CN100475027C - Reproducing method for cultivating high-frequency plant with somatic cell culture of Sudan grass - Google Patents
Reproducing method for cultivating high-frequency plant with somatic cell culture of Sudan grass Download PDFInfo
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- CN100475027C CN100475027C CNB2005101227884A CN200510122788A CN100475027C CN 100475027 C CN100475027 C CN 100475027C CN B2005101227884 A CNB2005101227884 A CN B2005101227884A CN 200510122788 A CN200510122788 A CN 200510122788A CN 100475027 C CN100475027 C CN 100475027C
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Abstract
The present invention relates to a Sudan grass somatic cell culture high-frequency plant regeneration technique, belonging to the field of biological technology, providing practical tissue culture technique for Sudan grass cell engineering and gene engineering. The granular embryonic callus is applicable for developing agrobacterium mediated gene conversion work. The young ear of Sudan grass is explant material, the callus induction culture medium and successive transfer culture medium are basic culture medium + 2.4 - D 3mg/L + KT 0.2 mg/L; and the green seedling differentiation culture medium is basic culture medium + l- BA 2mg/L +NAA 0.01 mg/L. The induction frequency of granular callus is 80%-90%, the callus green seedling differentiation frequency is up to above 60% and the transplanted survival rate of regeneration plant is about 70%-80%.
Description
One, technical field
The present invention relates to the technical method that the Sudan grass Somatic Cell Culture obtains the high-frequency plant regeneration, belong to biological technical field, be exclusively used in Sudan grass cell culture and test-tube plantlet micropropagation, and be applicable to the agrobacterium-mediated transformation genetic transformation.
Two, technical background
Sudan grass (Sorghum sudanese) is a warm season annual gramineae herbage.Sudan grass is easy to plantation, seedling growth is fast, good palatability, wide adaptability, plantation is all arranged all over the world, and China reaches Hainan in the south, and north all can be cultivated to the Inner Mongol, be area, middle and lower reach of Yangtze River herbage in topmost summer, account for more than 70% of this zone warm season pasture growing area.Existing domestic and international Sudan grass kind major defect takes place serious for the growing period leaf spot.Adopt routine techniques seed selection disease-resistanting leaf spot kind not only the cycle long, workload is big, and because the nearly edge antigen-like material of shortage, breeding for disease resistance is made slow progress.Simultaneously, development along with industry restructuring and phytophagy livestock and poultry breeding industry, people and animals strive ground to rural areas of south China in recent years, the contradiction of striving grain is especially outstanding, and the existing a large amount of beach resource of southern coastal provinces and cities, there is the research report to obtain some salt tolerant forage crop and obtained resistant gene of salt, but the kind that really obtains the large tracts of land promotion and application is few, its main cause may be owing to only pay attention to the quality and yield that forage crop is ignored in the screening of salt resistance resource, shortage high-quality, high yield, good palatability, the warm season gramineous forage grass that salt resistance is strong.Disease resistance and the salt resistance of utilizing biotechnology to improve the Sudan grass kind have important practical significance.And can be successfully biotechnology applications in the Sudan grass breeding, at first must set up the technical system of Sudan grass tissue culture high-frequency plant regeneration.
There are some researches show that Sudan grass is the sorghum crop, compare that it is more difficult relatively to obtain regeneration plant by tissue culture with other plant.Up to now, utilize both at home and abroad Sudan grass children fringe cultivate the report that obtains regeneration plant only have 1 example (Su Xuehui etc. the young fringe of three kinds of gramineous forage grasss is cultivated and [J] takes place somatic embryo, Sichuan Teachers College's journal (natural science edition), 1993,14 (4): 346-348).The somatic embryo that the document lays particular emphasis on Annual Ryegrass, Sudan grass and do not have an awns barnyard grass is studied, only reported the influence of hormone simply to Sudan grass children's fringe callus induction rate and bud differentiation rate (30%～50%), do not report different hormones to the influence of callus state and the bud differentiation rate and the planting percent of different conditions callus, especially do not relate to the inducing of graininess callus, differentiation and plant regeneration.And the graininess callus is to obtain the high-frequency regeneration plant, and the prerequisite of the agrobacterium-mediated transformation genetic transformation that application operating is easy, transformation efficiency is higher, genetic stability is good.
