CN102524064A - Rejuvenation and rooting culturing method for genetic transformation seedlings of Jatropha curcas - Google Patents
Rejuvenation and rooting culturing method for genetic transformation seedlings of Jatropha curcas Download PDFInfo
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Abstract
The invention relates to a rejuvenation and rooting culturing method for genetic transformation seedlings of Jatropha curcas, which comprises the following steps of: a preparation step of carrying out impregnation treatment on an explant by Agrobacterium tumefaciens, then carrying out kanamycin-resistant callus induction and culturing by a differential medium to obtain regeneration buds serving as resistant buds for later use; a good seedling culturing step of cutting down the resistant buds from kanamycin-resistant callus tissues, cutting off browning positions and transferring the obtained product into a good seedling culture medium P to carry out good seedling cultivation; a rooting culturing step of selecting the resistant buds which is 2 to 3cm higher after being subjected to good seedling cultivation and respectively have 4 to 6 leaves, cutting off the callus tissues at the roots of the resistant buds, soaking the resistant buds in rooting powder solution with the concentration of 10 to 20mg/L for 8 to 15 minutes, and transferring the resistant buds into a rooting culture medium R to carry out rooting cultivation until rooted seedlings reach the technical requirements for taking out of a tank and exercising; and a seedling exercising step of opening a cover to exercise the rooted seedlings which reach the technical requirements for taking out of the tank and exercising and transplanting the rooted seedlings into a greenhouse. According to the method disclosed by the invention, the delicate growth state of the genetic transformation seedlings of Jatropha curcas can be obviously improved, the rooting rate is improved and a good foundation is laid for molecular breeding of Jatropha curcas.
Description
Technical field
The present invention relates to Jatropha curcas plant genetic transformation technology field, relate in particular to a kind of rejuvenation of Jatropha curcas genetic transformation seedling and the cultural method of taking root.
Background technology
Jatropha curcas (Jatropha curcas), the perennial woody oil plants, Euphorbiaceae leprosy Pterostyrax originates in America, is distributed widely in the tropical and subtropical zone area.Jatropha curcas is drought-resistant barren, and seed oil content is one of at present important bioenergy plant up to 60%.
Yet at present manioca exists planting area narrow, problem such as yield poorly, and the traditional breeding method cycle is long, the kind that obtain a merit and genetic stability needs 8-10.In recent years, the fast development of plant gene engineering technology is being brought into play important effect aspect the germplasm improvement of many grains and economic crops.Therefore, become improvement manioca resistance, improve output, improve the important means of proterties such as oil.
Agrobacterium tumefaciens-mediated transformation is one of the most frequently used plant gene method for transformation.Agrobacterium tumefaciems contains Ti-plasmids, and one section T-DNA district is arranged on it.Genes of interest is inserted into the T-DNA district through transforming, after infecting plant wound entering cell, can genes of interest be inserted in the Plant Genome, stably entail the offspring through reduction division.
The research work of manioca is started late, and about the report of Jatropha curcas tissue culture and genetic transformation technology also seldom, and it is generally lower to transform the rooting rate of seedling.Researchers such as Li Meiru with the manioca cotyledon as explant; Phosphine oxamate is as selective agent; Set up Agrobacterium tumefaciens mediated genetic conversion system, transforming the seedling rooting rate is 78% (Li M., Li H.; Jiang H.; Et al. Transformation of an Agrobacterium-mediated cotyledon disc transformation method for Jatropha curcas. Plant Cell Tissue and Organ Culture, 2008,92 (2): 173-181).And researchers such as Kumar with the manioca blade as explant; Hygromycin is as selective agent; In the genetic transformation of the Agrobacterium tumefaciens mediated manioca of setting up; The rooting rate that transforms seedling has only 40% (Kumar N.; Anand K.G.V., Pamidimarri D.V.N.S. et al. Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants. Industrial Crops and Products, 32:41 – 47).Researchers such as Pan with the manioca blade as explant; Kanamycin is as selective agent; Utilize during agrobacterium-mediated transformation transforms manioca; The 120 strain regeneration plants that obtain have only 1 strain take root (Pan J., Fu Q., Xu F. Agrobacterium tumefaciens-mediated transformation of biofuel plant Jatropha curcas using kanamycin selection. African Journal of Biotechnology; 9 (39), 6477-6481).
