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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.
Little information is available on the reproductive behavior of Hydrangea macrophylla (Thunb. Ex J.A. Murr.) Ser. The objectives of this study were to investigate time of stigma receptivity, viability of pollen from sterile flowers, and self-incompatibility in this popular ornamental shrub. Pollen germination and pollen tube growth in styles were examined using fluorescence microscopy. Stigma receptivity was examined in cross-pollinations made from 1 day before anthesis to 8 days after anthesis. Maximum stigma receptivity for the two cultivars examined occurred from anthesis to 4 days after anthesis. Viability of pollen from sterile flowers was evaluated through pollen staining and observations of pollen tube growth. No significant difference in percent stainable pollen between fertile and sterile flowers was observed in any of the six taxa examined. Pollen germination and pollen tube growth were studied in cross-pollinations made using pollen from fertile and sterile flowers of two cultivars. For both cultivars, pollen tubes from fertile and sterile flowers grew to the same length and had entered ovules by 72 hours after pollination. Self-incompatibility was evaluated by comparing pollen germination and pollen tube growth in cross- and self-pollinations. In the five taxa examined, self pollen tubes were significantly shorter than cross pollen tubes in flowers that were examined 72 hours after pollination. This finding indicates the presence of a gametophytic self-incompatibility system in H. macrophylla.
Clethra alnifolia L., a native deciduous shrub cultivated as an ornamental, was recently hybridized with C. pringlei S. Wats. The purpose of this hybridization was to combine the cold hardiness and adaptability of C. alnifolia with the ornamental foliage of C. pringlei. While most of the C. alnifolia × C. pringlei hybrids more closely resembled C. alnifolia than the paternal species, a `Hokie Pink' × C. pringlei hybrid (NA71586) with foliage that flushes red like C. pringlei was recovered. The objectives of this study were to analyze cytologically the F1 and produce a F2 population from NA71586. Chromosome counts from root tips cells indicated that NA71586 has 32 chromosomes. Since the chromosome number of C. alnifolia is 2n = 32 and that of C. pringlei was found to be 2n = 16, NA71586 appears to have developed following fertilization of a C. alnifolia egg with an unreduced male gamete from C. pringlei. Both `Hokie Pink' and C. pringlei exhibited primarily bivalent pairing in pollen mother cells (PMCs). Over half of the PMCs from NA71586 contained 16 bivalents, indicating substantial homology within the C. alnifolia genome. It was theorized that C. alnifolia is either an autotetraploid that exhibits bivalent pairing or a segmental allotetraploid produced from hybridization of species with similar genomes. More than 700 F2 progeny were obtained from self-pollination of NA71586. Although many of the F2 progeny resembled NA71586, variation in foliage color, size and shape was apparent in the population.
Japanese snowbell (Styrax japonicus Sieb. & Zucc.) is an outstanding small ornamental tree that is underused in the U.S. One of the reasons this Asian native is not more widely planted is that it is subject to spring freeze damage. The objectives of this study were to determine if there was variability within S. japonicus for time of budbreak and if this variability could be used for selecting plants better adapted to areas of the country that frequently experience late spring freezes. During Spring 1999 and 2000, budbreak was evaluated weekly in 224 open-pollinated seedlings. While weather conditions varied greatly between the 2 years, there was good consistency between 1999 and 2000 data. There was a 4-week difference between the earliest and latest plants to break dormancy. Based on the 1999 and 2000 data, 28 plants were selected and propagated. A replicated trial involving these selections and three cultivars was carried out in 2002, 2003 and 2004. All of the selections broke bud later and suffered less freeze damage than the cultivars `Emerald Pagoda' and `Carillon', but many performed similarly to `Pink Chimes'. Variation in height, width, caliper and canopy shape was observed among the selections. There is an opportunity to utilize the genetic variability in S. japonicus for developing cultivars with reduced susceptibility to spring freeze damage.
The objectives of this study were to evaluate self-fertility and to determine the effectiveness of pollinations made over a 4-day period in Japanese snowbell, S. japonicum Sieb. & Zucc. Pollen germination and pollen tube growth were observed in stained styles following cross- and self-pollinations made from 1 day before to 2 days after anthesis. One month after pollination, fruit set averaged 40% in cross-pollinations and 14% in self-pollinations. Two months later, about one-third of the fruit resulting from cross-pollinations had aborted and only one fruit remained from the self-pollinations. This study demonstrated that stigmas of S. japonicum are receptive for at least 4 days and that flowers should be emasculated prior to making controlled cross-pollinations.
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).
Breeding efforts in Clethra alnifolia L., an ornamental shrub native to the Eastern U.S., are hindered by a lack of information on the reproductive behavior of this species. The objective of this study was to evaluate self-compatibility, time of stigma receptivity, and the relationship between time of pollen shed and stigma receptivity in C. alnifolia. Stigma receptivity and changes in floral morphology were monitored over a 7-day period beginning at flower opening. Pollen germination and pollen tube growth in styles were examined following self- and cross-pollinations using fluorescence microscopy. Seed set and germination were compared following self- and cross-pollinations. Anthers began to dehisce in `Hummingbird' and `Ruby Spice' the day after flowers opened, but stigmas did not become fully receptive to pollen until 2 days later. An increase in the length of pistils was observed following flower opening. Maximum elongation of pistils occurred at approximately the same time stigmas became receptive and could be utilized as an indicator of receptivity. While self-pollen tubes appeared to grow slightly slower than cross-pollen tubes, there was no indication of a self-incompatibility system acting at the stigmatic or stylar level in C. alnifolia. Self-pollinations of `Hummingbird' and `Ruby Spice' produced fewer seeds than did cross-pollinations of these cultivars. Germination of all seed obtained from this study was too poor to allow a comparison of germination rates of the self- and cross-pollinated seed. However, because a few self-progeny were obtained, emasculation is recommended when making controlled pollinations. The presence of a late acting self-incompatibility system or early-acting inbreeding depression was proposed as being responsible for the lower seed set following self-pollination.
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.
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).
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.