Mesophyll cells are desirable targets for studying responses to pathogens or pathogen-induced toxins. Based on host-pathogen or host-toxin interaction studies at the cellular level it can be determined whether a toxin can be used as a selective agent. Suspension cells are suitable selection units for in vitro selection of potentially useful somaclonal variants. Protocols for the isolation of muskmelon mesophyll and suspension cells were developed in order to study the effects of roridin E, a toxin produced by Myrothecium roridum, on leaf spot tolerant and sensitive muskmelon cultivars. Viable mesophyll cells were obtained by exposing leaf tissue to 1% cellulysin and 5% macerase in B5 medium with 0.4M sucrose for one hour. Viable suspension cells were maintained a medium consisting of MS salts, 3% sucrose, 3 (μM thiamine·HCl, 555 μM myo-Inositol, 28 μM kinetin and 9 μM IAA. Fluorescein diacetate was used to determine viability over time. Membrane stability was monitored by measuring changes in the fluorescence of cells stained with Merocyanine 540 (MC 540), an optical probe for changes in transmembrane electrical potential (PD).
P. Healey, T.J Ng, and F.A. Hammerschlag
Hector G. Nunez-Palenius, Daniel J. Cantliffe, Harry J. Klee, and Don J. Huber
Embryo abortion and empty seeds after self-pollination occur in some transgenic (ACO antisense) `Galia' male parental lines. An embryo-rescue system in this melon was developed to save potential viable embryos. To obtain the best and reliable embryo-rescue technique, several parameters were used including an improved (five new supplements) nutrient medium (named E-21) from the E-20A basic medium (Sauton and Dumax de Vaulx, 1987), an inoculation system (removing the embryo from the seed or intact seed), and the use of different fruit harvesting dates of the wild type and a transgenic `Galia' male parental line. Fruits of wild type (WT) and transgenic (ACO gene in antisense orientation) `Galia' male parental line were harvested at 4, 10, 17, 24, and 30 days after pollination (DAP). Fruits were surface sterilized by dipping in a 20% commercial bleach solution for 30 minutes. Subsequently, seeds were removed from fruit under sterile conditions. These seeds were either used to dissect the embryos or placed directly with the hilum facing E-20A or E-21 medium. Seedlings from all treatments were transferred to E-21 elongation medium, incubated 4 weeks, and transferred to soil to evaluate growth. The efficiency of this technique was greater when the time after pollination (4, 10, 17, 24, and 30 DAP) to rescue the embryos was increased. Thus, 30 DAP was the best time to rescue the embryos. The number of rescued embryos using E-21 medium was greater than with E-20A. We did not find any significant differences in survival efficiency rate between WT and transgenic embryos. We have obtained a competent embryo-rescue technique for WT and transgenic `Galia' male parental line, which can be applied to rescue valuable GMO hybrid-melon embryos.
To investigate the relationship between cell size and sugar accumulation, fruit of the melon was heated during the early stage of the growing period. The minimum air temperature in the heating apparatus was ≈10 °C higher than the ambient air temperature, and the weight of the heated fruit was greater than that of the control fruit. The number of rectangular parallelepiped (7-mm-long sample serially collected beginning at one end of the 10-mm-wide strip removed from the 10-mm-thick disk at the maximum transverse diameter of the fruit to the opposite end) with cells larger than 200 μm in the heated fruit at 17 days after anthesis (DAA, the end of heating treatment) was much larger that of the control fruit. The mean cell size in the heated fruit at 17 DAA was larger than that of the control fruit. Mean sucrose content of the heated fruit on 40 DAA was larger than the level in the control fruit. Higher fruit temperatures in melons covered with heating apparatus results in the predominance of larger cells and increased accumulation of sucrose in the fruit.
V. V. Meglic, T. F. Horejsi, J. E. Staub, and J. D. McCreight
The genetic diversity of 400 U.S. melon germplasm plant introductions was assessed using 35 enzyme systems. Polymorphisms were observed at 24 putative loci (Ac, Acp1, Acp4, Ak2, Ak3. Ak4, Fdp1, Fdp2, Fdp4, Gpi, Idh, Mdh2, Mdh4, Mdh5, Mdhb, Mpi1, Mpi2, Pgd1, Pgd2, Pgm, Pep-g1, Pep-1a, Pep-pap, Skdh) representing 17 different enzymes. Sixteen loci demonstrated simple Mendelian inheritance. Multivariate analyses aided in reduction of data using 16 loci and linkage relationships were observed among the plant introductions. Two of 16 loci (Pgd1 and Acp1) segregated independently. Fourteen loci were assigned into three linkage groups (A-C): A Fdp1, Fdp2, Acp4, Skdh; B Mdh2, Mdh4, Mdh5, Mdh6, Pep-g1, Pgm; C Mpi2, Ac, Idh.
Entin Daningsih, Laurie Hodges, and James R. Brandle
Experiments were conducted during summer seasons from 1991 to 1994 to find out the effect of winds on early growth of muskmelon. A randomized complete-block design with sheltered and exposed areas as treatments was used. Sensors for air temperature and relative humidity (model HMP35C or model XN217, Campbell Scientific) were placed at canopy height and 3-cup anemometers (model 12102, R.M. Young, Traverse City, Mich.) were 50 cm aboveground. All sensors were connected to CR10 automatic data loggers and recorded hourly average data. Using regression analysis, we found that the accumulative windspeed frequency below threshold (<4 m–s–1) can be used to predict both accumulative hourly heat units of air temperature (GDHT) with R2's more than 0.85 and total muskmelon fresh and dry weight and leaf area index at early growth. Predicted models using accumulative hourly windspeed frequency have R2's >0.80 in sheltered areas. Adding vapor pressure deficit to the model improves the prediction of muskmelon early growth, especially in exposed areas.
