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In Vitro Plant Regeneration from Hypocotyl and Cotyledon Explants of BARI Sarisha-13 (brassica Napus L.)

Sabrina Shameen Alam
Department of Genetic Engineering & Biotechnology University of Chittagong, Bangladesh

Mohammad Al- Forkan
Department of Genetic Engineering & Biotechnology University of Chittagong, Bangladesh

Laila Khaleda
Department of Genetic Engineering & Biotechnology University of Chittagong, Bangladesh

Abstract/ Executive Summary:

The aim of the present study was to develop an efficient in vitro regeneration system for BARI sarisha-13 (Brassica napus L.) where hypocotyls and cotyledonary petioles obtained from in vitro grown seedlings were used as explants for indirect regeneration. Single and combinatorial effects of plant growth regulators (PGRs) and additives were observed. Varying concentrations of hormones was found to change efficiency of callus, shoot and root formation. Increasing 2, 4- dichlorophenoxyacetic acid (2,4-D) from 0.5 mgl^-1 to above and 6, benzyl amino purine (BAP) concentration from 2 mg^-1 to 3 mg^-1 and NAA from 0.1 mg^-1 to 0.5 mg^-1 decreased callus and shoot formation, respectively. Roots were obtained when naphtalene acetic acid (NAA) was used. Effects of kinetin, AgNO₃, casein hydrolysate (CH), proline, indole 3-butyric acid (IBA), etc were also seen in different stages of regeneration. Percentage and photographs of responsive explants was taken after 24 days from inoculation.

Keywords: BARI-13, In vitro regeneration, PGRs

Location: Department of Genetic Engineering & Biotechnology, University of Chittagong, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Mustard

Objectives:

To develop a genotype independent in vitro tissue culture system for the local Brassica variety, BARI-13.

Methodology:

BARI Sharisha -13 (B. napus L.) was used for in vitro callogenesis and regeneration. Healthy seeds were collected from Bangladesh Agricultural Research Institute (BARI), Hathazari Substation, Chittagong. Half strength MS medium was used for seed germination whereas different combinations and concentrations of PGRs (Plant Growth Regulators) and additives were used for callus induction, shooting and rooting. MS media for callogenesis were supplemented with various concentration of 2,4-D and 30 gl^-1 (w/v) sucrose. BAP was also used at a concentration of 0.5 mgl^-1 in addition to 2, 4-D. Additives such as proline and CH and media with or without AgNO₃ were also used in some combinations. For the development of shoots from the calli, BAP, NAA and Kinetin were used with MS media. For the induction of roots IAA and IBA were used. pH was adjusted at 5.8 for all media. The incubation temperature was maintained at 26±2ºC in the culture room and a 16 hour photoperiod was maintained. Regenerated in vitro plants were successfully transplanted to soil.Sterilization of explants and media: Work was performed under the hood of laminar airflow. Collected BARI-13 seeds were immersed in 70% Ethanol for 10 mins. with occasional shaking followed by rinsing with double distilled water. Seeds were then placed in 0.1% HgCl₂ solution for another 10 minutes with periodical shaking and washed five to seven times. Seeds were then placed on 70 mm sterile filter paper and left for 10 minutes to remove excess water. All the media and glass wares were sterilized by autoclaving 20 mins. at 121ºC.

Source of Information: Asian Journal of Agriculture and Food Science (ISSN: 2321 – 1571), Volume 02 – Issue 01, February 2014

Article Link (Online Journal): http://www.ajouronline.com/index.php?journal=AJAFS&page=article&op=view&path[]=867&path[]=513

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Increasing 2, 4- dichlorophenoxyacetic acid (2,4-D) from 0.5 mgl^-1 to above and 6, benzyl amino purine (BAP) concentration from 2 mg^-1 to 3 mg^-1and NAA from 0.1 mg^-1 to 0.5 mg^-1 decreased callus and shoot formation in BARI-13, respectively. Roots were obtained when naphtalene acetic acid (NAA) was used. Effects of kinetin, AgNO₃_, casein hydrolysate (CH), proline, indole 3-butyric acid (IBA), etc were also seen in different stages of regeneration. The protocol can be used as an in vitro regeneration system which can be transmitted in the field in future.

Knowledge Management: Journal