Ninety-eight percent of cotyledons of Cucumis metuliferus (PI 482439) regenerated shoot buds in 5 weeks on MS medium with 10 μm BA. Regeneration rates on agar gelled medium and liquid/membrane system were compared after weekly transfers of tissue from liquid/membrane to agar during the 5 weeks of regeneration culture. Number of shoots and buds increased from six (on agargelled medium) to nine per cotyledon when explant on liquid/membrane system for 3 or 4 weeks was transferred to agar gelled medium. Shoot development, rooting, and survival in greenhouse was adequate regardless of whether regeneration was initiated on agar or liquid/membrane system. Tetraploid regenerants were found to be about 9% of the almost 400 regenerants screened. Pollen morphology was a quick and definitive screening technique for tetraploid plants. Frequency of tetraploid plants was similar after 5 weeks of induction on either agar or liquid/membrane system. This frequency decreased by a factor of 10 following transfer of explants from membrane to agar after the first week of induction. Timing of this transfer plays a critical role in eliminating tetraploid variants.
Jeffrey Adelberg and Matthew Cousins
Geophytes store carbohydrates in modified underground shoot systems protected by a broad array of biologically active chemistry. In vitro formation of storage organs requires months in the lab instead of years in the field, when water and nutrients are correctly supplied. Liquid and agar systems in large and small vessels were compared for sugar and water use with turmeric (Curcuma longa) as a model plant. Small jars on a shaker were compared with large, flat-bottomed vessels containing thin films of liquid media, intermittently tilted at slight inclines that allow the advantages of liquid phase transfer with gentle agitation. Liquid culture in small vessels on a shaker yielded the most plants and liquid culture on a thin-film rocker in a large vessel yielded the largest plants. Increased and improved biomass (fresh and dry) in liquid culture compared to agar was based on greater sugar use. When large vessels of liquid media were grown for 5 and 6 months on a rocker, 400 mL of media yielded 150 to 200 g (fresh weight) of plants. Similarly, 13 to 16 g (dry weight) of plant tissue was derived from 24 g of sugar. Plants were about one-third rhizome by fresh mass. Rhizomes had greater dry and fresh weight than leaves or roots, indicating solute actively accumulated in the rhizome. The rhizomes had normal morphology, characteristic pigments and fragrance, and rhizome extracts had strong antioxidant potential. The gentle rocking action of plantlets in sugar-containing liquid medium was demonstrated to produce functional storage organs.
Jeffrey Adelberg and Joe Toler
Micropropagation of black-stemmed elephant ear (C. esculenta (L.) Schott `Fontanesii')' and upright elephant ear (A. macrorrhizos G. Don) were compared in semi-solid agar media and agitated, liquid thin-film bioreactor vessels at four explant densities (33, 100, 165, and 330 explants/L of media) using two growth regulator combinations: 1) 1 μm benzylaminopurine (BA)—growth medium, and 2) 3 μm BA plus 3 μm ancymidol—multiplication medium. The thin-film liquid system outperformed agar culture for most measured responses. Some exceptions were relative dry weights at higher explant densities and multiplication rate of Colocasia. When the thin-film liquid system was compared to agar culture, Alocasia explants produced their greatest biomass and had the least residual sugar at the highest explant density. Alocasia explants multiplied most rapidly and had the greatest relative dry weight on liquid media at the low explant densities. Alocasia plants were larger in growth medium than multiplication medium and larger in liquid medium than agar medium. When compared to agar, Colocasia in the thin-film liquid system produced the greatest biomass at the highest explant density in growth medium, had the greatest relative dry weight at the lowest explant density, and used the most sugar at the highest explant density. Alocasia and Colocasia would likely produce greater fresh and dry weight at the highest explant density if additional sugar were supplied during thin-film culture. Greater growth in thin-film culture of Alocasia and Colocasia is due in part, to greater availability of sugar in liquid compared to agar medium.