Summary of the invention
Embodiment of the invention technical problem to be solved is, a kind of rejuvenation of Jatropha curcas genetic transformation seedling and the cultural method of taking root are provided, and with the obvious slim and frahile growth conditions of Jatropha curcas genetic transformation seedling that improves, improves rooting rate.
For solving the problems of the technologies described above, the present invention provides following technical scheme: a kind of rejuvenation of Jatropha curcas genetic transformation seedling and the cultural method of taking root comprise the steps:
Preparation process, select for use suitable explant by the Agrobacterium tumefaciems dip dyeing treatment after, again through kanamycin-resistant callus tissue induce and differential medium to cultivate back acquisition regeneration bud subsequent use as resistant buds;
The strong seedling culture step cuts resistant buds get off from resistant calli, cuts brownization position, is transferred among the strong seedling culture base P to carry out strong seedling culture;
The culture of rootage step; Picking is through after the strong seedling culture high 2-3 centimetre and the resistant buds of 4-6 sheet leaf is arranged, and excision base portion callus was with the rooting powder solution immersion of 10-20mg/L 8 ~ 15 minutes; Be forwarded to and carry out culture of rootage among the root media R, reach jar specification requirement of refining seedling until the seedling of taking root;
Refining seedling step to reaching jar seedling of taking root of the specification requirement of the refining seedling refining seedling of uncapping, is transplanted to the greenhouse.
Further, the explant of selecting for use in the preparation process is the stem section that has 4-6 sheet true leaf, and its length is 2-3cm.
The Agrobacterium tumefaciems of using in the said preparation process further, is to carry in the Agrobacterium tumefaciems bacterial strain that contains the genes of interest carrier any one as follows: EHA105, LBA4404, GV3101, MP90.
Further, the kanamycin-resistant callus tissue that uses in the said preparation process is induced and differential medium contains 1-3 mg/L phosphine oxamate or 20-50 mg/L kanamycin or 1-10 mg/L hygromycin.
Further; The prescription of strong seedling culture base P is: MS minimal medium+30g/L sucrose+0.2-1.5 mg/L 6-benzyl aminopurine+0.005-0.1 mg/L indolebutyric acid+1-5 mg/L silver nitrate+100-300 mL/L Coconut Juice+5-10 g/L agar+1-3 mg/L phosphine oxamate or 20-50mg/L kanamycin or 1-10mg/L hygromycin+50-150 mg/L Ticarcillin/Clavulanate Acid, pH=5.6-6.0.
Further, the condition of culture of strong seedling culture is: temperature 24-28 ℃, and intensity of illumination 1000-2000 lx, light application time 12-18 hour/day, incubation time was 3-5 week, per two all subcultures are once.
Further, root media R prescription is: 1/2MS minimal medium+0.01-0.1 mg/L indole-3-acetic acid+0.01-0.1 mg/L methyl+0.05-0.25 mg/L indolebutyric acid+10-15 g/L sucrose+4-8 g/L agar+1-3 mg/L phosphine oxamate or 20-50 mg/L kanamycin or 1-10 mg/L hygromycin+50-150 mg/L Ticarcillin/Clavulanate Acid.
Further, in the culture of rootage step, condition of culture is: temperature 24-28 ℃, and intensity of illumination 1000-2000 lx, light application time 12-18 hour/day, per two all subcultures were once.
Further, resistant buds is cultivated in root media R and was begun to take root in 7-10 days, reaches jar specification requirement of refining seedling in 25-35 days.
Further, take root that the root of seedling is long can to refine seedling for 3-5 cm.
Through adopting technique scheme, the present invention has following beneficial effect at least:
1. improve the growth conditions that transforms seedling.Conversion seedling through after the Agrobacterium tumefaciems dip-dye usually can be very thin more thin and weak, and rooting rate can be lower, especially obtains to transform seedling through kanamycin and hygromycin selection; Blade is less, and stem is thin and delicate, the plant jaundice; And the present invention makes the conversion seedling leaf dark green through increasing the strong seedling culture step, and the stem section is sturdy; It is vigorous to grow, and helps follow-up culture of rootage.
