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Brent Tisserat and Robert Silman

An inexpensive ultrasonic fogging system is presented that aids in the establishment of tissue culture shoots in soil under greenhouse conditions. In addition, ultrasonic fogging may be coupled to CO2 nutrient enhancement via bubbling CO2 into the water reservoirs prior to fogging to improve growth and morphogenesis responses of shoots. A list and cost of items for the system and its assembly is given. Transplanted tissue culture shoots of basil (Ocimum basilicum L.), hosta (Hosta sp.), mint (Mentha sp.), and thyme (Thymus vulgaris L.) were tested with this fogging system with and without CO2 nutrient elevation and compared to the growth of shoots grown under a misting system with and without CO2 nutrient elevation. In all cases, ultrasonic fogging enhanced survival rates, growth (fresh weights) and morphogenesis (axially shoots, leaves and roots) vs. that occurring in the misting system. For example, thyme and mint shoots exhibited 2- and 5-fold increases, respectively, in fresh weights under ultrasonic fogging with CO2 compared to misting systems with CO2. Associated with enhanced survival and morphogenesis was an overall enhancement of shoot and leaf size and overall maturation responses. This is also reflected in enhanced secondary products obtained from shoots grown under ultrasonic fogging compared to shoots grown in misting systems.

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Brent Tisserat and Robert Silman

A comparative study was undertaken to determine the influence of lighting, carbohydrate concentrations and ultra-high levels of CO2, i.e., >10,000 ppm, on sterile culture growth. Past CO2-sterile studies have confirmed that elevation of CO2 to as high as 1000 ppm resulted in beneficial growth. Within special constructed chambers, tissue cultures were given a variety of CO2 levels for 12–16 hours/day using artificial lighting and natural sunlight. Several different plants (lettuce, beans, pine) and plant culture types were grown in CO2-enriched environments, ranging from 350 to 50,000 ppm. In almost all cases, plant tissue cultures not only tolerated but exhibited enhanced growth using ultra-high levels of CO2. For example, lettuce cultures were found to grow 2 to 4 times faster under ultra-high CO2. levels than under normal atmospheric CO2 levels, i.e., 350 ppm. Natural sunlight was found to be suitable for sterile culture growth. Modes of administration of CO2 in vitro and gas permeability of various culture vessels are presented.

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Brent Tisserat, Robert Silman, and Karen Ray

Ultra-high levels of CO2, i.e., >10,000 ppm, enhance tissue culture growth and offers a relatively simple and inexpensive method to improve plant productivity in vitro. Growth responses employing ultra-high CO2 levels differ considerably in the literature. Unfortunately, various culture vessels and systems have been employed, making comparisons difficult. In this study, the influence of the vessel container size, medium volume, and various CO2 concentrations (0 to 50,000 ppm) was studied on the growth obtained from lettuce and spearmint cultures. All three of these factors influence growth responses from plants cultured in vitro. Vessel types tested included: culture tubes, Magenta containers, 1-quart jars, 0.5-gallon jars, and 1-gallon jars having culture volumes of 55, 365, 925, 1850, and 3700 ml, respectively. Increasing the size of the culture vessel resulted in an increase growth regardless of the CO2 level tested. For example, fresh weight of spearmint increases of >250% can be obtained in by employing a 1-quart jar compared to using a culture tube. Increasing medium volume using various vessel types, especially using high concentrations of CO2, resulted in dramatic growth increases. For example, a >100% increase in fresh weight could be obtained by increasing the medium volume from 50 ml to 100 ml within a 1-quart jar. These studies suggest that plant growth promoted by supplemental CO2 is limited by the culture vessel size and medium volume. Differences in growth responses obtained in past CO2 studies could be related to vessel type and medium volume as well as the CO2 levels employed. Future in vitro studies should consider these factors in the evaluation of the influence of Ultra-high CO2 levels on plant growth. Peculiar growth responses, especially pertaining to rooting and shooting exhibited by cultures grown in ultra-high CO2 levels will also be discussed.

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Brent Tisserat, Christopher Herman, Robert Silman, and Rodney J. Bothast

A continuous CO2 flow system was used to study the growth of carrot (Daucus carota L.), citrus (Citrus macrophylla L.), kale (Brassica oleracea L.), lettuce (Lactuca sativa L.), radish (Raphanus sativus L.), and tomato (Lycopersicum esculentum L.) cultures in vitro under photoautotrophic, photomixotrophic, and heterotrophic conditions. Lettuce plantlets were grown on Murashige and Skoog medium with 0%, 0.3%, 1%, and 3% sucrose within flow chambers containing 350, 750, 1500, 3000, 10,000, 30,000, and 50,000 μL·L−1 CO2. Increasing the levels of CO2, especially at the ultra-high levels (i.e., ≥3,000 μL·L−1 CO2), increased fresh weight, shoot length, leaf number, leaf length, leaf width, root number, and root length for plantlets grown regardless of sucrose levels tested compared to plantlets grown at normal atmospheric CO2 levels, i.e., 350 μL·L-1. For example, fresh weights of lettuce plantlets grown on medium containing 0% or 3% sucrose increased 11- and 13-fold, respectively, when supplemented with 30,000 μL·L-1 CO2 compared to growth of lettuce plantlets grown on the same media without CO2 enrichment. Similar fold increases in growth responses were obtained with carrot, citrus, kale, radish, and tomato plantlets grown in atmospheres enriched with high CO2 levels, elevated from 3000 to 30,000 μL·L-1. Optimum CO2 concentration varied among species, suggesting a species-related response. Varying the rate of CO2 application between 250, 500, 1500, or 2000 mL·min-1 did not effect the rate of growth of lettuce plantlets. The passive diffusion continuous flow-through system presented in this paper is inexpensive, easily constructed, and allows for testing ultra-high CO2 levels on plant culture growth in vitro.