One, summary of the invention
Technical problem the object of the present invention is to provide Sudan grass children fringe to cultivate the technology that obtains graininess embryo callus and high-frequency plant regeneration, overcome that jowar belongs to the white of inducing generation in the crop cultivation, translucent, the sticking green seedling differentiation rate of shape callus is low, should not be used for the defective of agrobacterium-mediated transformation genetic transformation.
Technical scheme
Sudan grass somatic culture high-frequency plant reproducing technology is characterized in that,
(1) material
Selecting for use field Sudan grass in 7～September grain husk to spend the young fringe of idiophase is explant material.
(2) callus induction and differential medium
Minimal medium: MS macroelement, trace element, B
5Vitamin, activated carbon 0.05%, sucrose 3%, the consumption of agar strip are 0.8%;
Callus inducing medium and subculture medium: additional 2,4 dichlorophenoxyacetic acid (is called for short 2,4-D) 3mg/L and kinetin (being called for short KT) 0.2mg/L;
Green seedling differential medium: additional 6-benzylaminopurine (being called for short 6-BA) 2mg/L and methyl (being called for short NAA) 0.01mg/L;
Medium is adjusted pH value to 5.8 with sodium hydroxide and hydrochloric acid before autoclaving.
(3) cultural method
Get the Sudan grass grain husk and spend the long young fringe of idiophase 1～5cm, under aseptic condition, wrap in leaf sheath outside the young fringe with the thorough wiping of 70% alcohol, and strip out young fringe, be cut into 2～3mm plurality of sections, put into the thimerosal 20min that is made into 1: 1 volume ratio with sterile water and hydrogen peroxide (original liquid concentration is 30%), with aseptic water washing 3 times, young fringe segment is inoculated on the inducing culture.Culturing room's temperature is 26 ℃～28 ℃, cultivates illumination every day 16h under scattered light, callus changes the subculture medium shoot proliferation over to behind 20～25d, change differential medium behind the 20d over to, about 25d, obtaining highly is the complete test-tube plantlet of 5～10cm, root system development.
Beneficial effect the present invention compared with prior art has following advantage and good effect:
The present invention is a material with the young fringe of Sudan grass, under isolated culture condition, by adding 0.05% activated carbon, solved Sudan grass a large amount of pigments in tissue culture procedures effectively and separated out and cause the callus induction failure, or the difficult problem that worsens rapidly of callus.Callus induction rate of the present invention between 80%～90%, green seedling differentiation frequency reaches more than 60%, for Sudan grass cell engineering and agrobacterium-mediated transformation genetic transformation engineering provide basic assurance.
Spend the young fringe of idiophase (from cob 1.5～5cm) by choosing grain husk, callus inducing medium and subculture medium all adopt minimal medium+3.0mg/L 2,4-D+0.2mg/L KT, the induction frequency of Sudan grass graininess embryo callus is 80%～90%, green seedling differential medium adopts minimal medium+2.0mg/L 6-BA+0.01mg/LNAA, and the green seedling differentiation rate of graininess callus reaches more than 60%.The graininess callus still keeps higher green seedling differentiation frequency through the successive transfer culture in continuous 4-5 generation, and translucent, sticking shape callus is through then completely losing the ability of plant regeneration behind the successive transfer culture.
White or light yellow, dry, the graininess callus that use the inventive method to cultivate can remain white (or light yellow), nutty structure and plant regeneration ability in the incubation altogether at the thick toxin of pathogen; At agroinfection with altogether can keep nutty structure substantially in the incubation, for carrying out the Sudan grass cell engineering and agrobacterium-mediated transformation genetic transformation work provides important basic condition.
