Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Agrobacterium-mediated Transformation in Potato

M.M.H. Molla

K.M. Nasiruddin
Biotechnology Division, BARI, Gazipur-170

M. Al-Amin
Biotechnology Division, BARI, Gazipur-170

M.S. Haque
Biotechnology Division, BARI, Gazipur-170

Maniruzzaman
Biotechnology Division, BARI, Gazipur-170

Abstract/ Executive Summary:

Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBI121 having one reporter gene (gus) and selectable marker gene (nptII) resistant to kanamycin with internode explant was used in this study. Transformation was done by optimizing two important parameters viz. infection time and co-cultivation period. Most of the explants produced shoots within 18-21 days on 5 mg L^-1 zeatin riboside and 50 mg L^-1 kanamycin supplemented MS medium through organogenesis. Explants infection for 30-minute with 3-day co-culture produced maximum 8.27 and 6.42 shoots in Asterix and Diamant varieties, respectively within 18-21 days. Stable integration and expression of the transgenes were confirmed by histochemical and molecular analysis. DNA from well established rooted plants confirmed nptII and gus positive through PCR analysis and resulted transformation rate 28.97% and 24.37% in Asterix and Diamant varieties, respectively. 100% shoots from putative transformed plants of Diamant and Asterix varieties produced roots on ½MS medium supplemented with 50 mg l^-1 cefotaxim, 50 mg L^-1 kanamycin and 0.5 mg L^-1IBA.

Keywords: Transformation, Potato, NptII gene, Gus gene, Agrobacterium

Location: Tuber Crops Research Centre, Bangladesh Agricultural Research Institute, Gazipur

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Potato

Objectives:

To optimize the Agrobacterium-mediated genetic transformation protocol, so that the gene of interest can be inserted efficiently in the economically important potato varieties of Bangladesh.

Methodology:

