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

Regeneration and Agrobacterium-mediated Genetic Transformation of Two Indigenous Potato Varieties of Bangladesh

R. H. Sarker
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Barkat Murtaja Mustafa
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Maximum shoot regeneration in two indigenous potato (Solanum tuberosum L.) was observed on MS semi-solid medium supplemented with 1.0 mg/l BAP and 0.1 mg/l GA3. Among the two varieties, namely Lal Pakri and Jam Alu the former showed best response in terms of number of shoots/explant, nodes/shoot and shoot length. Half strength of MS containing 0.1 mg/l IAA was found to be best for root induction from the excised shoots. Two strains of Agrobacterium namely, LBA4404 containing pBI121 and EHA105 having plasmid pCAMBIA1301 were used for transformation. Among the two strains, LBA4404 with the vector pBI121 showed better response. In the nodal segment of Lal Pakri, the transformed zone coincided with the regeneration point. Regeneration from transformed tissue was obtained on MS supplemented with 1.0 mg/l BAP and 0.1 mg/l GA3. Histochemical GUS assay showed the expression of GUS gene in the leaf tissues of the transformed shoot.

Keywords: Micropropagation, Indigenous, Potato, Genetic transformation, Agrobacterium

Location: Bogra and Mymensingh districts, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Potato

Objectives:

To optimize various factors influencing reproducible transformation protocols in two indigenous potato varieties using two strains of Agrobacterium containing different vectors.

Methodology:

Two indigenous potato (Solanum tuberosum L.) varieties, namely Lal Pakri and Jam Alu were used in the present study. The seed tubers of these two varieties were collected from the local growers of Bogra and Mymensingh, respectively. The tubers were kept in an incubator at 18⁰C for sprouting. About 4.0 - 5.0 cm long sprouts were cut and washed in distilled water. They were surfaced sterilized with 0.1 % HgCl₂ solution (w/v) for 15 min followed by several washes with sterilized distilled water. About 1.0 cm long sprout segments containing one to two nodes were used as explants and cultured on agar solidified MS medium supplemented with various combination of BAP, Kn and GA3 for induction and proliferation of multiple shoots. pH of the medium was adjusted to 5.8 before autoclaving. The culture vessels containing the explants were incubated in the growth room under 16/8 hrs light/dark cycle at 25 ± 2⁰C. For induction of roots, regenerated shoots (3 - 4 cm long) were excised and transferred to half strength of MS medium supplemented with different concentrations of IAA, IBA and NAA. Agrobacterium tumefaciens strain LBA4404 harboring the binary plasmid pBI121 and strain EHA105 containing the plasmid pCAMBIA1301 were used in the present study. Both the plasmids contain the GUS reporter gene. The plasmid pBI121 contains the plant selectable marker gene nptll conferring resistance to kanamycin and the plasmid pCAMBIA1301 contains the plant selectable marker gene conferring resistance to hygromycin, hptII. Leaf, internodal and nodal segments from in vitro grown planlets were used in the genetic transformation. The Agrobacterium suspension was centrifuged at 10000 rpm for 10 min and the precipitate was resuspended in appropriate volume of MS medium so that the density of bacterial suspension becomes 0.8 - 1.0 (at 600 nm). The cut explants were then immersed in the resuspended Agrobacterium suspension for different periods of time, blotted dry and cocultured for three days in the dark on MS medium with 1 mg/l BAP and 0.1 mg/l GA3. After coculture the explants were washed in cefotaxime (500 mg/l) to kill the Agrobacterium. They were then cultured on regeneration medium. Following cocultivation the explants were subjected to transient GUS histochemical assay according to Jefferson (1987) to monitor the efficiency of infection. The cocultivated explants were incubated for 24 - 48 h at 37⁰C in the substrate X-gluc (5-bromo, 4-chloro, 3-indolyl-glucuronide) and subsequently bleached with 70% ethanol before scoring for transient GUS expression.

Source of Information: Plant Tissue Cult. 12(1) : 69-77, 2002 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Transformed tissue were obtained from leaf, nodal and internodal segments. In both the potato varieties leaf explants showed best transformation ability followed by internodal and nodal segments. Histochemical GUS assay revealed the expression of GUS reporter gene in the leaf, basal callus and nodal segments in Lal Pakri. It was evident that the transformed sectors of the axillary buds coincided with the area of regeneration. Regenerated shoots from the transformed tissues showed numerous blue sectors in the leaf and newly developed shoot bud. However, future experiments are required to regenerate fully transformed plantlets showing stable integration of GUS and nptII gene. The results of the present investigation demonstrated the development of efficient in vitro regeneration systems in two local potato varieties, namely Lal Pakri and Jam Alu. Encouraging results of transformation experiments have shown that it may be develop disease resistant varieties of potato through genetic transformation.

Knowledge Management: Journal