CN102524064B - 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 Genetic Transformation in Higher Plants technical 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), perennial woody oil plants, Euphorbiaceae Jatropha, originates in America, is distributed widely in tropical and subtropical zone area.Jatropha curcas is drought-resistant barren, and seed oil content, up to 60%, is one of at present important bioenergy plant.
But at present manioca exists planting area narrow, the 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 bringing into play important effect aspect the germplasm improvement of much grain and economic crops.Therefore, become the important means of the proterties such as improvement manioca resistance, raising output, improvement oil.
Agrobacterium tumefaciens-mediated transformation is one of the most frequently used plant gene method for transformation.Agrobacterium tumefaciems contains Ti-plasmids, its Shang Youyiduan T-DNA district.Genes of interest is inserted into the T-DNA district through transformation, by infecting plants wound, enters after cell, genes of interest can be inserted in Plant Genome, by reduction division, stably entail offspring.
The research work of manioca is started late, also little about the report of the cultivation of Jatropha curcas tissue and genetic transfoumation, and the rooting rate of transformation seedlings is generally lower.The researchers such as Li Meiru are using manioca cotyledon as explant, phosphine oxamate is as selective agent, set up Agrobacterium tumefaciens mediated genetic conversion system, transformation seedlings 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 the researchers such as Kumar are using 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 of transformation seedlings only has 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).The researchers such as Pan are using manioca blade as explant, kanamycin is as selective agent, utilize during agrobacterium-mediated transformation transforms manioca, the 120 strain regeneration plants that obtain only have 1 strain (the Pan J. of taking root, 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 present 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 obviously to improve the slim and frahile growth conditions of Jatropha curcas genetic transformation seedling, improves rooting rate.
For solving the problems of the technologies described above, the 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, after selecting suitable explant by Agrobacterium tumefaciems dip dyeing treatment, then it is standby as resistant buds after kanamycin-resistant callus tissue induction and differentiation medium culture, to obtain regeneration bud;
Strong seedling culture step, resistant buds is cut from resistant calli, cut brownization position, be transferred in strong seedling culture base P and carry out strong seedling culture, the formula of described 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;
Culture of rootage step, picking after strong seedling culture high 2-3 centimetre and have the resistant buds of 4-6 sheet leaf, excision base portion callus, soak 8 ~ 15 minutes with the rooting powder solution of 10-20mg/L, be forwarded to and in root media R, carry out culture of rootage, until take root, seedling reaches the technical requirement of tank hardening, described root media R formula is: 1/2MS minimal medium+0.01-0.1 mg/L indole-3-acetic acid+0.01-0.1 mg/L methyl α-naphthyl acetate+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,
Hardening step, to reaching the seedling of taking root of technical requirement of the tank hardening hardening of uncapping, transplants to greenhouse.
Further, the explant of selecting in preparation process is the stem section with 4-6 sheet true leaf, and its length is 2-3cm.
The Agrobacterium tumefaciems of using in described preparation process further, is to carry as follows any one in the Agrobacterium tumefaciems bacterial strain that contains genes of interest carrier: EHA105, LBA4404, GV3101, MP90.
Further, the kanamycin-resistant callus tissue induction and differentiation medium using in described preparation process contains 1-3 mg/L phosphine oxamate or 20-50 mg/L kanamycin or 1-10 mg/L hygromycin.
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, every two weeks subcultures are once.
Further, in 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, every two weeks subcultures were once.
Further, resistant buds is cultivated and is started to take root for 7-10 days in root media R, within 25-35 days, reaches the technical requirement of tank hardening.
Further, take root that the root of seedling is long can carry out hardening for 3-5 cm.
By adopting technique scheme, the present invention at least has following beneficial effect:
1. improve the growth conditions of transformation seedlings.Transformation seedlings after Agrobacterium tumefaciems is contaminated conventionally can be more very thin thin and weak; rooting rate can be lower; especially through kanamycin and transformation seedlings that hygromycin selection obtains, blade is less, and stem is thin and delicate; plant jaundice; and the present invention is by increasing strong seedling culture step, make transformation seedlings leaf dark green, stem section is sturdy; grow vigorous, be conducive to follow-up culture of rootage.
