimum 29.7% is applied, up to 52.4%, average out to 45.9%, it can be seen that embodiment 3 is worst.
3rd, embryoid multiplication and grown cultures：
Above-mentioned steps 2 are cultivated to obtained material to be transferred on embryoid multiplication and growth medium, are first put into 6 ± 2 DEG C
Artificial climate incubator low temperature 6~8d of light culture, then it is transferred to 26~28d of culture under 600~800lx 16h/d low-lights, culture
Temperature is the same, counts embryoid formation rate.Its result is referring to table 1.
4th, the seedling-growing rapid breeding culture of Multiple Buds：
The explant that the embryoid Multiple Buds that above-mentioned steps 3 obtain are cut into 0.5cm sizes is transferred in differentiation
28~30d statistics differentiation rates are cultivated on seedling-growing rapid breeding culture medium, under 2000~2500lx 16h/d intensities of illumination；
5th, strengthening seedling and rooting culture：
The crowd shoots switching that above-mentioned steps 4 are obtained is on strengthening seedling and rooting culture medium, 2000~2500lx 16h/d light
The regeneration plant taken root is formed according to 26~30d of culture under intensity,.The rooting rate of regeneration plant is counted, passes through this method rooting rate
Reach 100%.(Fig. 5：1-2)
6th, Transplantation of Regenerated Plantlets and survive；
By the Transplantation of Regenerated Plantlets taken root into greenhouse small flower, basin local soil type point and weight percent are：Vermiculite：Peat
Soil：Garden mould=2：4：4 mixing；Small flower is put into shelter from the sun, watering 1~2 time daily of transplanting initial stage gradually subtracts after seedling survives
Lack irrigation times, survival rate is counted after 15~20d.Reach 100% by this method transplanting survival rate.(Fig. 6：1-2)
Reality is respectively adopted in each 600 explants of population (being each repeated 3 times) of 5 kinds of 8 populations in this experimental example
It applies and somatic embryo generation Fiber differentiation, embryoid multiplication and grown cultures is carried out in the combination of the culture medium in example 4~9, are grown thickly
Seedling-growing rapid breeding culture, strengthening seedling and rooting culture and the Transplantation of Regenerated Plantlets of bud survive.
By Comparison of experiment results, 4 culture medium of embodiment is combined as most preferred embodiment, and 6 culture medium of embodiment is combined as secondary
Good embodiment.
The experimental result of this experimental example is as follows：
Pass through microexamination (Fig. 1：1-6) it is clear that the generation of body embryo and embryoid growth and development process：Knot thread
Careless explant initial stage is initially formed callus, extends with incubation time, and some somatic differentiations are into embryo in callus
Cell, shows as that cytoplasm is dense, and the quick merisis of cell simultaneously forms some protrusions, has apparent boundary with surrounding cellular tissue
Limit, and then the formation of visible globular embryo, torpedo embryo or scultellum embryo etc. and develop into embryoid completely to entire callus and grow thickly
Bud (Fig. 2：1)；Embryoid Multiple Buds are transferred to culture (Fig. 2 on differentiation seedling-growing rapid breeding culture medium：2), it is possible to obtain
Substantial amounts of Multiple Buds (Fig. 3：1-4)；Embryoid and Multiple Buds can be obtained in the embodiment of above-mentioned 6 kinds of combinations, but frequency is
Difference, the best combination of embryoid formation rate effect are embodiments 4, followed by embodiment 5 (table 1)；
The different embodiments of table 1 compare the influence of 8 Zoysia japonica populations embryoid formation rates
Embodiment embryoid formation rate (%)
The different embodiments of table 2 compare the influence of 8 Zoysia japonica populations differentiation rates
Note：Korea lawn grass (Z1), Zoysia sinica (Zz), manilagrass (Zg), Zoysia macrostachya (Zd), mascarene grass
(Zx) Qingdao Korea lawn grass (Zq), orchid draw No. III Korea lawn grass (Z3), Zoysia matrella cv.Huanan (Zh)
The best combination of differentiation rate effect is embodiment 8 and embodiment 12, followed by embodiment 9 and embodiment 13
(table 2) is consistent with embryoid formation rate result, but examines discovery：Embodiment 12, embodiment 13 are although differentiation rate
As embodiment 8, embodiment 9, but crowd shoots some vitrification phenomenons, so the combination that differentiation rate effect is best
Or embodiment 8, followed by embodiment 9；
The embryoid Multiple Buds of 8 different germplasm genotype, which are transferred on differentiation seedling-growing rapid breeding culture medium, to be cultivated,
Can obtain substantial amounts of Multiple Buds, effect it is best be 8 (Fig. 4 of embodiment：1-8)；It can be illustrated by table 2 and Fig. 4：As long as have
Embryoid is formed, and differentiation regeneration is just easy to.So as to also illustrate that the induction of body embryo and being differentiated to form for embryoid are these
The key core of item inventive technique.
It therefore deduces that：By the way that suitable explant, explant different development stage is selected to select suitable culture medium
And ingredient, the links such as the different culture medium combination of adjustment, adjustment condition of culture, these factors is made to be used cooperatively in due course, are led to
The mutual synergistic effect between them is crossed, farthest plays its complementary effect, so as to efficiently solve Korea lawn grass germplasm
The problem of genotype limits, drastically increases its embryoid formation rate and differentiation rate, obtains complete regenerated plant
Effect.
