Optimization of in vitro culture media for improvement in yield of Navara ancient Indian medicinal rice

Abstract

Medicinally important ancient Navara rice (GI Kerala, India 2007) is a very short duration (60-70 days) variety with a yield of only 0.5 tonnes/hectare costing ~ Rs. 400/kg. It is used for indigenous treatment for chronic diseases by local and oral consumption. In this study, scutellum-derived calli were generated from mature Navara seeds and these were inoculated on different CIM-1 to CIM-5 media supplemented with 2.5 mg/l 2,4-dichlorophenoxyacetic acid. Regeneration of calli on different regeneration media RI, RII and RIII media were performed. Regeneration of 30-day-old calli on RI media showed 30%, for RII it showed no regeneration and on RIII media only 12% regeneration was obtained. The addition of glutamine and proline showed a 30-40% improvement in somatic embryogenesis. The 74-88% callus induction frequency was obtained on CIM-1 to CIM-5. The fresh weight (mg) of 30-day-old calli is CIM-2 < CIM-3 < CIM-4 < CIM-1 << CIM-5 and corresponding size shows CIM-2-CIM-3 < CIM-5 < CIM-1 < CIM-4. A negative correlation between the callus fresh weight and the regeneration efficiency was observed. In CIM-5, 20-25 days 3.4-fold increase and 25-30 days a 1.7-fold increase in fresh weight of calli is noted. The 20-day-old calli transfer to RI media shows 80% regeneration frequency and 6-7 plantlets/callus, which are twofold higher as compared with 30-day-old calli. The somatic embryogenesis and its regeneration on synthetic media provide an alternative for biotechnological intervention for yield improvement, in turn cost reduction for Navara rice.

Keywords: Callus; Navara; Plant tissue culture; Regeneration.

Fig. 1

The schematic diagram of the procedure used for seed sterilization, callus induction, regeneration and plantlet formation of Oryza sativa var. Navara

Fig. 2

Influence of additives with media (CIM-1) in callus induction frequency of Navara (15 days). The callus induction frequency (CIF) was determined on media containing A (Glutamine, Proline, Tryptophan and Casein hydrolysate), B (No additive), C (Glutamine), D (Proline), E (Tryptophan), F (Casein hydrolysate), G (Glutamine, Proline), H (Glutamine, Tryptophan), I (Glutamine, Casein hydrolysate), J (Proline Tryptophan), K (Proline, Casein hydrolysate), L (Tryptophan, Casein hydrolysate), M (Glutamine, Proline, Tryptophan), N (Proline, Tryptophan, Casein hydrolysate), O (Glutamine, Proline, Casein hydrolysate) and P (Glutamine, Tryptophan, Casein hydrolysate). Asterisk represents the significant p < 0.001 = ***, p < 0.01 = ** and p < 0.05 = *

Fig. 3

Influence of additives with media (CIM-1) in regeneration efficiency of calli of Navara after 20 days of transfer on RI media (Bars = 5 mm). Where A (Glutamine, Proline, Tryptophan and Casein hydrolysate), B (No additive), C (Glutamine), D (Proline), E (Tryptophan), F (Casein hydrolysate), G (Glutamine, Proline), H (Glutamine, Tryptophan), I (Glutamine, Casein hydrolysate), J (Proline Tryptophan), K (Proline, Casein hydrolysate), L (Tryptophan, Casein hydrolysate), M (Glutamine, Proline, Tryptophan), N (Proline, Tryptophan, Casein hydrolysate), O (Glutamine, Proline, Casein hydrolysate) and P (Glutamine, Tryptophan, Casein hydrolysate)

Fig. 4

Scutellum-derived callus on CIM-3 media. a After 15 days post-inoculation is seen as semi-compact, irregular shape and are light in yellow colour, b after 30 days post-inoculation loose compact are dark yellow colour. c In regeneration condition after 7 days post transfer, green spots were seen, d after 30 days post transfer, the regenerating calli show browning along with greening. Bar = 30 mm in a, 40 mm in b and d and 1 mm in c

Fig. 5

The diameter of the calli (mm) of Navara measured by Meazure™ 2.0 software after a 10 days, b 15 days post-inoculation raised on CIM-1, CIM-2, CIM-3, CIM-4 and CIM-5 supplemented with 2.5 mg/l 2,4-D, 3% sucrose, 500 mg/l proline and 500 mg/l glutamine and incubated at 27 ± 2 °C, 80% RH, in dark

Fig. 6

Correlation between the fresh weight of callus and regeneration efficiency using first two components from PCA approaches. a Classification of CIM-1 to CIM-5 on the basis of calli fresh weight, size, CIA (callus induction ability), CBW (callus browning), WYC (white/yellow/cream colour callus) and GSF (green spot frequency) b correlation loading of calli fresh weight and CIA on GSF

Fig. 7

Response of early transfer of calli is in RI regeneration media. The different aged (20-days, 25-days and 30-days) callus transferred to RI regeneration media (Bar = 2 mm). The callus raised on CIM-1 supplemented with 2.5 mg/l 2,4-D, 3% sucrose, 500 mg/l proline and 500 mg/l glutamine and incubated at 27 ± 2 °C, 80% RH, in dark