As long ago as 1986, the UCI Arboretum received the go ahead from its governing body to create a cryogenic orchid seed bank as a hedge against extinction for wild orchid species (Koopowitz, 1986). Since that time we have not published any further reports and there has been little public discussion. The silence evinced little curiosity, but some orchidists did assume that the concept of an orchid gene bank had died somewhere along the way. This article is a recounting of some of the events over the past eight years which will bring orchhid hobbyists up-to-date with orchid gene-banking in general and specific progress in this area at the UCI Arboretum.
It should be pointed out at the onset that UCI does maintain a cryogenic seed bank for African species in several plant families and that we currently have a small federal grant to increase the number of holdings of bulbous plants (not orchids, unfortunately) in our freezers.
The idea of a cryogenic seed bank is quite simple. Freshly matured orchid seed is partially dried and then sealed in a container and stored at below freezing conditions. As orchid seed is so small, usually weighing less than a few hundred micrograms, enormous numbers of seed and hence large numbers of future individual plants can be stored in a relatively small space. This is far more economical than keeping a living plant collection. Despite the fact that this idea has been Promulgated widely, few such banks actually exist. The ability to freeze and thaw orchid seeds without loosing viability has been repeatedly demonstrated with a variety of orchid seeds. Most researchers used moderate subfreezing temperatures while a few used liquid nitrogen. Nearly all of these studies demonstrated that seeds could be frozen and germinated after withdrawal from the cold storage temperatures. One of the most important and influential studies was that of Bowling and Thompson who reported in 1972 that they had tested the ability of seeds of 30 different tropical orchid species to withstand freezing at -10C.
A few months after announcing our proposed gene bank we came across a very unsettling paragraph that was published in the Orchid Research Newsletter (Pritchard, 1986). This reported on some further investigations carried out on the seed stored by Bowling and Thompson. After three years in storage some of their seeds were found to be viable but when additional seeds were tested again in 1980, between eight and ten years after having been frozen, all were found to be dead. However, this information was not made public until Pritchard's 1986 report. When it was released the results seemed devastating. The brief report suggested that long-term storage might not be feasible and therefore one of the most powerful options for conserving orchid species was eliminated.
There were several possible explanations to account for the loss of viability in the stored seeds. It was known that some non-orchid seeds are very fragile following freezing and if hydrated too rapidly their cell membranes might be damaged. But, if that was the case, why did they get germination after three years but not after eight? Perhaps the two groups of seed were not rehydrated equally? There was also the possibility that the seeds were not maintained in airtight containers and had dehydrated beyond some critical point as they lay in the refrigerators. The seed had been placed in glass tubes with corks that were then covered with wax. During storage the wax might have fractured and broken the airtight seal. But nobody knew for sure. Perhaps the temperatures in the freezer had oscillated so much that seeds were alternately frozen and thawed enough times that in the process they had been fatally damaged. Finally, there was also the dismal prospect that perhaps orchid seed could not be stored in a frozen condition for any extended period of time.
To be useful a long-term seed bank must be able to hold seed for at least 50 years but preferably one or two centuries. In good conscience, we felt we could not continue to set up the gene bank with the prospects of only keeping the seed alive for a few years. So little research had been done on long-term longevity of orchid seeds that some research in that direction seemed necessary before embarking on an orchid gene banking scheme. In the mean time we did have samples of seed of Encyclia vitelline which were stored in a freezer at -40C. These were left over from our original research that had been done to convince ourselves that orchid seed could indeed be frozen and then retrieved (Koopowitz and Ward, 1984). At that time, it had been decided to leave several samples of seed as long as possible to see how well they would last.
Around the world, there are gene banks for the seed of agriculturally important plants and also for seeds of many wild plant species. The recommended storage conditions for that seed is at -18C with a moisture content of about 5%. Those conditions were determined after several series of experiments to determine the way seeds age under a variety of above freezing storage temperatures and then extrapolating to frozen conditions. The experiments had to be performed at above freezing temperatures because the aging kinetics of seeds suggested that one would have to run experiments for decades in order to get useful information from tests run at subfreezing temperatures. With orchids, we have the additional constraint that the forests might not last long enough for us to do the needed experiments. Species are going extinct as we try to decide what to do. Disa uniflora, is a useful experimental orchid for seed work because the seeds are larger than average and can be germinated without having to resort to aseptic flasking. In addition seed maturation and germination only take a matter of weeks. We decided to run some experiments on seed aging in Disa anticipating that it could be used to give clues about the behavior of orchid seeds in storage and hopefully to lay a solid foundation for orchid gene-banking. Perhaps it might also give us clues about what had happened to Bowling and Thompson's seeds that had caused their demise.
Among other things, our research (Thornhill and Koopowitz, 1992) showed that with repeated freezing and thawing there was a loss in viability. Half the seed would be dead after approximately 68 freeze-thaw cycles and all will be lost after 140 cycles. These numbers might not be important over the short term, but are significant for storage conditions that last hundreds of years. There will be times when seed will need to be transfered to new freezers, when there are brown-outs, when seed is accidently taken out of the freezer, etc. The gene banker needs to be aware of this and should keep records of the number of times seed samples are thawed and refrozen.
Most interesting was what happened to seed longevity when we extrapolated our data down to subfreezing conditions. Depending on the mathematical assumptions that one made, there were two possible extrapolation curves. At -10', one of these graphs indicated that half of the viability would be lost in three years following freezing, but the other curve suggested that half should not have been lost until after 30 years. Statistics can not tell one which is the correct curve. One needs experimental evidence to disprove one of the two curves.
Well, we had our frozen samples of Encyclia vitelline seed which had been in an old freezer for ten years. The compressors had been replaced on the freezer several times and the temperature inside was now -24C compared to the original -40C. In addition with brown-outs and many openings and closings of the freezer lid the seed had probably been freeze- thawed a large number of times. Could the seed tell us anything about which of the two extrapolation curves we could trust? We knew that the seed originally had a very high level of viability. If most of the seed was now unable to germinate we could not be sure if the lower extrapolation line was the explanation. However, if most of the seed was still able to germinate then there was a reasonable chance that the lower extrapolation curve was incorrect and the upper curve, with more extended longevities, might be closer to reality and therefore we could proceed with gene banking.
Some of the seed was carefully removed from the container and exposed to a moist atmosphere where it was very gradually rehydrated, after which it was sown on a general germination medium. Within a short time greening and swelling of the embryos was observed and then leaves started to form. Germination and viability was excellent. It looked as if nearly every seed had germinated and viability was probably in excess of 95 percent. Based on these results, subfreezing seed storage now seems possible again. We may never know exactly what happened to Bowling and Thompson's seeds but it seems unlikely that the loss of viability was due to inability of the seed to withstand long- term freezing.
Additional samples of the original Enc. vitellina seed, were placed in a newer freezer at -40'C, to await further testing in another 10 and then 20 years time, but there is no need to wait for those results before starting to assemble a frozen seed collection. A cryogenic orchid seed collection is now a viable option for conserving orchid species. At the UCI Arboretum we have decided to steam ahead and start freezing seeds of orchid species. But we need help, both in procuring the seeds and in getting the money to process the seed and maintain the bank.