Rokhshana Khatun
Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh
S.M. Shahinul Islam
Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh
M.A. Bari Miah
Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh
Androgenesis, Callus, Embryoids, Regeneration, Albinos, Oryza sativa L.
Crop-Soil-Water Management
Plant Material: Mature seeds of twenty five rice (Oryza sativa L.) cultivars namely BR-2, 3, 4, 6, 8, 9, 10, 11, 14, 15, 16, 19, 26, 29, 33, 35, 36, 45, Chini Shankar, IR-43, IR-54, Jira Shail, Jaya, Nayanmoni and Ratna were collected from BRRI, Regional Rice Research Center, Rajshahi and head office in Joydebpur, Gazipur, Bangladesh. Plants were grown in the field of the Institute of Biological Sciences, Rajshahi University during the growing seasons of June - November 2008. In vitro Micropropagation: Mature seeds were dehusked and surface sterilized primarily with 70% ethanol for one minute and rinsed them three - four times with sterile distilled water. Then seeds were sterilized in 45% Clorex supplemented with 1-2 drops of Tween-20 for 30 min by gentle shaking. Rinsed them five - six times with sterile distilled water and plated directly in semi-solid N6 [18] medium for callus induction, which supplemented with 2 mg/l 2, 4-D, 500 mg/l Casein hydrolysate, 3% sucrose and 0.7% agar. In the present study we have modified the medium to add 200 mg/l BAP, 550 mg/l L-proline and 500 mg/l LGlutamine with other components. After four weeks of incubation, data were taken on the basis of total callus induction frequency, embryogenic and non-embryogenic calli production. For this study only embryogenic calli were transferred to callus induction medium for multiplication. The pH adjusted to 5.6 - 5.8 before autoclaving. The Petri dishes were sealed with parafilm and incubated at 27±2°C in the dark for three - four weeks. Subculture was carried out once every two weeks. Anther Culture: For anther culture panicles were harvested when the microspores were at the early to mid uni-nucleated stage as observed by 1:3 acetocarmine staining test. Harvested spikes were subjected to cold pretreatment at 4°C. Selected panicles were sterilized with 70% ethanol for few seconds and transfer them to 0.1% mercuric chloride solution for three - five minutes. Finally rinsed five - six times by sterile distilled water and anther were carefully removed from the middle part of the panicles. An average of 30 - 40 anthers plated per Petri dishes (90 × 15 mm) containing liquid SK-3[19] medium for callus induction. Previously our group tested some induction media for anther culture of rice e.g. R2[20], N6 and SK-3 and found that SK-3 showed better embryoids induction than others. So why for this study SK-3 medium was considered. Around 1000 anthers were plated for each cultivar. The SK-3 medium was modified in which sucrose was substituted with maltose (60 g/l), in addition it contained NAA (2 mg/l), Lproline (200 mg/l) and L-glutamine (250 mg/l). Cultures were incubated at 28±2°C in dark for three - six weeks for embryo induction. Anther derived embryos were transferred to the regeneration medium (RRM, modified by MS), supplemented with 0.5 mg/l Kn, 1 mg/l NAA, 30g/l sucrose. Cultures were kept at 16/8h light/dark regime for better regeneration. Experiment for rice anther culture was carried out on transferring time: Cont. (0), T1 (10 days), T2 (20 days), T3 (30 days) and T4 (40 days). For each time point inoculated anthers with or without embryo like structures (ELS) were transferred from liquid induction medium to semi-solid regeneration medium. For this experiment IR-43 and Jaya cultivars were taken for their better performance on anther culture response with this study. Data Recording and Analysis: For in vitro regeneration data were recorded on total callus induction frequency for embryogenic status. Callus induction data were recorded after three weeks of inoculation and continued up to five weeks. Embryogenic calli were transferred to regeneration medium after four weeks and data on green plants were recorded up to next three-four weeks. Frequency of callus and green plants regeneration data were calculated according to the number of seeds produced calli per 100 and number of green plants per 100 embryogenic calli. For anther culture data were recorded on the basis of embryoids and regeneration by the following traits: ELS- expressed as the number of embryos per 100 anthers, green and albino plant regeneration per 100 ELS. For both cases well developed rooted plantlets were transferred to soil until maturity.
Journal of Applied Sciences Research, 6(11): 1705-1711, 2010
Journal