Plant Cell Reports
Plant Cell Reports ( 1986) 5: 446- 447
© Springer-Verlag 1986
Flavor formation in tissue cultures of garlic (Allium sativum L.) N. P. Malpathak and S.B. David Department of Botany, University of Poona, Pune 411 007, India Received February, 1986 / Revised version received July, 1986 - Communicated by F. Skoog
ABSTRACT Differentiated and undifferentiated cultures of garlic (Allium sativum L-) were analyzed for the study of flavor formation in cultures. Attempts were made to correlate alliin content with free and bound amino acid contents and with enzymes like phenylanine ammonialyase (E.C. 4.1.1.5) and alliinlyase (E.C.4.4.1.4) which play important roles in formation of the flavor percursor alliin. It was observed that in differentiating cultures showing shoot formation, there is an increase in alliin content as well as ~ in free and bound amino acid contents. Corresponding to this there was also an increase in the activity of phenylalanine ammonialyase in differentiating cultures. Alliin-lyase activity was found to be significantly different in differentiating and undifferentiated cultures. The significance of these results is discussed. INTRODUCTION In most of the work on flavor production in tissue cultures, it has been reported that synthesis of flavor occurs only when the cultures are in the state of morphological differentiation into shoots or roots. Freidborg (1971), Freeman et al. (1974) and Turnbull et al. (1981) demonstrated that redifferentiation of onion tissue cultures into roots or shoots was essential for the production of onion flavor. The major flavor precursor of garlic, called alliin (Stoll and Seebeck, 1951), is 2-propenyl (allyl)-L-cysteine sulphoxide as shown by Freeman and Whenham (1975). So far flavor formation in tissue cultures of garlic has not been studied. Hence studies were carried out using shoot forming differentiating cultures and undifferentiated cultures. An attempt is being made to find a correla= tion between alliin content, activities of enzymes like phenylalanine ammonia-lyase or alliin-lyase and free and bound amino acid content. MATERIALS AND METHODS Garlic bulbs were first dipped in 70% ethanol for two minutes and then sterilized with 5% sodium hypochlorite solution for ten minutes. Garlic cultures were initiated by placing discs of garlic bulb tissue on Murashige and Skoog's basal medium (1962) containing 11.4 ~M indole acetic acid (IAA), 10.8 ~M napthalene acetic acid (NAA), 9 NM 2,4dichlorophenoxy acetic acid (2,4-D), 9.3 ~M kinetin
OffFrint requests to." N. P. Malpathak
and 15% coconut water. The eight week old callus was subcultured on the same medium and part of this callus was subcultured on medium for shoot differentiation, which was Murashige and Skoog's basal medium supplemented with 11.4 ~M IAA, 10.8 ~M NAA, and 9.3 ~M kinetin. With 2-3 weeks small shoots were obtained on the callus mass. Shoot forming differentiated cultures and undifferentiated cultures were harvested after 60 days for analysis. The extraction and estimation of alliin were done according to the method of Schwimmer and Guadagni (1962) using differentiated cultures, along with shoots and undifferentiated cultures. Alliin content was estimated in terms of ~g pyruvate/mg protein. Specific acticity of alliin-lyase was estimated using a modified method of Schwimmer and Mabelis (1963) as given by Collin and Watts (1983). Specific activity was expressed as Dg pyruvate/min/ mg protein. For phenylalanine ammonia-lyase, extracts were prepared according to the method of Mahadevan and Sridhar (1982). Enzyme activity was measured by Podostdski and Brown's method (1974). Specific activity was expressed in terms of ~g cinnamic acid/ hr/mg protein. Amino acid extraction was carried out by the method of Khanna and Nag (1973), and the quantitative analysis was done by Everett and Street's method (1979) with isoleucine as standard. All the experiments are repeated at least three times, each with 3 replicates. The results of all the experiments were nearly the same. RESULTS AND DISCUSSIONS Alliin content was much higher in differentiating cultures as compared with undifferentiated ones (Table i). Similar results also have been obtained by Freeman et al. (1974) using onion cultures. Similarly, phenylalanine ammonia-lyase activity was higher in differentiating cultures (Table 2). Since this enzyme has been known to play a regulatory role in flavor production (Col lin and Watts, 1983) it is possible that alliin levels in differentiating and undifferentiated cultures of garlic are regulated by phenylalanine ammonia-lyase. However, the activity of alliin-lyase which is a specific enzyme for destruction of alliin in flavor production was found to be significantly different in
