Changes in biochemical constituents and induction of early sprouting by triadimefon treatment in white yam (Dioscorea rotundata Poir.) tubers during storage

Changes in biochemical constituents and induction of early sprouting by triadimefon treatment in white yam ( Dioscorea rotundata Poir.) tubers during storage first-author †E-mail:suriyagopi@yahoo.co.in †E-mail:suriyagopi@yahoo.co.in suriyagopi@yahoo.co.in Stress Physiology Lab, Department of Botany, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India Stress Physiology Lab, Department of Botany, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India The ability of triadimefon (TDM), a triazolic fungicide, to alter the biochemical constituents and thereby minimizing the days required for sprouting in white yam ( Dioscorea rotundata Poir.) tubers during storage under (30±2) °C in the dark, was studied. TDM at 20 mg/L was given to tubers by dipping the tubers in treatment solution containing 20 mg/L TDM on 10, 25 and 40 d after storage (DAS). Starch, sugars, protein, amino acid contents as well as protease and α-amylase activities were estimated on 15, 30 and 45 DAS from two physiological regions viz., apical and basal regions of the tubers. In normal conditions (control) sprouting occurred on 70 to 80 DAS. The starch content decreased, while protein, amino acid, sugar contents and protease and α-amylase activities were increased due to TDM treatment and led to early sprouting. Biochemical constituents Enzyme activities Dioscorea rotundata Sprouting Storage Triadimefon (TDM) INTRODUCTION 1 Roots and tubers were critical components in the diet during the early evolution of mankind and were the most important food crops of very ancient origin in the tropics and sub tropics, associate with human existence, survival and socio-economic history (Asha and Nair, 2002 2002 ). Yams ( Dioscorea spp., Dioscoreaceae) are the third most important tropical “root” crop after cassava and sweet potato (Fu et al., 2005 2005 ). White yam ( Dioscorea rotundata Poir.) plant produces tubers and bulbils, which are edible and have economic importance (Omonigho and Ikenebomeh, 2000 2000 ). The tubers of various species of Dioscorea constitute one of the stable carbohydrate foods for the people in many tropical countries (Akissoe et al., 2003 2003 ). Many different forms and cultivars of the edible yam species are available in different areas and it is likely that these differ in composition and nutritional values (Bhandari et al., 2003 2003 ). Dioscorea spp. also have medicinal properties as their tubers contain diosgenin, a biochemical precursor in the synthetic production of progesterone and other corticosteroids (Albrecht and McCarthy, 2006 2006 ). The wide spread cultivation and large-scale production of white yam are hindered by the comparatively long dormancy period of the tubers. To sprout the tubers, for producing seed material, minimum 70 to 80 d after storage (DAS) is required (Muthukumarasamy and Panneerselvam, 2000 2000 ). In order to develop a competent method for sprouting in tuber crops, application of plant growth regulator (PGR) is a previously established technique (Paul and Ezekiel, 2003 2003 ). During storage of tubers, especially at higher temperatures, ageing takes place gradually and this is associated with an increase in abscisic acid (ABA) content and a decrease in gibberellic acid (GA) content, as reported in potato by Burton et al.( 1992 1992 ). Increase in GA content of tubers is reported to inhibit little tuber formation and sprouting in potato (Ezekiel et al., 2000 2000 ). These reports show clearly that if GA content of tubers is decreased and ABA content increased, the sprouting process can be initiated in tubers. Some unknown factors other than ABA might also be involved in dormancy-release in tubers (Xu et al., 2006 2006 ). Triazole compounds have both fungitoxic and plant growth-regulating properties (Fletcher et al., 2000 2000 ). In addition, they can also protect plants against various environmental stresses (Voesenek et al., 2003 2003 ). Triazoles adversely affect the isoprenoid pathway, and alter the levels of certain plant hormones by inhibiting gibberellin synthesis, reducing ethylene evolution, and increasing cytokinin levels (Kamountsis and Chronopoulou-Sereli, 1999 1999 ). Some previous work carried out in our lab revealed the morphological and physiological changes associated with triazole-treatment in various plants, include the inhibition of plant growth, decreased internodal elongation, increased chlorophyll levels, enlarged chloroplasts, thicker