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In Vitro Callus Induction and Regeneration Potentiality of Aromatic Rice (oryza Sativa L.) Cultivars in Differential Growth Regulators

Zahida Yesmin Roly
Departmentof Genetic Engineering and Biotechnology, Faculty of Life and Earth Science, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Mahmudul Islam
Departmentof Genetic Engineering and Biotechnology, Faculty of Life and Earth Science, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Pallob Ebna Shaekh
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Science, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Saiful Islam Arman
Department of Pharmacy, Faculty of Science, Universityof Rajshahi, Rajshahi-6205, Bangladesh.

Shah Md. Shahik
Department of Genetic Engineering and Biotechnology, Faculty of Biological Science, University of Chittagong, Chittagong-4331, Bangladesh

Dipesh Das
Department of Genetic Engineering and Biotechnology, Faculty of Biological Science, University of Chittagong, Chittagong-4331, Bangladesh

Md. Mujjammil E Haamem
Department of Genetic Engineering and Biotechnology, Faculty of Biological Science, University of Chittagong, Chittagong-4331, Bangladesh

Md. Khalekuzzaman
Professor, Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Science, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

Aromatic rice (Oryza sativaL.) cultivars are strong aromatic rice cultivars which can thrive well in rice fields prone to flood, drought and other soil constraints. The present investigation was undertaken to determine a suitable media compositions for callus induction and regeneration using immature embryo of six aromatic grown rice cultivars of Bangladesh, namely, Chinigura, Kalijira, Radhuni Pagal, Modhumala, Kataribog and Mohonbhog. For callus induction different concentrations and combinations of 2, 4-Dichlorophenoxyaceticacid (2,4-D) along with NAA were evaluated. Maximum callus induction (97.22%) was observed in Kalijirawhen 2 mg/L of 2, 4-D and 0.5 mg/l NAA was used and less Modhumala (66.67%) and remaining cultivars showed moderate. For regeneration, initially different concentrations and combinations of 6-BenzylAminoPurine (BAP) and Indole-3-Acetic Acid (IBA) were tested. Maximum regeneration frequency (91.67%) was observed Kalijira when the optimum concentrations and combinations of 0.5 mg/l of BAP + 0.1 of mg/l IBA were used. Presently optimized regeneration method holds promise for facilitating the deployment of the agronomical important trait through genetic transformation for the improvement of these important food crops.

Keywords: Oryza sativa;callus;embryogenic;regeneration;hormones

Location: Bangladesh,

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

(1) to identify a suitable medium for callus induction using immature embryos of six aromatics rice cultivars (2) to determine a suitable media composition for plant regeneration (3) to find out the highly regenerable cultivar among six aromatic rice (Oryza sativa L.) cultivars from mature dehusked seeds.

Methodology:

