since 1984. He will tell you that libraries, banks, orchards, and liquid nitrogen tanks have more in common than meets the eye when it comes to storing the nation's treasured apple collection. 
Geneva, NY - McIntosh and Golden Delicious may be popular apples, but they are two very small fish in a very large apple gene pool that includes thousands of varieties. Phil Forsline has been curator of the apple collection at the USDA-ARS Plant Genetic Resources Unit (PGRU) at the New York State Agricultural Experiment Station since 1984. He will tell you that libraries, banks, orchards, and liquid nitrogen tanks have more in common than meets the eye when it comes to storing the nation's treasured apple collection.
Up until recently, Forsline's "library" consisted of a 25-acre farm where he and his staff maintain some 5,000 apple trees-representing 2,500 different varieties-planted in lots of two. New germplasm is usually seed-propogated, often wild material, from centers of diversity like Kazakhstan.
"The collection includes rare varieties, some of which date back to ancient Rome, and some of which come from Kazakhstan, where researchers think apples first evolved," said Forsline. It is the largest living collection of catalogued apple trees anywhere. Forsline has traveled all over the world looking for new and unusual apple varieties to expand the apple gene pool represented in the collection.
"Older varieties of plant material are important to maintain to preserve diversity in the gene pool," said Forsline. If a new strain of disease comes along that decimates current commercial varieties of McIntosh or Golden Delicious, for instance, older varieties might provide the desirable resistant genes for future apple breeding.
Such a collection takes up a lot of space and costs some $375,000 annually to preserve. "It takes $75 to $100 a year to maintain one tree in an orchard," said Forsline. "In addition to space, you have to take into consideration the labor involved in spraying, cultivating, pruning, monitoring, and the costs of distributing 3,000 accessions annually to users as scion wood."
The nation's living library of apple is on the brink of drastic change. Forsline can foresee a not-too-distant future where apple trees will be preserved as live 1 1/2-inch apple buds in five lots of 12 buds each, in airtight tubes, at -150°C (-196°F), in tanks of liquid nitrogen instead of rooted as trees in their entirety in orchards.
"We can preserve 660 accessions in each tank. It is conceivable that we could take out or replace up to five acres worth of orchard with just one liquid nitrogen tank," said Forsline. Cost savings per tree amount to $74 per year; it only costs $1 per bud per year to preserve it in one tank which takes up less space than a VW Bug. Electricity is only used to monitor temperature probes and fill techniques; nitrogen in liquid form is naturally one of the coldest elements known to man (-196°F).
Forsline has been working closely on the cryopreservation of the Station's apple collection with Leigh Towill, a research plant physiologist at the USDA-ARS Fort Collins, Colorado, laboratory, for three years. He also collaborates closely with Norm Weeden, Herb Aldwinckle, and Susan Brown at Geneva on other projects in apple germplasm.
"The first person to successfully freeze woody material was Dr. Sakai, from Japan," said Forsline. Apple was first "frozen" in 1981 by Dr. Cecil Stushnoff, who continues to cooperate closely with research scientists at Geneva and Fort Collins. Currently, 500 samples of apples are in storage in tanks of liquid nitrogen located at the USDA-ARS facility in Fort Collins. One hundred samples are in storage in the tank in Geneva which went on-line in January, 1995. Ultimately, PGRU's entire collection of apples will be stored in tanks in the "master" bank at Fort Collins. At least half of the collection will be stored in tanks in Geneva so that only material that is in high demand remains in the field.
Forsline explained cryopreservation technique as it has evolved to date. "Cold hardiness is one of the traits of apples that makes them suitable for cryopreservation," he noted.
Live apple buds are collected in the winter after a hard freeze, and cut into 1 1/2-inch, one-bud segments. Then, they are stored at -4°C (25°C) for two weeks to maintain cold acclimation. At the same time, they are dehydrated to 30% moisture.
The buds are then placed in lots of 12 in sealed plastic tubes where they will maintain 30% hydration. The tubes are labeled, inventoried by computer, and placed 20 to an aluminum box. Put in a programmable freezer in which the temperature drops one degree per hour to -30°C, they are held at that temperature for 24 hoursÑ"a preconditioning treatment."
At this point Susan Sheffer, PGRU Science Technician, or Forsline, don heavy Thinsulate gloves, to unlock and open the stainless steel MVE Cryogenic XLC 1830. They gently place the boxes on a lazy-susan located just above the liquid nitrogen in the tank. Neither the apple buds nor the tubes ever touch liquid; they are kept cool by the vapor phase (-180°C).
Among the first group of buds to be frozen at PGRU were Esopus Spitzenburg, an old variety from Thomas Jefferson's day; Snow, thought to be a parent of the modern-day McIntosh; and Lady, one of the oldest apples on record.
Although being used routinely, some parts of the procedure are still in the research and development stage. Since 1988, Forsline and his crew have been participating in a 30-year viability study with the USDA lab in Fort Collins. They rehydrate the frozen samples in moist peat moss, re-graft the buds onto rootstock, and force them in a greenhouse setting to prove recoverability and viability. Variables include season, timing, rates of pre-conditioning and hydration, rehydration, and re-grafting.
"Our research shows that at least 80% of the samples stored have at least a 50% chance of buds producing healthy trees after they're rehydrated and grafted onto rootstock," said Forsline.
Forsline and other curators within the National Germplasm Repository system hope to use the cryopreservation technique to preserve sour cherries and grapes. "So far, we have been successful with certain varieties of cherries, but only the most cold-hardy grapes," said Forsline.
Eventually, Forsline hopes to bring other liquid nitrogen tanks on-line in Geneva. He will be working on storage with others curators and researchers at the Station in the apple, grape, and stone-fruit programs.
Plant germplasm contains the genetic information that is preserved, evaluated, cataloged and then distributed to people with a valid use by the National Plant Germplasm System, a network of federal, state, and private organizations and research units (including PGRU). The system is coordinated by the Agricultural Research Service (ARS), which is the principal research agency of the USDA.
Contact: Linda McCandless
315-787-2417
e-mail: llm3@cornell.edu
Communications Services, Geneva, NY
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