CN101180948A - Method for establishing high-efficiency somatic cell embryogenesis regeneration plant of plantain banana and Brazilian banana - Google Patents
Method for establishing high-efficiency somatic cell embryogenesis regeneration plant of plantain banana and Brazilian banana Download PDFInfo
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Abstract
The invention discloses a method for regeneration plant culture of efficient somatic embryogenesis of plantains and Brazil banana. The method is that the suspended embryo cells are pre-cultured for 7 to 10 days in an M2 liquid medium without 2, and 4-D before inducing, and then the cell mass with relative identical size and growth status are filtrated by a filter screen and inoculated to an embryos induction medium. Pre-culture of the embryos can enhance the embryo inducing rate and the later-received plant conversion rate by about 1 times; the embryo inducing effect of the cell masses with a size range of 154 Mum to 900 Mum after the filtration by filter is the best; the inoculation amount of the plantain and the Brazil banana respectively are 0.4ml 20% SCV and 1ml 20% SCV. In addition, the ABA and the ascorbic acid are added into the culture medium of somatic embryo of the prior art in the initial stage of somatic embryo inducing, prevents the easy browning phenomenon of culture material of plantain during the inducing period of somatic embryo to a certain extent, and improves the embryogenesis frequency of the embryonic suspended cells of the plantain and Brazil bananas effectively.
Description
Technical field
The invention belongs to banana biotechnology breeding field, the method for particularly a kind of effective raising plantain (MusaABB paradisiaca Linn.cv.Da Jiao) and Brazilian any of several broadleaf plants (Musa AAA Cavendish cv.Baxi) embryonal suspension cell (ECS) body embryo occurrence frequency and successful regeneration plant.
Background technology
Banana (comprising plantain) is one of most important in the world economic crops and crops, also is the main food of many tropical and subtropical zones countries and regions, for nearly 500,000,000 populations provide staple food, is the fourth-largest grain source after paddy rice, wheat, corn.The gross yield of China's banana occupies the three/four of the whole world, extensively plant in Guangdong, province such as Fujian, Hainan, Guangxi and Yunnan, the production and marketing number ranks the hat of southern fruit always, is praised to be called one of China's southern four big fruit, and huge economic and social benefit is arranged.But present banana disease wildness, be faced with the serious threat of multiple diseases such as comprising fungi, bacterium, virus and physiological, wherein the banana blight that is caused by Fusarium oxysporum can infect most banana cultivars, has become China and even the destructive disease of banana in the world.The most popular Brazilian any of several broadleaf plants (Musa AAACavendish cv.Baxi) of China is one of the kind of susceptible banana blight just, and another cultivated species plantain (Musa ABB paradisiaca Linn.cv.Da Jiao) has been proved the good characteristic with No. 4 biological strains of resisting banana vascular wilt, but the market income is not high because its flavor taste is relatively poor.Therefore, the banana novel species of how cultivating resisting banana vascular wilt and having a high economic benefit is the pressing issues that present banana industry is faced.Yet great majority cultivation any of several broadleaf plants is the triploid that can't produce seed, is difficult to utilize traditional crossbreeding mode that its germplasm is improved.And the biotechnology breeding modes such as mutagenesis in vitro, transgenosis, somatic hybridization that comprise are expected to solve the problems referred to above that banana faces.Set up stable banana cells,primordial suspension system (ECSs) and then be the precondition of banana biotechnology breeding through the plant regeneration system of body embryogenesis path efficiently.Banana ECSs can obtain by number of ways at present, comprises that immature zygotic embryos is cultivated, scalps cultivates and the prematurity inflorescence is cultivated.With the prematurity inflorescence is that explant is to obtain banana ECSs and obtain more effective and repeated the best way of regeneration plant through body embryogenesis path, uses this method and successfully obtained regeneration plant from the banana of a plurality of kinds.But also there is weak point in this cultivating system: the inflorescence explant collection is taken out the difference of the flower bud restriction in season, different plants inflorescence physiological status, not equal many factors of weather all influenced the stability of this system when sample was gathered, and caused its frequency of embryonic callus induction to be usually less than 4%; Embryo callus is converted to desirable ECS efficient and body embryo generating ability thereof, plant conversion ratio (not being higher than 20% usually) is all very low.In view of above technical bottleneck, do not have as yet at present and set up Brazilian any of several broadleaf plants ECS and plantain ECS and the relevant report by body embryo generation regeneration plant thereof.
Summary of the invention
In order to overcome the deficiency that above-mentioned prior art exists, the object of the present invention is to provide a kind of method of setting up plantain and Brazilian any of several broadleaf plants high-efficiency somatic cell tire (being called for short the body embryo) generation regeneration plant.This method is set up the cells,primordial suspension system of plantain (Musa ABB paradisiaca Linn.cv.Da Jiao) and Brazilian any of several broadleaf plants (Musa AAA Cavendishcv.Baxi) first, and the method for its somatic embryo occurrence frequency of a kind of effective raising success regeneration plant is provided, for the biotechnology breeding of plantain and Brazilian any of several broadleaf plants lays the foundation.