2. rooting rate is high.Exist the problem of the difficulty of taking root in the manioca tissue culture, rooting rate is generally lower always.Conversion seedling state through after the Agrobacterium tumefaciems dip-dye is relatively poor; Rooting rate is lower, and in the method for the present invention, no matter through phosphine oxamate; Kanamycin still is the conversion seedling that hygromycin selection obtains; After strong seedling culture, rooting rate all can reach 80-90 %, for the transgenic breeding of manioca is had laid a good foundation.
3. effectively filter out transgenic seedling.Prior art all is to add selective agent in resistant calli stage and differential period, the resistant buds that obtains is changed over to the root media that does not contain selective agent; The present invention not only adds selective agent in resistant calli stage and differential period, and the stage of taking root has also been selected suitable selective agent concentration, helps avoid the escape of non-conversion seedling like this, effectively filters out the conversion seedling, reduces the evaluation work in later stage.
Embodiment
Below in conjunction with instance description embodiment of the present invention are detailed.Need to prove that following instance is illustrative, is not determinate, can not limit protection scope of the present invention with following instance.
The present invention provides a kind of rejuvenation of Jatropha curcas genetic transformation seedling and the cultural method of taking root, and step is following:
Preparation process, select for use suitable explant by the Agrobacterium tumefaciems dip dyeing treatment after, again through kanamycin-resistant callus tissue induce and differential medium to cultivate back acquisition regeneration bud subsequent use as resistant buds;
The strong seedling culture step cuts resistant buds get off from resistant calli, cuts brownization position, is transferred among the strong seedling culture base P to carry out strong seedling culture;
The culture of rootage step; Picking is through after the strong seedling culture high 2-3 centimetre and the resistant buds of 4-6 sheet leaf is arranged, and excision base portion callus was with the rooting powder solution immersion of 10-20mg/L 8 ~ 15 minutes; Be forwarded to and carry out culture of rootage among the root media R, reach jar specification requirement of refining seedling until the seedling of taking root;
Refining seedling step to reaching jar seedling of taking root of the specification requirement of the refining seedling refining seedling of uncapping, is transplanted to the greenhouse.
Wherein, in the said preparation process, the explant of use is preferably the little seeds of a tung oil tree stem section that has 4-6 sheet true leaf, and its length is 2-3cm; And used Agrobacterium tumefaciems is to carry in the Agrobacterium tumefaciems bacterial strain that contains the genes of interest carrier any one as follows: EHA105, LBA4404, GV3101, MP90, and the genes of interest carrier can be pBI121-FT or pCAMBIA1301 etc.The kanamycin-resistant callus tissue that uses induce and differential medium in contain 1-3 mg/L phosphine oxamate or 20-50 mg/L kanamycin or 1-10 mg/L hygromycin.
In the strong seedling culture step; The prescription of strong seedling culture base P is: MS minimal medium+10-30g/L sucrose+0.2-1.5 mg/L 6-benzyl aminopurine+0.005-0.1 mg/L indolebutyric acid+1-5 mg/L silver nitrate+100-300 mL/L Coconut Juice+5-10 g/L agar+1-3 mg/L phosphine oxamate or 20-50mg/L kanamycin or 1-10mg/L hygromycin+50-150 mg/L Ticarcillin/Clavulanate Acid, pH=5.6-6.0.The strong seedling culture condition is: cultivation temperature 24-28 ℃, and intensity of illumination 1000-2000 lx, light application time 12-18 hour/day, incubation time was 3-5 week, per two all subcultures are once.
And in the culture of rootage step, root media R prescription is: 1/2MS minimal medium+0.01-0.1 mg/L indole-3-acetic acid+0.01-0.1 mg/L methyl+0.05-0.25 mg/L indolebutyric acid+10-15 g/L sucrose+4-8 g/L agar+1-3 mg/L phosphine oxamate or 20-50 mg/L kanamycin or 1-10 mg/L hygromycin+50-150 mg/L Ticarcillin/Clavulanate Acid.The culture of rootage condition is: cultivation temperature 24-28 ℃, and intensity of illumination 1000-2000 lx, light application time 12-18 hour/day, per two all subcultures were once.Usually, resistant buds is cultivated in root media R and was begun to take root in 7-10 days, reaches jar specification requirement of refining seedling in 25-35 days.
