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Barbara M. Reed

Cultures of 49 Pyrus species and cultivars and one Pyronia (Pyrus × Cydonia hybrid) selection were screened in vitro to determine a rooting method suitable for a wide range of germplasm. Auxin treatment was required for rooting in most cases. Eighteen of the 50 accessions rooted with a 15 sec. 10 mM indole-3-butyric acid (IBA) dip followed by growth on medium with no growth regulators (NCR). Medium with 10 μM IBA for one week followed by NCR medium produced 12 rooted accessions, but NCR medium alone produced little or no rooting. A 15 sec. dip in 10 mM naphthaleneacetic acid (NAA) followed by NCR medium was tested on 29 accessions which rooted poorly on the other three treatments. Twice as many (28%) rooted on NAA as on either IBA treatment (14% each). Additional treatments combining IBA with darkness or higher temperature were also tested and were successful for some cultivars. P. calleryana, P. koehnei, P. pashia, P. hondoensis, P. ussuriensis, P. betulifolia, P. regelii, P. pyrifolia hybrid cv. Shinseiki and the Pyronia selection failed to root. Twenty two of the 32 P. communis cultivars rooted on at least one treatment.

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Barbara M. Reed

In vitro cold storage of Rubus germplasm was investigated using several environmental conditons and types of storage containers. Shoot cultures of Rubus species and cultivars were grown in either tissue culture bags or 20 × 150 mm glass tubes and compared for plant condition and survival under various storage conditions. Cultures stored at 10 C in the dark were in poor condition after 6 months. Cultures kept at 4 C were in much better condition and had higher survival rates after 18 months when stored with a 12 h daylength rather than total darkness. Overall there were no differences in survival or condition between cultures in tubes and bags. Contamination rates were 15% in tubes and 0% in bags. Plants in tissue culture bags could be stored for 9 months at 25 C with 16 h light when the nitrogen level of the MS medium was reduced to 25% and the medium volume was increased from 10 to 20 ml per bag. Genotype differences were apparent under all conditions tested. The best storage condition for Rubus germplasm was 4 C with 12 h light. Plastic tissue culture bags were preferred over tubes due to lower contamination rates.

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Barbara M. Reed

Cold hardening is an effective method for conditioning meristems for cryopreservation. ABA plays a role in hardening and produces increased hardiness in suspension cultured cells. This study was designed to determine if growth, in vitro, on ABA (5×10-5 M) for one week, would substitute for one week of cold hardening, and if ABA would provide additional conditioning when added in combination with cold hardening treatments. In vitro plantlets of Rubus spp. were grown for one week with or without cold hardening and with or without ABA. Meristems from these plants were frozen at 0.8C* min-1 to -35 C, then plunged into LN2, thawed, and plated on recovery medium. One month after thawing, cold-hardened plants with and without ABA treatment had recovery rates of up to 83%. Survival of plants grown at room temperature ranged from zero to 8% and zero to 28% for plants grown on ABA at room temperature. At the rates tested, ABA is less effective than cold hardening in conditioning apical meristems of in vitro Rubus plants for cryopreservation and provides no additional protection to cold-hardened meristems.

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Barbara M. Reed

Four tissue-cultured mints, Mentha arvensis L., M. spicata L., M. suaveolens Ehrh. hybrid, and M. suaveolens cv. Variegata, were evaluated for survival during storage in media containing three concentrations of N and in four light and temperature regimes. Shoots were placed in plastic, five-chamber, tissue-culture bags on Murashige and Skoog medium (1962) containing 25%, 50%, and 100% of the normal N concentration (MS-N) and stored at 4 °C and –1 °C in darkness, at 4 °C with a 12-hour photoperiod, and at 25 °C with a 16-hour photoperiod. Shoots of all four genotypes stored at 25 °C were in excellent condition after 6 months but required subculture after 18 months. Condition ratings of stored shoots varied with genotype and N concentration. Cultures survived longest at 4 °C with a 12-hour photoperiod on 50% MS-N. Under this regime, all four genotypes were rated in good condition at 30 months but declined to poor condition by 36 months. Based on these data, I recommend that mint cultures be stored on MS medium with 50% MS-N at 4 °C with a 12-hour photoperiod. This regime should provide a minimum of 24 to 36 months of storage before subculture is required. Cold-sensitive genotypes could be stored for 18 months at 25 °C on 50% MS-N medium.