The above is only the preferred embodiment of the present invention, it should be pointed out that：Come for those skilled in the art
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. limit high-frequency somatic embryo regeneration culture method without germplasm genotype, which is characterized in that comprise the following steps：
1）Selection and the growth of sterile sprout domestication culture；
2）Leaf bud former base somatic embryo inducement is cultivated：It chooses aseptic seedling eustipes part and starts the leaf bud former base sprouted as outer
Implant cuts leaf bud former base and is transferred to the induction that somatic embryo is carried out on somatic embryo inducement culture medium, 28 ± 2d of light culture, institute
State somatic embryo inducement culture medium, including modified MS medium, 2~4mg/L 2,4-D, 0.1~0.2mg/L NAA, 0.05~
0.1 mg/L 6-BA, 40~50g/L sucrose, 0.6~0.7% agar, pH value are 5.8 ~ 6.0；
3）Embryoid is proliferated and grown cultures；
4）The seedling-growing rapid breeding culture of Multiple Buds；
5）Strengthening seedling and rooting culture；
6）It Transplantation of Regenerated Plantlets and survives.
2. no germplasm genotype limitation high-frequency somatic embryo regeneration culture method according to claim 1, which is characterized in that described
Step 1）Selection and the growth of sterile sprout domestication incubation step are as follows：The half tender stem band 2~4 in growth period is taken to save stem section work of crawling
It for sterilization treatment material, is cleaned through flowing water, disinfection, sterile material is seeded on sterile sprout culture medium and carries out sterile bud
The growth domestication culture of sprouting of seedling, wherein, the sterilizing stem with bud of inoculation includes 2~3 sections；28 ± 2 DEG C of cultivation temperature daytime,
20 ± 2 DEG C of night.
3. no germplasm genotype limitation high-frequency somatic embryo regeneration culture method according to claim 1, which is characterized in that described
Step 3）Embryoid is proliferated and grown cultures step is as follows：By step 2）The leaf bud former base somatic embryo of culture is transferred to embryoid
In multiplication and growth medium, 6 ± 2 DEG C of low temperature artificial climate incubator 6~8d of light culture are first put into, then is transferred to intensity of illumination and is
26~28d, 28 ± 2 DEG C of cultivation temperature daytime are cultivated under 600~800lx low-light intensity, 20 ± 2 DEG C of night counts embryoid
Formation rate.
4. no germplasm genotype limitation high-frequency somatic embryo regeneration culture method according to claim 3, which is characterized in that described
Step 3）Embryoid is proliferated and growth medium is MS+0.5 ~ 1mg/L+0.05 ~ 0.1mg/L of NAA 6-BA+0.1 ~ 0.2
+ 0.7% agar of mg/L 2,4-D+30g/L sucrose.
5. no germplasm genotype limitation high-frequency somatic embryo regeneration culture method according to claim 1, which is characterized in that institute
State step 4）The seedling-growing rapid breeding incubation step of Multiple Buds is as follows：By step 3）It is big that obtained embryoid Multiple Buds cut into 0.5cm
Small explant switching cultivates 28~30d on differentiation seedling-growing rapid breeding culture medium under 2000~2500lx intensities of illumination
Count differentiation rate.
6. no germplasm genotype limitation high-frequency somatic embryo regeneration culture method according to claim 5, which is characterized in that institute
State step 4）The seedling-growing rapid breeding culture medium of Multiple Buds is MS+1 ~ 2mg/L 6-BA+0.02 ~ 0.05mg/ L NAA+30g/L sugarcanes
Sugared+0.7% agar.
7. no germplasm genotype limitation high-frequency somatic embryo regeneration culture method according to claim 1, which is characterized in that described
Step 5）Strengthening seedling and rooting incubation step is as follows：By step 4）Obtained Multiple Buds switching on strengthening seedling and rooting culture medium, 2000~
The rooting rate of 26~30d statistics regeneration plants is cultivated under 2500lx intensities of illumination.
8. no germplasm genotype limitation high-frequency somatic embryo regeneration culture method according to claim 1, which is characterized in that described
Strengthening seedling and rooting culture medium is+0.7% agar of 1/2 MS+ 20g/L white sugar.
9. no germplasm genotype limitation high-frequency somatic embryo regeneration culture method according to claim 1, which is characterized in that described
Step 6）Transplantation of Regenerated Plantlets and to survive step as follows：By step 5）The obtained Transplantation of Regenerated Plantlets taken root is to greenhouse small flower
In, basin local soil type point and weight percent are：Vermiculite：Peat soil：Garden mould=2：4：4 mixing；Small flower is put into shelter from the sun, is transplanted
Initial stage watering 1~2 time daily, irrigation times are gradually decreased after seedling survives, survival rate is counted after 15~20 d.
10. high-frequency somatic embryo regeneration culture method is limited without germplasm genotype according to claim 1 ~ 9, which is characterized in that
The kind of the germplasm genotype is gramineous grass, is preferably the Korea lawn grass of different population that is wild or being bred as.
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