A tank mix of fish oil plus liquid lime sulfur has proven to be an effective chemical thinner for apples in the bloom and postbloom periods. This combination was labeled for use as a chemical thinner in Washington State in 2003. There are several concerns with fish oil when used in this thinning mixture. Phytotoxicity is one concern. Apple growers have a reluctance to utilize this oil because of its expense and repulsive odor. Research to date has been conducted using oil from a single small source in Washington State. Shipping fish oil across the country is expensive and the consistency and purity of fish oil from other sources is unknown. Fish oil may function as a surfactant and penetrant, and it may also have a direct thinning effect. The objective of these studies was to evaluate the efficacy of several surfactants and oils in combination with lime sulfur for thinning apples. Lime sulfur has been less effective as a thinner when used alone than when used with oil in our studies. Regulaid, LI-700, and Silwet L-77 were shown to be less effective than oils for achieving thinning. Vegetable oil has been very effective in the thinning combination, while petroleum oils have been effective in some eastern U.S. trials, but less effective in the west. Tank mixing fish oil with lime sulfur has remained among the best treatments in our trials, while vegetable oil also shows promise.
Many fruit growers in the Pacific Northwest region prefer to use a sprinkler system to produce high-quality fruit and to establish a cover crop in the orchard. However, water shortage mandates the use of more efficient methods of irrigation, such as drip. In this long-term experiment, the effects of seven irrigation systems for `Fuji' and two irrigation systems for `Gala' on five rootstocks on tree growth, water use, fruit quality, and mineral nutrients were studied. All forms of drip systems used less water than full micro-sprinkler (SP). Partial root drying sprinkler (PS) used 50% less water than SP. Trees with partial root drying drip and deficit drip had to receive 65% of full drip to survive. Each `Fuji' tree with SP used about 5397 L of water in 2004 and 5833 L in 2005, while each tree with full drip used 2403 L in 2004 and 3438 L in 2005. Thus, trees with full drip used 41% to 55% less water than those with SP system without any reduction in fruit quality. This leads to a major savings in the cost of fruit production. Fruit weight in trees with full drip was always greater than those with PS or deficit drip. Fruits with SP system had lower soluble solids than those with PS. Fruits from trees with partial drip had a higher starch degradation than those with other systems. Leaf minerals, particularly N and K, were affected by irrigation systems. `Pacific Gala' trees on B.9 rootstock were more precocious than those on Supporter-4 rootstock. In general, `Pacific Gala' on RN-29 had better tree performance and fruit quality than those on other rootstocks. The calculation of water requirements on a tree-use basis provided an excellent guide for drip irrigation.
Nearly 350 germplasm accessions representing 25 Allium species were evaluated for damage by onion maggot (OM) [Delia antiqua (Meigen)] in field experiments in 1989. In 1990, 188 additional accessions and breeding lines were evaluated, and 36 entries from the 1989 evaluation were re-evaluated. In both years, there were no significant differences in OM damage to seedlings among accessions within the species tested. However, differences among species were highly significant. Allium cepa L. (bulb onion) seedlings had consistently high OM damage. Species with significantly less seedling damage than A. cepa included: A. altaicum Pall., A. angulosum L., A. galanthum Kar. & Kir., A. pskemense B. Fedtsch., A. scorodoprasum L., A. ampeloprasum L. (leek), A. fistulosum L. (bunching onion), A. schoenoprasum L. (chive), and A. tuberosum Rottl. ex Spr. (garlic chive). Some species sustaining minimal damage as seedlings were nonetheless heavily damaged as mature plants by a later generation of OM. Allium cepa cultivars that were well-adapted to local conditions were heavily damaged as seedlings, but their bulbs were less damaged than those of poorly adapted A. cepa germplasm. Allium ampeloprasum seedlings and mature plants sustained low injury throughout both growing seasons.