Jin-Feng Chen and Jeffrey Adelberg
Perry E. Nugent and Jeffrey Adelberg
Thirteen triploid lines of melon (Cucumis melo L.) were derived from crosses involving five tetraploid and seven diploid lines. Fruit characters were assessed. When allowed to open pollinate in field plots with adjacent diploid pollinators, eight triploid genotypes were sterile or nearly sterile (<1% viable seed). Five triploid genotypes were partially fertile, indicating viable pollen grains were present. Cytological analysis performed on progeny of a partially fertile triploid plant fertilized by open pollination indicated euploid female gametes were common. Triploid hybrids between tetraploid `Miniloup' and several other diploid parents had vegetative and fruit characteristics intermediate to the parents. Most triploid genotypes yielded round fruit in contrast to their diploid parent whose fruit were oval to oblong and the tetraploid parent that had oblate fruit. Sugar levels of some triploid hybrids were as high as diploid parents.
Jeffrey Adelberg, Maria Delgado and Jeffrey Tomkins
Two tetraploid and two diploid genotypes of Hemerocallis spp. were micropropagated on an orbital shaker in Murashige and Skoog liquid medium in a factorial combination of two sucrose concentrations (90 mm and 180 mm), two 6-benzylaminopurine (benzyladenine) concentrations (0.32 μm and 3.2 μm), at two densities (57 explants/L and 171 explants/L), in the presence (0.32 μm) and absence of α-cyclopyl-α-[4-mehtoxyphenyl]-5-pyrimididinmethanol (ancymidol). There were linear relationships between fresh weight and water use (R 2 = 0.800, P < 0.0001), dry weight and sucrose use (R 2 = 0.636, P < 0.0001), and relative dry weight (dry weight/fresh weight = relative dry weight) to concentration of sucrose residual in medium after culture (R 2 = 0.553, P < 0.0001). Eighty-five percent of the water used and 74% of the sucrose used were incorporated as plant fresh weight and dry weight, respectively. A 1% increase in percent sucrose residual (mass/volume in spent medium) was correlated to an increase of 1.8% relative dry weight over the range 7% to 22% relative dry weight. In vessels with 90 mm initial sucrose, where the most growth had occurred (>15 g fresh weight), sucrose was depleted (<0.2% sucrose) and plantlets had the lowest relative dry weight (≈6.9%). In vessels from 180 mm initial sucrose, with similarly high fresh weight, plantlets had 12.0% relative dry weight with 2.1% sucrose residual in medium. Fresh weight, dry weight, or relative dry weight of plantlets in the laboratory did not correlate with subsequent survival or growth in the greenhouse. Plantlets grown without ancymidol at the lower benzyladenine concentration acclimatized to the greenhouse with 93% survival. However, greenhouse survival of plants grown with ancymidol and a higher level of benzyladenine was only 4%. ‘Barbara Mitchell’ was the largest plant in the laboratory, but often had poorest growth in the greenhouse. When optimizing a liquid micropropagation protocol for larger vessels, sucrose and water requirements may be directly related to targeted biomass yield, but each genotype needs to be handled independently with ex vitro validation of plant vigor.
Jeffrey Adelberg, B.B. Rhodes and Halina Skorupska
Watermelon (Citrullus Lanatus (Thunb.) Matsum and Nakai) and muskmelon (Cucumis melo) were regenerated from immature cotyledons cultured on MS medium containing 10 μM BA. Small population of watermelon and muskmelon regenerants contained tetraploids as variants. The tetraploid individuals were recognized by morphological features including enlarged leaves, tendrils, male flowers, and variable pollen grains. After self-pollination, seed lots reflected differences in size expected from tetraploid parents.. Cytological data from root tips of R1 populations will be presented.