Two, description of drawings
Fig. 1 children fringe is induced the generation callus
Fig. 2 appearance is drying, tight, graininess callus
The differentiation of Fig. 3 callus is sprouted and leaf
The regeneration plant of Fig. 4 Sudan grass children fringe cultured in vitro
Five, embodiment
1. materials and methods
1.1 material
Sudan grass strain 2098,1996 years is introduced from Japan.
1.2 condition of culture
Minimal medium: MS macroelement, trace element, B
5(minimal medium is promptly by MS macroelement, trace element (Murashige T for vitamin, Skoog F.A revised medium from rapid growth andbioassays with tobacco tissue cultures[J] .Physiol plant, 1962,15:473～497) and B
5Vitamin (Gamborg O L, Miller R A, Ojima K.Nutrient requirements of suspensioncultures of soybeanroot cells[J] .Exp.Cell Res., 1968.50:151～158) cooperation forms, and is a kind of medium commonly used of Plant Tissue Breeding.)
With MS is minimal medium, and additional 0.05% activated carbon, 3% sucrose and 0.8% agar, and specific design is as follows:
Inducing culture SI
1: minimal medium+2,4-D 2mg/L+KT 0.2mg/L+NaCl 1%;
Inducing culture SI
2: minimal medium+2,4-D 3mg/L+KT 0.2mg/L;
Inducing culture SI
3: minimal medium+2,4-D 4mg/L+KT 0.2mg/L;
Subculture medium SI
4: minimal medium+2,4-D 3mg/L+KT 0.2mg/L;
Subculture medium SI
5: minimal medium+2,4-D 4mg/L+KT 0.2mg/L;
Differential medium SG
1: minimal medium+6-BA 2mg/L+NAA 0.01mg/L;
Differential medium SG
2: minimal medium+CPPU 2mg/L+NAA 0.01mg/L.
1.3 cultured in vitro method
Get the Sudan grass tassel in 2098 booting stage, under aseptic condition, wrap in leaf sheath outside the young fringe, and strip out young fringe with the thorough wiping of 70% alcohol.Children's spike length degree is cut into 2～3mm plurality of sections less than 5cm; If the super 5cm of young spike length degree that strips out, then get in the bottom 5cm of young fringe, be cut into 2～3mm plurality of sections equally, then segment put into the thimerosal 20min that is made into 1: 1 volume ratio with sterile water and hydrogen peroxide, with aseptic water washing 3 times, young fringe segment is inoculated on the inducing culture, callus changes the subculture medium shoot proliferation over to behind 20～25d, changes differential medium behind the 20d over to, about 25d after, after seedling is practiced in the 2d greenhouse, move into and be equipped with in the Turnover Box of field soil.Culturing room's temperature is 26 ℃～28 ℃, and light intensity is 2000Lux, illumination every day 16h.
2 results
2.1 inducing and breed of callus
2.1.1 the young fringe puberty is to the influence of evoked callus
The Sudan grass children has bigger influence to inducing of callus a fringe puberty, and when the young fringe of get is in initial differential period, promptly young spike length degree is inoculated in inducing culture during less than 1.5cm, and fringe shape no change is not induced the generation callus.When children's spike length degree is 1.5cm～5cm, after being inoculated in inducing culture 2～3d, seeing has a small amount of young fringe to begin to occur deformation, obvious deformation (Fig. 1) appears in young fringe segment of part or Xiao Hua base portion about 7d, a large amount of young fringe generation deformation are arranged about 15d, and 10～17d segment otch and surface produce callus successively.Length is the young fringe of 5～15cm, and the small ear within its base portion 5cm is inoculated on the inducing culture, and a small amount of young fringe generation deformation is arranged behind 5～6d, and inoculation back 17～25d produces callus; Small ear more than cob joint 5cm, fringe deformation can take place in part, but has only only a few to produce callus.The young fringe of length more than 15cm, the segment within its base portion 5cm also has only the deformation of only a few generation fringe and produces callus.As seen Sudan grass children fringe is cultivated the young fringe of generally getting length 1.5～5cm, is cut into the segment of 2～3mm, and the effect of its callus induction is best.