2. rooting rate is high.Manioca tissue exists the difficult problem of taking root in cultivating always, and rooting rate is generally lower.Transformation seedlings state after Agrobacterium tumefaciems is contaminated is poor, rooting rate is lower, and in method of the present invention, no matter through phosphine oxamate, the transformation seedlings that kanamycin or hygromycin selection obtain, after strong seedling culture, rooting rate all can reach 80-90 %, for the transgenic breeding of manioca is had laid a good foundation.
3. Effective selection goes out transgenic seedling.Prior art is all to add selective agent in resistant calli stage and differential period, and the resistant buds obtaining is proceeded to the not root media containing 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 like this escape of non-transformed seedling, and Effective selection goes out transformation seedlings, reduces the evaluation work in later stage.
Embodiment
Below in conjunction with example, describe in detail and describe embodiment of the present invention.It should be noted that, following example is illustrative, is not determinate, can not limit protection scope of the present invention with following example.
The invention provides a kind of rejuvenation of Jatropha curcas genetic transformation seedling and the cultural method of taking root, step is as follows:
Preparation process, after selecting suitable explant by Agrobacterium tumefaciems dip dyeing treatment, then it is standby as resistant buds after kanamycin-resistant callus tissue induction and differentiation medium culture, to obtain regeneration bud;
Strong seedling culture step, cuts resistant buds get off from resistant calli, cuts brownization position, is transferred in strong seedling culture base P and carries out strong seedling culture;
Culture of rootage step, picking after strong seedling culture high 2-3 centimetre and have the resistant buds of 4-6 sheet leaf, excision base portion callus, soaks 8 ~ 15 minutes with the rooting powder solution of 10-20mg/L, be forwarded in root media R and carry out culture of rootage, until the seedling of taking root reaches the technical requirement of tank hardening;
Hardening step, to reaching the seedling of taking root of technical requirement of the tank hardening hardening of uncapping, transplants to greenhouse.
Wherein, in described preparation process, the explant of use is preferably the little seeds of a tung oil tree stem section with 4-6 sheet true leaf, and its length is 2-3cm; And Agrobacterium tumefaciems used is to carry as follows any one in the Agrobacterium tumefaciems bacterial strain that contains genes of interest carrier: EHA105, LBA4404, GV3101, MP90, and genes of interest carrier can be pBI121-FT or pCAMBIA1301 etc.In the kanamycin-resistant callus tissue induction and differentiation medium using, contain 1-3 mg/L phosphine oxamate or 20-50 mg/L kanamycin or 1-10 mg/L hygromycin.
In strong seedling culture step, the formula 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.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, every two weeks subcultures are once.
And in culture of rootage step, root media R formula is: 1/2MS minimal medium+0.01-0.1 mg/L indole-3-acetic acid+0.01-0.1 mg/L methyl α-naphthyl acetate+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.Culture of rootage condition is: cultivation temperature 24-28 ℃, and intensity of illumination 1000-2000 lx, light application time 12-18 hour/day, every two weeks subcultures were once.Usually, resistant buds is cultivated and is started to take root for 7-10 days in root media R, within 25-35 days, reaches the technical requirement of tank hardening.
According to current technical merit situation, the root of the seedling of taking root is long can carry out hardening for 3-5 cm.
Below in conjunction with example, describe in detail and describe embodiment of the present invention.It should be noted that, following example is illustrative, is not determinate, can not limit protection scope of the present invention with following example.
