Enzyme Digestion Time Trials for C. sativa l. Chromosome Squash

Hemp (Cannabis sativa L.) is a versatile agronomical crop in the US used for its fiber, seed, and oil that is growing with renewed interest as a versatile and high yeild crop^[3]. Recent research has shown that unfertilized female plants produce significantly higher concentration of cannabinoids, a major by-product of hemp production ^[4]. However, methods such as female-only cloning and feminization compounds can be very expensive. Additionally, tetraploid and triploid varieties have been shown to grow with greater vigor and higher fiber content than their common diploid counterparts ^[2]. Though there is also drawback of producing polyploid varieties due to a lack of common bio-engineering tools and protocols to examine polyploidy in hemp.

This research focuses on a traditional method known as chromosome squash (CS) which is an important technique providing karyotypes and confirming polyploids ^[1]. This technique however, takes much technique and tailoring for each specific plant species used, and new methods have to be adapted with each variety. Tests were performed on the proper enzyme digestion time of shoot apical meristem samples of C. sativa. Cells were paused in metaphase stage using a cell cycle suspension compound, 8-hydroxyquinoline (8HQ). Samples were then subjected to digestion agents of 6% pectinase and 6% cellulose for time trial periods ranging from 20 minutes to 5 hours. CS was then performed on each time trial and observed under a microscope using 4’,6-diamidino-2-phenylindole (DAPI) identifying adequate digestion times for samples. Good samples are described as those that have DNA condensed into clear distinguishable chromosomes that are adequately separated and identifiable. Trials point to a proper digestion time ranging from 40 minutes to an hour that contained properly dispersed cells with identifiable chromosome sets. Trials below this time were not adequately digested nor lysed and trials over this time had cells that were unidentifiable after squash. Further research should be done on the concentration of digestive agents and amending the cell cycle suspension protocol to have an increased number of cells in the metaphase stage.

This research contributes to the identification and confirmation of high-market value polyploid varieties that will aid in accelerating hemp research. Identification of superior triploid varieties will intensify the hemp industry with naturally sterile plants that have both the benefit of unfertilized femineity and polyploidy vigor.