To increase the global acceptability of chickpea by improving its nutritional quality, seed RFO (Raffinose Family Oligosaccharides) concentration needs to be reduced without affecting their role during seed development and positive impact on human health. To achieve this objective, the key regulating step(s) of RFO biosynthesis needs to be identified. The three main objectives of the thesis were: (1) to optimize an analytical method to determine soluble sugars concentration in chickpea seeds including RFO, (2) to determine chickpea genotypes with contrasting seed RFO concentration, and (3) to optimize and validate RFO biosynthetic enzyme activity assays. These three objectives of the thesis provided basis of the fourth objective. For the first objective, a modified HPAEC-PAD (High performance anion exchange chromatography with pulsed amperometric detector) based gradient approach was optimized to study the concentration and composition of soluble sugars in chickpea seeds. The optimized method separated all the soluble sugars within 20 min of run time with higher accuracy, sensitivity and precision compared to previously reported methods. Therefore, the optimized method was utilized to study the natural variation in RFO concentration of 171 chickpea genotypes. Sucrose (0.60 - 3.59 g/100 g) and stachyose (0.18 − 2.38 g/100 g) were predominant among soluble sugars and RFO, respectively. Analysis of variance revealed a significant impact (P ≤ 0.001) of genotype (G), environment (E), and their interaction (G×E) on seed RFO concentration in chickpea. A significant positive correlation was observed between substrate and product concentration in RFO biosynthesis. Raffinose, stachyose and verbascose showed moderate broad sense heritability (0.25 − 0.56) suggesting the quantitative nature of the RFO trait in chickpea seeds. Desi (ICC 1163, ICC 1471, ICC 9562, ICCV 07115, ICCV 07116 and ICCV 07117) and kabuli (ICC 5270, ICC 10674, ICC 16216, ICC 16528, ICCV 3 and ICCV 91302) chickpea genotypes with high and low RFO concentrations (high RFO genotypes are underlined) were identified. RFO biosynthetic enzymes activities were optimized for substrate and protein concentration, temperature (25 °C), time (10 min for galactinol synthase and 60 min for other biosynthetic enzymes) and pH (7.0). These assays were validated at different seed developmental stages of two released varieties: CDC Vanguard and CDC Frontier. Simultaneously, RFO accumulation at different seed developmental stages was also studied. During 18 - 38 DAF (days after flowering), about a 75 % decrease in seed moisture was observed coinciding with the accumulation of RFO providing desiccation tolerance to maturing seeds. The initial substrates viz. myo-inositol and sucrose were observed throughout seed development process having maximum accumulation at 18 - 20 (0.50 – 0.57 g/100 g) and 20 - 22 (9.94 - 11.17 g/100 g) DAF that decreased afterwards supporting the biosynthesis of galactinol and raffinose, respectively. Galactinol is considered as the universal galactosyl donor, it showed the highest concentration at 30 DAF and this was later utilized for increased RFO accumulation till 36 DAF. Activity of RFO biosynthetic enzymes was observed 2 - 6 days prior to first detection of their corresponding products whereas the highest activities were determined 2 - 4 days prior to maximum accumulation of RFO. However, maximum GS (galactinol synthase) activity was observed at 36 DAF but this did not correspond to amount of galactinol accumulation in seeds. This indicated that galactinol was synthesized in higher amount even after 30 DAF but directed towards RFO biosynthesis thus could not necessarily accumulate in seeds. A galactinol independent pathway was also found operative in chickpea seeds. These results suggested that substrate concentration and GS activity might be the possible factors regulating seed RFO concentration in chickpea. The fourth objective utilized the information, material and methods from the previous three objectives. Chickpea genotypes with contrasting RFO concentration were compared for seed size and weight, germination capacity and RFO biosynthesis (accumulation and biosynthetic enzymes activities during seed development). Sucrose concentration showed a significant positive (r = 0.728, P ≤ 0.05) correlation with seed size/weight. RFO concentration was a facilitator of seed germination rather than regulating factor. Higher accumulation of myo-inositol and sucrose in high RFO genotypes during early seed developmental stages suggested that initial substrates concentrations may influence seed RFO concentration. High RFO genotypes expressed about 2 - 3 fold higher activity for all RFO biosynthetic enzymes compared to those with low RFO concentration. The enzyme activity data corresponded with the accumulation of individual RFO during chickpea seed development. In conclusion, regulating galactinol synthase activity is a potential strategy to reduce seed RFO concentration in chickpea. The present study can be extended to study RFO biosynthesis at the transcript level and the influence of RFO biosynthetic enzymes on seed size and weight, germination, RFO concentration, yield, and stress tolerance.