Example 1
This example is with Agrobacterium tumefaciems GV3101 mediation, gus gene to be proceeded to the wild manioca of Yuanmou of Yunnan Province to cultivate, and each processing step is specific as follows:
Preparation process, by explant through carry contain genes of interest carrier pCAMBIA1301 Agrobacterium tumefaciems GV3101 contaminate after, then on the kanamycin-resistant callus tissue induction and differentiation medium that contains 2 mg/L phosphine oxamates cultivate obtain regeneration bud standby as resistant buds;
Strong seedling culture step, resistant buds is carefully cut from resistant calli, cut brownization position, be transferred in strong seedling culture base P and carry out strong seedling culture, described strong seedling culture base P formula is: mg/L phosphine oxamate+50, g/L agar+2, mL/L Coconut Juice+7, mg/L silver nitrate+200, mg/L indolebutyric acid+1, mg/L 6-benzyl aminopurine+0.005, MS minimal medium+30g/L sucrose+0.2 mg/L Ticarcillin/Clavulanate Acid, pH=5.6-6.0, condition of culture is: 24 ℃ of temperature, intensity of illumination 2000 lx, light application time 16 hours/day.Incubation time is surrounding, and every two weeks subcultures once;
Culture of rootage step, picking after strong seedling culture high 2-3 centimetre and have the resistant buds of 4-6 sheet leaf, 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 soaks 10 minutes, be forwarded in root media R, root media R formula is: mg/L phosphine oxamate+50, g/L agar+2, g/L sucrose+7, mg/L indolebutyric acid+10, mg/L methyl α-naphthyl acetate+0.05, mg/L indole-3-acetic acid+0.01,1/2MS minimal medium+0.01 mg/L Ticarcillin/Clavulanate Acid, condition of culture is: 24 ℃ of temperature, intensity of illumination 2000 lx, light application time 16 hours/day, every two weeks subcultures once, usually, 7-10 days start to take root, within 25-35 days, can reach the requirement of tank hardening,
Hardening step, the 25-35 days that will grow, the seedling of taking root of the long 3-5 cm of the root hardening of uncapping, transplants to greenhouse.
Example 2
This example is with Agrobacterium tumefaciems EHA105 mediation, gus gene to be proceeded to the wild manioca of Yuanmou of Yunnan Province to cultivate, and each processing step is specific as follows:
Preparation process, chooses suitable explant after carrying the Agrobacterium tumefaciems EHA105 dip-dye that contains genes of interest carrier pBI121-Hyg, cultivates acquisition regeneration bud standby as resistant buds on the kanamycin-resistant callus tissue induction and differentiation medium that contains 5 mg/L hygromycin;
Strong seedling culture step, resistant buds is carefully cut from resistant calli, cut brownization position, be transferred in strong seedling culture base P and carry out strong seedling culture, strong seedling culture base P formula is: mg/L hygromycin+150, g/L agar+5, mL/L Coconut Juice+7, mg/L silver nitrate+200, mg/L indolebutyric acid+5, mg/L 6-benzyl aminopurine+0.1, MS minimal medium+30g/L sucrose+1.5 mg/L Ticarcillin/Clavulanate Acid, pH=5.6-6.0, condition of culture is: 24 ℃ of temperature, intensity of illumination 2000 lx, light application time 16 hours/day, incubation time is surrounding, every two weeks subcultures once,
Culture of rootage step, picking after strong seedling culture high 2-3 centimetre and have the resistant buds of 4-6 sheet leaf, 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 20 mg/L that obtain soaks 10 minutes, be forwarded in root media R, root media R formula is: mg/L hygromycin+150, g/L agar+5, g/L sucrose+7, mg/L indolebutyric acid+15, mg/L methyl α-naphthyl acetate+0.25, mg/L indole-3-acetic acid+0.1,1/2MS minimal medium+0.1 mg/L Ticarcillin/Clavulanate Acid, condition of culture is: 26 ℃ of temperature, intensity of illumination 1800 lx, light application time 16 hours/day, every two weeks subcultures once, generally within 7-10 days, start to take root, within 25-35 days, can reach the requirement of tank hardening,
Hardening step, the 25-35 days that will grow, the seedling of taking root of the long 3-5 cm of the root hardening of uncapping, transplants to greenhouse.
