Randomly amplified polymorphic DNA (RAPD) technique is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. It has been widely used for genetic mapping, plant and animal breeding programs, and DNA fingerprinting. However, there is no single set of RAPD-PCR conditions that can be applied to all situations. In order to adjust reaction component concentrations within suggested ranges for efficient amplification during the use of RAPD in detection of genetic variation of genus Camellia, crucial factors, such as concentrations of MgCl2 and DNA, annealing temperature (37 to 44 °C), and the use of an AmpliTaq® DNA polymerase and Stoffel fragment were examined. Five camellia cultivars, `Winter's Beauty', `Pink Icicle', `Polar Ice', `Winter's Hope', and `Snow Flurry', were under investigation. Clear and reproducible amplification products were produced with 3.0 μM MgCl2 and 30 ng template DNA/25 μL reaction mixer at annealing temperature 37 °C and 40 °C, compared with MgCl2 at 1.5, 2.0, and 2.5 μM. When annealing temperature increased, the RAPD-PCR stringency was increased, as expected. Stoffel fragment was found to provide highly reproducible results.
Lianghong Chen and Mack Nelson
V. M. Gingas, J. C. Kamalay, and S. C. Domir
Suspension cultures of five elm selections (U. americana A, 680, 8630 and Del 2 and U. pumila S) exhibiting a range of susceptibility responses to the Dutch Elm Disease fungus (Ophiostoma ulmi) have been successfully established for future elicitor/phytoalexin studies. Calli initiated from foliar tissues of mature, greenhouse-grown trees cultured on a solid modified MS medium containing 2,4-D and BA were adapted to a liquid modified MS medium containing BA and either IAA or NAA. Cells were grown in either the presence or absence of light with continuous agitation. Uniform, rapidly dividing cell cultures were achieved when friable white or tan calli were grown in the medium containing 1 mg/l each NAA and BA in darkness. Cultures yielding an abundance of phenolic compounds exhibited decreased cell uniformity and proliferation. Increased phenolic production was associated with the presence of phenolics in the initial callus tissue, exposure to light and the use of IAA as the auxin source.
Theresa Bosma, John Dole, and Niels Maness
Marigold flower pigments can be extracted and used as a natural source of food colorants in the poultry and dairy industry. These pigments impart an orange color to egg yolks and a yellowish color to dairy products. We examined four African marigold cultivars for their ability to be commercially grown and harvested mechanically. `E-1236' yielded the highest quantity of lutein (22 kg/ha), a carotenoid pigment, using a spectrophotometer for quantification. `E-1236' and `A-975' were the earliest flowering cultivars, 11 June 1998 for transplants and 9 July 1998 for direct-seeded, at 8 weeks after sowing regardless of field establishment method. `E-1236' produced the greatest number of flowers in a production season, both as transplants (68 flowers/plant) and direct-seeded (57 flowers/plant) at 363,290 plants/ha. Transplants resulted in two more harvests in a single season than direct-seeded plants. Subsequently, more flowers and petal material were produced for pigment extraction than with direct-seeded plants. A one-time application of ammonium nitrate (28.02 kg/ha) at mid-season did not significantly effect flower number, flower weight, or pigment yield. Experiment was repeated in 1999 with four cultivars, two field establishment methods, seven harvest dates, and five nitrogen applications.
Ruwanthi C. Wettashinghe and Ellen B. Peffley
Random amplified polymorphic DNA (RAPD) are genetic markers that facilitate selection in plant breeding. To obtain clear reproducible, and repeatable RAPD bands, four DNA extraction protocols and two Taq polymerases were compared using thirteen TG1015Y (Allium cepa) genotypes. Protocols for DNA extraction followed those of a modified Tai and Tanksley, 1989 (PMBR); a modified Dellaporta et al., 1983 (PMBR); a modified Guillemunt et al., 1992 (PMBR); and extracted with a plant tissue DNA isolation kit from Gentra System (Minneapolis). The modified Guillemunt protocol was selected due to ease of extraction and cost effectiveness. Polymerases compared were Taq and Taq Stoffel fragment. Results are based on three separate amplifications and electrophoretic assays. PCR amplifications of Stoffel fragment produced more scorable and reproducible RAPD bands compared to bands produced using Taq polymerase.