Soon O. Park* and Kevin M. Crosby
Mature fruit size and shape are important traits of most melon types. Our objective was to identify RAPD markers associated with major QTL affecting fruit weight, length, diameter and shape by means of bulked segregant analysis in an F2 population from the ananas melon cross of Deltex (larger fruit size) × TGR1551 (smaller fruit size). Clear separations for fruit weight, length, diameter, and shape between Deltex and TGR1551 were observed. Continuous distributions for fruit weight, length, diameter and shape were found in the F2 population indicating quantitative inheritance for the fruit traits. Significant positive correlations were detected between fruit weight and shape traits (r = 0.73 to 0.80). A significant positive correlation was observed between fruit weight and glucose (r = 0.35) or fructose (r = 0.25), whereas no correlation was noted between fruit weight and sucrose or total soluble solids. Two small and large bulks for fruit weight and shape were developed from F2 plants. A total of 240 primers were used to simultaneously screen between the small and large bulks, and between Deltex and TGR1551. Twenty-six RAPD markers were polymorphic for the small and large bulks. Ten markers were found to be significantly and consistently associated with fruit size and shape traits on the basis of simple linear regression. Of the 10 markers associated, four displayed an amplified DNA fragment in the small bulk, while six showed an amplified DNA fragment in the large bulk. The associated marker OJ07.350 explained 15% to 27% of the phenotypic variation for the fruit traits. These markers associated with QTL for melon fruit size and shape are expected to be useful in melon breeding programs for modifying fruit size.
Wayne A. Mackay, Timothy J Ng, and Freddi A. Hammerschlag
Studies examining exposure methods and callus type were conducted to develop an in vitro selection system using roridin E as a selection agent. Vacuum infiltration of callus with the toxin solution was the only successful selection method at the concentrations tested. Primary callus (callus originating directly from the explant) was not sensitive to roridin A or E at the concentrations used. Secondary callus (callus produced from primary callus) exhibited a differential response to roridins A and E similar to that of detached-leaf assays. Electrolyte leakage studies of callus were not conclusive in establishing the membrane as the site of toxin action or useful for screening tolerance in vitro. A small percentage of callus from tolerant and susceptible cultivars survived repeated exposure to roridin E at 50 μg·ml-1.
Samuel Mendlinger and Dov Pasternak
The increasing salinity of both irrigated lands as well as irrigation water in many parts of the world have emphasized the importance of having appropriate breeding strategies for developing salt tolerant cultivars. In a program to breed for salt tolerance (high yield and good quality at 5,000 ppm salinity) in melons, several breeding strategies were tried. The only systems that succeeded was using combining abilities in a hybrid program. We found that salinity did not effect the number of fruit or fruit quality but only fruit weight. Fruit weight of hybrids grown in fresh water was controlled by dominant genes (h2=0.09) whereas the same hybrids grown under salinity had fruit weight control by additive genes (h2=0.54) Therefore, we were capable of breeding tolerant hybrids from non-tolerant parents.
S. Jenni and K.A. Stewart
As part of a program to develop a model for growth and development of muskmelon produced under various mulch and row cover combinations, all perfect flowers of five plants were tagged and measured on a daily basis. The polar and equatorial lengths of the inferior ovary were recorded from anthesis to fruit maturity. Mature fruits were harvested at full slip. The location of each flower on the plant was coded. Flowers and subsequent fruits were classified according to the following phenological stages: immature, close to mature, close to full bloom, full bloom, wilted, past full bloom, close to fruit, fruit. The netting was visually evaluated and rated on a scale of 0 to 5, the highest value representing maximum netting. At fruit maturity, the weight, polar and equatorial length of each fruit were measured and mature and immature seeds counted. The number of fruits that reached maturity was directly proportional to the total number of perfect flowers produced on a plant. Fruit weight during growth could be predicted from the polar and equatorial lengths.
David W. Wolff
We conducted a field screen of 130 melon cultigens to identify potential sources of host-plant resistance to Monosporascus cannonballus. Seed were sown in Speedling trays with inoculated or non-inoculated media. Plants were transplanted into a field known to be highly infested with Monosporascus cannonballus. Noninoculated plots were planted in rows that were fumigated with Telone II. Cultigens were arranged in a randomized complete block with three replications in each treatment (fumigated, nonfumigated). A disease symptom rating (1 = complete death to 5 = no symptoms) was taken at 78 and 90 days post-transplanting. Disease symptoms were most severe and occurred earliest in the inoculated, nonfumigated plots. Natural infection by Monosporascus occurred in the fumigated plots as over 95% of root samples collected contained perithecia. At the second rating date, 108 of the 130 cultigens tested were classified as moderately to highly susceptible (rating < 2.5). The four most resistant genotypes had a second rating equal or close to 4.0 (`Galia', `Deltex', `Rocky Sweet', and `Charlynne'). A group of 14 genotypes showed moderate resistance with a second rating of 3.0. Included in this group were `Morning Ice', `Doublon', `Israeli', `MR-1', `Santa Clause', and `Primo'. The physiological stress of a concentrated fruit set increases severity of vine decline symptoms.