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Cryopreservation and Seed Storage:
Meet the science and the scientist
by Kathy Waddington
Durban biologist Professor Patricia Berjak's many-year journey of discovery has
taken her through the rigours of finding the best methods for seed storage and
minimising destructive fungi, all the while making breakthroughs in
understanding that have resulted in accolades from world authorities. Now she
has reached an apex where her work borders on what was until recently reserved
for the realms of science fiction: cryopreservation. She recently received the
Silver Medal of the South African Botanist Society, awarded earlier this year.
Prof Berjak spoke to Kathy Waddington.
 What
if . . . KwaZulu Natal's sugarcane crops were to be wiped out in a natural
disaster? Or Brazil's coffee crops? If the avocado pear were to become extinct?
Hardly likely, you say? Many plant species face extinction around the world, and
one close to home is the pepper-bark tree (Warburgia salutaris),
practically extinct in the wild because of the demand, and lethal debarking
frequently employed, for its use in traditional medicine - and because it hardly
ever produces viable seeds.
Depredation of natural plant resources is an ever-escalating problem
throughout Africa, aggravated by land clearing for agriculture, erosion,
commercial forestry, settlement of an ever-burgeoning population, and
indiscriminate harvesting of many species. Not to mention severe climatic
changes, with increased floods and droughts around the world creating havoc with
food production; a world in which millions are already starving.
This represents a real threat to the gene pools of various species and the very
existence of many.
These are issues several University of Natal scientists, both biologists and
environmentalists, tackle from many different angles.
Among the leaders - provincially, nationally and globally - in terms of
'finding solutions' is the Biological Sciences Division of the School of Life
and Environmental Sciences, and in particular a woman who has devoted much time
and energy through research and teaching, to ensuring the safe storage of seeds
and other genetic material.
Professor Patricia Berjak's many-year journey of discovery has taken her
through the rigours of finding the best methods for seed storage and minimising
destructive fungi, all the while making breakthroughs in understanding that have
resulted in accolades from world authorities. Now she has reached an apex where
her work borders on what was until recently reserved for the realms of science
fiction: cryopreservation. And for all this work, she has received her most
recent honour, the Silver Medal of the South African Botanist Society, awarded
earlier this year.
Berjak is quick to deflect the glory: it's teamwork, she says, and over the
years many outstanding biological scientists have contributed to the success she
and her colleagues enjoy today. Not least is TA Villiers, the biologist-tutor
who introduced the young student to the fascination of seeds and electron
microscopy, sealing her fate as a leading research scientist.
Even before she obtained her PhD degree she was poised at the cutting edge of
plant science, with the first of her discoveries making plant biologists sit up
and take notice.
Having traversed a somewhat winding academic route that took in first
Biochemistry, then medical research, the electron microscope had suddenly
revealed a new and exciting world of mystery. Her first breakthrough came when
she established by electron microscopy and cytochemistry, that plant cells do
contain lytic compartments responsible for 'recycling', quite serendipitously
complementing the almost simultaneous publication of biochemical evidence by a
renowned researcher, Philippe Matile.
Her PhD work established more, however. Why, she was tasked to discover, do
seeds lose viability in storage? And, using the intuition that has marked her
innovations along the way, she reached an outcome that was both original and at
a tangent from current thinking of the day. While others were looking at the
chromosome level for the primary cause of the damage that resulted in lost
viability, Berjak established that membrane damage was the key feature.
This work, which addressed the problem of maize seed deterioration in air-dry
storage, was to become a major chapter in the authoritative book, Seed
Viability, by EH Roberts, then the foremost name in research on seeds - and one
of Berjak's external examiners for her PhD. It also generated five papers in New
Phytologist, as well as a presentation at a key conference in the UK -
altogether affording her work rapid recognition by the leading world figures in
the discipline.
Other revolutionary findings include linking free-radical-mediated
deterioration to seed cell damage and development of a quenching treatment,
described in a paper published in the journal, Science.
Then, her intuitive skills at the fore again, she came to suspect that fungi
were responsible for more than making available for the living, carbon and
nitrogen and other elements of the dead. This opened yet a new chapter in
Berjak's career when she demonstrated their pathological qualities, particularly
to the embryos of dry seeds.
