In vitro xylem vessel elements formation from banana embryogenic cells and expression analysis of vessel development-related genes

Abstract

Tracheary elements formation was observed in banana embryogenic cells cultured in brassinolide-supplemented medium after a period of 10 days. The highest frequency of tracheary elements observed after 10 days was up to 31.88 %. The auxin, 2,4-D was found to be inhibitor of tracheary elements formation in brassinolide-supplemented medium and at a concentration of 1.5 µM of 2,4-D, the frequency of tracheary elements formation was reduced to zero. The appearance of secondary wall in these xylem vessel elements was observed by toluidine blue staining and lignin autofluorescence under fluorescence as well as laser confocal microscopy. Similar to lignin autofluorescence imaging, the calcofluor white staining also revealed development of characteristic secondary wall in these xylem vessel elements indicating deposition of cellulose along with lignin during secondary wall formation. The xylem vessel elements induced comprised of fiber-like elements (FLEs) and tracheary elements with deposition of secondary wall. Expression analysis of lignin biosynthesis pathway genes indicated that important genes like CCR, CCoAOMT and COMT were induced as early as fifth day of brassinolide (BL) treatment. Changes in transcript level of key master regulators of xylogenesis during tracheary elements formation showed that expression of vascular-related NAC domain containing transcription factors (VND) were up-regulated with induction of VND1–3 and VND6–7 expression as early as third day of brassinolide treatment. This study of in vitro tracheary elements formation in embryogenic cell suspension cultures is useful in understanding the genes involved in secondary wall thickening in banana.