Purpose of the present invention realizes by following proposal: a kind of method of setting up plantain and Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant comprises the steps:
(1) inducing of embryo callus: be explant with plantain or Brazilian any of several broadleaf plants 1～12 comb prematurity male inflorescence respectively, be inoculated in and induce embryo callus on the callus inducing medium.
(2) foundation of the cells,primordial suspension system (ECS) of stable homogeneous: yellow fine granularity embryo callus of the plantain that picking step (1) induces or the light yellow fine granularity embryo callus of Brazilian any of several broadleaf plants, change the M2 liquid nutrient medium respectively over to, place suspension culture on 90～110rpm shaking table got final product in 3～4 months comparatively disperse, the plantain embryonal suspension cell or the Brazilian any of several broadleaf plants embryonal suspension cell of homogeneous.
(3) inducing of somatic embryo: elder generation changes above-mentioned embryonal suspension cell over to and does not contain 2 before the body embryonal induction, the pre-cultivation 7～10 days in the M2 liquid nutrient medium of 4-D, with after screen filtration filters out the consistent cell mass of size and growth conditions, therefrom drawing 0.2～2ml 20%SCV (settled cell volume leaves standstill the cell precipitation volume of gained after 10 minutes) is inoculated in and contains 1mgL
-1ABA and 5mgL
-1In the body embryonal induction medium of ascorbic acid, place 28 ± 1 ℃ of dark to carry out inducing of somatic embryo; The white globular embryo that induces changed over to continue in the body embryonal induction medium of removing ABA and ascorbic acid to cultivate, obtain ripe body embryo until body embryonic development maturation.
(4) sprouting of body embryo and plant regeneration thereof: the ripe body embryo that step (3) is induced changes in the body embryo germination medium; Place 28 ± 1 ℃ of dark culturing, treat that leaf sheath is extracted out after, it is gone to continues in the MR strong plantlets and rootage medium to cultivate, further develop into healthy and strong banana plantlet.
Describedly do not contain 2, the consisting of of the M2 liquid nutrient medium of 4-D: MS minimal medium+4.1 μ molL
-1Vitamin h+680 μ molL
-1Glutamine+100mgL
-1Malt extract+130mmolL
-1Sucrose, pH5.3, autoclave sterilization.
Consisting of of described body embryonal induction medium: SH (Schenk and Hildebrandt 1972) a large amount of and trace element and molysite+MS vitamin+4.5 μ molL thereof
-1Vitamin h+680 μ molL
-1Glutamine+2mmolL
-1Proline+100mgL
-1Malt extract+1.1 μ molL
-1NAA+0.5 μ molL
-1Kinetin+44 μ gL
-1Zeatin+29mmolL
-1Lactose+130mmolL
-1Sucrose+1mgL
-1ABA+5mgL
-1Ascorbic acid+2gL
-1Gelrite, wherein ABA and ascorbic acid are configured to 10mgml in advance respectively
-1Mother liquor adds pH5.8 to, temperature is reduced to mixing in all the other medium components about 45 ℃ behind autoclave sterilization, is sub-packed in the culture dish of diameter 9.5cm 20～25ml/ ware behind the filtration sterilization.
The described 20%SCV suspension cell of step (3) is to prepare as follows: draw about 5ml suspension cell in the 15ml graded tube, after leaving standstill 10 minutes, abandon supernatant, use the body embryonal induction medium that does not add coagulating agent Gelrite to be diluted to 5 times of volumes the cell precipitation volume of gained.
In order to realize the present invention better, be the cell mass of 154 μ m～900 μ m through screen filtration gained cell mass preferred size scope in the step (3).
Described plantain cell inoculation amount the best is 0.4ml 20%SCV; Brazil's any of several broadleaf plants cell inoculation amount the best is 1ml 20%SCV.
Compared with prior art, the present invention has following advantage and beneficial effect:
(1) adopt pre-incubated mode to reduce banana embryonal suspension cell external source 2 before the body embryonal induction, the content of 4-D can promote the body embryo occurrence frequency of suspension cell.Compare with the method for directly carrying out the body embryonal induction without pre-cultivation of bibliographical information, the former body embryonal induction rate and subsequent plant conversion ratio all improve about 1 times than the latter and (see Table 1).