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Barbara M. Reed

Micropropagated shoots of 49 Pyrus species and cultivars and one selection of Pyronia veitchii (Trabut) Guillaumin were evaluated to test their responses to several in vitro rooting techniques. Auxin treatment was required for rooting in most cases. Eighteen of 50 accessions rooted ≥50% with a 15-second, 10-mm IBA dip followed by growth on medium with no growth regulators (NGR). Twelve accessions rooted on a medium with 10 μm IBA applied for 1 week followed by NGR medium for 3 weeks; NGR medium alone was effective for only two accessions. Twenty-eight accessions rooted poorly with IBA treatments; an additional treatment of a 15-second dip in 10 mm NAA followed by NGR medium produced ≥50% rooting for eight genotypes. Root production increased for 10 of 19 especially recalcitrant genotypes by 10 μm IAA treatments in darkness or at 30C and NAA dip treatments. Of rooted shoots, 73% survived acclimation in the greenhouse. Selections of Pyrus betulifolia Bunge, P. calleryana Decne., P. hondoensis Kikuchi and Nakai, P. koehnei C. Schneider, P. pashia Buch.-Ham. ex D. Don, P. pyrifolia (Burm.f.) Nakai cv. Shinseiki, P. regelii Rheder, P. ussuriensis Maxim., and the Pyronia veitchii selection failed to root in any of the treatments. Twenty-five of 32 P. communis L. cultivars and three other species rooted on at least one of the treatments. Chemical names used: 1-naphthaleneacetic acid (NAA), 1H-indole-3-butyric acid (IBA), 1H-indole-3-acetic acid (IAA).

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Barbara M. Reed

Fragaria germplasm, stored at 4C as cold-hardened in vitro plantlets, was rated for condition on a 0–5 scale at 9, 12, and 19 months. Plants with ratings ≥2 were healthy enough to remain in storage. Benzyladenine (BA) at 0, 1, 2.5, and 5 μM and storage in darkness or with a 12-h photoperiod were examined for a group of four genotypes. Means of plant condition ratings over all treatments and genotypes were best (3.4) at 9 and 12 months and declined to 2.2 at 19 months. Increasing concentrations of BA were correlated with higher condition ratings compared to those without BA, and plants stored with a 12-h photoperiod had higher ratings than those stored in the dark. At 9 and 12 months, plants grown with BA and a 12-h photoperiod had the highest ratings. Five different Fragaria genotypes were used to study the effect of photoperiod and cold-hardening on condition ratings of stored plantlets. Cold-hardened plants had higher condition ratings (2.1) than those stored without cold-hardening (1.8). Plants stored with a 12-h photoperiod had higher mean condition ratings (2.2) than those stored in the dark (1.7). Storage with cold-hardening and a 12-h photoperiod resulted in improved plant condition. The extent of improvements was genotype-dependent.

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Barbara M. Reed

Apical meristems of four pears (Pyrus communis L. cv. Beurre Hardy, P. koehnei Schneider, P. cossonii Coss. and Dur., and a hybrid, P. dimorphophylla Makino × P. fauriei Schneider) were tested for their ability to survive immersion in liquid nitrogen. Plantlets were grown in vitro at 25C or cold-hardened for 1 week at – 1C before cooling at rates of 0.1, 0.3, 0.5, and 0.8 C/rein to –40C, followed by plunging the vials into liquid nitrogen. Vials were thawed for 1 min at 40C. A cryoprotectant mixture of polyethylene glycol, glucose, and dimethylsulfoxide (DMSO) was used. Regrowth of meristems ranged from 0% to 61% for plants grown at 25C and from 5% to 95% for cold-hardened plants. Cold-hardening significantly improved the recovery rates of all species tested. Survival rates increased as cooling rates decreased. Survival rates were not linked to the geographic origin of the species tested.

