Protocols for Micropropagation of Woody Trees and Fruits

ORGANOGENESIS AND CRYOPRESERVATION OF RADIATA PINE
moisture content adversely. If the optimum moisture content is not realised, cotyledon
response post-cryopreservation may be uneven.
Following storage in liquid nitrogen, vials are thawed rapidly in sterile water
heated to 40°C for 2 min (please note safety considerations with regard to thawing
material from the liquid phase). They are then treated in the same way that noncryopreserved
cotyledons are treated, as described earlier for adventitious-axillary
shoot production. It has been observed that cryopreserved cotyledons do take approximately
24 hours longer to begin to green in comparison to control cotyledons.
3. CONCLUSION AND FUTURE PROSPECTS
The methods described here for organogenesis have been developed over several
decades and are used successfully to amplify elite clonal material for afforestation.
In general, genotype capture is high, and organogenic approaches can be used to
amplify material from zygotic embryos, embryos from embryogenic tissue and fieldgrown
material. The in-vitro shoots readily initiate roots and form good root systems.
Improving methods of meristem cryopreservation will ensure the juvenility of selected
material while field testing takes place. Hybrid approaches, combining somatic embryogenesis,
organogenesis and nursery cuttings, offer possibilities of reducing costs
and increasing effectiveness of existing propagation methods.
There are also wider implications of this work, including investigating fundamental
aspects of maturation in Pinus. Some maturation is beneficial for improved
stem form, while excessive maturation compromises root initiation and early growth
rate. The ability to control maturation in vitro would be very beneficial. Accelerated
maturation would be very useful in breeding programmes, especially where markeraided
selection was employed. Importantly, some induced maturation may help
ascertain wood quality attributes, which only become apparent in trees with increased
age, and also shorten breeding cycles if early flowering was induced.
We also need to ask hard questions with regard to our methodologies if we are
to continue using organogenesis in combination with adventitious shoot induction,
embryogenesis and nursery propagation. We know that shoots of cotyledonary
adventitious-axillary origin show increased maturation in comparison to epicotylaxillary,
but why? If shoots are induced adventitiously from primary needles instead
of cotyledons, do they show signs of increased maturation? Is the increased maturation
simply a factor of cell division? In radiata pine, the origin of embryogenic
tissue is the early zygotic embryo, which is encouraged into a state of prolonged
cleavage polyembryony before further media modifications induce embryo maturation.
If we apply organogenesis protocols to embryos from embryogenic tissue,
there has already been a massive amount of cell division beyond the ‘rejuvenating’
meiotic event of fertilisation. These organogenesis protocols provide powerful tools
to investigate these issues further.
Acknowledgements. The authors would like to acknowledge the contribution of current and
former colleagues in developing the tissue-culture methods presented here for Pinus radiata.
These include Lynette Grace, Keiko Gough, Margaret Sigley, Dolina Skudder, Cathie Reeves,
Susan van der Maas, Mike Dibley, Lyn Holland, Jenny Aitken, Kathryn Horgan, Dale Smith
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