Jeffrey Adelberg, Maria Delgado and Jeffery Tomkins
Two tetraploid and two diploid varieties of daylily were micropropagated on a shaker in MS liquid medium containing high and low sugar levels (3% and 6% sucrose), 2 BA levels (0.32 and 3.2 μm), at two densities (57 and 171 explants/L), in the presence (0.32 μm) and absence of ancymidol. Biomass and media use were partitioned for the four genotypes and 32 cultural conditions with three replications (4 × 2 × 2 × 2 × 2 × 3). Genotype greatly effected f resh weight, dry weight, media, sugar and water use, but ploidy had little effect. Vessels at high density (171 explants/L) produced 1.8× more fresh weight, 1.4× more dry weight, used 1.6× more media and sugar than low density (57 explants/L). Plants from low density were 1.7× larger, 2× greater dry weight, and used 2× more sugar and media, than from high-density culture (per explant). Doubling the initial sugar level increased dry weight and sugar use 1.3×. There was a linear relation between sugar residual and percentage of dry weight (R 2 = 0.55, P < 0.0001), where a 1% increase in °Brix raised percentage of dry weight 1.8 units over the range of 9% to 22%. Ancymidol and BA had less effect on plant size, sugar and media use than genotype or plant density. Greenhouse survival was reduced by including ancymidol (90% to 30%) and increased BA concentration (85% to 35%). Lab plant density and initial sugar concentration had no apparent effect on greenhouse growth. `Barbara Mitchell' had greatest mass, used more sugar and media than the other varieties, yet had least greenhouse growth. Nutrient use with `Barbara Mitchell' was linearly correlated (R 2>80%) to lab growth for seven of 12 ions. P and Fe supply was inadequate to support optimal growth, as indicated by low residual in media (>1% of MS formulation).
Jeffrey Adelberg, Kazuhiro Fujiwara, Chalermpol Kirdmanee and Toyoki Kozai
Growth and net photosynthetic rates of shoots of a triploid melon clone, `(L-14 × B) × L-14', were observed over 21 days following transfer from a multiplication MS medium containing 3% sucrose and 10 μM BA to a shoot development medium containing 1 μM BA at varying levels of sucrose in the medium (0%, 1%, and 3%), and light (50, 100, and 150 PPF) and CO2 (500, 1000, and 1500 ppm) in the headspace. Largest numbers of shoot buds were observed in media with 3% sucrose. Increased light and CO2 had a positive interactive effect. Fresh and dry weights were greatest at highest levels of sucrose, light, and CO2. Although there was less growth in the absence of sucrose, fresh or dry weight of shoot buds grown without sucrose in the media still doubled over the 21 days of culture. Net photosynthetic rates of buds were negative 4 days after initiation of culture and approximately zero after 20 days of treatment. When transferring buds to fresh, sugar-free media, net photosynthetic rates became highly positive. Buds that had been cultured in the absence of sucrose and at highest light levels had the highest net photosynthesis rates upon transfer to fresh, sugar-free media.
Jeffrey Adelberg, Robert Pollock, Nihal Rajapakse and Roy Young
Two varieties of Cattleya orchids (C. Loddigessi `Elen' × C. Loddigessi Alba `Extra' and Brassolaeliocattleya. Mem. `Helen Brown' Sweet Afton) were micropropagated in sealed, three-dimensional polypropylene vessels with microporous, semi-permeable membrane films to allow diffusion of water, dissolved nutrients, and gas to plant material inside the vessels. During tissue culture on sugar-containing media, chance contaminants were eliminated on the vessels outer surface using 5% bleach solution. Proper decontamination treatment did not effect carbohydrate content or subsequent growth of tissues contained within the vessels. Plantlets remaining in membrane vessels were shipped (7 days at 14–30°C) from Japan to the United States in the dark in a plastic tray and arrived without changes in fresh or dry weight of whole plantlets. However, shoot dry weight did increase significantly. Sucrose, glucose, and fructose reserves established on sugar-containing media were greater in root than shoot tissue and were largely expended during shipping concurrent with increased shoot dry weight. It is likely carbohydrate catabolism provided energy for these CAM plantlets to continue carbon fixation, resulting in positive net carbon assimilation in the dark shipping environment. Changes in starch concentrations during shipping were not significant. Plantlets grew photoautotrophically in hydroponic culture in the greenhouse, following transport in the same sealed membrane vessels. Carbohydrate concentration of plantlets following hydroponic culture was not significantly different than after the shipping process. Sealed-membrane vessels for micropropagation, decontamination, shipping and greenhouse growth were useful for culture of Cattleya to facilitate scale-up of materials handling and international commerce of tissue-cultured plants.