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Barbara M. Reed

Cold storage is important for managing in vitro germplasm collections. Strawberry shoot cultures can typically be held at 4 °C for 9 to 24 months before they require repropagation. Concentration of BA in the storage medium, pre-storage cold acclimatization (CA), and exposure to a photoperiod during storage were studied to determine conditions for improved strawberry culture storage. Fragaria shoot cultures stored at 4 °C were rated for plantlet condition on a 0-5 scale at 9, 12, and 19 months. Four species were CA and stored on medium with 0, 1, 2.5, or 5 μm BA either in darkness or under a 12-hour photoperiod. Mean ratings over all treatments and genotypes were best at 9 and 12 months (3.4) and declined at 19 months (2.2). BA in the storage medium significantly improved ratings for two species at 9 and 12 months, but ratings were not significantly different at 19 months. At 19 months of storage, shoot cultures stored with a photoperiod were rated significantly better (P ≤ 0.05) than those grown in darkness. Five Fragaria genotypes stored on medium without BA were used to study the effect of photoperiod and CA on ratings of stored plantlets. CA-shoot cultures stored for 9 or 12 months were rated significantly better than non-CA cultures. After 12 and 19 months storage, three of the five genotypes stored under a 12-hour photoperiod had significantly higher ratings than those stored in the dark (P ≤ 0.01), but by 19 months CA was nonsignificant. Overall, the addition of a photoperiod improved the condition of Fragaria shoot cultures stored at 4 °C. Chemical name used: N 6-benzyladenine (BA).

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Barbara M. Reed

Medium-term in vitro cold storage of Rubus germplasm was investigated using various temperatures, photoperiods, and storage containers. Shoot cultures of several Rubus taxa were grown either in tissue-culture hags or 20 × 150-mm glass tubes. Cultures stored at 10C in darkness were in poor condition after 6 months. Overall survival and condition ratings were significantly better for bags than tubes when cultures were kept at 4C. Contamination was present in 14% of the tubes, but only 3% of the bags. Addition of a 12-hour photoperiod to 4C storage significantly improved both condition ratings and survival percentages of many individual genotypes. Evaluation of the 250-accession germplasm collection after 12 months at 4C (dark) showed 92% of accessions in bags and 85% in tubes in suitable condition to remain in storage. Storage of cold-sensitive genotypes in tissue-culture bags at 25C with a 16-hour daylength was extended to 9 months when the MS-medium nitrogen level was reduced to 25% of standard concentration. Survival of `Mandarin' raspberry stored for 9 months improved from 40% at 4C (100% N) to 90% at 25C (25% N). Results of these studies suggest that most Rubus germplasm can be stored safely at 4C with 12 hours of light. Plastic tissue-culture bags are preferred over tubes due to higher survival and lower contamination rates. Storage at 25C on reduced-nitrogen medium is an alternative method for cold-sensitive genotypes.

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Xiaoline Yu and Barbara M. Reed

Adventitious shoots were regenerated from stem segments or leaf discs of hazelnut (Corylus species) in vitro shoot cultures. Five to 10% of stem segments of `Nonpareil' regenerated adventitious shoots on modified MS medium and NCGR-COR medium supplemented with 200 mg·1-1 glutamine and combination of 1 or 5 μM thidiazuron (TDZ) and 0.1 μM naphthaleneacetic acid (NAA). Callus derived from stem segments of `Nonpareil', `Tonda Gentile Romana', and `Willamette' and leaf discs of `Dundee' cultured on medium with TDZ and NAA also produced shoots (buds) after transfer to NCGR-COR medium or modified MS medium with benzyladenine (BA) and indole-3-butyric acid (IBA). Adventitious roots were produced from leaf discs and stem segments on medium with NAA alone or with high levels of IBA or NAA combined with low levels of BA. Regenerated shoots of `Nonpareil' and `Willamette' were multiplied, rooted, and acclimatized in the greenhouse. This provides a starting point for improving the plant regeneration frequency to a level useful for genetic manipulation.