Developmental anatomy and cryopreservation of structures created during indirect organogenesis in a Eucalyptus grandis / Eucalyptus urophylla hybrid
Date
2009-09-03T10:43:50Z
Authors
Simelane, Phindile Garish
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Abstract
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.