CN100473276C - High frequency plant stem regeneration method by cell-culture of Xiangcao grass - Google Patents
High frequency plant stem regeneration method by cell-culture of Xiangcao grass Download PDFInfo
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Abstract
A technique for high-frequency regrowth of grassiness by somatocyte culture features that the granular embryonic calli are used for culture, the improved culture medium MS is used as basic one, the inducing culture medium contains additionally 2.4-D and KT, the secondary culture medium contains additionally 2,4-D and 6-BA, and the differential culture medium contains additionally CPPU, NAA or KT, and IAA.
Description
One, technical field
The present invention relates to the technical method that the napier grass Somatic Cell Culture obtains the high-frequency plant regeneration, belong to biological technical field, be exclusively used in napier grass Somatic Cell Culture, test-tube plantlet micropropagation and somatic mutants screening, and be applicable to the agrobacterium-mediated transformation genetic transformation.
Two, technical background
Napier grass (Pennisetum Purpureum Schumach) is a grass family Pennisetum herbaceos perennial, originate in Tropical Africa, be the high yield herbage that is suitable for the torrid zone and subtropical zone plantation, can be used for herbvore livestock and poultry cultivation, highway slope protection, medium-to-high grade paper pulp and artificial board production, the napier grass that studies show that in recent years still is a kind of desirable biomass energy crop, and cultivated area has the trend of rapid expansion both at home and abroad.But because napier grass is a short day crop to the illumination sensitivity, can only normally blooms in 28 ° of areass to the south of north latitude and finish growing process and survive the winter naturally, in the middle and lower reach of Yangtze River and the same latitude area mainly do annual utilization, can not flowering naturally solid.Flowering naturally or artificial induction napier grass bloom, all differ, be easy to shattering and germination rate is extremely low owing to seed maturity, on producing, can only adopt the vegetative method of kind of stem that seedling is provided, and lack the napier grass kind cold-resistant, that salt resistance is strong, influence its large tracts of land popularizing planting.
Xuan Piao etc. are explant material with the axillalry bud and the terminal bud of Pennisetum crossbreed hybrid giant napier, set up fast traditional font by callus induction and green seedling differentiation pathway to be, but the frequency of hybrid giant napier axillalry bud and terminal bud evoked callus only are 10% that green seedling differentiation rate is 5%.Do not see the report of relevant napier grass tissue culture high-frequency plant regeneration technique both at home and abroad up to now as yet.This research is explant material with the young fringe of napier grass kind N51, attempt to verify different hormone combinations to the callus induction that is in different development stage children fringe and the influence of plant regeneration ability, in the hope of setting up napier grass Somatic Cell Culture high-frequency plant regeneration technique system, lay the foundation for realizing napier grass batch production test-tube plantlet production and utilizing biotechnology to cultivate cold-resistant, salt tolerant new varieties.
Three, summary of the invention
Technical problem the objective of the invention is to set up napier grass children fringe and cultivates the technology that obtains graininess embryo callus and high-frequency plant regeneration; overcome jowar genus crop and in tissue culture, induce white, translucent, the sticking shape callus of generation; green seedling differentiation rate is low; callus should not be used for defectives such as breeding of scale test-tube plantlet and molecular biology breeding through forfeiture plant differentiation capability behind the successive transfer culture.
Technical scheme
Napier grass Somatic Cell Culture high-frequency plant regeneration technique is characterized in that,
(1) material
The napier grass children fringe of selecting for use handled for 3 all branched ear original hase idiophases through short-day 7～September is an explant material.
