Saving Endangered Plants by Deep Freezing

© Marinda Louw

With a career spanning more than three decades, Patricia Berjak has devoted her professional life to the study of seeds the basis of commercial agriculture and an important storehouse of floral biodiversity for future generations. Berjak was named as the 2004 Distinguished Woman Scientist by the South African Department of Science and Technology and has been awarded the Silver Medal of the South African Association of Botanists.  Taking an early career turn away from animal biochemistry, she is passionate about cell biology and the survival of seeds in particular.

Her current focus includes saving the genetic material of plants used in traditional medicine. Together with her colleague (who is also her husband) Norman Pammenter and various graduate students, she is working towards the long-term storage of what are known as 'recalcitrant' seeds.

In scientific circles, seeds are divided into orthodox and recalcitrant. Orthodox seeds are dry, and can be stored for long periods of time under the correct conditions, before the addition of water allows them to germinate and grow into mature plants. Maize seeds are a good example of orthodox seeds.

Recalcitrant Seeds

Recalcitrant seeds are wet, and are destroyed when they dry out. They cannot be stored in the normal low-temperature, low-relative humidity conditions that keep orthodox seeds in a state of suspended animation. Recalcitrant seed species include mangos, avocados, coconuts, rubber trees, cocoa trees, and many plant species that are extensively used in African traditional medicine.

Coupled with the overharvesting of plants for 'muthi' purposes, many indigenous species are faced with extinction in the wild, and the loss of genetic variability that allows species to adapt to different conditions.Over the course of several years of re- search, Berjak and her students have made strides in saving the seeds of recalcitrant species. They discovered that the seeds are metabolically active from the time they are shed by a plant, starting germination processes in their cells, where only electron microscopy and biochemistry can detect what is happening.

These cellular changes continue even if the seed begins to dry out, but the seed be- comes more and more sensitive to water loss as germination progresses. Eventually, the germination process gets so far that the loss of even a small amount of water kills the emerging plant, and the unique genetic makeup of the seed is lost. Prevention of germination could be one way of extending the storage life of these wet seeds.

To prolong storage time they tried to reduce the amount of  water  in  the seeds so that germination could not occur, but essential metabolic   processes could carry on. However, this was not viable as it actually reduced the storage time. Current work has suggested that this is because a slight amount of dehydration actually stimu- lates the metabolic processes that begin germination. Another method of preventing germination and lengthening seed life is by lowering the storage temperature so that all metabolic activity in the seed slows down. However, most recalcitrant seeds are very sensitive to chilling.

Despite this, research is focusing on storing recalcitrant seeds by freezing them in liquid nitrogen at -196°C. To achieve this practically is very difficult as the seeds are  usually large and always full of water. When the water freezes, it expands and  destroys the cells inside the seed. However, not all of the seed is actually needed in order to produce a new plant, so Berjak's research is now looking at ways to use just what's necessary  and to reducethe amount of water it contains,  to survive cryostorage.

Only the small 'embryonic axis' which makes up the root and shoot in a germinating seedling is needed, so this is cut out of the wet seed and then flash-dried by a technique developed by Berjak and her colleagues, so that it can be cooled in liquid nitrogen without being harmed.

They are working on finding out which techniques give the best survival rate for this highly invasive way of storing seeds, and have found temperate species do better than tropical ones. One of Berjak's colleagues, Joseph Kioko, has successfully dried and cryogenically stored the small seeds of the pepper bark tree (Warburgia salutaris), which has been harvested virtually to extinction in South Africa for traditional medicine.

Synthetic Seeds

While the drying and freezing challenge is underway, the researchers are also trying to find a convenient way of distributing and planting the once-frozen embryonic axes to produce viable plants without the benefit of their original food storage tissues. This will involve creating synthetic seeds, where individual axes are placed in a gel capsule containing what is necessary torecreate seed-like structures.

Seed-Destroying Fungi

Berjak is also interested in the role that fungi play in destroying seeds. Hidden within seeds, is a variety of fungal species that live off the seed tissues. These parts of the seed are intended to supply food to a growing root and shoot before photosynthesis can provide a seedling with the energy it needs to develop. Berjak had previously worked with orthodox maize seeds, and found that by placing maize seeds in a static electric field their life span could be dramatically extended.

This was thought to be because the charged field helped prevent free-radicals from forming and then destroying the seed embryo, but thirty years on from this discovery, her thoughts turned to whether or not the extended seed life was actually because the static field damaged the fungi living in the seeds. Further research into seed fungi found that if maize grains were placed in hot water for a short period of time, the amount of fungus in each seed could be reduced.

This meant that the seed losses sustained by poor rural people who store maize in warm, humid conditions could be reduced by hot water treatment followed by redrying of the seeds. This kind of treatment would also reduce the risk of accumulation in the seeds of cancer-causing chemicals that the fungi produce.The study of the fungal species that can survive the dry conditions inside an orthodox seed has now turned to the amazing plant that grows in the Namib desert - Welwitschia mirabilis.

This extraordinary plant has a phenomenally long taproot and only produces two leaves in its entire life, but is a haven for many desert animals. Welwitschia survival is endangered by a fungus that can survive the ultra-dry conditions found in the plant's seeds, and Berjak and her team hope to be able to solve the fungal problem and so improve the long-term survival of the seeds.

Recalcitrant seeds also are threatened by associated fungi so, between optimising  cryogenic storage of the embryonic axes and lowering their fungus levels, Berjak hopes to be able to preserve recalcitrant-seeded species in Africa. The ultimate ambition of Berjak and her research team to establish a cryobank, for which they are actively seeking financial backing: "It would be our way of combating the spectre of genetic erosion and extinction of the continent's most valuable and sought after plants."

By Melissa Wray

Kruger National Park - South African Safari