Aroma synthesis in cell and callus cultures of rice

24. Aroma synthesis in cell and callus cultures of rice

P. SUPRASANNA, T.R. GANAPATHI, N.K. RAMASWAMY, K.K. SURENDRANATHAN and P.S. RAo

Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Bombay 400

One of the emerging areas in plant biotechnology is the production of value added compounds in cell and tissue cultures. The biotechnological approach for flavour production offers advantages of non-dependency over the environmental influence and creation of non-exhaustible source of homogenous and well-defined products (Krings and Berger 1998).

Rice is a staple food for millions all over the world and a considerable number of rice varieties are cultivated across different regions. Among different rice varieties, Basmati rice is preferred for its characteristic aroma and high export value in the international market. Earlier studies have reported the characterization of aroma component of Basmati rice to be 2-acetyl-1-pyroline (Buttery et al. 1983). In vitro cultures offer a suitable system for the study of aroma synthesis. The main objectives of our investigation were to study the production of Basmati rice aroma in cell/callus cultures, and develop a system for continuous aroma production. In this report, we present our results on the over-production of aroma in Basmati rice callus cultures, characterization of its uniqueness in comparison to aroma from seed samples of Basmati and immobilization (encapsulation) of cell/callus cultures.

24. Aroma synthesis in cell and callus cultures of rice P. SUPRASANNA, T.R. GANAPATHI, N.K. RAMASWAMY, K.K. SURENDRANATHAN and P.S. RAo Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India One of the emerging areas in plant biotechnology is the production of value added compounds in cell and tissue cultures. The biotechnological approach for flavour production offers advantages of non-dependency over the environmental influence and creation of non-exhaustible source of homogenous and well-defined products (Krings and Berger 1998). Rice is a staple food for millions all over the world and a considerable number of rice varieties are cultivated across different regions. Among different rice varieties, Basmati rice is preferred for its characteristic aroma and high export value in the international market. Earlier studies have reported the characterization of aroma component of Basmati rice to be 2-acetyl-1-pyroline (Buttery et al. 1983). In vitro cultures offer a suitable system for the study of aroma synthesis. The main objectives of our investigation were to study the production of Basmati rice aroma in cell/callus cultures, and develop a system for continuous aroma production. In this report, we present our results on the over-production of aroma in Basmati rice callus cultures, characterization of its uniqueness in comparison to aroma from seed samples of Basmati and immobilization (encapsulation) of cell/callus cultures. Callus cultures were established from mature seed explants of rice cv. Basmati 370 on MS medium supplemented with 2,4-D (Fig. 1). The callus was found to exhibit typical Basmati aroma and upon heating, the same was found to be intense. Cell suspension cultures raised from this callus also contained the same aroma. Several precursor-like compounds or intermediates (such as L-proline, hydroxy-proline, acetyl CoA, sodium acetate and pantothenic acid), possibly related to flavour production pathway were tested, to see their effect on flavour production. Proline in the medium was found to enhance the aroma as compared to control (untreated Basmati callus) and other compounds, based on the organoleptic evaluation. Treated and untreated samples as well as samples from a non-Basmati rice variety were subjected to the evaluation in the heated and non-heated condition and in both cases, treatments with L-proline followed by acetyl CoA had a positive influence on aroma production. Encapsulation of callus tissues was carried out with 3% sodium alginate as per the procedure described earlier (Suprasanna eta!. 1996). The encapsulated beads incubated in liquid MS basal mediul for 2-3 weeks were found to possess the aroma in the medium. The authenticity of the aroma components was confirmed by determining the gas chromatographic profiles of the aroma concentrates extracted from Basmati rice seed (5 Kg) and Basmati rice callus (120 gm). Aroma concentrate from both had identical profiles (Fig. 2). The concentration of the principal aroma constituents in callus subjected to over-production process was at least 40-50 times more as compared to its concentration in rice seed samples. The number of peaks and pattern were identical in both cases confirming that addition of proline to the culture media resulted in aroma production in cell suspension cultures and callus. Fractions separated on silica gel were organoleptically evaluated for aroma and positive ones were eluted from TLC plates. Efforts are being made to identify the individual components responsible for aroma development in cell suspension and callus cultures as well as to optimize methods for large scale cell cultures for enhanced aroma production which can be used in the generation of aroma principle.

Callus cultures were established from mature seed explants of rice cv. Basmati 370 on MS medium supplemented with 2,4-D (Fig. 1). The callus was found to exhibit typical Basmati aroma and upon heating, the same was found to be intense. Cell suspension cultures raised from this callus also contained the same aroma. Several precursor-like compounds or intermediates (such as L-proline, hydroxy-proline, acetyl CoA, sodium acetate and pantothenic acid), possibly related to flavour production pathway were tested, to see their effect on flavour production. Proline in the medium was found to enhance the aroma as compared to control (untreated Basmati callus) and other compounds, based on the organoleptic evaluation. Treated and untreated samples as well as samples from a non-Basmati rice variety were subjected to the evaluation in the heated and non-heated condition and in both cases, treatments with L-proline followed by acetyl CoA had a positive influence on aroma production.

Encapsulation of callus tissues was carried out with 3% sodium alginate as per the procedure described earlier (Suprasanna eta!. 1996). The encapsulated beads incubated in liquid MS basal mediul for 2-3 weeks were found to possess the aroma in the medium.

The authenticity of the aroma components was confirmed by determining the gas chromatographic profiles of the aroma concentrates extracted from Basmati rice seed (5 Kg) and Basmati rice callus (120 gm). Aroma concentrate from both had identical profiles (Fig. 2). The concentration of the principal aroma constituents in callus subjected to over-production process was at least 40-50 times more as compared to its concentration in rice seed samples. The number of peaks and pattern were identical in both cases confirming that addition of proline to the culture media resulted in aroma production in cell suspension cultures and callus. Fractions separated on silica gel were organoleptically evaluated for aroma and positive ones were eluted from TLC plates. Efforts are being made to identify the individual components responsible for aroma development in cell suspension and callus cultures as well as to optimize methods for large scale cell cultures for enhanced aroma production which can be used in the generation of aroma principle.

Krings, U. and R.G. Berger, 1998. Biotechnological production of flavours and fragrances. App!. Microbiol. Biotechn. 49: 1-8.

Buttery, R.G., L.C. Ling and B.O. Juliano, 1983. Cooked rice aroma and 2 acetyl-1-pyroline. Jour. Agr. Food. Chem. 31: 823-826.

Suprasanna, P., T.R. Ganapathi and P.S. Rao, 1996. Artificial seeds in rice: encapsulation of somatic embryos derived from mature embryo derived callus cultures. Asia Pacific Jour. Mol. Biol. & Biotechnol.