Working with Associate Professor Michelle McLean, then a research student,
they showed that fungi, surmised to invade seeds only during dry storage, were
present, long before harvest, also demonstrating that each fungus
preconditioned the seeds for the next in succession. Work with another research
student, David Mycock (now an Associate Professor at Wits University) showed
unequivocally that fungi could be transmitted from infected seeds through the
growing plant to the next generation.
The impact of Berjak's work on storage problems of seeds that behave in a
predictable manner, or 'orthodox' seeds, was manifested when she was invited as
one of a select group of scientists to consider the conservation of seeds
considered unstorable, the 'recalcitrant' seeds, the behaviour of which is
unpredictable. Examples include seeds of the avocado, natural rubber, mango,
litchi . . .the English oak and the pepper-bark trees. That meeting, in the
UK in 1980, was to prove to be a pivotal point, heralding the phase that was to
establish her group as among the foremost researchers on seed recalcitrance in
the world.
Fired with enthusiasm, she returned with the knowledge that in sub-tropical
KwaZulu Natal, the stage was set for a high-tech approach with a readily
available seed source. So began her onslaught on recalcitrant seeds and, working
with NW Pammenter, her husband and major collaborator in this field, within
three years she presented the first major paper at an international congress.
This ascribed the 'problem' to a consequence of developmental metabolism that
would grade into that of germination as a post-harvest phenomenon in these
necessarily-wet seeds. Basically, it was known that recalcitrant seeds would not
tolerate drying, being killed in the process. Berjak reported that they were
'wet' for very good reason: they were in a state of 'invisible' germination and
their metabolic functions required that moisture. Slow drying would, inevitably,
kill them. At that congress a doyen of seed research, JD Bewley, commented that
"this was the first time anyone had provided a meaningful explanation of
the basis of recalcitrant seed behaviour".
Jill Farrant, now an Associate Professor at the University of Cape Town,
whose PhD studies were the first to document pre-shedding development of a
highly recalcitrant seed-type, then joined Berjak's team. The team, since
augmented by others, has placed South Africa firmly in the international arena
in understanding the biology of recalcitrant seeds, their improved handling and
the potential for their improved short-term storage, as well as long-term
conservation of the genetic resources of the many species concerned.
Out of this came Berjak's pioneering 'flash drying' work, involving the
removal of water as quickly as possible from embryonic axes excised from seeds.
Flash drying, which minimises the time factor during dehydration - when lethal
processes occur - was introduced to the National Seed Storage Laboratory, USA,
during a collaborative working visit by Berjak and Pammenter, and it is now in
routine use there. The technique affords the only possibility for long-term
conservation of seed-derived germplasm: cryostorage, i.e. storing in a frozen
state, usually in liquid nitrogen.
Developing the axis into plants then requires an initial bio-technological
operation in tissue culture. Cryostorage, a major research focus of James
Wesley-Smith now working with Berjak and Pammenter, has met with unprecedented
success . . . while colleagues in Europe have been unable to store English oak
seeds in the long-term or to cryostore the axes, Berjak and her team, using
flash drying, fast-freezing and cryostorage, have achieved a 75% success rate in
producing healthy seedlings.
And what of the pepper-bark tree? A tropical species, very different, very
difficult and on the verge of extinction, it is disappearing more quickly than
it can regenerate. There are few seedlings because the seeds are readily
parasitised unless carefully protected during development, while bark harvesting
kills the tree itself. It is propagated, but by cuttings, so the genetic base
has become extremely narrow.
"Every attempt to preserve excised axes failed," says Berjak, so,
with Joseph Kioko, who is preparing his PhD thesis, they tried freezing the
entire seed, which is small and soft enough to dry very rapidly.
And the triumph of the experiment means that the pepper-bark can now be saved
for posterity: seeds can be cryoconserved for planting programmes to replenish
the devastation this species has suffered. Berjak's passion now is to secure
funding for an accelerated research programme to create a cryopreservation
facility. "We could then offer a national service of developing cryostorage
protocols in South Africa that would allow us to conserve our genetic resources,
crop ancestral genotypes, as well as those of clonal material."
Expertise in, and experience with, the biotechnology involved in cryostorage
is clearly established in Durban, at the University of Natal. What is needed now
is funding to accelerate the research and to create the facility that could -
and should - be nurturing these resources for the future.
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