MSc

World is running towards 9 billion population by 2050 along with two serious issues in its hand– climate change and food insecurity. It can ultimately hamper the world to achieve Sustainable Development Goals (SGDs). High yielding and climate resilient crops are crucial towards accomplishing SDGs. Half of the world population relies on rice and it is also a model monocot crop which is widely used for the study of various crop improvement programs. Indica rice is the widely cultivated genotype across India. Improvement in quantitative and qualitative traits of rice is very important to battle against hunger and malnutrition. Micropropagation takes the lead towards crop improvement through various biotechnological approaches. In indica there is a research gap due to recalcitrant calli which is poor to regenerate under micropropagation conditions. The present study was undertaken to dissect the reason behind this limitation in indica.
In the current study, IR64 an indica genotype along with a check japonica genotype, Nipponbare were used. The manually dehusked surface sterilized seeds were inoculated in the callus induction media with various dosages of 2,4-D (0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg/L). The response of the seeds to 2,4-D was examined by callus induction frequency (%), callus size (mm), and fresh and dry weight of callus (g). The calli from 3 mg/L 2,4-D had maximum callus induction frequency in IR64 while in Nipponbare it was 100 % at 1.5 mg/L 2,4-D. The size, fresh weight and dry weight of the callus was maximum at 3 mg/L of 2,4-D in both genotypes. Calli from IR64 was compact and larger than that of Nipponbare. Considering all the dosages, calli from 3 mg/L 2,4-D performed best in both genotypes. The maximum size and weight might be the due to aggressive cell division and growth of the calli. This uncontrolled cell division can possibly affect the development of embryogenic calli. Hence calli from suboptimal concentration i.e., 2.5 mg/L of 2,4-D was selected for regeneration process.
Two to three week old calli from 2.5 mg/L 2,4-D were transferred to two regeneration media. The first media was supplemented with 0.05 mg/L NAA and 2, 2.5, 3 mg/L BAP, kinetin and the other with 0.5 mg/L NAA with same concentration of


cytokinins as in the first set of treatments. The differential response of the callus to the media was determined by recording the number of green calli, shoots, brown and hairy calli, chlorophyll and carotenoid content, secondary metabolites, and endogenous hormonal homeostasis. In general, green calli were more in the media with 0.5 mg/L NAA in both genotypes. IR64 had maximum number of green calli at 0.5 mg/L NAA with 3 mg/L BAP and Nipponbare at 0.05 mg/L NAA with 3 mg/L BAP. No shoots were regenerated from IR64 in both regeneration media types tested. In the media with 0.5 mg/L NAA shoots from Nipponbare were regenerated. Number of brown calli were more in IR64 than Nipponbare in both media. Maximum number of brown calli were found in the media with 0.05 mg/L NAA in IR64. Total number of hairy calli were higher in Nipponbare than in IR64 irrespective of the media. Chlorophyll content in IR64 was maximum at 0.05 mg/L NAA and in Nipponbare at 0.5 mg/L NAA. In both cases, media with BAP had maximum chlorophyll content. Carotenoid content was high in IR64 at 0.05 mg/L NAA. In the media with 0.5 mg/L NAA, higher carotenoid was found at BAP in IR64 and kinetin in Nipponbare. Secondary metabolites had both positive and negative roles in the regeneration of calli. Total phenols were significantly high at 0.05 mg/L in IR64 than at
0.5 mg/L NAA. Nipponbare had maximum phenol content at 0.05 mg/L NAA and 3 mg/L BAP and with no significant difference in other treatments for both the media types. Alkaloid content of calli was collectively less in the media with 0.5 mg/L NAA than 0.05 mg/L NAA. In IR64 the alkaloid content exhibited a two-fold increase at 0.05 mg/L NAA. Nipponbare maintained steady alkaloid content in almost all treatments in both media. As auxin and cytokinin ratios are critically important, in this study auxin content and IAA oxidase activity were determined. Higher auxin content in both genotypes has recorded at the media supplemented with 0.05 mg/L. In the media 0.5 mg/L NAA the auxin content was reduced in IR64 and also had improved auxin homeostasis.
After four weeks, shoots of Nipponbare were transferred to the ½ x MS media without NAA and 0.5 mg/L NAA. The efficacy of the rooting media was determined by observing root number, total root length, dry weight of roots and shoots. Media without hormone failed to produce roots however the media with 0.5 mg/L NAA had the positive


effect of rooting irrespective of the shooted calli from different treatments. The present study points that IR64 exhibited considerably good green calli number, better accumulation of chlorophyll content than Nipponbare. But better regeneration of Nipponbare may be attributed to its friable nature of the calli along with steady maintenance of the secondary metabolites and auxin content. The poor performance of IR64 might be due to higher levels of phenolics and alkaloids accumulation. These findings also give insights into the importance of auxin-cytokinin signaling crosstalk and auxin transport during shoot regeneration.