Example 3
This example is with Agrobacterium tumefaciems MP90 mediation, FT gene to be proceeded to the wild manioca of Yuanmou of Yunnan Province to cultivate, and each processing step is specific as follows:
Preparation process, chooses suitable explant after carrying the Agrobacterium tumefaciems MP90 dip-dye that contains genes of interest carrier pBI121-FT, obtains regeneration bud standby as resistant buds on the kanamycin-resistant callus tissue induction and differentiation medium that contains 20 mg/L kanamycin;
Strong seedling culture step, resistant buds is carefully cut from resistant calli, cut brownization position, be transferred in strong seedling culture base P and carry out strong seedling culture, strong seedling culture base P formula is: mg/L kanamycin+100, g/L agar+20, mL/L Coconut Juice+7, mg/L silver nitrate+200, mg/L indolebutyric acid+3, mg/L 6-benzyl aminopurine+0.05, MS minimal medium+30g/L sucrose+1.0 mg/L Ticarcillin/Clavulanate Acid, pH=5.6-6.0, condition of culture is: 25 ℃ of temperature, intensity of illumination 1500 lx, light application time 16 hours/day, incubation time is surrounding, every two weeks subcultures once,
Culture of rootage step, picking after strong seedling culture high 2-3 centimetre and have the resistant buds of 4-6 sheet leaf, 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 soaks 10 minutes, be forwarded in root media R, root media R formula is: mg/L kanamycin+100, g/L agar+20, g/L sucrose+7, mg/L indolebutyric acid+12, mg/L methyl α-naphthyl acetate+0.1, mg/L indole-3-acetic acid+0.05,1/2MS minimal medium+0.05 mg/L Ticarcillin/Clavulanate Acid, condition of culture is: 28 ℃ of temperature, intensity of illumination 2000 lx, light application time 16 hours/day, every two weeks subcultures once, usually, 7-10 days start to take root, within 25-35 days, can reach tank hardening degree,
Hardening step, the 25-35 days that will grow, the seedling of taking root of the long 3-5cm of the root hardening of uncapping, transplants to greenhouse.
The rooting rate of the resistant buds of above three example gained is added up as following table:
| Example sequence number | Survival resistant buds number () | Take root seedling () | Rooting rate (%) |
| Example 1 | 11 | 10 | 90.90 |
| Example 2 | 12 | 10 | 83.33 |
| Example 3 | 126 | 101 | 80.16 |
As seen from the above table, adopt cultural method of the present invention, the rooting rate of resistant buds can reach 80 ~ 90%, far above the rooting rate of prior art, practical.
Claims (8)
1. the rejuvenation of Jatropha curcas genetic transformation seedling and a cultural method of taking root, is characterized in that, step is as follows:
Preparation process, after selecting suitable explant by Agrobacterium tumefaciems dip dyeing treatment, then it is standby as resistant buds after kanamycin-resistant callus tissue induction and differentiation medium culture, to obtain regeneration bud;
Strong seedling culture step, resistant buds is cut from resistant calli, cut brownization position, be transferred in strong seedling culture base P and carry out strong seedling culture, the formula of described 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;
Culture of rootage step, picking after strong seedling culture high 2-3 centimetre and have the resistant buds of 4-6 sheet leaf, excision base portion callus, soak 8 ~ 15 minutes with the rooting powder solution of 10-20mg/L, be forwarded to and in root media R, carry out culture of rootage, until take root, seedling reaches the technical requirement of tank hardening, described root media R formula is: 1/2MS minimal medium+0.01-0.1 mg/L indole-3-acetic acid+0.01-0.1 mg/L methyl α-naphthyl acetate+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,
Hardening step, to reaching the seedling of taking root of technical requirement of the tank hardening hardening of uncapping, transplants to 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 in preparation process is the stem section with 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 described preparation process is to carry as follows any one in the Agrobacterium tumefaciems bacterial strain that contains genes of interest carrier: 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 induction and differentiation medium using in described preparation process 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 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 is 3-5 week, and every two weeks subcultures once.
6. the rejuvenation of Jatropha curcas genetic transformation seedling according to claim 1 and the cultural method of taking root, it is characterized in that: in culture of rootage step, condition of culture is: temperature 24-28 ℃, intensity of illumination 1000-2000 lx, light application time 12-18 hour/day, every two weeks 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: resistant buds is cultivated and started to take root for 7-10 days in root media R, within 25-35 days, reaches the technical requirement of tank hardening.
8. according to the rejuvenation of the Jatropha curcas genetic transformation seedling described in claim 1 or 7 and the cultural method of taking root, it is characterized in that: the root of the seedling of taking root is long can carry out hardening for 3-5 cm.
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