According to present technical merit situation, the root of the seedling of taking root length can be refined seedling for 3-5 cm.
Below in conjunction with instance description embodiment of the present invention are detailed.Need to prove that following instance is illustrative, is not determinate, can not limit protection scope of the present invention with following instance.
Instance 1
This instance is to change gus gene over to Yuanmou, Yunnan wild manioca with Agrobacterium tumefaciems GV3101 mediation to cultivate, and each processing step is specific as follows:
Preparation process, with explant after carrying the Agrobacterium tumefaciems GV3101 that contains genes of interest carrier pCAMBIA1301 and contaminating, again the kanamycin-resistant callus tissue that contains 2 mg/L phosphine oxamates induce and differential medium on cultivate that to obtain regeneration bud subsequent use as resistant buds;
The strong seedling culture step; Resistant buds is carefully cut from resistant calli; Cut brownization position, be transferred among the strong seedling culture base P and carry out strong seedling culture, said strong seedling culture base P prescription is: MS minimal medium+30g/L sucrose+0.2 mg/L 6-benzyl aminopurine+0.005 mg/L indolebutyric acid+1 mg/L silver nitrate+200 mL/L Coconut Juices+7 g/L agar+2 mg/L phosphine oxamates+50 mg/L Ticarcillin/Clavulanate Acids; PH=5.6-6.0; Condition of culture is: 24 ℃ of temperature, intensity of illumination 2000 lx, light application time 16 hours/day.Around incubation time was, per two all subcultures once;
The culture of rootage step, picking is through after the strong seedling culture high 2-3 centimetre and the resistant buds of 4-6 sheet leaf is arranged, excision base portion callus; The rooting powder solution of using the ABT-1 root-inducing powder of being developed and being sold by Beijing Ai Bidi research and development centre to prepare 10 mg/L that obtain soaked 10 minutes; Be forwarded among the root media R, root media R prescription is: 1/2MS minimal medium+0.01 mg/L indole-3-acetic acid+0.01 mg/L methyl+0.05 mg/L indolebutyric acid+10 g/L sucrose+7 g/L agar+2 mg/L phosphine oxamates+50 mg/L Ticarcillin/Clavulanate Acids, and condition of culture is: 24 ℃ of temperature; Intensity of illumination 2000 lx; Light application time 16 hours/day, per two all subcultures once, usually; 7-10 days begin to take root, and can reach a jar refining seedling requirement in 25-35 days;
Refining seedling step will grow 25-35 days, the seedling of taking root of the long 3-5 cm of the root refining seedling of uncapping, and transplanting is to the greenhouse.
Instance 2
This instance is to change gus gene over to Yuanmou, Yunnan wild manioca with Agrobacterium tumefaciems EHA105 mediation to cultivate, and each processing step is specific as follows:
Preparation process is chosen suitable explant after carrying the Agrobacterium tumefaciems EHA105 that contains genes of interest carrier pBI121-Hyg and contaminating, the kanamycin-resistant callus tissue that contains 5 mg/L hygromycin induce and differential medium on cultivate that to obtain regeneration bud subsequent use as resistant buds;
The strong seedling culture step carefully cuts resistant buds get off from resistant calli, cuts brownization position; Be transferred among the strong seedling culture base P and carry out strong seedling culture; Strong seedling culture base P prescription is: MS minimal medium+30g/L sucrose+1.5 mg/L 6-benzyl aminopurines+0.1 mg/L indolebutyric acid+5 mg/L silver nitrates+200 mL/L Coconut Juices+7 g/L agar+5 mg/L hygromycin+150 mg/L Ticarcillin/Clavulanate Acids, and pH=5.6-6.0, condition of culture is: 24 ℃ of temperature; Intensity of illumination 2000 lx; Around light application time 16 hours/day, incubation time be, per two all subcultures once;
The culture of rootage step; Picking is through after the strong seedling culture high 2-3 centimetre and the resistant buds of 4-6 sheet leaf is arranged, and excision base portion callus uses the rooting powder solution of 20 mg/L that obtained by the development of Beijing Ai Bidi research and development centre and the ABT-1 root-inducing powder preparation of selling to soak 10 minutes; Be forwarded among the root media R; Root media R prescription is: 1/2MS minimal medium+0.1 mg/L indole-3-acetic acid+0.1 mg/L methyl+0.25 mg/L indolebutyric acid+15 g/L sucrose+7 g/L agar+5 mg/L hygromycin+150 mg/L Ticarcillin/Clavulanate Acids, and condition of culture is: 26 ℃ of temperature, intensity of illumination 1800 lx; Light application time 16 hours/day; Per two all subcultures once, general 7-10 days begin to take root, and can reach a jar refining seedling requirement in 25-35 days;
Refining seedling step will grow 25-35 days, the seedling of taking root of the long 3-5 cm of the root refining seedling of uncapping, and transplanting is to the greenhouse.
