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Haploid Plantlet Regeneration Through Anther Culture in Oilseed Brassica Species

M. A. ALAM
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202

M. A. HAQUE
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202

M. R. HOSSAIN
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202

S.C. SARKER
Department of Agroforestry, Bangladesh Agricultural University, Mymensingh 2202

R. AFROZ
Scientific Officer
Bangladesh Agricultural Research Institute, Joydebpur, Gazipur 1701, Bangladesh.

Abstract/ Executive Summary:

Anther of five varieties of Brassica species, namely BARI Shariaha-7, Tori-7, Agrani, Daulat and Safal were cultured in vitro to observe their regeneration potentiality. Different concentrations and combinations of growth regulators were supplemented in MS medium. The range of callus induction was 12.50- 87.50 %. Maximum callus induction (75.00%) was observed on MS +4 mg/L 2, 4-D + 1.0 mg/L BAP. Among the genotypes, BARI Sharisha-7 showed the highest percentage of callus induction (60.42%). Among the treatments, highest percentage of shoot regeneration (75.00%) was observed on MS + 4 mg/L BAP + 1.0 mg/L NAA. BARI Sharisha-7 also showed the highest rate of plant regeneration (66.67%). Root induction was highest (75%) on half strength MS medium supplemented with 1.0 mg/L IBA and 0.5 mg/L NAA. The plantlets with sufficient roots thus obtained were transferred successfully to plastic pots and subsequently to the field. BARI Sharisha-7 and Tori-7 survived easily in the pots as well as in the field but Safal was very poor in survivability both in the pots and in the field.

Keywords: Brassica, Haploid, Anther culture, In vitro regeneration

Location: Bangladesh Agricultural University, Mymensingh

Start Date: 00-08-2006

End Date: 00-05-2007

Program Area: Variety and Species

Commodity: Mustard

Objectives:

To observe regeneration potentiality of Brassica species.

Methodology:

The experiment was carried out during the period from August 2006 to May 2007 at the Tissue Culture Laboratory and the Biotechnology and Genetic Engineering (BGE) Laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh. The seed materials of five varieties of indigenous Brassica species (BARI Shariaha-7, Tori-7, Agrani, Daulat and Safal) were collected from Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur and Bangladesh Institute of Nuclear Agriculture (B1NA), Bangladesh Agricultural University Campus, Mymensingh. Different media compositions were used to investigate the regeneration potentiality. MS medium supplemented with 1.0, 2.0 and 4.0 mg/L 2,4-D along with the constant addition of 1.0 mg/L BAP were used for callus induction. Likewise, MS medium supplemented with 2.0, 3.0 and 4.0 mg/L BAP along with the constant addition of 1.0 mg/L NAA were used for shoot initiation, while half strength MS (Murashige and Skoog, 1962) medium with supplementation of 1.0, 2.0 and 3.0 mg/L IBA with 0.5 mg/L NAA in each treatment were used for root initiation. Before culture, surface sterilization of flower buds was carried out in the Laminar Air Flow Cabinet. They were rinsed in 0.1% HgCI₂ for one minute, and then thoroughly washed with sterilized distilled water for three times. Then the following culture methods were employed in the present study: Flower buds of all varieties were collected at the late uninucleate stage in the morning before 10 a.m. Flowers of the Brassica varieties whose bud condition were used as explant (anther and filament) source are presented in plate 1 & 2. The developmental stages of the microspores were determined by studying microscopic slides prepared according to the acetocarmine squash method. Collected flower buds were wrapped in aluminum foil and kept in a refrigerator at 5°C for 24 hours. Anthers were aseptically removed from flower buds using fine tweezers and inoculated into 8cm petridishes each containing 10ml MS medium with different hormone concentrations viz., T_1-MS medium containing l mg/L 2,4-D + 1.0 mg/L BAP, T₂-MS medium containing 2 mg/L 2,4-D + 1.0 mg/L BAP and T₃_- MS medium containing 4 mg/L 2,4-D + 1.0 mg/L BAP with four replications. Cultures were maintained in an incubator at 250 ± 1°C temperature in complete dark condition for callus induction and checked to record the response. Six to seven weeks after inoculation of anthers, the regenerated calli attained convenient size. Then they were removed aseptically from the existing medium to a sterilized culture vessel inside the laminar Air Flow Cabinet. The calli were cut into few pieces and again placed them into the small vials with shoot regeneration media. Sub-culture was done on the MS media containing different concentrations of BAP and NAA. The vials were incubated at 25±1°C with 16 hours photoperiod. The sub-cultured calli contained to proliferate and differentiate into shoots when these shoots grew 2-3 cm in length. They were rescued aseptically from the cultured vials and were separated from each other and again cultured on vials with freshly prepared root induction medium to induce root. The vials containing plantlet were incubated under continuous light. Day to day observation was carried out to note the response of the growing plantlets. For the preparation of pots garden soil, sand and cowdung in the ratio of 1:2:1 were mixed properly and autoclaved for one hour in 1210 C at 1.6 kg/cm2 . Then the soil mixtures was allowed to cool in normal temperature and poured into 10 cm plastic pots for growing the plantlets at in vivo condition. When the plantlets reached 6-10 cm in length with sufficient root system then they were taken out from the vials. Medium attached to the roots were gently washed out with running tap water. The plantlets were then transplanted to pots containing the potting mixture. Immediately after transplantation, the plants along with the pots were covered with polythene bag to prevent desiccation. To reduce sudden shock, the pots were kept in growth room for 7-15 days under controlled environment.

Source of Information: Bangladesh J. Agril. Res. 34(4) : 693-703, December 2009, ISSN 0258-7122

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In this experiment, in vitro regeneration potentiality of five Brassica varieties has been observed and an efficient as well as reproducible protocol for regeneration of the genotypes has been developed using anther as explants. Since genetic engineering of crop plants relies on the development of efficient methods for the regeneration of viable haploid plantlets (using anther), this protocol can be followed for genetic manipulation for improvement of Brassica species. Considering the findings, further investigation is required for the callus induction and subsequent haploid production of the five varieties of Brassica by changing the type of media, hormonal composition and by trying additional growth regulators rather than those were used.

Knowledge Management: Journal