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Development of Somaclone from BRRIdhan 47 and Optimize the Effect of Different Hormones to Achieve High Frequency of Plant Regeneration by Seed Culture

Enamul Hoque
Principal Scientific Officer
Biotechnology Division, BRRI Head Office, Gazipur 1701.

Nilufar Yasmin Shaikh
Senior Scientific Officer
Biotechnology Division, BRRI Head Office, Gazipur 1701.

Shahanaz Sultana
Senior Scientific Officer
Biotechnology Division, BRRI Head Office, Gazipur 1701.

Abstract/ Executive Summary:

Somaclonal variation is a useful source for introducing genetic variations that could be of value to plant breeders. Somaclonal variations have been created as the potential weapon for developing new rice varieties with desirable characters. The aim of the study was to create somaclonal variants for lower seed shattering of BRRIdhan 47 and optimize the effect of different hormones to achieve high frequency plant regeneration from embryogenic callus by seed culture. Seeds of BRRI dhan 47 were sterilized with 70% ethanol, Clorox 50%, Tween 20 and distilled water. These seeds were placed into callus induction medium consisting of MS basal salts (Murashige and Skoog, 1962) with 2.0 mgl^-1 (9µM) of 2,4-dichlorophenoxy acetic acid (2,4-D), Sucrose (30 gl^-1) and 0.8% agar. 15 days old calli were sub-cultured in the same media for 7 days. Then calli were transferred to regeneration media consisting of MS salts with four different hormone combinations: R-1: 2 mg/L BAP, 1 mg/L NAA and 1 mg/L Kinetin; R-2: 1 mg BAP and 0.5 mg/L NAA; R-3: 5 mg/L BAP and 1 mg/L NAA and R-4: 1 mg/L NAA and 1 mg/L Kinetin. These calli were kept in 16/8h light /dark for 30d. Regenerated plantlets at 8-10 cm height were planted in soil pots for hardening. Data on callus %, regeneration %, no. of green plants, and no. of albino plants were collected. The percentage of callus induction was found 71%. Regeneration % . Regeneration media R-1 and R-2 were performed better regeneration efficiency than R-3 and R-4. 79, 74, 22 and 14 plants were obtained from R-1, R-2, R-3 and R-4 media respectively. Regenerated plants were grown in glass house and seeds were harvested at maturity stage. These harvested seeds will be multiplied and used for further evaluation under salinity condition to observe the seed shattering. Future works are needed to utilize the great potential of development of somaclonal variation technology for rice development.

Keywords: Rice, Somaclone, Seed culture, Somaclonal variants.

Location: Biotechnology Division, BRRI Head Office, Gazipur 1701

Start Date: 01-07-2011

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

To create somaclonal variants for lower seed shattering from BRRI dhan47.

Methodology:

Mature healthy seeds of BRRI dhan 47 were manually de-husked. The seeds were sterilized with 70% ethanol for 1 minute. Then the seeds were sterilized with Clorox 50% and 2-3 drops of Tween 20 by shaking for 20 minutes. Again washed with Clorox 50% by shaking for 20 minutes, followed by 2-3 washing with sterile distilled water. About 450 seeds were blot dried under laminar air flow (LAF) on sterile tissue paper and placed into callus induction medium (CIM) consisting of MS basal salts (Murashige and Skoog, 1962) with 2.0 mgl^-1 (9µM) of 2,4-dichlorophenoxy acetic acid (2,4-D), Sucrose (30 gl^-1) were used as carbon source, and 0.8% agar was used as gelling agent. The medium (pH 5.8) were autoclaved 15 min at 121ºC. 10 seeds were placed in each of the petridish. Calli were sub-cultured in the same media after 15 days. Calli were transferred to regeneration media consisting of MS salts with four different hormone combinations: R1: 2 mg/L BAP, 1 mg/L NAA and 1 mg/L Kinetin; R2: 1 mg BAP and 0.5 mg/L NAA; R3: 5 mg/L BAP and 1 mg/L NAA and R4: 1 mg/L NAA and 1 mg/L Kinetin. These calli were kept in 16/8h light /dark for 30d. After 12-15 days green spots appeared on the callus surface and within 1 month plantlets were regenerated. Plantlets at 8-10 cm height were taken out from the culture tubes. These plantlets were planted in pots. Then seeds were harvested at maturity stage. Data of callus %, regeneration %, no. of green plants, and no. of albino plants were collected.

Source of Information: Proceedings of the BRRI Annual Internal Review for 2011-12, BRRI.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The percentage of callus induction was found 71%. Regeneration media R1: MS salts with 2 mg/L BAP, 1 mg/L NAA and 1 mg/L Kinetin and R2: MS salts with 1 mg BAP and 0.5 mg/L NAA; was performed better regeneration than R3: MS salts with 5 mg/L BAP and 1 mg/L NAA; and R4: MS salts with 1 mg/L NAA and 1 mg/L Kinetin. 79 and 74 plants were obtained from R1 and R2 media, 22 and 14 plants were obtained from R3 and R4 media. Total 189 regenerated plantlets of 8-10 cm height were taken out from the culture tubes. These plantlets were planted in pots for hardening. Survived 20 plants were grown in glass house. Then seeds were harvested at maturity stage. These harvested seeds will be multiplied for further study.

Knowledge Management: Report/Proceedings