Instance 3
This instance is to change the FT gene over to Yuanmou, Yunnan wild manioca with Agrobacterium tumefaciems MP90 mediation to cultivate, and each processing step is specific as follows:
Preparation process is chosen suitable explant after carrying the Agrobacterium tumefaciems MP90 that contains genes of interest carrier pBI121-FT and contaminating, the kanamycin-resistant callus tissue that contains 20 mg/L kanamycin induce and differential medium on to obtain regeneration bud subsequent use as resistant buds;
The strong seedling culture step carefully cuts resistant buds get off from resistant calli, cuts brownization position; Be transferred among the strong seedling culture base P and carry out strong seedling culture; Strong seedling culture base P prescription is: MS minimal medium+30g/L sucrose+1.0 mg/L 6-benzyl aminopurines+0.05 mg/L indolebutyric acid+3 mg/L silver nitrates+200 mL/L Coconut Juices+7 g/L agar+20 mg/L kanamycin+100 mg/L Ticarcillin/Clavulanate Acids, and pH=5.6-6.0, condition of culture is: 25 ℃ of temperature; Intensity of illumination 1500 lx; Around light application time 16 hours/day, incubation time be, per two all subcultures once;
The culture of rootage step, picking is through after the strong seedling culture high 2-3 centimetre and the resistant buds of 4-6 sheet leaf is arranged, excision base portion callus; The rooting powder solution of using the ABT-1 root-inducing powder of being developed and being sold by Beijing Ai Bidi research and development centre to prepare 15 mg/L that obtain soaked 10 minutes; Be forwarded among the root media R, root media R prescription is: 1/2MS minimal medium+0.05 mg/L indole-3-acetic acid+0.05 mg/L methyl+0.1 mg/L indolebutyric acid+12 g/L sucrose+7 g/L agar+20 mg/L kanamycin+100 mg/L Ticarcillin/Clavulanate Acids, and condition of culture is: 28 ℃ of temperature; Intensity of illumination 2000 lx; Light application time 16 hours/day, per two all subcultures once, usually; 7-10 days begin to take root, and can reach a jar refining seedling degree in 25-35 days;
Refining seedling step will grow 25-35 days, the seedling of taking root of the long 3-5cm of the root refining seedling of uncapping, and transplanting is to the greenhouse.
More than the rooting rate statistics of resistant buds of three instance gained like following table:
| The instance sequence number | Survival resistant buds number () | Take root seedling () | Rooting rate (%) |
| Instance 1 | 11 | 10 | 90.90 |
| Instance 2 | 12 | 10 | 83.33 |
| Instance 3 | 126 | 101 | 80.16 |
Can find out that by last table adopt cultural method of the present invention, the rooting rate of resistant buds can reach 80 ~ 90%, and is far above the rooting rate of prior art, practical.