2.1.2 hormone combinations is to the influence of young fringe callus growth conditions and evoked callus
According to morphologic observation, the callus on the inducing culture can be divided into 3 types: I callus quality is soft, and water stain shape is creamy white; The densification of II callus quality is white in color or faint yellow, graininess; III callus quality is fluffy, is faint yellow, green or brown, and sheet appears in part callus surface.
Different hormone combinations is induced the effect difference to young fringe callus.Containing 2, among the inducing culture SI1 of 4-D 2mg/L+KT0.2mg/L+NaCL1%, the callus that young fringe produces all is soft basically, and the water stain shape that is creamy white belongs to the I class, and young fringe callus of induce rate is 30%～50%; Adding 2, among the inducing culture SI2 of 4-D 3mg/L+KT0.2mg/L, the callus that young fringe segment produces is a graininess, and it is hard that quality is crisp, is white in color with faint yellow, belongs to II class (Fig. 2), and callus lures the rate of healing to reach 80%～90%; Adding 2, among the inducing culture SI3 of 4-D 4mg/L+KT 0.2mg/L, callus is fluffy state, and propagation is exceedingly fast, and belongs to the III class, and callus induction rate reaches 70%～80%.
2.1.3 the successive transfer culture of callus
Callus is transferred in the subculture medium cultivate 25～30d in inducing culture after, I class callus is additional 2, and 4-D 3mg/L+KT 0.2mg/L subculture medium SI4 and additional 2 is among the subculture medium SI5 of 4-D 4mg/L+KT0.2mg/L, poor growth, and the death that runs down.When the 1st successive transfer culture, select II class callus and change over to additionally 2, among the 4-D 3mg/L+KT 0.2mg/L subculture medium SI4, initial 10～15d propagation is very fast; Growth rate slows down behind the 20d; If do not shift subculture, callus begins to transfer yellow to by white behind the 25d, and stops growing gradually, and is therefore comparatively suitable with 20～25d successive transfer culture 1 time.II class callus is transferred to and adds 2, and among the subculture medium SI5 of 4-D 4mg/L+KT 0.2mg/L, callus propagation is rapid, but can become extremely fluffy III class by granular II class.III class callus changes additional 2 over to, behind the 4-D 3mg/L+KT 0.2mg/L subculture medium SI4, the part callus then can be converted into granular II class by the III class of puffy, the conversion ratio of the callus that different young fringes are induced is also different, 4 callus that young fringe is induced, the conversion ratio is respectively 12.5% (2/16), 33.33% (4/12), 50% (5/10) and 52.94% (9/17), and mean value is 36.36%.Adding 2, in the 4-D 4mg/L+KT 0.2mg/L subculture medium, callus can continue to keep fluffy state.
2.2 the differentiation of plant
Select the graininess callus behind the successive transfer culture, place the differential medium of different hormone combinations, callus begins increase behind about 5d, occurs green bud point then; About 15d, bud begins elongation, and grows complete blade (Fig. 3), and this differentiation synchronously of bud and foundation forms complete plantlet (Fig. 4).The embryo callus differentiation capability is stronger, and diameter is the every callus lines of 3～4mm, renewable plant 5～6 strains.
Result of study also shows, seedling greening-rate and spend the influence that the seedling rate not only is subjected to the differential medium hormone combinations in vain, but also be subjected to the influence of callus growth state.
Differentiation rate at each differential medium medium green seedling, II class callus is all than II class and the compound callus height of III class, this may be owing to have only eugonic graininess callus ability seedling differentiation, III class callus then can only be after being converted into the II class could seedling differentiation, the callus that while is transformed by the III class, the seedling rate of spending in vain in identical differential medium all are higher than the callus of II class originally.
When NAA concentration is 0.01mg/L in the differential medium, add CPPU 2mg/L and 6-BA 2mg/L and all can make graininess callus seedling differentiation, adopt continuously the green seedling differentiation rate of II class callus of SI4 subculture medium all be higher than via SI5 change the SI4 successive transfer culture over to and II class callus, Bai Miao leads then opposite (seeing Table 1).
