Immature, green female cones of Pinus patula Scheide et Deppe. were collected on a weekly basis during the South African summer months from December 1991 to February 1992 and from December 1992 to March 1993. Embryogenic tissue was initiated from excised megagametophyte explants containing immature zygotic embryos. Embryogenic induction was achieved using both MSG (BECWAR, NAGMANI & WANN 1990) and DCR (GUPTA & DURZAN 1985) media. The highest induction frequency was obtained on DCR1 (Douglas-fir Cotyledon Revised) medium supplemented with 0.5 mg 1 ¯¹ BA and 3.0 mg 1 ¯¹ 2,4-D, using L-glutamine as the major nitrogen source. Embryogenic tissue was translucent-to- white and mucilaginous in nature, composed of elongated, suspensor-like cells. The tissue was extruded from the micropylar end of the female gametophyte. In comparison, nonembryogenic tissue was produced from the gametophytic tissue itself and consisted of small, compact, spherical cells, crystalline in nature. Anatomical studies of developing patula seed demonstrated that the production of embryogenic tissue from the immature explants co-incided with the period, approximately two weeks after fertilization and with the occurrence of cleavage polyembryony in the developing zygotic embryos. Embryogenic tissue was maintained in culture by a recapitulation of the cleavage process. Transfer of the embryogenic tissue to DCR2 medium containing 1.3 mg 1 ¯¹ ABA resulted in tissue maturation and in the subsequent development of somatic embryos. Presence of ABA in the culture medium stimulated the development of cotyledonary initials in the apical region of the embryos. Elongated embryos, possessing small cotyledons, were rooted (50 to 60 %) on MSG6 medium containing no plant growth regulators. Somatic plantlets were successfully hardened-off under greenhouse conditions. Liquid culture methods were found to be a useful means of rapidly increasing the volume of embryogenic suspensor masses. Maturation , in terms of somatic embryo development and the production of cotyledonary initials, though , was not obtained in suspension. Reestablishment onto agar-solidified medium (DCR2) was required before maturation could occur. ABA is also responsible for stimulating reserve deposition and mobilization. In this regard, lipid accumulation in the developing somatic embryos was quantified and found to be significantly lower than in developing zygotic embryos. Similarly, non-matured embryogenic tissue contained less lipid deposits than matured (ABA-treated) tissue, indicating the requirement for ABA during maturation. Quantification of the lipids deposits is useful in determining the potential for somatic embryos to acclimatize to ex vitro conditions since their further growth and development is based on their ability to accumulate storage reserves. Somatic embryogenesis was found to be a useful method of propagation, producing plantlets with seedling-like qualities. This development has important consequences for the production of clonal plantlets in the Forestry Industry. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1994.

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

Summary: The future mass propagation of elite families of Pinus patula by cuttings is a realistic method of deployment if the short-term performance of cuttings and seedlings are confirmed at harvesting. This will impact significantly on the future outlook of forestry in South Africa as softwood yields are improved substantially through the introduction of material of high genetic value in commercial plantings. This, however, will require significant changes in future silviculture and other management practices as foresters and plantation staff learn to regenerate, maintain, and schedule the harvesting of cutting stands according to a different set of demands as a result of the change in plant type. Contrary to operational experience, cutting survival was similar to seedling survival in all field studies. This indicates that factors other than those that were studied and reported on, such as planting techniques, may be contributing to mortality. Also, due to the different root structure of cuttings they may be more fragile. The similar survival observed in these trials, therefore, may have been due to the close supervision given to the planting operations by the research staff. Although survival was similar, both plant types survived unacceptably poorly in the majority of studies with an average stocking of approximately 50% at one year. It is therefore anticipated that commercial stands will require several blanking operations in order to achieve an acceptable stocking in excess of 85% by the following planting season. The reduction in expected profitability as a result of blanking costs, delayed establishment, and the loss of improved genetic plant material, indicates that this is an area that still requires further research irrespective of what plant type is being planted. The pathogen, Fusarium circinatum, was commonly isolated from the planting stock before and after planting in two studies. Due to its virulent nature, it was assumed that mortality on the trees on which F. circinatum was isolated was principally due to this pathogen. At planting all plants were observed to be healthy and free of disease indicating that this pathogen maybe carried from the nursery to the field in a cryptic form, either inside or outside the plant tissue , which results in the death of the newly planted tree. In two field studies, where F. circinatum was commonly isolated, the application of Benomyl fungicide and to some extent the biological control agent Trichoderma harzianum at planting appeared to improve survival although this improvement was not significant. Laboratory studies, designed to determine alternatives to Benomyl fungicide, indicated that three fungicides (Octave, Folicur and Tilt), three sterilants (Sporekill®, Prasin®and Citex®) , as well as a biological control agent (T.harzianum), were all highly successful in controlling F. circinatum colony growth in vitro. It is recommended that these products undergo nursery testing , where the plant material is inoculated with F. circinatum spores, in order to test their efficacy and possible phytotoxicity in vivo before commercial application. Post-planting survival was also affected by site climate . Greater temperature extremes, as well as lower humidity and less rainfall resulted in poor survival. Plant dimension at planting was found to interact with site quality where it was a significant factor on a poor quality site. Optimal cutting dimensions at planting was a root collar diameter of 2.8 - 3.2 mm, and a stem height greater than 7 cm at planting for cuttings produced in cavities 90 ml in volume. Optimal seedling dimensions at planting were a root collar diameter of 1.8 - 2 mm, and a stem height of 10 - 15 cm for seedlings produced in cavities 80 ml in volume. In a separate study, plant morphological criteria influenced medium-term growth, where greater root mass and thicker cutting root collar diameters at planting improved field growth performance for seven years after planting. A greater root mass at planting was achieved by raising cuttings in containers that could support greater medium volume. From the study it was concluded that cuttings should be raised for an approximate period of 9 months in container cavities no smaller than 80 ml in volume and possess an oven-dry root mass of 0.3 - 0.5 g at planting. In addition to similar survival, the cuttings in this study grew either similarly to, or in some cases out-performed, the seedlings that were used as a control. Several other published studies indicate that hedge maturation poses the greatest threat to the success of softwood cutting deployment. This is especially true in clonal forestry and methods to maintain juvenility, such as cold storage of shoots and cryopreservation, require further research before clonal plantations of P. patula can be realised. In the studies carried out on family hedges in this report, the effect of donor hedge maturation was found to influence nursery management practice and the characteristics of rooted cuttings. The nursery data indicates that rooting efficiency, root system quality, and stem size and form, all decline with increasing hedge age particularly from two years after the date of sowing. A decline in root system quality was particularly apparent and was observed prior to a decline in rooting efficiency. If field trials indicate poorer performance from older hedges , it may be necessary to determine whether the causes are purely ontogenetic, morphological, or both before drawing final conclusions about hedge longevity. Until such results are known, it is recommended that P. patula cuttings should be propagated from seedling donors maintained as hedges , approximately 15 cm high, for a period not more than three years from the date of sowing. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.