447 Table i.
Change in all±in content and free and bound amino acid contents in differentiating and undifferentiated cultures. All±in Free amino @g-pyruvate/ acids mg protein) @ g / m g mg protein)
i. Differ5.1± 0.2 entiating cultures
46.1±1.4
2. Undiffer- 2.1± 0.i entiated callus
37.5-+1.1
Bound amino acids (Dg/mg protein
ACKNOWLEDGEMENTS N. P. Makpathak gratefully acknowledges the receipt of Senior Research Fellowship from C.S.I.R., New Delhi.
58.1± 1.7 REFERENCES 54.7± 1.3
Cell±n, H. A., Watts, M. (1983) INN: Evans D. A., Sharp, W. R., Ammirato, R. N., Yamada, Y. (ed) Handbook of plant cell cultures, Vol. i. Macmillan Publishing Co., New York. pp. 729-747.
Mean of 3 replicates± S.E. Everett, N. P., Street, H. E. (1979) 409-411. Table 2.
Changes in specific activity of enzymes in differentiating and undifferentiated cultures.
Phenylalanine ammonia-lyase ( g cinnamic aeid/hr/mg protein)
Alliin-lyase ( g pyruvate/ min/mg protein)
J. Exp. Bot. 30:
Freeman, G. G., Whenham, R. J., MacKenzie, I. A., Davey, M. R. (1974) Plant Sci. Lett. 3:121-125. Freeman, G. G., Whenham, R. J. (1975) Agric. 26:1869-1886.
J. Sci. Fed.
Freidborg, G. (1971)
Physiol. Plant. 25:436-440.
Granroth, B. (1970) 152:1-71.
Ann. Acad. Sci. Fenn. Ser. A2
i.
Differentiating callus
0.46± 0.02
0.32-+ 0.01
Khanna, P., Nag, T. N. (1973) 310-311.
2.
Undifferentiating callus
0.15-+ 0.01
0.26-+ 0.01
Mahadevan A., Sridhar, R. (1982) Methods in Physiological Plant Pathology Ed. 2. Sirakami Pub., India pp. 146-149.
Mean of 3 replicates ±S.E.
differentiating and undifferentiated cultures (Table 2). Thus there is a possibility of the regulation of all±in content through its destruction. On the other hand free and bound amino acid content was increased in differentiating cultures (Table i). Some of the amino acids are known to be precursors in all±in synthesis (Granroth, 1970; Whitaker, 1976). We, therefore, feel that all±in content is regulated in differentiating and undifferentated callus through increased synthesis at a particular stage of cell development or through destruction. The question of how the all±in content increased during differentiation still remains unanswered.
ind. J. Exp. Bot. ii:
Murashige, T., Skoog, F. (1962) 473-497.
Physiol. Plant 15:
Podostdski, A. J., Brown, G. N. (1974) Plant Physiol. 54:41-43. Schwimmer, S., Guadagni, D. G. (1962) 27:94-97. Schwimmer, S., Mabel±s, M. (1963) Biophys. 100:66-73. Stoll, A., Seeback, E. (1951) 400.
d. Food Sci.
Arch. Biochem.
Adv. Enzymol. 11:377-
Turnbull, A., Galpin I. J.~ Smith, J. L., Collins, H. A. (1981) New Phytol. 87:257-268. Whitaker, J. R. (1976)
Adv. Food Res. 22:73-133.
