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Indirect Organogenesis and Somaclonal Variation in Four Rice Cultivars of Bangladesh

A.H. Kabir
Department of Botany, Rajshahi University, Bangladesh.

Istiak Mahfuz
Department of Zoology, Rajshahi University, Bangladesh.

M.A. Razvy
Department of Botany, Rajshahi University, Bangladesh.

M. Bulbul Ahmed
Department of Botany, Rajshahi University, Bangladesh.

M.F. Alam
Department of Botany, Rajshahi University, Bangladesh.

Abstract/ Executive Summary:

An investigation was made on the initiation and maintenance of callus from explants (mature seeds) of four rice cultivars, viz., BRRI-28, BRRI-29, BRRI-30 and BRRI-32. Mature seeds were used as explants. MS medium supplemented with 2 mgL of 2, 4-D was found suitable for inducing high -1 amount of embryogenic calli in the studied genotypes. In this hormonal concentration, maximum 86.11% callus was induced in BRRI-28, followed by 80.55% in BRRI-30. The range of shoot was 1-8 and the highest mean number of shoot were (5.8±0.67) obtained from calli by using MS medium containing 0.5 mgL BAP and 0.1 mgL IBA in BRRI-28. Maximum shoot induction (85.33%) was also observed -1 -1 in the same media component in BRRI-28. BRRI-28 has been found most suitable for in vitro culture among the four genotypes used. Plants regenerated in vitro were successfully established in soil and produced fertile seeds. The regenerated plants showed wider range of variability in comparing with different agronomic traits. The wider variability among the regenerated plants shows the use of in vitro culture technique for creating genotypic variability and subsequent selection for desirable variants.

Keywords: Callus induction, Panicle, Variation, Transplantation

Location: Regional Research Station of Bangladesh Rice Research Institute (BRRI), Rajshahi, Bangladesh.

Start Date:

End Date:

Program Area: Socio-economic and Policy

Commodity: Rice

Objectives:

The aim of this research was to characterize somaclonal variation in populations derived from callus, as well as to select and characterize somaclonal lines in four rice cultivars in Bangladesh.

Methodology:

Plant Materials: The experimental materials (BRRI-28, BRRI-29, BRRI-30, BRRI-32) were collected from the Regional Research Station of Bangladesh Rice Research Institute (BRRI), Rajshahi, Bangladesh. Mature rice grain embryos were used as the sources of primary explant. Mature seeds were used as explant. Mature seeds of the studied cultivars were dahusked carefully so that the embryo remains intact. In explant selection diseases and unhealthy seeds were avoided. Surface Sterilization: The dehusked seeds were surface sterilized with 70% ethanol for 3-5 minutes followed by treatment with savlon. After sterilization, the explant were washed 3-4 times with distilled water and placed on sterilized filter paper to remove excess water before inoculating them on to callus inducing or germinating medium. In this procedure dehusked seed were washed thoroughly with distilled water adding with few drops of 'Tween-80'. After this the seeds were transferred to sterilized conical flask containing 0.1% mercuric chloride and treated for three minutes with constant shaking inside the laminar air flow cabinet. After that the sterilized material were washed 7-8 times with sterile distilled water to remove the effect to HgCl. 2 Incubation: All the inoculated culture tubes and flasks were incubated in a growth chamber providing a special culture environment. The tubes were placed on the shelves of a culture rack in the growth chamber. The culture flasks and tubes kept in the dark at 25 - 26 C throughout the incubation period. The cultures 0 were checked time to time for monitoring the response. Callus Induction: Callus is a coherent but unorganized and amorphous tissue, formed by the vigorous division of plant cells. For callus induction MS medium supplemented with different concentration of 2, -D (1, 2, 3, 4 mgL ) either along or in combination with -1 other growth regulators (BAP, KIN). The primary callus induced from explants was sub-cultured for further proliferation and developing embryogenic nature. The frequency of explant forming embryogenic callus, degree of callus growth, color and texture were recorded after 6 - 9 weeks of culture. After 5-7 days of callus initiation, the primary tiny calli of explants were separated aseptically from the source of explants, so that no contact of parental tissue remained and set them again on the same medium for proliferation of calli without root-shoot differentiation. Repeated sub-culturing was done after every two weeks for maintenance and proliferation of the calli. The quantitative measurement of callus growth was estimated in terms of percentage of callus and degree of callus growth. Plant Regeneration: After desired passage time the developing embryogenic calli were selected and tested their regeneration ability using different kinds and concentrations of cytokinin and auxins. The cultures were incubated at 24 C- 26 C under white light 0 0 (28-34 PPF) for 14/10 hours light/dark condition. After 4-5 weeks differentiation of shoots and roots were observed. The number of calli producing plantlets and the total number of plants were counted for each treatment. Shoots having insufficient root system was not suitable to transfer into soil. So when the shoots attained a size of 3-5 cm they were aseptically separated from each other. After that they were transferred to half strength of MS salts and vitamins, supplemented with 2% sucrose without growth regulators for root proliferation. Transplantation: The regenerated plants with well developed sufficient root system were ready for transfer to soil. When the plantlets remained in culture they were brought out of the controlled environment of growth room and were kept in the room temperature for 2-3 days to bring then in the contact of normal temperature. The plantlets were then rescued very carefully from the culture tubes. Agar attached to the root was washed gently under running tap water. Immediately after that they were transplanted to small pots containing sterilized ground soil, sands and cowdung in the ratio of 1:2:1. The pots with plantlets were kept in shade place and necessary cultured management was undertaken for good growth and development of the plant. After 7 days, the pots with plants were transferred to direct sunlight and after 15-25 days plantlets were finally transferred in new pots.

Source of Information: Journal of Applied Sciences Research, 4(4): 451-458, 2008

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The regenerated plants showed wider range of variability in comparing with different agronomic traits. The wider variability among the regenerated plants shows the use of in vitro culture technique for creating genotypic variability and subsequent selection for desirable variants.

Knowledge Management: Journal