leaf tissue, increased root to shoot ratio, increased antioxidant potentials and enhancement in alkaloid production (Muthukumarasamy and Panneerselvam, 1997 1997 ; Muthukumarasamy et al., 2000 2000 ; Panneerselvam et al., 1997 1997 ; 1998 1998 ; Jaleel et al., 2006 2006 ). Triadimefon (TDM) [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-tria-zol-yl)-2-butanone], a triazole fungicide, having PGR properties, is reported to inhibit GA biosynthesis and increase ABA and cytokinin contents (Fletcher et al., 2000 2000 ). It is necessary to investigate the efficacy of this compound in the induction of early sprouting of white yam tubers during storage in order to minimize the days required for sprouting. Hence, the aim of the present work was to estimate the ability of TDM in early initiation of sprouting process and to analyse the changes in the biochemical constituents due to TDM treatment of white yam tubers during storage under (30±2) °C in the dark. MATERIALS AND METHODS 1 Plant materials and TDM 2 Tubers of Dioscorea rotundata cv. Sree Priya were obtained from Central Tuber Crop Research Institute (CTCRI), Kerala, India and planted in Botanical Garden of Annamalai University. The tubers were harvested after the death of aerial parts and stored in plastic trays in the dark at room temperature (30~32 °C) and relative humidity (RH) 80%~85%. TDM as BAYLETON 25 WP was obtained from Bayer (India) Ltd., Mumbai. TDM treatment 2 In the preliminary experiments, four concentrations [10, 15, 20 and 25 mg/(L tuber)] of TDM were prepared and used for treatment of tubers to determine the optimum concentration at which maximum sprouting occurred. Among these concentrations, 20 mg/(L tuber) was found to enhance the sprouting process and at lower [10 and 15 mg/(L tuber)] and higher [25 mg/(L tuber)] concentrations, there was no significant effect. Therefore 20 mg/(L tuber) was used for this study. The treatment was given by dipping the tubers in TDM solution for 15 s, at three time intervals i.e. 10, 25 and 40 DAS. For each dipping one litre of the solution was used and the dipping was done in three installments with 30 min gap to facilitate maximum absorption of the chemical. The control tubers were dipped in distilled water. Analysis of biochemical constituents 2 The biochemical constituents were analysed at three storage periods viz., 15, 30 and 45 DAS in the two physiological regions i.e., apical (1 cm below the tip) and basal (1 cm above the base) regions of the tubers. Starch and sugars 2 Cylinders of the tuber tissue were removed by using cork borer and homogenized in 80% ethanol (v/v). The homogenate was refluxed over a water bath and centrifuged. The residue was extracted twice more with 80% ethanol. The supernatant liquids were combined and used for estimation of soluble sugars by the method of Dubois et al.( 1956 1956 ). The remaining pellets were used for the estimation of starch by the method of McCready et al.( 1950 1950 ). Both estimations were carried out at room temperature (30~32 °C). Protein and total free amino acid 2 The protein content was determined by the method of Bradford ( 1976 1976 ). Total free amino acid was extracted and estimated by following the method of Moore and Stein ( 1948 1948 ). Protease (EC 3.4.2.2) 2 The protease activity was determined by the method described by Prisco et al.( 1975 1975 ), using glycine as standard. The enzyme activity was expressed in terms of enzyme units/mg protein [U/(mg protein)]. One U is defined as μmoles of α-NH 2 released per mg protein per hour. α-amylase (EC 3.2.1.1) 2 The tuber tissues were excised and 1 g of fresh mass was ground with a pestle in an ice-cold mortar with 10 ml of distilled water at 4 °C. The homogenate was centrifuged at 4 °C for 30 min at 15000 r/min. The supernatant was used to assay α-amylase activity. The protein content in the supernatant was estimated according to Bradford ( 1976 1976 ), using bovine serum albumin as standard. The activity of α-amylase (EC 3.2.1.1) was assayed by the method of Bernfeld ( 1955 1955 ), using β-limit dextrin as substrate. The enzyme extract was heated at 70 °C for 5 min with 3 mmol/L CaCl 2 to inactivate β-amylase as described by Tarrago and Nicolas ( 1976 1976 ). Statistics 2 The experiment was carried out in completely randomized design (CRD). Each treatment was analyzed with at least seven replicates and a standard deviation ( SD ) was calculated. The data were expressed in mean± SD of 7 replicates. RESULTS AND DISCUSSION 1 The two physiological regions (apical and basal) were demarcated in yam tubers as described