The influence of the culture chamber size and medium volume on the growth rates of shoot tips of peas, lettuce, kidney beans, and spearmint were determined after 8 weeks of incubation. Cultures were grown in a variety of culture chambers including culture tubes, baby food jars, Magenta GA-7 containers, 1-pint Mason jars, 1-quart Mason jars used with and without an automated plant culture system (APCS), 0.5-gal Mason jars with and without an APCS, Bio-safe chambers with an APCS, and polycarbonate culture chambers with an APCS having culture chamber volumes of 55, 143, 365, 462, 925, 1850, 6000, and 16,400 ml, respectively. Plans are presented for the construction of various culture chambers used in an APCS. The APCS consisted of a peristaltic pump, media reservoir containing 1 liter of liquid nutrient medium, and a culture chamber. Cultures grown with an APCS consistently produced higher fresh weights than cultures using any of the agar culture systems tested. Growth rates varied considerably depending on the plant species and culture system tested. Peas, lettuce, and spearmint exhibited flowering only when grown in the APCS. A cost comparison using the APCS versus various conventional tissue culture systems is presented.
Methods to enhance sweetgum (Liquidambar styraciflua L.) in vitro axillary shoot formation and shoot establishment into soil are presented. Sweetgum shoots grown in an automated plant culture system (APCS) produced 400 to 500 shoots via axillary branching compared to only 40 shoots produced within Magenta vessels containing agar medium after 8 weeks of incubation. Vitrification was observed in as many as 80% of the axillary shoots produced in the APCS. A continuous carbon dioxide (CO2)-flow-through system was tested on both vitrified and non-vitrified sweetgum shoots transferred from the APCS to soil. One- and two-cm-long vitrified shoots were grown within CO2-flow-through system chambers and subjected to 350, 1500, 3000, 10,000, or 30,000 μL·L–1 (ppm) CO2 for 4 weeks. Administering 10,000 μL·L–1 CO2 improved culture survival and enhanced overall shoot and root growth compared to shoots grown under ambient atmosphere (i.e., 350 μL·L–1 CO2).
Comparative tests were conducted to determine the influence of the culture vessel size and medium volume on the growth rates of shoot tips of peas (Pisum sativum cv. `Wando'), lettuce (Lactuca salvia) Kidney beans (Phaseolus vulgaris). Culture vessels employed included: culture tubes, baby food jars, Magenta rectangular containers, 1 -pint Mason jars, 1 -quart Mason jars, l-quart Mason jars employed with an automated plant culture system (APCS), 1/2-gallon Mason jars with an APCS, BioSafe containers with an APCS, and mega-culture chambers with an APCS. The APCS consisted of a peristaltic pump, media reservoir containing 1 liter of nutrient medium, and a culture chamber (<925 mm3). High positive correlations occurred comparing culture weight, leaf length and plant height with culture chamber volume, media volume and culture chamber height. APCSs consistently gave higher growth rates and exhibition of mature morphogenetic responses such as flowering and fruiting than growing plants on agar culture systems. Cost analysis comparing APCSs and conventional tissue culture systems is presented.
Brent Tisserat and Steven Vaughn
The influence of a wide range of CO2 levels on the growth, morphogenesis, and secondary metabolite production in vitro was evaluated. Shoots of thyme (Thymus vulgaris L.) and a spearmint–peppermint cross (Mentha spicata × Mentha piperita) were grown on MS medium with and without 3% sucrose under 350, 1500, 3000, 10,000, and 30,000 μL CO2/L for 8 weeks. Dichloromethane extracts from leafs were analyzed using GC-MS techniques. Prominent peaks were identified by comparison with known standards. Highest growth (i.e., fresh weight) and morphogenesis responses (i.e., leafs, shoots and roots) were obtained when shoots were grown under 10,000 μL CO2/L regardless of whether or not sucrose was included in the medium. Ultra-high CO2 concentrations (3000 μL CO2/L) stimulated secondary metabolite production regardless of whether or not the medium contained sucrose. However, the combination of certain ultra-high CO2 levels (e.g., 3000 to 10,000 μL CO2/L) and the presence of sucrose in the medium resulted in shoots producing the highest levels of secondary metabolites. These results suggest that in vitro photosynthesis, which is stimulated by ultrahigh CO2 levels, may enhance secondary metabolite production.