Claims (10)
1. the rejuvenation of a Jatropha curcas genetic transformation seedling and the cultural method of taking root is characterized in that step is following:
Preparation process, select for use suitable explant by the Agrobacterium tumefaciems dip dyeing treatment after, again through kanamycin-resistant callus tissue induce and differential medium to cultivate back acquisition regeneration bud subsequent use as resistant buds;
The strong seedling culture step cuts resistant buds get off from resistant calli, cuts brownization position, is transferred among the strong seedling culture base P to carry out strong seedling culture;
The culture of rootage step; Picking is through after the strong seedling culture high 2-3 centimetre and the resistant buds of 4-6 sheet leaf is arranged, and excision base portion callus was with the rooting powder solution immersion of 10-20mg/L 8 ~ 15 minutes; Be forwarded to and carry out culture of rootage among the root media R, reach jar specification requirement of refining seedling until the seedling of taking root;
Refining seedling step to reaching jar seedling of taking root of the specification requirement of the refining seedling refining seedling of uncapping, is transplanted to the greenhouse.
2. the rejuvenation of Jatropha curcas genetic transformation seedling according to claim 1 and the cultural method of taking root is characterized in that: the explant of selecting for use in the preparation process is the stem section that has 4-6 sheet true leaf, and its length is 2-3cm.
3. the rejuvenation of Jatropha curcas genetic transformation seedling according to claim 1 and the cultural method of taking root is characterized in that: the Agrobacterium tumefaciems of using in the said preparation process is to carry in the Agrobacterium tumefaciems bacterial strain that contains the genes of interest carrier any one as follows: EHA105, LBA4404, GV3101, MP90.
4. the rejuvenation of Jatropha curcas genetic transformation seedling according to claim 1 and the cultural method of taking root is characterized in that: the kanamycin-resistant callus tissue that uses in the said preparation process is induced and differential medium contains 1-3 mg/L phosphine oxamate or 20-50 mg/L kanamycin or 1-10 mg/L hygromycin.
5. the rejuvenation of Jatropha curcas genetic transformation seedling according to claim 1 and the cultural method of taking root; It is characterized in that: the prescription of strong seedling culture base P is: MS minimal medium+10-30g/L sucrose+0.2-1.5 mg/L 6-benzyl aminopurine+0.005-0.1 mg/L indolebutyric acid+1-5 mg/L silver nitrate+50-300 mL/L Coconut Juice+5-10 g/L agar+1-3 mg/L phosphine oxamate or 20-50mg/L kanamycin or 1-10mg/L hygromycin+50-150 mg/L Ticarcillin/Clavulanate Acid, pH=5.6-6.0.
6. according to claim 1 or the rejuvenation of 5 described Jatropha curcas genetic transformation seedlings and the cultural method of taking root; It is characterized in that: the condition of culture of strong seedling culture is: temperature 24-28 ℃; Intensity of illumination 1000-2000 lx; Light application time 12-18 hour/day, incubation time was 3-5 week, and per two all subcultures once.
7. the rejuvenation of Jatropha curcas genetic transformation seedling according to claim 1 and the cultural method of taking root is characterized in that: root media R prescription is: 1/2MS minimal medium+0.01-0.1 mg/L indole-3-acetic acid+0.01-0.1 mg/L methyl+0.05-0.25 mg/L indolebutyric acid+10-15 g/L sucrose+4-8 g/L agar+1-3 mg/L phosphine oxamate or 20-50 mg/L kanamycin or 1-10 mg/L hygromycin+50-150 mg/L Ticarcillin/Clavulanate Acid.
8. according to claim 1 or the rejuvenation of 7 described Jatropha curcas genetic transformation seedlings and the cultural method of taking root; It is characterized in that: in the culture of rootage step, condition of culture is: temperature 24-28 ℃, and intensity of illumination 1000-2000 lx; Light application time 12-18 hour/day, per two all subcultures once.
9. the rejuvenation of Jatropha curcas genetic transformation seedling according to claim 1 and the cultural method of taking root is characterized in that: resistant buds is cultivated in root media R and was begun to take root in 7-10 days, reaches jar specification requirement of refining seedling about 25-35 days.
10. according to claim 1 or the rejuvenation of 9 described Jatropha curcas genetic transformation seedlings and the cultural method of taking root, it is characterized in that: the root length of the seedling of taking root can be refined seedling for 3-5 cm.
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