(2) before the body embryonal induction, use the screen cloth in two kinds of different apertures that the banana embryonal suspension cell is carried out sieves and select size and the consistent cell mass of growth conditions, can realize the synchronization of body embryonal induction to a certain extent, thereby improve the body embryonal induction frequency of suspension cell very significantly.Experimental result shows that the body embryonal induction rate of the plantain cell mass of size between 154 μ m～900 μ m on average exceeds 7.2 times than the cell mass of all the other two kinds of different magnitude range, and the more remarkable effect that this obtains on Brazilian any of several broadleaf plants on average can exceed 20.5 times (seeing Table 2).
(3) adopt different cell inoculation amounts to carry out inducing of banana body embryo, the result shows that the inoculum concentration of plantain cell is that the inoculum concentration of 0.4ml 20%SCV, Brazilian any of several broadleaf plants cell can obtain optimum efficiency when being 1ml 20%SCV, on average can exceed 2.8 times (seeing Table 2) than all the other inoculum concentrations.Also obtained similar effects in the test of the banana variety of this laboratory beyond plantain and Brazilian any of several broadleaf plants, promptly the body embryo induce the cell inoculation amount that has a best, different banana varieties is slightly variant.
(4) add 1mgL at the body embryonal induction simultaneously on just aspire to body embryonal induction medium
-1ABA and 5mgL
-1Ascorbic acid, this has prevented the phenomenon of plantain easy brownization of culture during the body embryonal induction to a certain extent, the while can make the body embryonal induction rate of plantain and Brazilian any of several broadleaf plants embryonal suspension cell all improve more than 1 times and (see Table 3).
The pre-influence of cultivating of table 1 to embryonal suspension cell body embryonal induction and plant conversion
| Banana variety | Cell before the body embryonal induction is handled | Body embryonal induction rate (individual/ml SCV) | Plant conversion ratio (%) |
| Plantain Brazil any of several broadleaf plants | Cultivate through cultivating without pre-cultivation in advance through pre-the cultivation without pre- | 1600±67 950±44 18600±405 12500±276 | 20±3 10±2 35±4 17±3 |
Experiment repeats 3 times, and the gained data are mean+/-standard error.
Table 2 cell mass size and inoculum concentration are to the influence of body embryonal induction
| The cell inoculation amount of banana variety and inductor embryo (ml 20%SCV) | Body embryo occurrence frequency (individual/ml SCV) | ||
| Cell mass size＞900 μ m | 154 μ m＜cell mass size＜900 μ m | Cell mass size＜154 μ m | |
| Plantain 0.2 0.4 0.6 0.8 1.0 2.0 Brazilian any of several broadleaf plants 0.5 1.0 2.0 | 140±18 120±13 100±13 60±8 30±6 20±5 800±66 1500±76 500±32 | 1500±76 2000±87 1000±42 400±36 250±31 90±26 6000±76 25000±153 11000±126 | 240±26 320±31 200±36 80±26 100±15 50±12 400±36 700±53 200±25 |
Experiment repeats 3 times, and the gained data are mean+/-standard error.
Table 3 ABA and ascorbic acid are to the influence of banana body embryonal induction and brownization of culture degree
| Banana variety | Medium | Body embryonal induction rate (individual/ml SCV) | Brownization of culture degree |
| Plantain Brazil any of several broadleaf plants | Body embryonal induction medium (M) M+1mg.L of prior art -1ABA+5mg.L -1Body embryonal induction medium (M) M+1mg.L of ascorbic acid prior art -1ABA+5mg.L -1Ascorbic acid | 950±44 2500±79 12500±276 28500±361 | The no browning of serious brownization slight brownization of slight brownization part |
Experiment repeats 3 times, and the gained data are mean+/-standard error.
Description of drawings
Fig. 1 is the foundation of plantain cells,primordial suspension system and through the procedure chart of body embryogenesis path regeneration plant.
Fig. 2 is the foundation of Brazilian any of several broadleaf plants cells,primordial suspension system and through the procedure chart of body embryogenesis path regeneration plant.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Various medium required for the present invention is as follows:
Callus inducing medium: MS minimal medium (Murashige and Skoog, 1962)+4.5 μ molL
-1Vitamin h+18 μ molL
-12,4-D+5.4 μ molL
-1NAA+5.7 μ molL
-1IAA+87mmolL
-1Sucrose+7gL
-1Agar, pH 5.8.
M2 liquid nutrient medium: MS minimal medium+4.5 μ molL
-12,4-D+4.1 μ molL
-1Vitamin h+680 μ molL
-1Glutamine+100mgL
-1Malt extract+130mmolL
-1Sucrose, pH5.3.
Do not contain 2, the M2 liquid nutrient medium of 4-D: MS minimal medium+4.1 μ molL
-1Vitamin h+680 μ molL
-1Glutamine+100mgL
-1Malt extract+130mmolL
-1Sucrose, pH5.3.
