Medium- and long-term storage of the Pycnanthemum (mountain mint) germplasm collection.

The United States of America collection of mountain mint (Pycnanthemum Michx.) is held at the USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon as seed, potted plants and tissue cultures and a long-term storage collection is preserved at the USDA-ARS National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado. The clonal collection is comprised of 34 accessions as potted plants that are duplicated with 31 accessions stored as in vitro cultures at 4 degrees C in tissue culture bags for medium-term storage at NCGR and as cryopreserved shoot tips in liquid nitrogen at NCGRP for long-term storage. This study reports on these two models of preservation of mountain mint at the U.S. National Plant Germplasm System. In vitro plants required 2 to 7 months for propagation on MS medium without growth regulators before storage at 4 degrees C. Plants remained in storage with good vigour in bags on 1/2x nitrogen MS medium without growth regulators for a mean of 2.08 y. An encapsulation-dehydration protocol was successful for cryopreservation of shoot tips from cold acclimated in vitro plants. Post-cryo viability, indicated by shoot tips with developed leaves and roots, ranged from 60 to 100 % for 27 accessions and 40 to 50 % for the other four. The encapsulation-dehydration cryopreservation method proved suitable for long-term preservation of the 31 Pycnanthemum accessions. These alternative storage forms allow for active use of the collection as well as base storage for clonally propagated accessions.

Micropropagation of pear (Pyrus sp.).

Elements of micropropagation include establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets. The wide genetic variation in Pyrus makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced dormant shoots or from scions grafted onto seedling rootstocks to impose juvenility. Clean shoots are recovered after testing for contaminants at the initiation stage on ½ strength Murashige and Skoog 1962 medium (MS), at pH 6.9 for 1 week or by streaking on nutrient agar. Although pear species and cultivars are cultured on several well-known media, MS is the most commonly used. Our studies showed that multiplication and growth of shoots are best on Pear Medium with higher concentrations of calcium chloride, potassium phosphate, and magnesium sulfate than MS medium and 4.4 µM N(6) benzyladenine. Pear shoots are often recalcitrant to rooting; however, a 5 s dip in 10 mM indole-3-butyric acid or naphthalene acetic acid before planting on basal medium without plant growth regulators is effective for many genotypes. Pear shoots store well at 1-4°C, and can hold for as long as 4 years without reculture. Cryopreservation protocols are available for long-term storage of pear shoot tips. Acclimation of in vitro-rooted or micrografted shoots in a mist bed follows standard procedures.

Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage.

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.

Cold storage and cryopreservation of hops (Humulus l.) shoot cultures through application of standard protocols.

The USDA-ARS National Clonal Germplasm Repository (NCGR) stores the global diversity of Humulus for the US Plant Germplasm System as trellised plants in a field genebank. In vitro storage and cryopreservation are now considered excellent ways to provide medium and long-term storage for plant collections. Developing a new cryopreservation or cold storage protocol for every accession or genus of large multi-crop collections can be a very time consuming and long-term activity. We propose that standard cold storage and cryopreservation techniques used for other temperate crop genera would be successful for additional crops with few modifications. This study was initiated to determine if a large collection of hops germplasm could be successfully stored with techniques developed for unrelated genera. In this study we characterized the response of diverse Humulus genotypes to in vitro storage under low light at 4 degree C following techniques used for strawberry and mint plants, and cryopreservation in liquid nitrogen by slow cooling with a pear protocol. The average storage time without transfer for the 70 genotypes evaluated was 14 +/- 3.5 months with a range of 6 to 26 months. Mean recovery of cryopreserved shoot tips of accessions with 1-wk cold acclimation was 41 +/- 18 percent and increased to 54 +/- 13 percent with 2-wk cold acclimation. This demonstrates that application of a well-tested standard technique can provide a quick start for storing additional germplasm collections.