Selim Morshed
Plant Genetic Engineering Lab., Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
Abu Baker Siddique
Plant Genetic Engineering Lab., Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
S. M. Shahinul Islam
Plant Genetic Engineering Lab., Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
Maize, Callus induction, Immature and mature embryo, In vitro regeneration, Somatic embryogenesis
Bangladesh Agriculture Research Institute (BARI), Gazipur, Institute of Biological Sciences, University of Rajshahi, Bangladesh.
Variety and Species
For this study mature seeds of four maize varieties viz. Barnali, Mohar, Khoi bhutta and Shuvra were collected from the Bangladesh Agriculture Research Institute (BARI), Gazipur, Bangladesh. For cultivation, seeds were sown in the research field of Institute of Biological Sciences, University of Rajshahi, Bangladesh. From the plants grown in research field, the young and mature cobs were collected. The young cobs and mature seeds were used as the source of explants i.e. immature embryos (IE) and mature embryos (ME) respectively. Both types of explants were sterilized with 70% (v/v) ethanol for 1.5 minutes and in sodium hypochlorite (NaOCl) for 25 minutes. Then they were sterilized with 0.1% (v/v) mercuric chloride (HgCl2) for 5 minutes and washed with double distilled water. Culture media, inoculation and callus induction After sterilization, immature and mature seeds were inoculated on callus induction media (CIM); and after sealing with paraflim, petri dishes were kept under dark conditions at 25 ± 2°C.Three basal media, such as MS, N6 and 6N1 were individually supplemented with four different concentrations of 2, 4-D (0.5, 1.0, 1.5 and 2.0 mg/l). The other supplements of CIMs were 2.87 mg/l L-proline, 0.1 g/l casein hydrolysate, 2% sucrose and 0.01 g/l silver nitrate (AgNO3) as constant. Eight percent agar was used as gelling agent and pH 5.8 was adjusted for all the media. Maintenance and formation of embryogenic callus After inoculation, 10 days (d) old calli were transferred to CIM for their maintenance, proliferation and embryogenic callus formation. The calli age of 3-4 weeks (w) after inoculation were visually observed and the qualitative features on color and texture were recorded; along with the weight was determined as quantitative features. The frequencies of callus induction (CI) were calculated by using formula. The calli age of 3-4 w, derived from immature and mature embryos were transferred separately to regeneration media (RM) for plant regeneration. Three basal media, such as MS, N6 and 6N1 were individually supplemented with different concentrations and combinations of IAA (0.0, 0.5, 1.0, 1.5 mg/l) and/or BAP (0.5, 1.0, 1.5, 2.0 mg/l). Regenerated shoots, length of 2 cm were transferred to RM for shoot elongation. After 4 w of transfer, the lengths of shoots were measured and the shoot lengths were determined by deducting 2cm from the measured length. Regenerated shoots, height of 3-4 cm were transferred to root induction media (RIM). In this case, the basal media MS, N6 and 6N1 were individually supplemented with IAA (0.0, 0.5, 1.0, 1.5 mg/l) and/or BAP (0.5, 1.0, 1.5, 2.0 mg/l). The well rooted plant lets were transferred to plastic cups containing autoclaved soil and sealed with polythene bags. After acclimatization, plants were transferred to natural condition for further study and seed collection. The average or mean values were computed from five replicates with standard error (SE) and each experiment was repeated thrice. Analysis of variance (ANOVA) and list significant difference (LSD) was done by SPSS 16.0 software.
International Journal of Agriculture Innovations and Research Volume 3, Issue 3, ISSN (Online) 2319-1473
Journal