Cryopreservation of Pinus patula Scheide et Deppe embryogenic tissue.

Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.

Embryogenic tissue of Pinus patula Scheide et Deppe was initiated from immature green female cones during the months of November 1996 to February 1997 and December 1997 to January 1998. Tissue was maintained on MSG3 medium (BECWAR, NAGMANI and WANN 1990) supplemented with maltose. A comparison of various sugars as a carbohydrate source for maintaining embryogenic tissue showed that maltose was far superior to sucrose and the other sugars tested. Embryogenic tissue was successfully cryopreserved for up to 8 weeks using 0.3 M sorbitol and 5 % DMSO. Recovered tissue initially underwent a lag phase in tissue regrowth, but by the end of 5 weeks post-thaw, tissue proliferation was as vigorous as the unfrozen, untreated control. Fluoresceine diacetate (FDA) staining revealed that the embryonal head survived cryopreservation, but the highly vacuolated suspensor tissue had ruptured and died. Embryogenic tissue from two different families and four genotypes were successfully cryopreserved using this protocol. A comparison of commonly used cryopreservation techniques was conducted. It was found that the slow addition of the cryoprotectants over two days slowed the recovery rate of the tissue and increased the chances of contamination. Embryogenic tissue did not respond well to cryopreservation using a combination of the cryoprotectants PEG, glucose and DMSO (10-8-10%). Only a small proportion of the tissue survived, and initial tissue regrowth took up to 5 weeks. Embryogenic tissue was also set in gel and immersed directly in liquid nitrogen in an effort to cryopreserve tissue using the process of vitrification. However, none of the tissue survived, possibly due to insufficient dehydration prior to immersion in liquid nitrogen. Tissue recovery was highest when the tissue was precooled to -70°C in a container filled with isopropyl alcohol placed in a static freezer prior to immersion in liquid nitrogen. Recovery of tissue was improved by suspending the tissue on polyester grids and removing the liquid medium prior to placing onto MSG3 medium. Recovered tissue was bulked up using suspension cultures, and then paced onto MSG5 (BECWAR, NAGMANI and WANN 1990) or 240 medium (PULLMAN and WEBB 1994) to mature. Mature embryos were isolated from both media and germinated. Somatic plantlets were successfully hardened-off under greenhouse conditions. The successful cryopreservation of a number of genotypes and lines, and the maturation of recovered tissue has been achieved. This technique is now being actively incorporated into P. patula somatic embryo research, enabling the long-term storage of juvenile reference tissue while field trials are carried out and evaluated.