Aromatic rice varieties have become popular owing to their characteristic aroma and flavor. Among these rice varieties, Basmati rice is preferred for its unique aroma, hence, several new varieties were bred to produce new aromatic varieties (Mittal et al. 1996). Biological formations of aroma in vitro and studies on its enhancement have been of great interest to rice researchers. Buttery et al. (1983) and Lorieux et al. (1996) reported the characterization of aroma component of Basmati rice variety as 2-acetyl-1-pyrroline (2AP), but chemically synthesized 2AP did not contribute to the flavor that the natural aroma in rice varieties yielded. In a first report, Suprasanna et al. (1998) found that L-proline supplementation could yield an increase in aroma production in cell cultures of Basmati rice. Recently Yoshihashi et al. (2002) confirmed the role of proline as a precursor in the aroma formation in var. Khao Dawk Mali 105. In this report, a comparative response of aromatic and non-aromatic rice varieties to L-proline supplementation , alginate encapsulation and subsequent aroma synthesis are presented.

The following nine rice cultivars were used for the initiation of callus cultures: Basmati 370 (Bas), Pusa Basmati 1 (PB-1), Pusa 33 (P33), Kasturi (Kas), Indrayani (Ind), Manas Sarovar (MS), Jeera Rice (JR), IET 5656 (IET) and Kala Namak (KN). Seeds from each variety were dehusked, treated with 0.1% mercuric chloride for 6 min, and then rinsed with sterile distilled water for 3 - 4 times. Seeds were soaked overnight, and then inoculated onto callus induction medium (CIM): Murashige and Skoog's, (1962) medium with 2,4-D (2 mg/l), L-tryptophan (50 mg/l), sucrose 3% (w/v) and agar (0.8%). The cultures were incubated at 25 + or - 2C for about 2 - 3 weeks under an illumination of 1000 lux. Calli formed from the scutellar region was subcultured on CIM and regularly subcultured thereafter at an interval of 4 weeks. During the second subculture, the calli were transferred to CIM containing 500mg/l of L-proline or L-glutamine. Callus samples were checked for aroma (typical of Basmati rice) under both thermal and non- thermal condition using an organoleptic (sensory) evaluation (Hori et al., 1994). Normal aroma was given a value of 75, high aroma is scored 100 and no aroma was scored 0. After four weeks, callus cultures were scored for embryogenic response. Callus tissues were encapsulated in 3% sodium alginate to prepare beads according to the procedure of Suprasanna et al. (1996). The beads were incubated in liquid CIM medium with or without
proline. Subsequently, after removing the beads, the medium was checked for aroma by organoleptic evaluation.

All cultivars exhibited variation in callusing frequency (Bas 95%, Kas 100%, PB1 79%, Ind 35%, KN 29%, JR 30%, P33 77%, IET 50% and MS 52%). Subcultured calli of MS, Ind, PB1 and Bas were used for the aroma experiments. The cultures on medium with proline had higher aroma compared to those on medium without proline (Fig. 1). Sensory (organoleptic) evaluation confirmed the evolution of aroma. Proline addition enhanced the aroma of callus cultures of aromatic rice, PB1 and Bas, but not the aroma of non-aromatic rice, suggesting that proline was related to aroma synthesis and its formation was inherent to aromatic varieties. On the other hand, addition of glutamine did not yield any appreciable aroma compared to control callus. Although non-aromatic varieties exhibited an increase in embryogenic response, they

had no aroma. This suggests that proline has a dual role through osmotic adjustments towards aroma and/or embryogenesis.

Callus tissues of Basmati encapsulated in sodium alginate resumed their growth within few days of culture in a liquid medium supplemented with proline. Organoleptic evaluation of the spent medium exhibited an intense aroma, implying that aroma constituents were released into the medium, particularly under normal non-thermal condition. Generally, rice aroma is generated upon heating, however, the present observations indicate that rice aroma can be released into the medium under non-thermal condition, and hence, the system may be useful for continuous generation of aroma.

Proline is generally supplemented to the culture medium to enhance embryogenic response (Suprasanna et al. 1994, Holme et al. 1997). In this study, callus cultures were more embryogenic and produced more aroma on proline medium compared to those grown on medium without proline. Proline mediated stimulation of aroma has been observed in rice. Suprasanna et al. (1998) suggested that proline could be associated with the flavor production pathway . In a recent study, tracer experiments have established that proline was the nitrogenous source of 2AP, and proline acted as a precursor for aroma (Yoshihashi et al. 2002). Since flavor in aromatic varieties is generally influenced by water, salinity and temperature stress, flavor must be related to the accumulation of proline that plays a key role in aroma synthesis. Further studies should be made on the identification of key enzymes and related genes in the aroma pathway in rice.

References

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Holme, I.B., K. Peter and J. Hansen, 1997. Embryogenic callus formation, growth and regeneration in callus and suspension cultures of Miscanthus X ogiformis Honda Giganteus as affected by proline. Plant Cell Tiss. Org. Cult. 50: 203-210.

Hori, K., R.B.R.A. Purboyo, M. Jo, S. Kim, Y. Akinaga, T.Okita and M. Kang. 1994. Comparison of sensory evaluation of aromatic rice by consumers in East and Southeast Asia. J. Consumer Stud. Home Econ. 18: 135-139.

Loriex, M., M. Petrov., N. Huang, E. Guiderdoni and A. Ghesquire, 1996. Aroma in rice: genetic analysis of a quantitative trait. Theor. Appl. Genet. 93: 1145-1151.

Mittal, U,K., K. Preet, D. Singh, K.K. Shukla and R.G. Saina, 1995. Variability for aroma in some land races and cultivars of scented rice. Crop Improvement 22: 109-112.

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Suprasanna, P., Ganapathi, T.R. and P.S. Rao, 1996. Artificial seeds in rice (Oryza sativa L.): encapsulation of somatic embryos from mature embryo callus cultures. Asia Pacific J. Mol. Biol. & Biotechnol. 4: 90-93.

Suprasanna, P., Ganapathi, T.R., Ramaswamy N.K., Surendranathan, K.K. and P.S. Rao, 1998. Aroma synthesis in cell and callus cultures of rice. Rice Genet. Newsl. 15: 123-125.

Yoshihashi T., N.T.T. Huong and H. Inatomi, 2002. Precursors of 2-acetyl-1-pyrroline, a potent flavor compound of an aromatic rice variety. J. Agr. And Food Chem. 50: 2001-2004.