Body embryonal induction medium: SH (Schenk and Hildebrandt 1972) a large amount of and trace element and molysite+MS vitamin+4.5 μ molL thereof
-1Vitamin h+680 μ molL
-1Glutamine+2mmolL
-1Proline+100mgL
-1Malt extract+1.1 μ molL
-1NAA+0.5 μ molL
-1Kinetin+44 μ gL
-1Zeatin+29mmolL
-1Lactose+130mmolL
-1Sucrose+1mg.L
-1ABA+5mg.L
-1Ascorbic acid+2gL
-1Gelrite, pH5.8.
Body embryo germination medium: MS macroelement and trace element and molysite thereof+Morel and Wetmore vitamin (Morel and Wetmore 1951)+0.2 μ molL
-16-BA+1.1 μ molL
-1IAA+87mmolL
-1Sucrose+2gL
-1Gelrite, pH 5.8.
MR strong plantlets and rootage medium: MS minimal medium+87mmolL
-1Sucrose+0.1% active carbon+7gL
-1Agar, pH5.8.
Embodiment 1
Set up the method for plantain high-efficiency somatic cell embryogenesis regeneration plant, comprise the steps:
(1) banana variety chooses.The kind of selecting for use is the plantain (Musa ABB paradisiaca Linn.cv.Da Jiao) of one of China's main breed, No. 4 biological strains of resisting banana vascular wilt.
(2) the plantain embryo callus induces.Top bud after getting the plantain result mid-August divests outside bract and male flower to from inflorescence top 4～6cm part, handles about 5min with 75% alcohol disinfecting, and sterile water washs 3 times.Under aseptic condition, further divest peripheral petal to 1～1.5cm, with the bud apical meristem is that benchmark cuts the immature male inflorescence of 1～12 comb and is inoculated on the callus inducing medium, cultivated 5～6 months down in 28 ± 1 ℃ of dark conditions, during upgrade 1～2 subculture according to circumstances; Induce the yellow fine granularity embryo callus of plantain.
(3) foundation of the plantain cells,primordial suspension system (ECS) of stable homogeneous.About yellow fine granularity embryo callus 2 grams that picking step (1) induces, change in the 100mL conical flask that contains 30ml M2 liquid nutrient medium, place 28 ± 1 ℃, dark culturing on 90～110rpm shaking table.Inhale weekly in first month of cultivating and to abandon about 2/3 old culture fluid, replenish with fresh M2 liquid nutrient medium, remove the bulky grain that is difficult for dispersion with 900 μ m aperture sieve net filtrations simultaneously, thereafter per 2 weeks are changed a subculture, behind the subculture one month, remove bigger cell mass with 154 μ m aperture sieve net filtrations, continue to cultivate get final product about January comparatively disperse, the plantain cells,primordial suspension system of homogeneous.
(4) the plantain somatic embryo induces.Before the body embryonal induction, earlier the plantain embryonal suspension cell is changed over to and do not contain 2, the pre-cultivation 7 days in the M2 liquid nutrient medium of 4-D, with after 154 μ m aperture sieve net filtrations, get the cell mass less than 154 μ m that filters gained, absorption 0.2ml 20%SCV (leaving standstill the cell precipitation volume of gained after 10 minutes) is inoculated in and contains 1mgL
-1ABA and 5mgL
-1In the body embryonal induction medium of ascorbic acid, place 28 ± 1 ℃ of dark to carry out inducing of body embryo.The white globular embryo that will induce after one month changes the body embryonal induction medium of removing ABA and ascorbic acid over to and continues to cultivate about February until body embryonic development maturation, obtains ripe body embryo; Body embryonal induction rate is about 380/ml SCV.
(5) sprouting of plantain body embryo and plant regeneration.The ripe body embryo that step (4) is induced changes in the germination medium.Sprout and initial culture placed 28 ± 1 ℃ of dark culturing, treat that leaf sheath is extracted out after, it is gone in the MR strong plantlets and rootage medium in 30 μ mol m
-2s
-1Light intensity (white fluorescent lamp) continues under the 16h/8h photoperiod condition to cultivate about January, further develops into healthy and strong plantain plantlet; The plant conversion ratio is about 14%.
Embodiment 2
Set up the method for plantain high-efficiency somatic cell embryogenesis regeneration plant, comprise the steps:
(1) banana variety chooses.The kind of selecting for use is the plantain (Musa ABB paradisiaca Linn.cv.Da Jiao) of No. 4 biological strains of resisting banana vascular wilt.
(2) the plantain embryo callus induces.Induce the yellow fine granularity embryo callus of plantain with embodiment 1 step (2).