previously (Isamah et al., 2000 2000 ). Sprouting occurs mainly in the apical region. The starch content varied in the two physiological regions of white yam tubers during storage (Fig. 1a 1a ). The highest content was seen in early storage periods (15 DAS). It declined slightly at 30 DAS and decreased further towards the onsetting of sprouting (45 DAS in TDM treated and 70 to 80 DAS in control tubers). This result is in accordance with reports of Faboya and Asagbra ( 1990 1990 ). TDM at 20 mg/L concentration reduced the starch content in both apical and basal regions of the tubers on 15, 30 and 45 DAS. A significant decrease was noted in the basal region of the tubers on 45 DAS under TDM treatment. The decrease in starch content can be correlated to the necessity of starch breakdown to initiate sprouting. Sprouting was initiated due to TDM treatment on 45 DAS. Reduced starch content is the prerequisite to initiate the sprouting process (Ravindran and Wanasundera, 1992 1992 ). Reduced starch content is reported in potato during sprouting (Davies and Ross, 1984 1984 ). The breakdown of starch is essential to enhance sprouting in order to utilize and translocate the food reserves from tuber (Edelman et al., 1968 1968 ). Values are mean± SD of 7 replicates A variation in the sugar content was seen in apical and basal regions of the tubers on all storage periods except 15 DAS. The sugar content increased due to TDM treatment, which facilitate sprouting (Fig. 1b 1b ). An increase of sugar content during sprouting was observed in potato by Isherwood and Burton ( 1975 1975 ). A decrease in starch and an increase in sugar content were reported in Crocus sativus corms during sprouting (Chrungoo and Farooq, 1985 1985 ). Sprouting behaviour of Lilium bulblets cultivated under low temperature conditions showed rapid starch hydrolysis accompanied by accumulation of sugars (Miller and Langhans, 1990 1990 ). The accumulation of sugars could influence the sprouting and bud growth in tubers (Xu et al., 2006 2006 ). The protein content was highly increased due to TDM treatment in both basal and apical regions of white yam tubers on 15, 30 and 45 DAS (Fig. 2a 2a ). At the time of sprouting, the protein content increased to a maximum extent at the apical region but it declined at the basal region. Protein synthesis was proved to be one of the essential processes associated with sprouting (Jayakumar et al., 1993 1993 ). The amino acid content was more or less the same in apical and basal regions of the tubers (Fig. 2b 2b ). A rapid increase was seen on 45 DAS in TDM treated tubers, which is helpful to initiate sprouting. Increased amino acid content was reported in white yam tubers during sprouting by Splittstoesser and Rhodes ( 1973 1973 ). Values are mean± SD of 7 replicates The protease activity is directly related to sprouting. Increased protease activity was found in TDM treated white yam tubers during storage (Fig. 3a 3a ). The increase in protease activity and amino acid content with a concomitant reduction in the protein content of the basal region was observed during sprouting. Izundu and Fasidi ( 1991 1991 ) reported similar observation of Dioscorea dumetorum . Stored protein in the tuber is hydrolyzed in the form of amino acid by the enzyme protease during the time of sprouting (Muthukumarasamy and Panneerselvam, 1996 1996 ). Values are mean± SD of 7 replicates The α-amylase activity was increased due to TDM treatment. The extent of increase was high in basal region, and was higher than that in the apical region during sprouting (Fig. 3b 3b ). The higher α-amylase activity coincides with the higher sugar content in the tubers during sprouting (Ikediobi and Oti, 1983 1983 ). Increased α-amylase activity was reported by Panneerselvam ( 1987 1987 ) in sprouting rhizomes of Curcuma and tubers of Dioscorea . The dormancy period of Dioscorea tubers decreased due to TDM treatment. Enhanced breakdown of starch and production of sugars and early initiation of sprouting are the evidence for this. Related observations were made in TDM treated potato, where the little tuber production was increased due to TDM treatment (Paul and Ezekiel, 2003 2003 ). This result can also be correlated with the findings of Li and Zhang ( 1999 1999 ) who reported increased adventitious bud development in triazole treated maize. From the results of this investigation, it can be concluded that the application of TDM can be used for inducting and enhancing of sprouting in white yam tubers by reducing the dormancy period. 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