Plant Materials - Diamant and Asterix varieties were collected from Tuber Crops Research Centre, Bangladesh Agricultural Research Institute, Gazipur and sprouts were collected from the tuber and cultured in vitro. The internode explants from 21 days old in vitro plantlets were used as plant materials in this experiment. Agrobacterium Strain and Plasmid - Agrobacterium tumefaciens strain LBA4404 with the binary plasmid pBI121 was used for transformation for above mentioned two varieties of potato. The binary vector pBI121 has the backbone of pBIN19. It contains a reporter gene GUS (β-glucurunidase) NOS promoter and terminator driven by a CaMV35S. In addition it has a selectable merker gene nptII. It encodes for neomycin phosphotransferase that confers kanamycin resistance. Two type of culture media YMB (Yeast extract Mannitol Broth) and LB (Luria Broth) were used for the Agrobacterium strain. The first medium was used to maintain Agrobacterium stock and the second one was for the infection of explants. For maintenance, on single colony from previously maintained Agrobacterium stocks was streaked into freshly prepared Petri dish containing YMB medium having kanamycin (50 mg L^-1). The Petri dish was sealed with Para film and kept in the incubator at 28ºC at least 48 hours. This was then kept at 4ºC to check over growth. Such culture of Agrobacterium strain was thus ready to use for liquid culture. The culture was sub-cultured at each week in fresh prepared medium to maintain the stock. For infection, from this Agrobacterium stock single streak was taken in an inoculation loop and was inoculated in a conical flask containing liquid LB medium with 50 mg L^-1 kanamycin. The culture was allowed to grow over night inside the digital incubating shaker (Vision Scientific Co. ltd) at 28ºC @ 250 rpm to get optimum population of Agrobacterium. Plant Induction Media For preparation of explants, instant Murashige and Skoog (MS) medium including vitamins and minerals (4.4 g L^-1) (Duchiffa, Netherlands) was used to prepare explants of potato for transformation work. MS medium supplemented with different concentration of ZR in addition with gibberellic acid (GA3) (0.2 mg L^-1) and indole-3-acetic acid (IAA) (0.01 mg L^-1) concentration based on Cearly and Bolyard was used for direct shoot regeneration of potato. Instant MS medium (4.4 g L^-1) supplemented with 5 mg L^-1 zeatin riboside (ZR), 1 mg L^-1 IAA, 3 mg L^-1 GA₃ and 50 mg L^-1 acetosyringone, 20 g L^-1sucrose and 2 g L^-1gelrite were used for co-cultivation medium. Filter sterilized ZR was added inside laminar air flow cabinet. Following co-culture the explants were washed several times with sterile distilled water with gentle shaking until no opaque suspension was seen. The infected explants were finally washed for 3 minutes in MS liquid medium supplemented with 400 mg L^-1 cefotaxime. MS medium supplemented with 5 mg L^-1 ZR, 1 mg L^-1 IAA, 3 mg L^-1GA₃, 50 mg L^-1 kanamicin, 200 mg L^-1 carbenecilline, 20 g L^-1 sucrose and 2 g L^-1 gelrite were used for post cultivation and shoot differentiation. To eliminate untransformed tissues, the regenerating explants were sub-cultured after 2-3 times on fresh regeneration medium initially with 50 mg L^-1 kanamycin. The concentration of kanamycin was increased at every 2 weeks when fresh subculture was made until its level reached 100 mg L^-1. During each subculture the dead and deep brown tissues were discarded and green shoots were subcultured in a fresh medium containing the next higher concentration of kanamycin. ½MS, 50 mg L^-1cefotaxime, 50 mg L^-1 kanamycin and 0.5 mg L^-1 indole-3-butyric acid (IBA) were used for rooting. Infection and Co-cultivation - The Agrobacterium tumefaciens LBA4404 harboring binary vector pBI121 grown in liquid LB media were used for infection either co-cultivation maintaining the optical density of OD600=0.6 to get suitable and sufficient infection of explants. Freshly excised explants were wounded and immersed in a culture of the above bacteria solution for 20, 30 and 40 minutes with gentle shaking and then transferred them to co-cultivation medium. For infection the explants were dipped in the Agrobacterium suspension for 20, 30, 40 minutes and co-cultivation for 2, 3 and 4 days. Infection medium were prepared by adding liquid bacterial culture with liquid MS in 1:1 ratio and acetosyringone (phenolic compound, inducer of Agrobaterium) 50 mg L^-1 was added to activate the vir genes. Methods of GUS (β-glucuronidase) Histochemical Assay - Randomly selected five co-cultured explants from each of the treatments were examined for GUS histochemical assay. Co-cultured explants were immersed in fixation solution (formaldehyde (750μl, 0.5M MES (2 mL), menitol (5.47g) followed by Xgluc (5-bromo-4-chloro-3-indolyl glucuronide 50mM phosphate buffer having pH 7.8) solution and incubated at 37ºC for 24-48h. A characteristic blue color would be the expression of GUS (β- glucuronidase) gene in the plant tissue. For control treatment, GUS assay was done with the explants from normal plant. Leaves, shoots and roots were assayed from randomly selected plants. Explants and plant parts were dipped into GUS solution for 24-48 hours at 37ºC. After 24-48 h treatment, explants were soaked with 70% ethanol to remove chlorophyl. Then the de-greened explants/plant parts were observed under a stereomicroscope (Olympus, Japan). The experiment was laid out in factorial completely randomized design having seven replications and each petri dish containing 7 (seven) explants were considered as single replication.. Data were recorded on responsive explants (%) , days required for shoot initiation (days), number of shoots per explant, length of visible shoot at 21 days (cm), number of visible leaves at 21 days, diameter of visible shoot at 21 days (mm) and regeneration frequency, percentage of GUS positive and transformed plants. Data were analyzed using MSTAT-C statistical package. Differences among the means were compared following DMRT test at 5% and 1% level of significance. The analysis of variance for different characters was performed and means were compared by the Duncan’s Multiple Range Test (DMRT). The percentage data were subjected to appropriate transformation like arcsine and square root wherever applicable according to Gomez and Gomez.

Source of Information: Thai Journal of Agricultural Science 2011, 44(2): 93-102

Article Link (Online Journal): www.thaiagj.org

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Explants infection for 30-minute with 3-day coculture produced maximum shoots in Asterix and Diamant variety, respectively within 18-21 days in 5 mg L^-1 zeatin riboside and 50 mg L^-1 kanamycin supplemented MS medium. DNA from well established rooted plants confirmed nptII and GUS positive through PCR analysis.

Knowledge Management: Journal