(3) foundation of the plantain cells,primordial suspension system (ECS) of stable homogeneous.About embryo callus 2 grams that picking step (1) induces, change in the 100mL conical flask that contains 30ml M2 liquid nutrient medium, place 28 ± 1 ℃, dark culturing on 90～110rpm shaking table.Inhale weekly in first month of cultivating and to abandon about 2/3 old culture fluid, replenish with fresh M2 liquid nutrient medium, remove the bulky grain that is difficult for dispersion with 900 μ m aperture sieve net filtrations simultaneously, thereafter per 2 weeks are changed a subculture, behind the subculture one and a half months, remove bigger cell mass with 154 μ m aperture sieve net filtrations, continue to cultivate get final product about January comparatively disperse, the plantain cells,primordial suspension system of homogeneous.
(4) the plantain somatic embryo induces.Before the body embryonal induction, earlier the plantain embryonal suspension cell is changed over to and do not contain 2, the pre-cultivation 8 days in the M2 liquid nutrient medium of 4-D, subsequently successively through 900 μ m and 154 μ m aperture sieve net filtrations, get filter gained less than 900 μ m and greater than the cell mass of 154 μ m, draw 0.4ml20%SCV (leaving standstill the cell precipitation volume of gained after 10 minutes) and be inoculated in and contain 1mgL
-1ABA and 5mgL
-1In the body embryonal induction medium of ascorbic acid, place 28 ± 1 ℃ of dark to carry out inducing of body embryo.The white globular embryo that will induce after one month changes over to and continues in the body embryonal induction medium of removing ABA and ascorbic acid to cultivate about February until body embryonic development maturation, obtains ripe body embryo; Body embryonal induction rate is about 2760/ml SCV.
(5) sprouting of plantain body embryo and plant regeneration.With embodiment 1 step (5) the ripe body embryonic development of plantain is become healthy and strong plantain plantlet; The plant conversion ratio is about 22%.
Embodiment 3
Set up the method for plantain high-efficiency somatic cell embryogenesis regeneration plant, comprise the steps:
(1) banana variety chooses.The kind of selecting for use is the plantain (Musa ABB paradisiaca Linn.cv.Da Jiao) of No. 4 biological strains of resisting banana vascular wilt.
(2) the plantain embryo callus induces.Induce the yellow fine granularity embryo callus of plantain with embodiment 1 step (2).
(3) foundation of the plantain cells,primordial suspension system (ECS) of stable homogeneous.About embryo callus 2 grams that picking step (1) induces, change in the 100mL conical flask that contains 30ml M2 liquid nutrient medium, place 28 ± 1 ℃, dark culturing on 90～110rpm shaking table.Inhale weekly in first month of cultivating and to abandon about 2/3 old culture fluid, replenish with fresh M2 liquid nutrient medium, remove the bulky grain that is difficult for dispersion with 900 μ m aperture sieve net filtrations simultaneously, thereafter per 2 weeks are changed a subculture, subculture after two months, remove bigger cell mass with 154 μ m aperture sieve net filtrations, continue to cultivate get final product about January comparatively disperse, the plantain cells,primordial suspension system of homogeneous.
(4) the plantain somatic embryo induces.Before the body embryonal induction, earlier the plantain embryonal suspension cell is changed over to and do not contain 2, the pre-cultivation 10 days in the M2 liquid nutrient medium of 4-D, with after 900 μ m aperture sieve net filtrations, get the cell mass greater than 900 μ m that filters gained, absorption 2.0ml 20%SCV (leaving standstill the cell precipitation volume of gained after 10 minutes) is inoculated in and contains 1mgL
-1ABA and 5mgL
-1In the body embryonal induction medium of ascorbic acid, place 28 ± 1 ℃ of dark to carry out inducing of body embryo.The white globular embryo that will induce after one month changes over to and continues in the body embryonal induction medium of removing ABA and ascorbic acid to cultivate about February until body embryonic development maturation, obtains ripe body embryo; Body embryonal induction rate is about 46/ml SCV.
(5) sprouting of plantain body embryo and plant regeneration.With embodiment 1 step (5) the ripe body embryonic development of plantain is become healthy and strong plantain plantlet; The plant conversion ratio is about 11%.
Be illustrated in figure 1 as the foundation of plantain cells,primordial suspension system and through the process of body embryogenesis path regeneration plant, wherein: the plantain prematurity male inflorescence explant (bar=1.5mm) of A, evoked callus; B, the yellow particle shape embryo callus (bar=8mm) that induces by plantain prematurity male inflorescence; The initial suspension culture (bar=13mm) of C, plantain embryo callus; D, behind 900 μ m and 154 μ m aperture sieve net filtrations, cultivate the plantain cells,primordial suspension system (bar=11mm) of the homogeneous dispersion of gained more than 3 months successively; Initial the inducing (bar=15mm) of E, plantain embryonal suspension cell body embryo; F, the globular embryo (bar=6mm) that after cultivating 20 days on the body embryonal induction medium, obtains; G, upgrade the body embryo (bar=17mm) that continues to cultivate gained after January behind the body embryonal induction medium; H, the ripe body embryo (bar=6mm) of cultivation after three months; Body embryo (bar=8mm) in I, the sprouting; J, sprouting body embryo change the plantain plantlet (bar=12mm) that obtains after January in the MR medium over to.