The influence that table 1 different hormone combinations medium leads the seedling greening-rate and the Bai Miao of Sudan grass II class callus differentiation
To the II class callus of continuous employing SI4 subculture medium, than high by 22.53% in the differential medium of interpolation 6-BA, Bai Miao leads low 26.80% in the differential medium medium green seedling differentiation rate of adding CPPU; Than high by 11.08% in the differential medium that adds 6-BA, white seedling differentiation rate then hangs down 43.10% in the differential medium that adds CPPU for the II class callus that gets change the SI4 successive transfer culture over to via SI5, green seedling differentiation rate.
2.3 the transplanting of regeneration plant
Regeneration plant is grown in triangular flask about 20d, can transplant.At first, remove the cotton plug that seals, add running water, be advisable a little more than medium with the water surface, winter behind 25 ℃ intelligent greenhouse hardening 2d, with running water rinse well the regeneration plant root with medium, transplant to the Turnover Box that field soil is housed, survival rate can reach 70%～80%.
3. conclusion
(spike length is less than 15cm to get the Sudan grass grain husk flower branch florescence, get part from cob 1.5～5cm) young fringe, in callus inducing medium, add 3.0mg/L 2,4-D and 0.2mg/L KT, induction frequency reaches 80%～90%, still adds 3.0mg/L 2,4-D+0.2mg/L KT in the subculture medium, the callus growth rate is fast, and keeps the ability of plant regeneration; Add 2.0mg/L 6-BA+0.01mg/LNAA in the callus differential medium, the green seedling differentiation rate of callus through 3～5 generations of successive transfer culture remains on more than 60%, and transplanting survival rate reaches 70%～80%; Selecting in successive transfer culture that appearance is white in color, dry, tight, granular callus, is to improve the effective ways that its regeneration plant ability, minimizing are spent the seedling rate in vain.
Claims (1)
1, Sudan grass Somatic Cell Culture plant regeneration method is characterized in that,
1) material
The young fringe of selecting Sudan grass for use is an explant material;
2) callus induction and differential medium
With MS is minimal medium, and additional 0.05% activated carbon, 3% sucrose and 0.8% agar;
Callus inducing medium and subculture medium: additional 2,4 dichlorophenoxyacetic acid 3mg/L and kinetin 0.2mg/L;
Green seedling differential medium: additional 6-benzylaminopurine 2mg/L and NAA 0.01mg/L;
Medium is adjusted pH value to 5.8 with sodium hydroxide and hydrochloric acid before autoclaving;
3) cultural method
Get the long young fringe of Sudan grass 1～5cm, under aseptic condition, wrap in leaf sheath outside the young fringe with the thorough wiping of 70% alcohol, and strip out young fringe, be cut into 2～3mm section, put into sterile water and the original liquid concentration thimerosal 20min that to be 30% hydrogen peroxide be made into 1: 1 volume ratio, with aseptic water washing 3 times, young fringe segment is seeded on the callus inducing medium, culturing room's temperature is 26 ℃～28 ℃, and illumination every day 16h cultivates under scattered light, obtain white or light yellow behind 20～25d, dry, the graininess callus, change subculture medium shoot proliferation 20d over to, change on the green seedling differential medium, form the good whole plant of root system development behind the 25d.
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| CN103444519A (en) * | 2013-10-10 | 2013-12-18 | 林平 | Seed breeding method for sugar sorghum hybrid sudangrass breed |
| CN104126510B (en) * | 2014-07-28 | 2016-10-26 | 江苏省农业科学院 | A kind of method of the external evoked tracheid of arabian cron |
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| 物理因子对3种野生禾本科植物体外胚胎发生的影响. 林红等.广东农业科学，第6期. 1997 |
| 物理因子对3种野生禾本科植物体外胚胎发生的影响. 林红等.广东农业科学，第6期. 1997 * |
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