Molecular DNA markers based on the RAPD (random amplified polymorphic DNA) assay are gaining use in germplasm assessment. RAPD markers are simple, relatively inexpensive, and highly informative. We used five primers to assess 26 Brassica oleracea breeding lines from the IVF and nine accessions from the PGRU. The test array included eight subspecies of B. oleracea. We generated 90 RAPD markers and were able to unambiguously discriminate among all 35 test entries, but could not separate subspecies within B. oleracea. Genetic similarity between subspecies ranged from 0.629 to 0.738. Average similarity within accessions was 0.96, confirming the suspected homogeneity of breeding lines. Nevertheless, significant genetic diversity was found among kohlrabi, broccoli, and cabbage accessions. Similarity analysis of breeding lines and hybrids confirmed their pedigree relationships. Interestingly, B. o. subsp. costata `Couve Nabica' showed closer similarity to B. napus subsp. oleifera `Jet Neuf' than to other B. o. materials and B. o. subsp. italica `Packman' showed higher similarity to some cabbages than to other broccolis. Results provide further evidence that diversity assessment using RAPDs is broadly applicable and useful in germplasm conservation and utilization.
We discuss a series of studies in our units employing molecular genetic markers in collection management, primarily for identity and diversity assessment and partitioning of genetic variation. Isozymes, random amplified polymorphic DNAs (RAPDs), and simple sequence repeat DNAs (SSRs) have been used for these purposes. We analyzed a range of Brassica oleracea accessions at six isozyme loci. Unique isozyme profiles (or fingerprints) were found for 40% of the individual genotypes within accessions. While isozymes were extremely valuable for partitioning genetic variability between and among subspecies, they failed to identify accessions and subspecies. Furthermore, relationships found did not correspond to those predicted by taxonomy. In a study of three species of Chinese vegetable brassicas using 112 RAPD markers, we were able to unambiguously distinguish all 52 accessions studied, despite some intra-accession variability. In addition, cluster analysis correctly grouped all individuals of the same species, but below that rank, taxonomic groupings occasionally broke down. RAPD profiles were found that unambiguously distinguished the three Brassica species from one another, but, for subspecies, no such profiles were found. In another RAPD study of B. oleracea subsp. capitata (cabbage), a closely related set of cultivars were not distinguishable, although more distantly related cultivars were. We had disappointing results with a RAPD study of Vitis accessions. DNA was extracted from the leaves of 23 greenhouse-grown and 52 field-grown vines. Twelve of the 23 greenhouse vines were rooted cuttings collected from 12 of the field-grown vines. Unfortunately, the RAPD profiles of all vines grown in the same location (whether greenhouse or vineyard) were more similar to one another than were profiles from the same clone grown in the two different locations. We are studying whether this result is due to physiological differences in plants growing under different conditions, to differences between PCR reagent lots, to pathogen infestation, or to DNA sample contamination. In a study of 23 accessions representing 15 Vitis species and three species hybrids, we used six different SSR markers to identify individual genotypes. We were able to unambiguously distinguish all genotypes, except two that were identical at all six loci. Review of planting records revealed that the two genotypes were probably the same grape clone. SSR results were not congruent with known taxonomic relationships or geographic origin of genotypes. The SSR polymorphisms found in even this small subset of the Vitis collection in principle make possible the identification of more than 130 trillion different genotypes. This high level of polymorphism, however, makes our particular SSR loci of limited use for identification of species and for the determination of genetic relationships. Molecular genetic markers offer a powerful, efficient approach to assessing questions of identity, relationship, and diversity in germplasm collections, but markers need to be selected based on their suitability for the particular task.