Embodiment 4
Set up the method for Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant, comprise the steps:
(1) banana variety chooses.The kind of selecting for use is China extensive and the most popular Brazilian any of several broadleaf plants (Musa AAA Cavendish cv.Baxi) of cultivation at present.
(2) Brazilian any of several broadleaf plants embryo callus induces.Top bud after getting Brazilian any of several broadleaf plants result mid-August divests outside bract and male flower to from inflorescence top 4～6cm part, handles about 5min with 75% alcohol disinfecting, and sterile water washs 3 times.Under aseptic condition, further divest peripheral petal to 1～1.5cm, with the bud apical meristem is that benchmark cuts the immature male inflorescence of 1～12 comb and is inoculated on the callus inducing medium, cultivated 5～6 months down in 28 ± 1 ℃ of dark conditions, during upgrade 1～2 subculture according to circumstances.Induce the light yellow fine granularity embryo callus of Brazilian any of several broadleaf plants.
(3) foundation of the Brazilian any of several broadleaf plants cells,primordial suspension system (ECS) of stable homogeneous.About light yellow fine granularity embryo callus 2 grams that picking step (1) induces, change in the 100mL conical flask that contains 30ml M2 liquid nutrient medium, place 28 ± 1 ℃, dark culturing on 90～110rpm shaking table.Inhale weekly in first month of cultivating and to abandon about 2/3 old culture fluid, replenish with fresh M2 liquid nutrient medium, remove the bulky grain that is difficult for dispersion with 900 μ m aperture sieve net filtrations simultaneously, thereafter per 2 weeks are changed a subculture, behind the subculture one month, remove bigger cell mass with 154 μ m aperture sieve net filtrations, continue to cultivate get final product about January comparatively disperse, the Brazilian any of several broadleaf plants cells,primordial suspension system of homogeneous.
(4) Brazilian any of several broadleaf plants somatic embryo induces.Before the body embryonal induction, earlier Brazilian any of several broadleaf plants embryonal suspension cell is changed over to and do not contain 2, the pre-cultivation 7 days in the M2 medium of 4-D, with after 154 μ m aperture sieve net filtrations, get the cell mass less than 154 μ m that filters gained, absorption 0.5ml 20%SCV (leaving standstill the cell precipitation volume of gained after 10 minutes) is inoculated in and contains 1mgL
-1ABA and 5mgL
-1In the body embryonal induction medium of ascorbic acid, place 28 ± 1 ℃ of dark to carry out inducing of body embryo.The white globular embryo that will induce after one month changes over to and continues in the body embryonal induction medium of removing ABA and ascorbic acid to cultivate about February until body embryonic development maturation, obtains ripe body embryo; Body embryonal induction rate is about 720/ml SCV.
(5) sprouting and the plant regeneration of Brazilian any of several broadleaf plants body embryo.The ripe body embryo that step (4) is induced changes in the germination medium.Sprout and initial culture placed 28 ± 1 ℃ of dark culturing, treat that leaf sheath is extracted out after, it is gone in the MR strong plantlets and rootage medium in 30 μ mol m
-2s
-1Light intensity (white fluorescent lamp) continues under the 16h/8h photoperiod condition to cultivate about January, further develops into healthy and strong Brazilian any of several broadleaf plants plantlet; The plant conversion ratio is about 24%.
Embodiment 5
Set up the method for Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant, comprise the steps:
(1) banana variety chooses.The kind of selecting for use is Brazilian any of several broadleaf plants (Musa AAA Cavendish cv.Baxi).
(2) Brazilian any of several broadleaf plants embryo callus induces.Induce the light yellow fine granularity embryo callus of Brazilian any of several broadleaf plants with embodiment 4 steps (2).
(3) foundation of the Brazilian any of several broadleaf plants cells,primordial suspension system (ECS) of stable homogeneous.About embryo callus 2 grams that picking step (1) induces, change in the 100mL conical flask that contains 30ml M2 liquid nutrient medium, place 28 ± 1 ℃, dark culturing on 90～110rpm shaking table.Inhale weekly in first month of cultivating and to abandon about 2/3 old culture fluid, replenish with fresh M2 liquid nutrient medium, remove the bulky grain that is difficult for dispersion with 900 μ m aperture sieve net filtrations simultaneously, thereafter per 2 weeks are changed a subculture, behind the subculture one and a half months, remove bigger cell mass with 154 μ m aperture sieve net filtrations, continue to cultivate get final product about January comparatively disperse, the Brazilian any of several broadleaf plants cells,primordial suspension system of homogeneous.
