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

In Vitro Plantlet Regeneration and Agrobacterium Mediated Genetic Transformation of Wheat (triticum Aestivum L.)

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

Ashapurno Biswas
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Different explants such as mature and immature embryos, seeds, endosperms, leaves, shoot bases and root tips of four local wheat (Triticum aestivum L.) varieties, namely Sourav, Gourav, Kanchan and Protiva were evaluated for their in vitro callus induction and regeneration of plantlets as well as Agrobacteriummediated genetic transformation. Among the explants immature embryo was found to be the best for callus induction and shoot regeneration. Profuse callus developed when MS was supplemented with 5.5 mg/l 2,4-D. Maximum shoot regeneration was obtained on MS medium containing 0.5 mg/l BAP, 0.5 mg/l Kn and 25 mg/l tyrosine. Majority of regenerated shoots (90%) developed roots on half strength of MS without any hormonal supplement. Plantlets were success-fully established in soil. No remarkable variation was observed among the wheat varieties regarding their ability for callus induction and shoot regeneration. Two strains of Agrobacterium, namely LBA4404 containing pBI121 and EHA105 having plasmid pCAMBIA1301 were used in transformation experiments. Among the two strains EHA105 was found to be compatible to the aforesaid four local varieties of wheat. Maximum transformation efficiency, monitored by transient GUS histochemical assay, was obtained in callus derived from immature embryos with a bacterial suspension of optical density 0.75 at 600 nm.

Keywords: Regeneration, Wheat, Transformation, Agrobacterium

Location: The Plant Breeding and Tissue Culture Laboratory, Botanical garden, Department of Botany, University of Dhaka.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Wheat

Objectives:

To developing Agrobacterium mediated transformation protocols for the said wheat varieties cultivated in Bangladesh.

Methodology:

Seeds of four local wheat (Triticum aestivum L.) varieties, namely Sourav, Gourav, Kanchan and Protiva were collected from the Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur. Different explants such as mature and immature embryos, seed, endosperm, leaf, shoot base and root tip were used for the induction of callus and subsequent development of shoots. Explants like root tip, shoot base and leaf were collected from the aseptically grown two - three-day-old seedlings. Immature embryos were collected from the spike after 14 - 18 days of anthesis. Both mature and immature seeds were washed thoroughly under running tap water and then rinsed several times in distilled water. Then they were dipped in 70% ethanol (v/v) for two - three min followed by washing with distilled water. Finally surface disinfection was done with 0.1% HgCl₂ solution (w/v) for 5 min followed by several washes with sterilized distilled water. Then the seeds were dissected. Excised embryos were cultured in tubes. In the transformation experiments Agrobacterium tumefaciens strain LBA4404 harboring the binary plasmid pBI121 and strain EHA105 containing the plasmid pCAMBIA1301 were used. Both plasmids contain the GUS reporter gene as well as the nptII gene conferring resistance to kanamycin. Following cocultivation the explants were subjected to transient GUS histochemical assay to monitor the efficiency of infection. The cocultivated explants were incubated for 24 - 48 hrs 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(2) : 155-165, 2002 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

It was found that EHA105 having an OD 0.75 at 600 nm when used for infection under 5 min vacuum showed response. For good transformation efficiencies the bacteria had to be subsequently shaken with explant in infection media for 50 min followed by a cocultivation of 96 hours. Transformed cells were obtained from all the explants. Immature embryos of all the four wheat varieties showed the best transformation ability. The in vitro regeneration procedure described here provides a rapid and reproducible protocol for four locally grown wheat varieties. The protocol for successful transformation established in the present study can now be used for developing transgenic wheat plants for incorporation of disease resistance and other agronomically important traits. Among the two strains EHA105 was found to be compatible to the aforesaid four local varieties of wheat. Maximum transformation efficiency, monitored by transient GUS histochemical assay, was obtained in callus derived from immature embryos with a bacterial suspension of optical density 0.75 at 600 nm.

Knowledge Management: Journal