The USDA–ARS active collection of Malus includes over 2500 accessions maintained as field-grown trees at the Plant Genetic Resources Unit (PGRU), Geneva, N.Y. Nearly 30% of this collection is presently cryopreserved as dormant buds at the National Seed Storage Laboratory, Fort Collins, Colo., as a backup security collection. Successful bud-grafting recovery rates (≥40%) after one to four years of cryogenic storage have been documented for over 675 of 750 accessions tested. However, current protocols dictate budwood collection at PGRU from late December through early March, when buds are thought to be optimally acclimated for desiccation and slow freezing to –30°C, our pretreatment for cryopreservation. This causes a processing bottleneck. Our observations suggest temporary storage of budwood at –4°C after field harvest is possible, but we had not tested this directly. Therefore, we collected budwood from four accessions representing different levels of cold tolerance on six dates from January to March, 1995. Dormant buds were processed for cryopreservation monthly after storage in sealed bags at –4°C for 1 to 6 months. Recovery rates ranged from 55% to 100%. Neither collection date nor length of storage at –4°C affected rate of recovery. These results suggest we can significantly increase the throughput and efficiency of our cryopreservation efforts, thereby enhancing management and security of the Malus collection.
To comprehend genetic identity and relatedness in Malus germplasm held in situ and ex situ, we are employing simple sequence repeat (SSR) DNA fragment information in combination with passport and horticultural data. SSRs offer certain advantages for characterizing large arrays of germplasm efficiently. They are abundantly dispersed throughout plant genomes and are exceedingly polymorphic. In addition, they can be PCR-amplified and detected by automated fluorescence-based technology. A size-fractionated DNA library of M. ×domestica cv Golden Delicious was screened to identify SSR loci. Eight loci were found to be reliably informative and were used to prepare locus-specific primer pairs. Characterization of the 75 M. ×domestica accessions included in the core subset of the USDA-ARS Malus germplasm collection revealed six of the eight loci were polymorphic within the array. The number of alleles per locus ranged from two to 21. Throughput was enhanced by multiplexing, allowing simultaneous use of two or three primer pairs. With improved genetic characterization of Malus germplasm, we intend to better develop and relate the core subset to the rest of the collection and to in situ Malus genetic resources. SSR markers appear to be an efficient and reliable tool to expedite this process.
In 2002 in New York State, we collected king fruit of `Gala' and `Red Delicious' on fruiting spurs from 0 to 66 days after full bloom (DAB). In 2003 in Washington State, we collected king fruit of these cultivars from 14 to 62 DAB. At each collection we determined radial cell number across the fruit cortex and developmental stage of the embryo and endosperm in seeds. Fruit diameter was slightly greater in Washington fruit than in New York fruit until about 40 DAB; thereafter, New York `Delicious' outgrew Washington `Delicious', while `Gala' in the two climates (and two different years) grew identically. The New York fruits had a much earlier rise in fruit growth rate and maintained a slightly higher rate throughout the period. The cortex thickness of Washington fruit was greater than that of New York fruit for both cultivars. Most rapid cell division in the cortex occurred between 10 and 28 DAB and, by 40 DAB, most cell proliferation had ceased. The Washington fruit formed more cells across the radius than did New York fruit. Cortex thickness increased with respect to increase in cortex cell number about 30% to 40% faster in Washington fruit than in New York fruit. Developmental stages of embryos and endosperm followed a sigmoid time pattern for both cultivars in both states. By 60 DAB, embryos and endosperm reached their maximum stage of development. In both cultivars and states, cell divisions were nearly completed by the time the embryo and endosperm approached stage 3: for embryos this is the heart-shaped stage, for endosperm it is near completion of cell wall formation. The completion of wall formation in the endosperm, the near completion of cortex cell division, and the generation of the cotyledons and apical meristems in the embryo are highly correlated processes. We saw no evidence that endosperm development precedes embryo development.
Genetic variation and relationships in genetic resources collections can be assessed using molecular genetic markers. We examined the applicability of the RAPD assay for quick, cost-effective, and reliable use in improving collection management. Fourteen accessions of Brassica oleracea spp. capitata `Golden Acre' (cabbage) were screened using nine decamer oligonucleotide primers. We obtained 110 reproducible fragments, of which 80 were polymorphic, ranging in size from 370-1730 bp. Individual accessions were readily distinguished. A cluster analysis of genetic distances generated by bootstrapping reflected all known genetic relationships, except one. Bulking strategies were also investigated. RAPD markers can be applied to gene bank management to measure variation, identify accessions, and establish genetic similarity at the intra-specific level addressing the needs of both curators and users.