An immense increase in human population along with diminished lands necessitates the increase of rice production since, it serves the human population as a staple food. Though rice hybrids (RH) are showing considerable yield enhancement over inbreds in terms of both quality and quantity, farmers' adoption of hybrid rice technology has been much slower than expected because of several constraints such as seed cost and quality. Doubled haploid (DH) technology was considered useful for the development of inbred lines from rice hybrids in a single generation. Androgenesis shows its significance in development of DHs in rice which requires an efficient method to establish the production of large population. To start the anther culture, anthers are the main component of androgenesis to be isolated from unopened spikes. However, the duration of spikes availability for anther culture coupled with the segregation of rice hybrids in the next generation requires the main crop be ratooned to reduce the cost of cultivation. Therefore, the efficiency of the androgenic method was tested in main crop using a quality indica rice hybrid, 27P63 and its ratooned ones. The effects of various factors such as cold temperature pre-treatment of boots, treatment duration, and different combination of plant growth regulators (PGR) on callus response along with shoot regeneration were tested for development of DHs from both ratooned and non-ratooned plants. The N6 medium supplemented with 2.0 mg/L 2,4-D (2,4-dichlrophenoxy acetic acid), 0.5 mg/L BAP (6-benzylamino purine), and 30 g/L maltose was found to be most effective for callusing as compared to MS (Murashige and Skoog) medium. The N6 media inducted calli showed maximum response rate for green shoot regeneration in MS media supplemented with 0.5 mg/L NAA (1-napthaleneacetic acid), 0.5 mg/L Kn (Kinetin; 6-furfurylaminopurine), 1.5 mg/L BAP and 30 g/L sucrose after 2 weeks of culture. The pre-treatment of spikes at 10 °C for 2 d followed by a 7th and 8th d were found to be most effective for callusing as well as for regeneration, producing a total of 343 green plants from ratooned and main rice hybrid, 27P63. Morpho-agronomic trait-based assessment of ploidy status revealed 94.46% diploids, 3.49% polyploids, 0.58% mixploids, and 1.45% haploids. Microsatellite markers could authenticate all 324 fertile diploids as true DHs. Though this study shows a reduction in generation of DHs from ratooned plants as compared to the main crop, manipulation of chemical factors could optimize the method to enhance the production of considerable number of DHs. Utilization of ratooned of hybrid rice in androgenesis would save time and cost of cultivation.
Keywords: androgenesis; doubled haploid; hybrid rice; ratoon; regeneration; spike.