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Development of Efficient Callus Initiation of Malta (citrus Sinensis) Through Tissue Culture

Fazle Azim
Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology, Bangladesh

M.M. Rahman
Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology, Bangladesh

Shamsul H. Prodhan
Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology, Bangladesh

Saif U. Sikdar
Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology, Bangladesh

Nayem Zobayer
Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology, Bangladesh

M. Ashrafuzzaman
Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology, Bangladesh

Abstract/ Executive Summary:

The effects of different hormonal concentration on shoot formation and callus induction were studied on BARI Malta-1 (Citrus sinensis). Seeds containing seed coat and without seed coat was treated by Murashige and Skoog (MS) media supplemented with 6-benzyl adenine (BA) and Kinetin (KIN). Removal of seed coat showed an early response for shoot formation. The highest (70%) shoot formation was obtained from seeds without seed coat treated with MS basal media + BA 1.0 mg/l while KIN showed no response for shoot formation in any supplemented concentration. However, in case of callus induction internodes and apical shoot tips were used as explants and 2, 4-dichlorophenoxy acetic acid (2, 4-D) was used as callus inducing hormone. MS basal media supplemented with 2, 4-D, 2.0 mg/l showed highest (68%) callus induction.

Keywords: Malta, , In vitro, Seed shoot formation, Callus induction

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Sweet orange

Objectives:

To develop an efficient callus initiation system of Malta (Citrus sinensis) through tissue culture which might be used in genetic transformation system and/or efficient and suitable regeneration protocol of malta in future.

Methodology:

This experiment was conducted at the Plant Genetic Engineering Laboratory of the Department of Genetic Engineering and Biotechnology, Shahjalal University of Science & Technology (SUST), Sylhet, Bangladesh. Collection of explant: The young malta (Citrus sinensis) plants were collected from BRAC, Gazipur, Dhaka, Bangladesh before starting this experiment and grown in the pot. The malta seeds were collected from local nursery of Sylhet. The types of explants were seeds, internodes and apical shoot tips. The internodes and apical shoot tips were aseptically excised and cultured on the callus induction medium. Explant sterilization: Seeds, internodes and shoot tips of C. sinensis were washed by using detergents for 2 minutes. Then explants were immersed for 15 minutes with 2 or 3 drops of Tween-20. In order to remove all traces of detergents and Tween-20 from the surface, explants were washed by sterile-distilled water for 3-4 times. Shoot formation media: Seed regeneration media were prepared by mixing all the components as the callus induction media except hormones. In case of hormone BA varying in concentration from 1.0 mg/l to 2.0 mg/l was used. The other steps i.e., agar melting, autoclaving etc. were similar to callus induction media preparation. Explants were inoculated and data were recorded. Callus induction media: For callus induction popular callus inducing hormone 2, 4-D was used. MS media supplemented with 2, 4-D varying in concentration from 1.0 mg/l to 3.0 mg/l were prepared separately in conical flasks. The 3% sucrose was added to each conical flask. The PH was adjusted to 5.6-5.8. Then 0.7% agar was added. The agar was melted by boiling at 110ºC for 2-3 minute. After melting the agar, the media were poured into the test-tubes and autoclaved at 121ºC and 15 psi for 15 minute. After autoclaving the media were allowed to cool down and coagulate in the Laminar Air Flow Cabinet. Inoculation of explant: Shoot tips and internodes were inoculated in callus induction media. For shoot formation of seeds, some seeds were inoculated with seed coat and some were not. The cultures were incubated at 25±2°C with 16 h of photoperiod with the light intensity of 2000 lux under cool-white fluorescent lamps. All the treatments were conducted with 5 replicates.

Source of Information: ISSN: 2224-0616 Int. J. Agril. Res. Innov. & Tech. 1 (1-2): 64-68, December, 2011 Available at http://www.ijarit.webs.com

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Removal of seed coat showed an early response for shoot formation. C. sinensis contain two layers of seed coat. Some seed were inoculated with seed coat and some were without seed coat. The shoot formation percentage was higher in seeds without seed coat. 2, 4-D, 4.0 mg/l and 2, 4-D 3mg/l showed highest percent callus induction. But 2, 4-D, 2.0 mg/l showed a good response. However, we also found that 2, 4-D, 1.0 mg/l and 2, 4-D, 3.0 mg/l also showed a moderate response. However, in case of callus induction internodes and apical shoot tips were used as explants and 2, 4-dichlorophenoxy acetic acid (2, 4-D) was used as callus inducing hormone. MS basal media supplemented with 2, 4-D, 2.0 mg/l showed highest (68%) callus induction.

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