(2) medium
Minimal medium: MS macroelement, trace element, B
5Vitamin, sucrose 3%, the consumption of agar strip are 0.8%;
Callus inducing medium: additional 2,4 dichlorophenoxyacetic acid (is called for short 2,4-D) 4.0mg/L and kinetin (being called for short KT) 0.05mg/L or 0.1mg/L;
Callus subculture medium: add 2,4-D 3.0mg/L and 6-benzylaminopurine (being called for short 6-BA) 0.2mg/L
Callus differential medium: the additional phenyl ureas basic element of cell division (being called for short CPPU) 2.0mg/L and methyl (being called for short NAA) 0.01mg/L or KT 0.5mg/L and heteroauxin (being called for short IAA) 0.5mg/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 branched ear original hase idiophase 2～5cm, under aseptic condition, wrap in leaf sheath young fringe outside, strip out young fringe, be cut into section sheet that 2～3mm grows with the thorough wiping of 70% alcohol.Young fringe segment is inoculated on the callus inducing medium, behind 20～25d callus is changed over to the callus subculture medium.Change green seedling differential medium behind successive transfer culture 20～25d over to, green bud or graininess callus change fresh differential medium continuation differentiation culture again over to behind 20～25d.The seedling of 3 leaf sizes changes strong root medium over to, 15d grow up to root system development good complete plantlet.Culturing room's temperature is 26 ℃～28 ℃, and light intensity is 2000lux, illumination every day 16h.Winter and early spring test-tube seedling transplanting to the greenhouse, but other in season direct transplanting to the land for growing field crops.
Beneficial effect the present invention compared with prior art has following advantage and good effect:
The present invention adopts the method for dark place reason artificial induction ear differentiation, (the young fringe from cob 2～5cm) is a material to choose the young fringe of napier grass branched ear original hase idiophase, pass through hormone regulating and controlling, under isolated culture condition, solved effectively napier grass in tissue culture procedures owing to induce water stainization of callus of generation or lose the difficult problem of plant differentiation capability because of forming a large amount of hairy root.The inductivity of graininess embryo callus of the present invention between 70%～80%, green seedling differentiation frequency is between 44～51%.
White or light yellow, dry, the graininess callus that use the inventive method to cultivate are coerced the ability that can remain white or light yellow, nutty structure and high-frequency plant regeneration in the screening process at salt tolerant (NaCl6～18 ‰) somatic mutants; Callus can keep nutty structure substantially in agriculture bacillus mediated genetic transformation process, provides important basic condition for carrying out the breeding of napier grass Celluar and Molecular Biology.
Four, description of drawings
Fig. 1 children fringe is induced the callus of generation
The regeneration plant that Fig. 2 children fringe cultured in vitro obtains
The plant that the screening of Fig. 3 salt-tolerant mutant obtains
Fig. 4 agrobacterium-mediated transformation changes resistant gene of salt (BADH) plant
Five, embodiment
1 materials and methods
1.1 material
Napier grass kind N51, (public, 1985 from U.S.'s introduction) planted the stem vegetative propagation and preserved, and the land for growing field crops is expanded numerous.Cover 15 hours (daylight 9 hours) of dark place reason with black plastic film, after 3 weeks were managed in the dark place, the young fringe of getting napier grass carried out cultured in vitro.
1.2 medium
Minimal medium is by MS mineral salt and B
5Organic principle is formed, and adds the hormone combinations of different proportionings, additional 3% sucrose and 0.8% agar, and the pH value is 5.8.
Interpolation is provided with different hormone combinations (seeing Table 1) to callus inducing medium with the callus subculture medium.The hormone combinations that green seedling differential medium adds is: SG
1, additional CPPU 2.0mg/L, NAA 0.01mg/L; SG
2, additional KT 0.5mg/L, IAA 0.5mg/L.Strong root medium is the Rr:1/2MS medium, adds NAA 0.5mg/L.
The hormone combinations of table 1 callus induction and subculture medium
1.3 cultural method
The clip plant first half removes blade, wraps in the leaf sheath and the stem of young fringe outside under aseptic condition with 70% alcohol wipe, strips out young fringe.Children's spike length degree is directly inoculated less than 2cm; Children's spike length degree 2～5cm is cut into the long fringe section of 2～3mm; Children's spike length degree surpasses 5cm, then gets young fringe base portion 5cm with interior and two parts in addition, is cut into the fringe section that 2～3mm grows equally.
Children's fringe is inoculated on the callus inducing medium, behind 20～25d callus is changed over to the callus subculture medium.Change green seedling differential medium behind callus subculture 20～25d over to, green bud or graininess callus change fresh differential medium continuation differentiation culture again over to behind 20～25d.The seedling of 3 leaf sizes changes strong root medium over to, growth 15d, and test-tube plantlet moves into simple plastic greenhouse after seedling is practiced in the 1d greenhouse.
Culturing room's temperature is 26 ℃～28 ℃, and light intensity is 2000lux, illumination every day 16h.