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.
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.
Brent Tisserat and Paul Galletta
Plans are presented for the construction of a three-channel reversible peristaltic pump that can operate at a rate of 45 ml/minute per channel. The cost of this peristaltic pump is about $84 compared to $620 for a commercially available peristaltic pump of similar design and capability. An electronic control scheme for pump operation is presented also.
Brent Tisserat and Amy Stuff
The effects of aqueous solutions applied as foliar spray and drench applications of glycerol were tested on the ‘Chantenay’ carrot (Daucus carota L.) family Apiaceae, corn (Zea mays L.) family Poaceae, and spearmint (Mentha spicata L.) family Lamiaceae under greenhouse conditions. Foliar sprays and drenches were administered to carrots at concentrations of 0, 1, 3, 5, 10, 25, or 50 ml·L−1. Fresh weights, dry weights, and taproot diameter from carrot seedlings sprayed with a solution containing 5 mL·L−1 (50 mm) glycerol increased 105.6%, 158.4%, and 53.8%, respectively, when compared with untreated carrots. Foliar sprays were administered to corn at concentrations of 0, 0.1, 0.3, 0.5, and 1 ml·L−1 and spearmint at concentrations of 0, 1, 5, and 10 mL·L−1. Growth responses increased in corn and spearmint by using certain glycerol concentrations. Fresh weights, dry weights, and shoot length from corn seedlings sprayed with a solution containing 0.5 mL·L−1 (5 mm) glycerol increased 83.5%, 154.6%, and 90.9%, respectively, when compared with untreated corn. Fresh weights, dry weights, and shoot length from mint plants sprayed with a solution containing 5 mL·L−1 (50 mm) glycerol increased 46.6%, 68.7%, and 102.5%, respectively, when compared with untreated plants. Glycerol applications can stimulate growth responses in diverse plant species.
Brent Tisserat and Paul D. Galletta
Flowering and fruit production were obtained from cultured shoot tips of `California Wonder', `Super Cayenne', and `Zippy' peppers grown in a liquid Murashige and Skoog (MS) medium without growth regulators. Thirty-day-old pepper shoot tips, derived from seedlings germinated sterilely, were cultured in a 6-liter polycarbonate container coupled to an automated plant culture system (APCS). Plants were given ten daily, 30-minute, compressed-air applications at 300 ml/minute. These shoots began flowering after an additional 60 to 90 days in culture and flowered continuously thereafter. About 5% to 10% of the flowers set fruit. Maximum fruit size obtained was ≈25% to 75% of the size of fruit produced on plants grown in vivo. In contrast, no flowering occurred from shoot tips grown in 25 × 150-mm culture tubes containing agar medium and subcultured every 8 weeks to fresh medium. Immature fruit excised from plantlets grown in the APCS were cultured separately on agar medium containing 0.0, 0.1, 0.3, or 1.0 mg BA/liter with and without 0.1 mg NAA/liter. Isolated fruit grew best on MS medium with BA only and poorest on medium without growth regulators. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); 1-naphthaleneacetic acid (NAA).
Brent Tisserat and Paul D. Galletta
Some cultivars of mandarin (Citrus reticulata Blanco), sweet orange [Citrus sinensis (L.) Osb.], and trifoliate orange [Poncirus trifoliata (L.) Raf.] were found to have adventitious juice vesicles originating from their exocarp (peel). Several hundred green vesicles may be initiated from curvilinear stylar peel depressions of immature fruit. These vesicles develop similarly to juice vesicles from the endocarp except that, as the fruit matures, exocarp adventitious vesicles die prematurely and degenerate into a fruit surface blemish. Evidence suggests that juice vesicles and oil glands are homologous and merit reconsideration in ontological studies.