(4) Brazilian any of several broadleaf plants somatic embryo induces.Before the body embryonal induction, earlier Brazilian any of several broadleaf plants embryonal suspension cell is changed over to and do not contain 2, the pre-cultivation 8 days in the M2 medium of 4-D, subsequently successively through 900 μ m and 154 μ m aperture sieve net filtrations, get filter gained less than 900 μ m and greater than the cell mass of 154 μ m, draw 1.0ml20%SCV (leaving standstill the cell precipitation volume of gained after 10 minutes) and be inoculated in and contain 1mgL
-1ABA and 5mgL
-1In the body embryonal induction medium of ascorbic acid, place 28 ± 1 ℃ of dark to carry out inducing of body embryo.The white globular embryo that will induce after one month changes over to and continues in the body embryonal induction medium of removing ABA and ascorbic acid to cultivate about February until body embryonic development maturation, obtains ripe body embryo; Body embryonal induction rate is about 28500/ml SCV.
(5) sprouting and the plant regeneration of Brazilian any of several broadleaf plants body embryo.With embodiment 4 steps (5) the ripe body embryonic development of Brazilian any of several broadleaf plants is become healthy and strong Brazilian any of several broadleaf plants plantlet; The plant conversion ratio is about 37%.
Embodiment 6
Set up the method for Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant, comprise the steps:
(1) banana variety chooses.The kind of selecting for use is Brazilian any of several broadleaf plants (Musa AAA Cavendish cv.Baxi).
(2) Brazilian any of several broadleaf plants embryo callus induces.Induce the light yellow fine granularity embryo callus of Brazilian any of several broadleaf plants with embodiment 4 steps (2).
(3) foundation of the Brazilian any of several broadleaf plants cells,primordial suspension system (ECS) of stable homogeneous.About embryo callus 2 grams that picking step (1) induces, change in the 100mL conical flask that contains 30ml M2 liquid nutrient medium, place 28 ± 1 ℃, dark culturing on 90～110rpm shaking table.Inhale weekly in first month of cultivating and to abandon about 2/3 old culture fluid, replenish with fresh M2 liquid nutrient medium, remove the bulky grain that is difficult for dispersion with 900 μ m aperture sieve net filtrations simultaneously, thereafter per 2 weeks are changed a subculture, subculture after two months, remove bigger cell mass with 154 μ m aperture sieve net filtrations, continue to cultivate get final product about January comparatively disperse, the Brazilian any of several broadleaf plants cells,primordial suspension system of homogeneous.
(4) Brazilian any of several broadleaf plants somatic embryo induces.Before the body embryonal induction, earlier Brazilian any of several broadleaf plants embryonal suspension cell is changed over to and do not contain 2, the pre-cultivation 10 days in the M2 medium of 4-D, with after 900 μ m aperture sieve net filtrations, get the cell mass greater than 900 μ m that filters gained, absorption 2.0ml 20%SCV (leaving standstill the cell precipitation volume of gained after 10 minutes) is inoculated in and contains 1mgL
-1ABA and 5mgL
-1In the body embryonal induction medium of ascorbic acid, place 28 ± 1 ℃ of dark to carry out inducing of body embryo.The white globular embryo that will induce after one month changes over to and continues in the body embryonal induction medium of removing ABA and ascorbic acid to cultivate about February until body embryonic development maturation, obtains ripe body embryo; Body embryonal induction rate is about 930/ml SCV.
(5) sprouting and the plant regeneration of Brazilian any of several broadleaf plants body embryo.With embodiment 4 steps (5) the ripe body embryonic development of Brazilian any of several broadleaf plants is become healthy and strong Brazilian any of several broadleaf plants plantlet; The plant conversion ratio is about 19%.