C.L. Boehm, I.K. Lee, G. Jung, H.C. Harrison, J. Nienhuis, M. Sass, and Moore Hall
Random amplified polymorphic DNA (RAPD) may have utility as genetic markers facilitating selection in ginseng crop improvement. This experiment determined chemical buffer and root tissue-type combinations that yield repeatable bands. The results allow further experiments using RAPD markers for estimating the genetic distance between ginseng landraces, selection for crop improvement, and extensive fingerprinting for use in determining the origin of tissue samples. This experiment determined mean band yields for all combinations of dry, fresh, and powdered root with cetyltrimethylammonium bromide, potassium/sodium ethyl xanthogenate, and urea buffers. The buffers were applied in replication to the tissue-types with other extraction protocol factors constant. Replications were amplified four times with four different primers using constant PCR and agarose gel electrophoretic protocols. Distinct bands were counted in each replication, and the summation of the replication repeats considered an observation. Least squares means for several response variables were analyzed. The most significant difference found was between buffers. The buffers ctab and urea were productive, and the pex was not. Significant difference was found when buffers were crossed with tissue. The applications of urea to fresh root, ctab to dry root, urea to dry root, and ctab to powdered root were productive. Based on these results we conclude 1) urea and ctab are productive when applied to all tissue-types, 2) dry root, which is easily collected and stored, yields sufficient DNA for analysis, and 3) powdered root, often the form of commercial products that might be tested for genetic origin, will yield sufficient DNA for analysis.
J. Michele Myers and Philipp W. Simon
We evaluated the efficiency of transformation in garlic for promoter activity, osmoticum effect and shaker speed using particle bombardment as the method of gene delivery. Callus was produced from root segments on a modified B-5 medium for four garlic clones. Suspension cultures were then established on a modified B-5 medium + 2,4-D using 6-month-old callus. Cells were collected by vacuum filtration and the Bio-Rad PDS-1000/He system was used to deliver genes. The activities of CaMV 35S, maize Adh1, and rice Act promoters were evaluated for transient expression using the β-glucuronidase (GUS) reporter gene. Osmotic conditioning of cells was performed by adding both mannitol and sorbitol to the medium. Osmoticum effect was evaluated for enhancement of transformation efficiency using GUS. The effect of shaker speed (120, 180 and 240 rpm) on cell type was evaluated for transformation efficiency using GUS. CaMV 35S promoter activity was much higher for garlic than either the maize Adh1 or rice Act promoters. Osmoticum did not enhance promoter activity, but differences in response to osmoticum among garlic clones were observed. Shaker speed did affect cell type, and transformation efficiency was greatly increased at higher shaker speeds. Confirmation of stable transformation and regeneration are in progress.
John M. Dole, Paul Fisher, and Geoffrey Njue
Several treatments were investigated for increasing vase life of cut `Renaissance Red' poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch.) stems. A vase life of at least 20.6 days resulted when harvested stems were placed directly into vases with 22 °C deionized water plus 200 mg·L-1 8-HQS (the standard floral solution used) and 0% to 1% sucrose without floral foam. Maturity of stems at harvest, ranging from 0 to 4 weeks after anthesis, had no effect on vase life or days to first abscised leaf. Pretreatments immediately after harvest using floral solution heated to 38 or 100 °C, or 1 or 10-min dips in isopropyl alcohol, had no effect, whereas 24 hours in 10% sucrose shortened vase life by 6.4 days and time to first abscised cyathium by 4.5 days. Stem storage at 10 °C decreased vase life, particularly when stems were stored dry (with only 0.8 days vase life after 3 weeks dry storage). Increasing duration of wet storage in floral solution from 0 to 3 weeks decreased vase life from 21.5 to 14.6 days. Placing cut stems in a vase containing floral foam decreased time to first abscised leaf by 3.7 to 11.6 days compared with no foam. A 1% to 2% sucrose concentration in the vase solution produced the longest postharvest life for stems placed in foam but had little effect on stems not placed in foam. A 4% sucrose concentration decreased vase life compared with lower sucrose concentrations regardless of the presence of foam. Holding stems in the standard floral solution increased vase life and delayed leaf abscission compared with deionized or tap water only, with further improvement when stem bases were recut every three days. Commercial floral pretreatments and holding solutions had no effect on vase life and days to first abscised cyathium but delayed leaf abscission.