Plant Cell Reports ( 1986) 5: 446- 447
© Springer-Verlag 1986
Flavor formation in tissue cultures of garlic (Allium sativum L.) N. P. Malpathak and S.B. David Department of Botany, University of Poona, Pune 411 007, India Received February, 1986 / Revised version received July, 1986 - Communicated by F. Skoog
ABSTRACT Differentiated and undifferentiated cultures of garlic (Allium sativum L-) were analyzed for the study of flavor formation in cultures. Attempts were made to correlate alliin content with free and bound amino acid contents and with enzymes like phenylanine ammonialyase (E.C. 4.1.1.5) and alliinlyase (E.C.4.4.1.4) which play important roles in formation of the flavor percursor alliin. It was observed that in differentiating cultures showing shoot formation, there is an increase in alliin content as well as ~ in free and bound amino acid contents. Corresponding to this there was also an increase in the activity of phenylalanine ammonialyase in differentiating cultures. Alliin-lyase activity was found to be significantly different in differentiating and undifferentiated cultures. The significance of these results is discussed. INTRODUCTION In most of the work on flavor production in tissue cultures, it has been reported that synthesis of flavor occurs only when the cultures are in the state of morphological differentiation into shoots or roots. Freidborg (1971), Freeman et al. (1974) and Turnbull et al. (1981) demonstrated that redifferentiation of onion tissue cultures into roots or shoots was essential for the production of onion flavor. The major flavor precursor of garlic, called alliin (Stoll and Seebeck, 1951), is 2-propenyl (allyl)-L-cysteine sulphoxide as shown by Freeman and Whenham (1975). So far flavor formation in tissue cultures of garlic has not been studied. Hence studies were carried out using shoot forming differentiating cultures and undifferentiated cultures. An attempt is being made to find a correla= tion between alliin content, activities of enzymes like phenylalanine ammonia-lyase or alliin-lyase and free and bound amino acid content. MATERIALS AND METHODS Garlic bulbs were first dipped in 70% ethanol for two minutes and then sterilized with 5% sodium hypochlorite solution for ten minutes. Garlic cultures were initiated by placing discs of garlic bulb tissue on Murashige and Skoog's basal medium (1962) containing 11.4 ~M indole acetic acid (IAA), 10.8 ~M napthalene acetic acid (NAA), 9 NM 2,4dichlorophenoxy acetic acid (2,4-D), 9.3 ~M kinetin
OffFrint requests to." N. P. Malpathak
and 15% coconut water. The eight week old callus was subcultured on the same medium and part of this callus was subcultured on medium for shoot differentiation, which was Murashige and Skoog's basal medium supplemented with 11.4 ~M IAA, 10.8 ~M NAA, and 9.3 ~M kinetin. With 2-3 weeks small shoots were obtained on the callus mass. Shoot forming differentiated cultures and undifferentiated cultures were harvested after 60 days for analysis. The extraction and estimation of alliin were done according to the method of Schwimmer and Guadagni (1962) using differentiated cultures, along with shoots and undifferentiated cultures. Alliin content was estimated in terms of ~g pyruvate/mg protein. Specific acticity of alliin-lyase was estimated using a modified method of Schwimmer and Mabelis (1963) as given by Collin and Watts (1983). Specific activity was expressed as Dg pyruvate/min/ mg protein. For phenylalanine ammonia-lyase, extracts were prepared according to the method of Mahadevan and Sridhar (1982). Enzyme activity was measured by Podostdski and Brown's method (1974). Specific activity was expressed in terms of ~g cinnamic acid/ hr/mg protein. Amino acid extraction was carried out by the method of Khanna and Nag (1973), and the quantitative analysis was done by Everett and Street's method (1979) with isoleucine as standard. All the experiments are repeated at least three times, each with 3 replicates. The results of all the experiments were nearly the same. RESULTS AND DISCUSSIONS Alliin content was much higher in differentiating cultures as compared with undifferentiated ones (Table i). Similar results also have been obtained by Freeman et al. (1974) using onion cultures. Similarly, phenylalanine ammonia-lyase activity was higher in differentiating cultures (Table 2). Since this enzyme has been known to play a regulatory role in flavor production (Col lin and Watts, 1983) it is possible that alliin levels in differentiating and undifferentiated cultures of garlic are regulated by phenylalanine ammonia-lyase. However, the activity of alliin-lyase which is a specific enzyme for destruction of alliin in flavor production was found to be significantly different in
