SCREENING OF SORGHUM (SORGHUM BICOLOR L.) GENOTYPES FOR BMRTRAIT BY HISTOCHEMICAL STAINING TECHNIQUE

Abstract

Reddish to pink coloration of brown-midrib (bmr) mutant tissues upon histochemical staining with Wiesners’ reagent has been associated with accumulation of cinnamaldehydes and acts as an indirect indicator of quality improvement in forage. Cinnamaldehydes are phenolic substrates associated with lignin biosynthesis pathway. This study was planned with an objective of understanding the relationship between Wiesner staining in stem, leaf midrib and forage quality traits. Twenty forage sorghum genotypes of both brown-midrib (bmr) and wild-type genotypes were used in the study. The genotypes were planted in a randomized complete block design. Histochemical staining on leaf and stem sections and stem forage quality traits were analyzed at flowering stage. The Wiesners reagent’s active ingredient, phloroglucinol (1.0% (w: v)), reacts best in an acidic (10.1
M HCl) environment, and was dissolved in four acid-ethanol (v/v) levels of 35/65, 30/70, 25/75, 20/80. Stained cross-sections images of leaf midrib and stem tissues of the genotypes were captured in flatbed scanner and the staining intensity was scored using traditional histochemical and digitized methods. Forage quality analysis was performed on grounded stem tissue samples using Near Infrared Spectroscopy (NIRS). The analysis of variance revealed significant difference among genotypes for the histochemical staining traits, percent in vitro organic matter digestibility (IVOMD), Metabolizable energy (ME mj/kg), and percent neutral detergent fiber (NDF). Orthogonal contrast analysis revealed genotypes carrying bmr loci (bmr6, bmr6bmr12 and bmr12) had significantly lower ADL (acid detergent lignin) and higher IVOMD than wild-type genotypes. Genotypes BN612, 59353, 59354, N605, N596 N600 had the highest IVOMD and lowest ADL, while wild-type genotypes IS26694, SSV-74, IS11861 and ICSV12006 had the lowest IVOMD and highest ADL. These genotypes had highly red-stained and faintly stained tissues,respectively based on traditional and digitized staining assessment. A highly significant positive correlation between the histochemical staining of leaf midrib and stem tissues was detected suggesting that similar substrates responsible for reaction with phloroglucinol were deposited in both organs. Furthermore, a significantly greater staining in bmr6>bmr6bmr12>bmr12>Bmr were detected. These findings shown promise for employing histochemical staining combined with high-through-put phenotyping in selection of bmr inbred lines of higher forage quality value. However, further studies are recommended using large number of bmr inbred lines to confirm the application of the histochemical staining technique for selection in early and later generation advancement of bmr inbred line development.