Raffinose family oligosaccharides (RFO) are major soluble carbohydrates of lentil (Lens culinaris Medik) seeds. When consumed by humans, RFO pass indigested through upper digestive tract as ¦Á-galactosidase enzyme required for RFO breakdown is not produced in humans. Consumption of lentils with high concentrations of RFO result in stomach discomfort, bloating and diarrhea due to bacterial fermentation of RFO in large intestine. This has lead to a relatively low use of lentils for human consumption. RFO are therefore considered anti-nutritional factors and development of lentil cultivars with reduced RFO concentrations is desired to improve lentil quality and consumption. To explore the possibility to develop lentil cultivars for low-RFO concentration, heritability of RFO trait and influence of environmental conditions on RFO must be known. In addition, RFO biosynthesis and accumulation in lentil seeds must be understood. However, very limited information is available on the above mentioned aspects of RFO in general and in lentil in particular. Therefore, the objectives of this study were: (1) to evaluate natural variation in RFO concentration and composition in commonly grown lentil cultivars and to determine the correlation between RFO concentration and other important seed constituents, (2) to investigate heritability and effect of environment on concentration and composition of lentil seed soluble carbohydrates, (3) to assess natural variation and diversity in RFO concentration in the genus Lens, (4) and to evaluate the association between galactinol synthase activity and accumulation of RFO in lentil seeds. Analysis of 22 lentil genotypes revealed significant (P ¡Ü 0.05) variation in total starch, amyolse, protein, total RFO and seed weight and seed colour. Stachyose was the major RFO in all lentil genotypes followed by raffinose and verbascose. A significant (P ¡Ü 0.05) inverse correlation was found between RFO and amylose concentration (r = -0.34); whereas RFO concentration and thousand seed weight correlated positively (r = 0.35). The analyses of variance of eleven cultivars grown at ten different environments showed that cultivar, environment and their interaction had significant effects on sugar concentration in lentil seeds. The high broad sense heritability of RFO (h2= 0.85) indicated that RFO concentration in lentil seeds is highly heritable and thus amenable to genetic improvement. An extensive evaluation of domesticated and wild species and subspecies of the genus Lens revealed significant (P ¡Ü 0.05) variation and diversity in RFO concentration and composition of individual oligosaccharides. Higher Shannon-Weaver diversity indices (SDI) for total RFO, raffinose and verbascose traits were observed in wild lentils compared to domesticated genotypes. Lens ervoides genotypes and some wild genotypes contained almost half the RFO concentration of cultivated lentils and therefore, wild genotypes may be useful for developing low-RFO lines. Higher verbascose and lower stachyose concentration was found in Lens ervoides genotypes, whereas higher raffinose and lower verbascose concentration is found in Lens nigricans genotypes. Study of galactinol synthase activity in developing seeds with varying RFO concentration showed no clear association between galactinol synthase activity and RFO concentration. The sucrose and galactinol concentration of developing seeds were also not associated with total RFO concentration of lentil seeds. This finding suggests a non-regulatory role of galactinol synthase in RFO biosynthetic pathway in lentil seeds. Together, all these findings are not only significant to devise strategies to develop lentil cultivars with reduced RFO concentration but also for understanding RFO biosynthesis in lentil seeds.

Raffinose family oligosaccharides (RFO) are major soluble carbohydrates of lentil (Lens culinaris Medik) seeds. When consumed by humans, RFO pass indigested through upper digestive tract as ¦Á-galactosidase enzyme required for RFO breakdown is not produced in humans. Consumption of lentils with high concentrations of RFO result in stomach discomfort, bloating and diarrhea due to bacterial fermentation of RFO in large intestine. This has lead to a relatively low use of lentils for human consumption. RFO are therefore considered anti-nutritional factors and development of lentil cultivars with reduced RFO concentrations is desired to improve lentil quality and consumption. To explore the possibility to develop lentil cultivars for low-RFO concentration, heritability of RFO trait and influence of environmental conditions on RFO must be known. In addition, RFO biosynthesis and accumulation in lentil seeds must be understood. However, very limited information is available on the above mentioned aspects of RFO in general and in lentil in particular. Therefore, the objectives of this study were: (1) to evaluate natural variation in RFO concentration and composition in commonly grown lentil cultivars and to determine the correlation between RFO concentration and other important seed constituents, (2) to investigate heritability and effect of environment on concentration and composition of lentil seed soluble carbohydrates, (3) to assess natural variation and diversity in RFO concentration in the genus Lens, (4) and to evaluate the association between galactinol synthase activity and accumulation of RFO in lentil seeds. Analysis of 22 lentil genotypes revealed significant (P ¡Ü 0.05) variation in total starch, amyolse, protein, total RFO and seed weight and seed colour. Stachyose was the major RFO in all lentil genotypes followed by raffinose and verbascose. A significant (P ¡Ü 0.05) inverse correlation was found between RFO and amylose concentration (r = -0.34); whereas RFO concentration and thousand seed weight correlated positively (r = 0.35). The analyses of variance of eleven cultivars grown at ten different environments showed that cultivar, environment and their interaction had significant effects on sugar concentration in lentil seeds. The high broad sense heritability of RFO (h2= 0.85) indicated that RFO concentration in lentil seeds is highly heritable and thus amenable to genetic improvement. An extensive evaluation of domesticated and wild species and subspecies of the genus Lens revealed significant (P ¡Ü 0.05) variation and diversity in RFO concentration and composition of individual oligosaccharides. Higher Shannon-Weaver diversity indices (SDI) for total RFO, raffinose and verbascose traits were observed in wild lentils compared to domesticated genotypes. Lens ervoides genotypes and some wild genotypes contained almost half the RFO concentration of cultivated lentils and therefore, wild genotypes may be useful for developing low-RFO lines. Higher verbascose and lower stachyose concentration was found in Lens ervoides genotypes, whereas higher raffinose and lower verbascose concentration is found in Lens nigricans genotypes. Study of galactinol synthase activity in developing seeds with varying RFO concentration showed no clear association between galactinol synthase activity and RFO concentration. The sucrose and galactinol concentration of developing seeds were also not associated with total RFO concentration of lentil seeds. This finding suggests a non-regulatory role of galactinol synthase in RFO biosynthetic pathway in lentil seeds. Together, all these findings are not only significant to devise strategies to develop lentil cultivars with reduced RFO concentration but also for understanding RFO biosynthesis in lentil seeds.