Somatic embryo development and phenotypic variation in an abscisic acid-independent line of Larix x eurolepis
Date
2018-08-02
Authors
Hay, Elizabeth Irene
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Abstract
The objectives of this thesis were to trace the developmental pathways of somatic embryos
of an abscisic-acid independent line of Larix x eurolepis. to catalogue the phenotypes of mature
embryos, to determine critical stages of development and to attempt to increase the number of
maturing somatic embryos. The low rate of maturation could not be entirely explained by
differences in phenotypes of early embryos, critical stages of development, or the lack of plant
growth regulators in the medium. In addition, the shape and epidermal type of the mature embryo
did not always determine the type of epicotyl produced, nor did it affect the rooting and mortality
rates. Six types of embryonal structures were evident in the aggregates of line 2086: (1) a smooth
(SEMLS) or (2) rough (REMLS) embryonal mass subtended by a cylindrical, compact, long
suspensor. (3) a rough embryonal mass subtended by a long, loose suspensor (REMLLS). (4) a
rough embryonal mass subtended by a suspensor arising from greater than one quarter of the
surface area of the embryonal mass (REMST). (5) a rough embryonal mass subtended by a short,
compact, cylindrical suspensor (REMSS). and (6) a cluster of meristematic cells which may or may
not have single suspensor cells attached (MC). For isolated embryonal structures of all types, to
continue development into a nodule or a mature embryo was the least common fate, while
proliferation and developmental arrest were more common. In general, the more organized
embryonal structure types (SEMLS and REMLS) had higher rates of maturation compared to the
other 4 types but the most common fate was still developmental arrest (74% SEMLS. 62%
REMLS), followed by proliferation (10% SEMLS. 30% REMLS), and nodule or embryo
development (16% SEMLS. 9% REMLS). REMLLS and REMST embryonal structures became
developmentally arrested or proliferated (43-47%) while the rate of nodules/mature embryos
production was 9-11%. Neither individual REMSS nor MC structures produced any nodules or
mature embryos, but REMSS had a lower rate of developmental arrest (81%) and a higher rate of
proliferation (19%) than MC (89% and 11% respectively). Embryos at more advanced stages of
development were less likely to die, become developmentally arrested or become nodules, but more
likely to become mature embryos than embryos at less advanced stages of development. A critical
stage of development appeared to be the focal zone stage at the formation of a complete polyphenol
band around the basal end of the embryonal mass. At this stage, the majority of immature embryos
became mature embryos (61%) while only 3% of the embryos died. 10% became developmentally
arrested, and 20% became nodules. The majority of mature somatic embryos were normally
proportioned with a smooth epidermis (43%) rather than vitrified (12%). normal with a rough
epidermis (12%) or misshapen (smooth or rough. 33%). The shape of the mature embryo was
associated with the type of epidermis, with mature somatic embryos with normal proportions more
likely to have smooth epidermis (78%) than a rough epidermis (22%) while mature embryos with
abnormal proportions were as likely to have a smooth epidermis as a rough epidermis. The shape
of the mature embryo was associated with the shape of the epicotyl produced. Normal-smooth,
mature embryos were more likely to produce normal-smooth epicotyls (73%) than twin epicotyls
(21%), vitrified epicotyls (2%) or misshapen epicotyls (5%) compared to vitrified mature embryos
(42% normal-smooth epicotyls, 34% twin epicotyls, 23% vitrified epicotyls, 1% misshapen
epicotyls) or misshapen mature embryos (22% normal-smooth epicotyls, 47% twin epicotyls, 7%
vitrified epicotyls, 24% misshapen smooth/rough embryos). The number of mature embryos
which germinated or died was not associated with either the epidermal quality or the shape of the
mature embryo. Few SEMLS or REMLS embryonal structures responded to auxin and cytokinin
treatments. There appeared to be a trend towards less developmental arrest and proliferation and
more nodules/mature embryos produced on media with no auxin compared to media with 2,4-D
and a trend towards more developmental arrest and fewer nodules/mature embryos on media
without BA compared to media with BA. Only nodules on media without plant growth regulators
produced roots or cotyledons. There was no effect of embryonal structure type (SEMLS or
REMLS), or sucrose concentration (58 μM or 174 μM) on the maturation of immature embryos,
but on media without ABA, fewer immature embryos proliferated or became developmently
arrested and more embryos became nodules or mature embryos than on medium with 6-24 μM
ABA.
Description
Keywords
Plant regulators, Somatic embryogenesis, Growth (Plants)