Richard S. Buker*, Jackie K. Burns, and Fritz M. Roka
Continuous canopy shakers (CCS) were developed in the late 90's and have been used to commercially harvest citrus in Florida. A viable mechanical harvester in Florida must be able to selectively remove mature `Valencia' fruit. A study was conducted to evaluate the effect of operating conditions on mature and immature fruit removal during the 2003 harvest season. The study was conducted in the southern flat woods and northern ridge areas. The study treatments were completely random and replicated four times. The CCS treatments were 145, 215, 230, and 245 cycles per minute (cpm) and a hand picked control. The harvest occurred on 17 and 19 June at the southern and northern sites, respectively. Mature fruit removal linearly increased from 95.7% to 97.9% between 145 and 245 cpm, respectively. Varying the operating ranges significantly influenced mature fruit removal in the southern flat woods site. The trees at the southern site were taller (>4m), and had a larger crop load. At the northern ridge site where trees were smaller, varying the CCS operating ranges did not significantly influence mature fruit removal. Immature fruit removal was influenced by the operating ranges. Immature fruit removal was increased at least 22% over hand picked controls. The results were interpreted to indicate the frequency of CCS is dependent on tree size. The initial selectivity of the CCS was not equal to hand picking.
Susan L. Barkley, Sushila Chaudhari, Jonathan R. Schultheis, Katherine M. Jennings, Stephen G. Bullen, and David W. Monks
There is a research gap with respect to documenting the effects of sweetpotato (Ipomoea batatas) seed root density and size on transplant yield and quality. Field studies were conducted in 2012 and 2014 to determine the effect of sweetpotato seed root (canner size) density [12, 24, 37, 49, 61, 73, and 85 bushels [bu (50 lb)] per 1000 ft2] on ‘Covington’ and ‘Evangeline’ slip production in propagation beds. Another field study was conducted in 2012 and 2013; treatments included canner, no. 1, and jumbo-size ‘Covington’ roots at 49 bu/1000 ft2, to determine the effect of seed root size on slip production. As seed root density increased in the propagation bed, transplant production increased with no change in slip quality as measured by node counts and slip length except for stem diameter. In 2012, the best marketable slip yield was obtained at root densities of 73 and 85 bu/1000 ft2. In 2014, marketable slip production of ‘Evangeline’ increased as seed root density increased at a greater rate than ‘Covington’. In 2014, the best seed root density for marketable slip production was 49 to 85 bu/1000 ft2 for ‘Covington’ and 85 bu/1000 ft2 for ‘Evangeline’. In 2012, potential slip revenues increased with an increase in seed root density up to 73 bu/1000 ft2. In 2014, revenue trend was similar for ‘Covington’ as 2012; however, for ‘Evangeline’, revenue was greatest at 85 bu/1000 ft2. Seed root size had no effect on marketable slip production when using a once-over harvest system. Results suggest growers would use a seed root density from 49 to 85 bu/1000 ft2 depending on variety, and any size roots for production of optimum marketable slips. Selection of optimum seed root density also depends on grower needs; e.g., high seed root density strategy will have a higher risk due to the upfront, higher seed costs, but potentially have higher profits at harvest time. Lower seed root density strategy would be a lower initial risk with a lower seed cost, but also potentially have lower net revenues.
Ruwanthi C. Wettasinghe and Ellen B. Peffley
Random amplified polymorphic DNA (RAPD) have potential as genetic markers that may facilitate selection in plant improvement. To obtain clear, reproducible, and repeatable RAPD bands, four DNA extraction protocols and two Taq polymerases were compared. DNA extraction followed modified Tai and Tanksley (PMBR), Dellaporta et al. (PMBR), and Guilllemant et al. (PMBR) protocols, and a plant tissue DNA isolation kit from Gentra Systems was used. The modified Guillemant protocol was selected because of ease of extraction and cost effectiveness. Genotypes studied were TG1015Y (Allium cepa). Polymerases compared were Taq and Taq Stoffel fragment. Results are based on separate amplifications and electrophoretic assays. PCR amplifications of Stoffel fragment produced more scorable and reproducible RAPD bands compared to bands produced using Taq polymerase.