2 results and analysis
2.1 inducing of callus
The length of napier grass children fringe has tangible influence (table 2) to the time that goes out to heal, the state and the induction frequency of callus.When young spike length degree is 2～5cm, behind inoculation 2～3d, there is a spot of young fringe deformation to occur, 6～8
Young fringe size of table 2 and hormone combinations are to the influence of callus induction rate
* inoculate back 15d median average
* inoculation back 30d median average
Obvious deformation appears in the most of young fringe segment of d, 10～15d produces white or light yellow graininess callus (Fig. 1) successively, a small amount of callus differentiates hairy root behind the 21d, callus is puffy or brownization, and the mean value of inducing culture 30d graininess callus is 44.9%.When the young fringe of get is in initial differential period, be that young spike length degree is during less than 2cm, behind the inoculation 10d, a small amount of young fringe begins to take place deformation, the most of young fringe deformation of 14～21d also produces white or faint yellow graininess callus, but most of callus water stain shape that is creamy white behind 25～30d, it is 16.1% that inducing culture 30d keeps the mean value of the callus of nutty structure.Length is the young fringe of 5～10cm, small ear within its fringe base portion 5cm is inoculated in inducing culture, the most of young fringe generation fringe deformation of 7～9d, 10～15d produces part graininess callus and root simultaneously, a large amount of graininess callus become puffy or brownization behind the 21d, and the mean value of graininess callus is 16% behind the 30d.Small ear more than cob 5cm, fringe deformation also takes place in most of young fringe about 7d, and 10～15d produces part graininess callus simultaneously and differentiates root, and the mean value of 30d graininess callus is 8.8%.This result shows that napier grass children fringe cultured in vitro is the most suitable with length 2～5cm.
3 kinds of hormone combinations that are provided with in this test go out to young fringe that more time and callus state do not have obvious influence.Behind children's fringe inoculation 15d, callus all presents white or light yellow graininess, and the graininess callus induction rate is all higher.Different hormone combinations shows the influence of callus state, behind the young fringe inoculation 30d, and the occurrence frequency difference of graininess callus, this influence is also relevant with the length of young fringe.Children's spike length degree is during less than 5cm, and 2, when 4-D concentration was 4.0mg/L, behind the inoculation 30d, the inductivity of graininess callus totally presented the trend that the rising with KT concentration improves; Children's spike length degree is 2～5cm, and when KT concentration was 0.05mg/L and 0.1mg/L, the induction frequency indifference of graininess callus all was the frequency of graininess callus in the inducing culture of 0.02mg/L apparently higher than KT concentration.During children's spike length degree 5～10cm, no matter fringe base portion or top, when KT concentration was 0.05mg/L, the inductivity of graininess callus was the highest.When KT concentration was 0.02mg/L in the inducing culture, callus easily produced cilium or hairy root, and this may be on the low side relevant with KT concentration.
2.2 the successive transfer culture of callus
Callus is transferred to subculture medium cultivate 25～30d in inducing culture after, and 3～5d in all subculture mediums is all dead for water stain shape callus; It is rapid that the puffy callus is transferred to the successive transfer culture basal growth, but behind 7～10d, part puffy callus produces a large amount of hair shape structure and adventive root, and most of puffy callus 15d runs down later on, loses the ability of green seedling differentiation.Select from inducing culture that color and luster is vivid, the graininess callus carries out successive transfer culture, when the hormone combinations of adding is 4.0mg/L2,4-D, 0.05mg/L KT promptly adopt NI
2When inducing culture carried out successive transfer culture, from whole brownization behind white in the different inducing cultures or the light yellow graininess callus subculture 10d, this may be owing to 4.0mg/L 2, the reason that 4-D concentration is too high.2,4-D concentration is reduced to 3.0mg/L or 2.0mg/L, the graininess callus is grown on subculture medium rapidly, behind the 10d, the part callus begins to become puffy, and the part callus begins to differentiate green bud point or hairy root, is 3.0mg/L2 adding hormone combinations behind the 15d, on the medium of 4-D, 0.2mg/L 6-BA, a small amount of green bud point seedling differentiation.The statistics of subculture 21d shows, from inducing culture NI
1, NI
3The graininess callus at 3.0mg/L 2,4-D, 0.2mg/L6-BA and 2.0mg/L 2, propagation is fast in 4-D, the 0.2mg/L 6-BA medium, the frequency of graininess callus is respectively 74.0% and 69.5%, apparently higher than hormone combinations is 3.0mg/L 2, the frequency of graininess callus on the subculture medium of 4-D, 0.05mg/L KT; From inducing culture NI
2The graininess callus, in 3 kinds of subculture mediums of this test, the frequency of graininess callus is lower, is 31.1%～43.6%,
Not isogeneous induction of table 3, subculture medium are to the influence of graininess callus
But green bud point differentiation rate is obviously the highest.Hormone combinations is 3.0mg/L 2, and in the medium of 4-D, 0.2mg/L 6-BA, green bud point differentiation rate is up to 35.4%, at 3.0mg/L 2,4-D, 0.05mg/L KT and 2.0mg/L2, in the subculture medium of 4-D, 0.2mg/L6-BA, green bud point of part even seedling differentiation.This result shows, callus keeps minute eliminating of the nutty structure of callus and green bud point to be subjected to outside the influence that the subculture medium hormone forms in the successive transfer culture process, and is also relevant with the hormone in medium component of the initial evoked callus of young fringe.
