Developmental anatomy and cryopreservation of structures created during indirect organogenesis in a Eucalyptus grandis / Eucalyptus urophylla hybrid

Simelane, Phindile Garish
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Callus was initiated from Eucalyptus grandis x urophylla axillary bud segments and cultured on a callus induction medium to regenerate shoots. At the gross morphological level two types of callus were described; a crystalline callus and compact glassy callus (yellow, white and brown) that gradually became nodular. A red callus was also identified in the late stages of development (between days 43 and 57). The crystalline and compacted callus sometimes formed dark spots on the surface which were possibly an indication of the accumulation of tannins. Histologically callus developed two types of cells; small compact morphogenic cells and loosely arranged large nonmorphogenic cells. The morphogenic cells were meristematic in appearance and presumed to be involved in the formation of shoot-bud structures. The meristematic activity produced vascular nodules (nodular structures) which differentiated into shoot-buds. The vascular tissue of callus was continuous with the developed shoot-buds. This showed that the shoots were initiated from the differentiation of callus and not from the original axillary bud merisitems. The process of shoot-bud development was monitored for 8 weeks with 39% of the material producing shoots. The induced shoots were then cultured on a root induction medium. Results indicated that root initiation occurred within 24 hours of culture. Roots were either formed from callus redifferentiation at the base of the shoot or from the shoot vascular cambium. Vascular connections that developed in the adventitious roots formed from parenchyma differentiation. Another connection was formed between the lateral root primordium and primary root. The total yield of shoots that formed roots was 74% over a four-week period. Although cells that formed the adventitious roots were identified, the initial stages of root initiation were not recorded. Callus that was 22 days old was cryostored using both slow and rapid freezing rates and the subsequent survival determined by tetrazolium chloride testing (viability) and the potential to regenerate shoots (post-thaw growth). The callus (slow freezing rate) that was pretreated by exposure to cryoprotectants and then dried for 20 minutes showed 55% viability compared with callus that was dried first and then exposed to cryoprotectants which had a 52% viability during slow freezing. The material that was rapidly frozen when was not viable. None of the cryo-treatments resulted in postthaw growth. The results indicate that the slow freezing method has the potential to be the method for cryostorage of callus of Eucalyptus.