Nutrient composition and degradation characteristics of anthocyanidin containing alfalfa transformed with Lc, C1 and Lc x C1 regulatory genes
Alfalfa (Medicago sativa L.) is rich in nutrients. However, utilization of its nutrients in ruminants is restricted due to rapid rumen degradation of protein. This may be prevented if adequate protein binding proanthocyanidins are present in the aerial part of the plant. Proanthocyanidins are synthesised by the flavonoid pathway. The Lc gene and C1 genes synthesise bHLH and MYB transcription factors associated with the flavonoid pathway regulation. The objective of this study was to investigate the influence of single gene (Lc and C1) and double gene (Lc x C1) transformation on chemical composition and degradation characteristics of protein and carbohydrate compared to non-transgenic (NT) parental plants. Samples were collected from plant populations of C1 genotype, two Lc genotypes (Lc1 and Lc3), two Lc x C1 genotypes (Lc1C1 and Lc3C1), NT and AC-Grazeland (ACGL); and maintained in a growth chamber. Plants were harvested at late-bud stage, freeze-dried and ground (1 mm). Chemical composition was determined by AOAC methods. Protein and carbohydrate sub-fractions were estimated according to Cornell Net Carbohydrate and Proyein System (CNCPS ver. 6.1). Anthocyanidin was extracted with aqueous acetone and hydrolyzed with butanol-HCl to measure spectrometric absorbance. The extractable anthocyanidin contents in Lc alfalfa and LcxC1 averaged 149 ±85 and 185±74 µg/g DM respectively. The single gene had a higher total carbohydrate (CHO; 70% vs. 68%; P=0.03) and non-fibre carbohydrate (NFC; 44% vs. 40%; P<0.001) than double gene transformed alfalfa. There was no difference (P>0.05) in structural carbohydrate (ADF and NDF) content. The single gene alfalfa had a lower CP (19% vs. 21%; P=0.02) than double gene transformed alfalfa. The profiles of protein (PA, PB1, PB2 and PB3), and carbohydrate (CA, CB1, CB2, and CB3) varied among different genotypes, resulting in different degradation profiles. Double gene genotypes had a higher (P<0.01) rumen degradable crude protein (RDCP) but lower (P<0.01) RD-CHO than single gene genotypes. This caused an increase (P<0.01) in total degradable N to CHO ratio in double gene transformation by 10 g N/kg CHO. In conclusion, the gene transformation influenced anthocyanidin accumulation in aerial parts as well as accumulation of nitrogenous compounds and non-structural carbohydrates thereby changing chemical composition and degradation characteristics. The single gene transformed alfalfa had a lower N to CHO balance than double gene alfalfa. The C1 gene influences nutrient composition of alfalfa differently, when co-expressed with two Lc lines (Lc1 and Lc3).
Keywords: Alfalfa, Lc and C1 genes, gene transformation