Improving germination in white spruce somatic embryos with desiccation and/or cold treatments
Date
2017-11-10
Authors
Pond, Sharon Elizabeth
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Abstract
Clonal propagation of white spruce (Picea glauca (Moench) Voss) through somatic
embryogenesis (SE) has important applications in tree improvement programs and will
help the forest industry to achieve maximum sustainable yield. The level of induction of
embryogenic tissue and the yield of mature embryos through SE has reached acceptable
levels using current protocols. However, a large percentage of these embryos produce
abnormal seedlings. This problem needs to be assessed and this was done in the work
described in this thesis.
Empirically derived, uncontrolled partial desiccation procedures are currently used to
improve germinaton. No systematic study has previously been done to correlate the
effects of controlled desiccation on germinant quality. My study looked at the effects of
controlled partial and complete desiccation of white spruce somatic embryos at four
stages of development on subsequent germinant quality. Both slow desiccation at 5°C
and flash desiccation at ambient temperature were examined. The effect of temperature
treatments as an alternate means of improving germinant quality and its effect on
desiccation tolerance were also examined. Dried somatic embryos are likely to suffer
imbibitional damage as they (unlike zygotic embryos) have no protective structures
surrounding them to regulate water uptake during imbibition. Therefore, the effects of
various rehydration methods were also examined.
Large numbers of mature embryos were required for our desiccation experiments.
Therefore, a method of squashing the embryogenic tissue into a polypropylene mesh was
developed. This method allowed embryogenic tissue to be easily transferred to fresh
medium and produced a flat mat of mature embryos that were more accessible for
harvesting.
The tolerance of the embryos to desiccation, and the level of desiccation required to
improve germinant quality, increased as the embryos matured. The largest improvement
in germinant quality was achieved by slowly desiccating 39-d embryos at 5°C for 7 days
over a 0.48 M NaCl solution with a water potential of -2 MPa and rehydrating them at
100% RH at a temperature of 5°C. This treatment produced approximately 84% normal
germinants. More severe desiccation caused increasing damage.
A temperature treatment of 5 and 10°C also improved germinant quality, producing 70-
80% normal germinants. The 5°C treatment can be used as a short-term storage method.
Germinant quality from untreated embryos increased with maturity until the embryos
became fully mature by 51 d, then quality quickly decreased. Mature 51-d embryos were
stored for 8 weeks at 5°C with no loss of germinant quality.
A 5°C temperature treatment for 4-8 weeks significantly improved the tolerance of 39-51
d embryos to flash desiccation (embryos were dried in a laminar flow hood and lost all
free cytoplasmic water within 15 minutes). This has important applications in the
development of synthetic seed. All of the 8-week cold stored 51-d embryos survived
flash desiccation and 58% of them produced normal germinants. The roots developed
desiccation tolerance faster than the cotyledons+hypocotyls.
Rehydration experiments showed that slowly and rapidly desiccated embryos responded
differently to the method of rehydration. Slowly desiccated embryos suffered less
imbibitional damage if they were indirectly rehydrated at 100% RH. Flash desiccated
embryos suffered less damage if they were rehydrated directly on germination medium.
This suggests that there is no one simple explanation for damage as a result of desiccation and imbibition.
Reduction of 2,3,5-triphenyltetrazolium chloride (TTC) was an effective test for
delineating damaged areas in rehydrated embryos, but actual germination tests were the
only way of accurately determining germinant quality.
The above treatments have significantly improved germinant quality.
Description
Keywords
White spruce