2.3 differentiation of calli
The graininess callus is selected bright in colour, white or light yellow graininess callus and is changed differential medium respectively over to the callus of green bud point behind 20～25d successive transfer culture, with the callus of green bud point at SG
1And SG
2Planting percent all reaches 100% on the differential medium.From the performance of graininess callus on different differential mediums in the different subculture mediums differ (table 4).At subculture medium is 3.0mg/L2,4-D, 0.2mg/L 6-BA (NC
2) on the graininess callus, change 2.0mg/L CPPU, 0.01mg/LNAA (SG over to
1) differential medium, the 21d planting percent reaches 36.4%; Change 0.5mg/L KT, 0.5mg/L IAA over to
The influence that the different subcultures of table 4, differential medium break up the graininess callus
Annotate: the mean of cultivating 21d
(SG
2) differential medium, 21d graininess callus planting percent only is 7.7%, but green bud differentiation rate is 30.8% and since the green bud in differential medium change identical differential medium over to after planting percent reach 100%, therefore from subculture medium NC
2The graininess callus change SG over to
2Final planting percent reach 38.5%, show SG
2To from differential medium NC
2The graininess callus also be the differential medium that suits.Subculture medium is NC
1And NC
3, after the graininess callus changed differential medium over to, the total differentiation rate of green bud and green Miao was all with SG
1Be higher than SG
2From subculture medium NC
3The graininess callus change differential medium SG over to
2, 21d, most of callus continue to keep nutty structure, and part graininess callus differentiates hairy root, and the part callus browning does not have the differentiation of green bud and seedling, and this may be because when successive transfer culture 2,4-D concentration is too low, influences the differentiation of calli ability.Graininess callus in the differential medium changes differential medium once more over to, and all worsening behind the 10d can not Cheng Miao.The seedling growing way on the differential medium a little less than, move into early spring and live after the simple plastic greenhouse slowly, and be difficult for tiller; There is the seedling of 3 leaves to change strong root medium over to, growth 15d, the well developed root system seedling is strengthened (Fig. 2).Test-tube plantlet is practiced seedling through the 1d greenhouse, transplant to simple plastic greenhouse, and alive fast, and transplanting survival rate reaches more than 95%, grows new tillering about the seedling 15d of robust growth.
Three. discuss
In gramineous plants tissue culture and genetic transformation research, Chinese scholars generally believes that young fringe, immature embryo and mature embryo are the most suitable explant materials, and the induction frequency of embryo callus and plant regeneration frequency are significantly higher than the explant material of other type.Xuan Piao etc. are explant material with the axillalry bud and the terminal bud of hybrid giant napier, set up fast traditional font by callus induction and green seedling differentiation pathway to be, but the frequency of hybrid giant napier axillalry bud and terminal bud evoked callus only are 10% that green seedling differentiation rate is 5%.The king is explant material with green grass or young crops etc. with stipes (band lateral bud), stem section, lobus cardiacus and the tender leaf of hybrid Chinese pennisetum (American pennisetum alopecuroides and napier grass crossbreed), by the culture medium prescription screening, sets up plant regeneration system.The present invention is an explant material with the young fringe of napier grass, set up the plant regeneration technique system of upper frequency, and launched somatic mutants screening and agriculture bacillus mediated genetic transformation work on this basis, obtained at present salt tolerant coerce the plant of screening and commentaries on classics resistant gene of salt BADH (Fig. 3, Fig. 4).