Plant material Immature zygotic embryo of Six aromatic grown rice varieties (Oryza sativaL.) of Bangladesh, namely, Chinigura, Kalijira, Radhuni Pagal, Modhumala, Kataribog and Mohonbhog were used as explants for study of in vitrocallus induction and regeneration. Mature seed of threeIndicarice genotypes (Oryza Sativa L.) Chinigura, Kalijira, Radhuni Pagal, Modhumala, Kataribog and Mohonbhog were collected from Bangladesh Rice Research Institute (BRRI), Regional office, Rajshahi, Bangladesh. Seeds were sown in the department field. Immature embryo isolation Immature embryos, unripe seeds were collected 12 to 15 days after anthesis from field-grown plants and surface-sterilized with HgCl2(0.1%) for 10 min. These seeds were thoroughly washed four to five times in sterile distilled water. Using fine scalpels and forceps, immature embryos were excised under a binocular microscope. Culture media and culture conditions basic medium (BM) was composed of MS salts and organic compounds, 30g/l sucrose and 8g/l agar. The pH was adjusted to 5.7 before adding the gelling agent and media were autoclaved for 20 min at 121⁰C and 1.07 kg/cm2. Petridishes with 25 ml of medium and sealed with Parafilm were used. The immature embryos were cultured aseptically on nutrient media in the dark at 25±1°C with the scutellum side facing upwards. Callus induction Ten immature embryos from isolated sterilized seeds were placed individually in each Petri dish containing 25 ml of modified MS with various concentrations of 2, 4-D and NAA. The seeds were incubated in the dark at 25 ± 2ºC. Only embryogenic calli were transferred to a fresh callus induction medium for multiplication. Sub-culturing of the callus was carried out once every two weeks. The callus was observed from 2nd to 7thweek. The percentage (%) of callus (total/embryogenic) induction frequency (CIF) for each group was calculated as follows: Selection of embryogenic callus Embryogenic callus of indica rice (Oryza sativa L.) cultivars namely Kalijira, Chinigura, Radhuni Pagal, Modhumala and Mohonbhog can be described as yellowish and granular callus, compact, greenish-yellow, granular calli. These types of EC were selected and used for regeneration.RegenerationThe green colour embryogenic calli were then transferred to fresh shoot regeneration medium; MS with BAP (2.0-2.5 mg/l), NAA (1.5 mg/l) and kinetin (1 mg/l) and incubated under light condition. The well-developed calli with shoot primordia were sub-cultured on MS shooting regeneration medium in a test tube and incubated at 27°C under continuous light. Healthy shoots with defined stem were transferred to MS rooting medium and incubated at 28°C under continuous light. The plantlets with a well-developed root system were planted in the pots containing autoclaved mud that was collected from rice fields. The plantlets were established in planted in several pots. BRRI), Regional Office, Rajshahi, Bangladesh. Seeds were sown in the department field. Immature embryo isolation Immature embryos, unripe seeds were collected 12 to 15 days after anthesis from field-grown plants and surface-sterilized with HgCl2(0.1%) for 10 min. These seeds were thoroughly washed four to five times in sterile distilled water. Using fine scalpels and forceps, immature embryos were excised under a binocular microscope. Culture media and culture conditions The basic medium (BM) was composed of MS salts and organic compounds, 30g/l sucrose and 8g/l agar. The pH was adjusted to 5.7 before adding the gelling agent and media were autoclaved for 20 min at 121⁰C and 1.07 kg/cm². Petridishes with 25 ml of medium and sealed with Parafilm were used. The immature embryos were cultured aseptically on nutrient media in the dark at 25±1°C with the scutellum side facing upwards. Callus induction Ten immature embryos from isolated sterilized seeds were placed individually in each Petri dish containing 25 ml of modified MS with various concentrations of 2, 4-D and NAA. The seeds were incubated in the dark at 25 ± 2ºC. Only embryogenic calli were transferred to a fresh callus induction medium for multiplication. Sub-culturing of the callus was carried out once every two weeks. The callus was observed from 2nd to 7^th week. The percentage (%) of callus (total/embryogenic) induction frequency (CIF) for each group was calculated.

Source of Information: Int J Appl Sci Biotechnol, Vol 2(2): 160-167 (2014)

Article Link (Online Journal): http://ijasbt.org&http://nepjol.info/index.php/IJASBT

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

For regeneration via callus different combination of auxin and cytokinins were tried. The combinations were BAP + IBA, BAP+NAA, BAP+IAA, KIN+NAA and KIN+IAA. The results from these treatments varied upon with cultivars. The combination of BAP+IBA was best for regeneration efficiency. The combination of 0.5 mg/l of BAP + 0.1 of mg/l IBA showed the highest percentage of plant regeneration (Chinigura, 83.33%, Kalijira, 91.67%, Radhuni Pagal, 83.33%, Modhumala, 66.67%, Kataribog, 66.67% and Mohonbhog, 66.67%). In respect of all cultivars, Kalijira gave the best result, where the remaining cultivars were comparatively low. This result was confirmed the earlier results of Agrawal et al., (2005). This shows the selection of genotype is a key factor for in Vitro study in rice. Variability of plant regeneration efficiency in rice genotypes is also reported by earlier workers (Khanna and Raina 1998; Lee et al.,2002).

Knowledge Management: Journal