Be illustrated in figure 2 as the foundation of Brazilian any of several broadleaf plants cells,primordial suspension system and through the process of body embryogenesis path regeneration plant, wherein: the Brazilian any of several broadleaf plants prematurity male inflorescence explant (bar=1.4mm) of A, evoked callus; B, the light yellow graininess embryo callus (bar=4.8mm) that induces by Brazilian any of several broadleaf plants prematurity male inflorescence; The cells,primordial suspension system (bar=12mm) of the homogeneous dispersion of C, Brazilian any of several broadleaf plants embryo callus suspension culture gained more than 3 months; The body embryo (bar=15mm) of D, Brazilian any of several broadleaf plants embryonal suspension cell gained after cultivating January on the body embryonal induction medium; E, the ripe body embryo (bar=13mm) of cultivation after three months; Body embryo (bar=16mm) in F, the sprouting; G, after cultivating January on the MR medium the Brazilian any of several broadleaf plants plantlet (bar=10mm) of gained.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spiritual essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1. a method of setting up plantain and Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant is characterized in that comprising the steps:
(1) inducing of embryo callus: be explant with plantain or Brazilian any of several broadleaf plants 1～12 comb prematurity male inflorescence respectively, be inoculated in and induce embryo callus on the callus inducing medium;
(2) foundation of the cells,primordial suspension system of stable homogeneous: yellow fine granularity embryo callus of the plantain that picking step (1) induces or the light yellow fine granularity embryo callus of Brazilian any of several broadleaf plants, change over to respectively in the M2 liquid nutrient medium, place suspension culture on 90～110rpm shaking table got final product in 3～4 months the plantain cells,primordial suspension system or the Brazilian any of several broadleaf plants cells,primordial suspension system of dispersion, homogeneous;
(3) inducing of somatic embryo: elder generation changes above-mentioned embryonal suspension cell over to and does not contain 2 before the body embryonal induction, the pre-cultivation 7～10 days in the M2 liquid nutrient medium of 4-D, with after screen filtration filters out the cell mass of size and growth conditions unanimity, from cell mass, draw 0.2～2ml 20%SCV and be the cell inoculation amount and be inoculated in and contain 1mgL
-1ABA and 5mgL
-1In the body embryonal induction medium of ascorbic acid, place 28 ± 1 ℃ of dark to carry out inducing of somatic embryo; The white globular embryo that induces changed over to continue in the body embryonal induction medium of removing ABA and ascorbic acid to cultivate, obtain ripe body embryo until body embryonic development maturation;
(4) sprouting of body embryo and plant regeneration thereof: the ripe body embryo that step (3) is induced changes in the body embryo germination medium; Place 28 ± 1 ℃ of dark culturing, treat that leaf sheath is extracted out after, it is gone to continues in the MR strong plantlets and rootage medium to cultivate, further develop into healthy and strong banana plantlet.
2. a kind of method of setting up plantain and Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant according to claim 1 is characterized in that: describedly do not contain 2, the consisting of of the M2 liquid nutrient medium of 4-D: MS minimal medium+4.1 μ molL
-1Vitamin h+680 μ molL
-1Glutamine+100mgL
-1Malt extract+130mmolL
-1Sucrose, pH5.3, autoclave sterilization.
3. a kind of method of setting up plantain and Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant according to claim 1 is characterized in that: the consisting of of described body embryonal induction medium: SH a large amount of and trace element and molysite+MS vitamin+4.5 μ molL thereof
-1Vitamin h+680 μ molL
-1Glutamine+2mmolL
-1Proline+100mgL
-1Malt extract+1.1 μ molL
-1NAA+0.5 μ molL
-1Kinetin+44 μ gL
-1Zeatin+29mmolL
-1Lactose+130m molL
-1Sucrose+1mgL
-1ABA+5mgL
-1Ascorbic acid+2gL
-1Gelrite, wherein ABA and ascorbic acid are configured to 10mgml in advance respectively
-1Mother liquor adds pH5.8 to, temperature is reduced to mixing in all the other medium components of 45 ℃ behind autoclave sterilization, is sub-packed in the culture dish of diameter 9.5cm 20～25ml/ ware behind the filtration sterilization.
4. a kind of method of setting up plantain and Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant according to claim 1, it is characterized in that: the 20%SCV suspension cell is to prepare as follows described in the step (3): draw the 5ml suspension cell in the 15ml graded tube, after leaving standstill 10 minutes, abandon supernatant, use the body embryonal induction medium that does not add coagulating agent Gelrite to be diluted to 5 times of volumes the cell precipitation volume of gained.
5. a kind of method of setting up plantain and Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant according to claim 1 is characterized in that: the magnitude range through screen filtration gained cell mass described in the step (3) is 154 μ m～900 μ m.
6. a kind of method of setting up plantain and Brazilian any of several broadleaf plants high-efficiency somatic cell embryogenesis regeneration plant according to claim 1, it is characterized in that: the inoculum concentration of described plantain cell is 0.4ml 20%SCV; The inoculum concentration of Brazil's any of several broadleaf plants cell is 1ml 20%SCV.
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| CN101869076A (en) * | 2010-07-06 | 2010-10-27 | 广东省农业科学院作物研究所 | Method for inoculating stem tip of banana sucker |
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| CN101869076A (en) * | 2010-07-06 | 2010-10-27 | 广东省农业科学院作物研究所 | Method for inoculating stem tip of banana sucker |
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| CN113016622A (en) * | 2021-04-29 | 2021-06-25 | 中国科学院华南植物园 | Rapid propagation method for high-quality seedlings of canna indica tissue culture |
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| CN113068613A (en) * | 2021-05-06 | 2021-07-06 | 中国科学院华南植物园 | Red scorpion-rich cauda banana tissue culture method |
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| CN114107165B (en) * | 2021-11-15 | 2024-02-06 | 中国热带农业科学院热带作物品种资源研究所 | Low-temperature preservation method of banana embryogenic cell suspension system |
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