Result of the test of the present invention shows that the different development stage of napier grass children fringe has the greatest impact to the inductivity of graininess callus.The inductivity of the young fringe graininess callus that 2～5cm is long is significantly higher than＜2cm or the long young fringe of 5cm.2,4-D concentration is 4.0mg/L, and KT concentration is 0.05mg/L or 0.1mg/L, and the inductivity of graininess callus is higher.In subculture medium 2, when 4-D concentration was identical with inducing culture, callus worsened rapidly behind the subculture, reduced in the subculture medium 2,4-D concentration, help the graininess callus maintenance and propagation.The height of shared ratio of graininess callus and green bud differentiation rate is not only relevant with the hormone combinations in the subculture medium during successive transfer culture, also is subjected to the influence of the hormone combinations in the initial inducing culture.Adopt identical subculture medium NC2, hormone combinations is 2 in inducing culture, and when 4-D 4.0mg/L and KT0.05mg/L (NI2), the conservation rate of graininess callus has only 40.9% during successive transfer culture, but green bud differentiation rate height is 35.4%; And hormone combinations is 2 in the inducing culture, and when 4-D 4.0mg/L and KT0.1mg/L (NI3), the conservation rate of graininess callus is up to 74.0% during successive transfer culture, and green bud differentiation rate has only 15.5%.Identical differential medium is respectively 50.2% and 43.99% from the last planting percent of inducing culture NI2 and NI3.Although a little less than NI2, graininess callus conservation rate height helps carrying out biotechnology breeding research during owing to successive transfer culture from the last planting percent of the graininess callus of inducing culture NI3; The early stage differentiation rate height of the callus that inducing culture NI2 induces, favourable to micropropagation.In the successive transfer culture process, select the maintenance that appearance is dry, tight, the graininess callus helps the propagation and the plant regeneration ability of callus.
Claims (1)
1, the renovation process of napier grass Somatic Cell Culture plant is characterized in that,
1) material
The young fringe of selecting napier grass for use is an explant material;
2) medium
Minimal medium: MS macroelement, trace element, B
5Vitamin, sucrose mass ratio 3%, agar strip mass ratio 0.8%;
Callus inducing medium: additional 2,4 dichlorophenoxyacetic acid 4.0mg/L and kinetin 0.05mg/L or 0.1mg/L;
Callus subculture medium: additional 2,4 dichlorophenoxyacetic acid 3.0mg/L and 6-benzyladenine 0.2mg/L;
Callus differential medium: additional CPPU 2.0mg/L and methyl 0.01mg/L; Or additional kinetin 0.5mg/L and heteroauxin 0.5mg/L;
Seedling is strengthened the root medium: 1/2 MS medium, additional methyl 0.5mg/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 napier grass 2～5cm, under aseptic condition, wrap in the leaf sheath and the stem of young fringe outside with the thorough wiping of 70% alcohol, strip out young fringe, be cut into the long fragment of 2～3mm, the segment of young fringe is seeded on the callus inducing medium, culturing room's temperature is 26 ℃～28 ℃, illumination every day 16h, under scattered light, cultivate, obtain white or light yellow behind 20～25d, dry, the graininess callus, after changing callus subculture medium propagation 20～25d over to, change on the callus differential medium, green bud or graininess callus change fresh differential medium continuation cultivation again over to behind 20～25d; The seedling that has 3 leaves changes strong root medium over to, and 15d grows up to the healthy and strong test tube plantlet of well developed root system.
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| CN102388742B (en) * | 2011-08-25 | 2013-01-23 | 江苏沿海地区农业科学研究所 | Method for rapidly planting Napier grass cutting seedlings in coastal mud flat saline-alkali lands |
| CN114931095B (en) * | 2022-05-25 | 2023-02-07 | 四川农业大学 | Method for regenerating plant by tissue culture of mature embryo of pennisetum americanum |
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| 杂交狼尾草不同外植体材料组织培养实验. 王凭青等.重庆大学学报（自然科学版），第28卷第6期. 2005 * |
| 矮象草的试管快繁技术研究. 江洪如等.中国重要会议论文全文数据库. 2003 * |
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