447 Table i.
Change in all±in content and free and bound amino acid contents in differentiating and undifferentiated cultures. All±in Free amino @g-pyruvate/ acids mg protein) @ g / m g mg protein)
i. Differ5.1± 0.2 entiating cultures
46.1±1.4
2. Undiffer- 2.1± 0.i entiated callus
37.5-+1.1
Bound amino acids (Dg/mg protein
ACKNOWLEDGEMENTS N. P. Makpathak gratefully acknowledges the receipt of Senior Research Fellowship from C.S.I.R., New Delhi.
58.1± 1.7 REFERENCES 54.7± 1.3
Cell±n, H. A., Watts, M. (1983) INN: Evans D. A., Sharp, W. R., Ammirato, R. N., Yamada, Y. (ed) Handbook of plant cell cultures, Vol. i. Macmillan Publishing Co., New York. pp. 729-747.
Mean of 3 replicates± S.E. Everett, N. P., Street, H. E. (1979) 409-411. Table 2.
Changes in specific activity of enzymes in differentiating and undifferentiated cultures.
Phenylalanine ammonia-lyase ( g cinnamic aeid/hr/mg protein)
Alliin-lyase ( g pyruvate/ min/mg protein)
J. Exp. Bot. 30:
Freeman, G. G., Whenham, R. J., MacKenzie, I. A., Davey, M. R. (1974) Plant Sci. Lett. 3:121-125. Freeman, G. G., Whenham, R. J. (1975) Agric. 26:1869-1886.
J. Sci. Fed.
Freidborg, G. (1971)
Physiol. Plant. 25:436-440.
Granroth, B. (1970) 152:1-71.
Ann. Acad. Sci. Fenn. Ser. A2
i.
Differentiating callus
0.46± 0.02
0.32-+ 0.01
Khanna, P., Nag, T. N. (1973) 310-311.
2.
Undifferentiating callus
0.15-+ 0.01
0.26-+ 0.01
Mahadevan A., Sridhar, R. (1982) Methods in Physiological Plant Pathology Ed. 2. Sirakami Pub., India pp. 146-149.
Mean of 3 replicates ±S.E.
differentiating and undifferentiated cultures (Table 2). Thus there is a possibility of the regulation of all±in content through its destruction. On the other hand free and bound amino acid content was increased in differentiating cultures (Table i). Some of the amino acids are known to be precursors in all±in synthesis (Granroth, 1970; Whitaker, 1976). We, therefore, feel that all±in content is regulated in differentiating and undifferentated callus through increased synthesis at a particular stage of cell development or through destruction. The question of how the all±in content increased during differentiation still remains unanswered.
ind. J. Exp. Bot. ii:
Murashige, T., Skoog, F. (1962) 473-497.
Physiol. Plant 15:
Podostdski, A. J., Brown, G. N. (1974) Plant Physiol. 54:41-43. Schwimmer, S., Guadagni, D. G. (1962) 27:94-97. Schwimmer, S., Mabel±s, M. (1963) Biophys. 100:66-73. Stoll, A., Seeback, E. (1951) 400.
d. Food Sci.
Arch. Biochem.
Adv. Enzymol. 11:377-
Turnbull, A., Galpin I. J.~ Smith, J. L., Collins, H. A. (1981) New Phytol. 87:257-268. Whitaker, J. R. (1976)
Adv. Food Res. 22:73-133.
