Hexaploid Development and Fertility Assessment, De Novo Regeneration, and Biolistic Transformation of Cannabis sativa L.
Trevor Pougnet, MS Seminar
Thursday, June 27, 2024, 10:30 am
(Under the direction of Dr. Hsuan Chen, Co-Chair and Dr. Kedong Da, Co-Chair)

Location: 121 Kilgore / Hybrid
Join Zoom Meeting: https://ncsu.zoom.us/j/92169390332?pwd=bksdkmiBRhU0dYPVJbfWlIOe3tqsPF.1
Meeting ID: 921 6939 0332
Passcode: 645460

 Pougnet.Abstr.pdf

Abstract:
Traditionally, polyploidization has been employed in Cannabis sativa L. for enhancing secondary metabolite production and yield. Previous studies have only investigated triploids and tetraploids. This study aimed to generate the first hexaploid hemp through colchicine treatment and evaluate its fertility via intra- and interploidy hybridizations. Hexaploid compatibility and fertility were assessed by performing intra- and interploidy crosses with diploids and tetraploids. Progenies were germinated to verify their ploidy by flow cytometry. This study also generated the first stable pentaploid hemp from interploidy hybridizations.

In addition, we investigated de novo regeneration primarily for its importance in genetic engineering. Successful regeneration requires optimizing several in vitro tissue culture factors like basal salt composition and plant growth regulators (PGRs). A reliable regeneration system has yet to be established in C. sativa. This study examined various effects of PGRs on callogenesis in leaf-petiole explants aiming to achieve de novo regeneration via indirect organogenesis or somatic embryogenesis. Kinetin and BAP produced the highest quality type II calli. One de novo regeneration event by indirect organogenesis was achieved.

The last study evaluated the potential for in planta transformation of C. sativa meristem explants using the Bio-Rad PDS-1000/He biolistic system. Biolistic transformation advantages such as transforming a wide range of plant species, direct DNA delivery, and high efficiency if optimized. However, biolistic transformation in C. sativa has not been reported in literature. This study aimed to transform in vitro meristem explants by particle bombardment. Two bombardment trials were conducted. The first trial investigated different DNA volumes in coating particles (microcarriers), while the second trial used a reduced recovery time and a fixed DNA-to-gold particle ratio. Both trials showed limited transient and stable expressions of GFP and GUS in leafy tissues but demonstrated key factors for further improvement.