Plant Posters
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In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-013-9614-4 2013 IN VITRO BIOLOGY MEETING ABSTRACT ISSUE Plant Posters P-2000 time-lapse videos capture. The use of gravity as a driving force for TIB operation has allowed the decoupling of gas and liquid Biosynthesis Enhancement of Stilbenoids in Hairy Root Cul- flow rates, making gas phase supplementation feasible, thus tures of Peanut. L. OGUTU, L. Nopo-Olazabal, and F. taking full advantage of the high mass-transfer coefficients Medina-Bolivar. Arkansas Biosciences Institute and Depart- achieved in these systems. Applications of this system include ment of Biological Sciences, Arkansas State University, supplementing O2 to improve growth rates and supplementing Jonesboro, AR. Email: [email protected], CO2 in the development of sugar-free photoautotrophic bioreac- [email protected] tors. Preliminary results growing N. benthamiana hairy root cultures in oxygen supplemented environments have shown that Resveratrol, a stilbenoid found in plants such as peanuts and these cultures are capable of utilizing a greater degree of available grapes, has a wide range of positive biological effects. Of sugars than controls. While experiments have shown comparable particular interest for this study are the hairy root cultures of amounts of tissue, this is believed to be caused by unequal peanut which have the ability to produce resveratrol and its stresses on each treatment due to periodic flow problems during prenylated analogs arachidin-1 and arachidin-3 upon elicitor prototype trouble-shooting. Photoautotrophic growth in the bio- treatment. Arachidin-1 has demonstrated higher antioxidant ac- reactor has also been demonstrated using seedless watermelon tivity and potentially higher metabolic stability than resveratrol tissue in elevated CO2 environments. Due to a lack of available based on in vitro studies. Based on these findings, we tested sugar, these systems are significantly more resistant to contami- different elicitation strategies to produce high levels of nation, and thus more readily deployed in the development of arachidin-1 in hairy root cultures. Stilbenoids were extracted economically important crops, including an ongoing effort to from the culture medium with ethyl acetate and analyzed by develop disease resistant cacao (chocolate tree). HPLC. The highest levels of arachidin-1 were obtained upon treatment with cyclodextrin and methyl jasmonate in combina- P-2002 tion with a putative precursor. The feasibility of this putative precursor to produce high levels of arachidin-1 in the presence Shoot Organogenesis from In Vitro Derived Leaf Tissues of of cyclodextrin suggested its participation in the biosynthesis of Piper longum. D. RANI and P. K. Dantu. Plant Biotechnology arachidin-1 and proposed a mechanism for the regulation of Laboratory, Department of Botany, Dayalbagh Educational stilbenoid production in the peanut tissue cultures. Institute (Deemed University), Dayalbagh, Agra 282110, INDIA. Email: [email protected], [email protected] P-2001 An efficient regeneration protocol from in vitro derived leaf Novel Temporary Immersion Bioreactor Allows the Manipula- explants via multiple shoot induction was developed for Piper tion Headspace Composition to Improve Plant Tissue Propaga- longum. Leaves from in vitro derived shoots were cultured on tion. M. CURTIS, S. Florez, and W. Curtis. The Pennsylvania Murashige and Skoog medium (MS) supplemented with as low State University. Email: [email protected] as 0.10 mg/l of TDZ and 40 mg/l ascorbic acid which at the end of four weeks differentiated an average of 18 shoot buds without A bioreactor system has been fabricated for plant tissue culture any intervening callus. When the leaf explants were cultured on propagation which utilizes low-cost plastic bags combined with MS supplemented with different combinations of BA and Kn, gravity driven flows as an approach to achieving scale-up. These highest shoot proliferation of 17 shoot buds per leaf explant. temporary immersion bioreactors (TIB) provide for complete Shoot buds developed in clusters in both the media and sustained media-tissue contacting while minimizing boundary layer mass- multiplication could be achieved by subculturing in the same transfer resistances and water-logging the plant tissue. Media medium every four weeks. Degree of callusing was directly flow to and from the bioreactor is facilitated via stepper motors proportional to the concentration of TDZ. High callusing with interfaced to a computer with LabVIEW. LabVIEW has also occasional root formation was observed on medium containing provided a means to monitor the reactors over the web utilizing BA and Kn. Shoot induction was observed in 100% of the PLANT POSTERS explants. Endophytic bacteria are a serious problem in the cul- their filamentous trichomes to PLL coated polypropylene or tures of P. longum as they make appearance in long term cultures nylon. To find an appropriate blending solution, leafy tissue of resulting in the loss of precious cultures. However, shoots dif- in vitro A.annua were blended in deionized water, half strength ferentiating cultures derived from in vitro leaves even after ten MS media, 15 g/L sucrose, 30 g/L sucrose and 16 g/L mannitol. subcultures did not show growth of endophytic bacteria. Rooting The blenderized tissue from each solution were then sieved into was readily achieved in medium supplemented with 0.25 mg/l four size categories (>1 mm, 0.5-1 mm, 0.35-0.5 mm, 0.15- IBA. Hardening and acclimatization process resulted in a pro- 0.35 mm) and cultured in 12-well petri dishes filled with solid duction of more than 90% healthy plantlets. Plants transferred to MS shooting medium. Regeneration ratio, defined as number of the field produced spikes that were found to contain piperine inoculated tissue pieces that regenerated new shoots over total when analyzed through HPLC. number of inoculated tissue, was observed after 6 weeks. Re- gardless of blending solution, the regeneration ratio increased as P-2003 inoculated tissue size increased. For tissue sized larger than 1 mm, 30 g/L sucrose blending solution had 48% regeneration Camelina sativa Protoplast Isolation, Purification, and Cul- ratio, which was 3 times higher than tissue blended in deionized ture. E. BABIKOW1,F.Ansary2, M. Chennavasin3,M. water. Both 15 g/L and 30 g/L sucrose solution had highest Reitzel2, K. Chowdhury4, A. Shuler2, S. Potlakayala2, and regeneration ratio of 4% on spray-able blenderized tissue sized S. Rudrabhatla2. 1Eastern Mennonite University, 1200 Park between 0.15-0.35 mm. In future experiments, blenderized ex- Rd, Harrisonburg, VA 22802; 2Penn State Harrisburg, 777 plants of A. annua will be propagated on PLL coated polypro- W. Harrisburg Pike Middletown, PA 17057; 3Penn State pylene or nylon strips hanging in mist reactors. Both the mist University Park, 201 Old Main, University Park, PA reactor and our attachment technology may offer opportunities 16802; and 4Claflin University, 400 Magnolia St., for at least partial automation of micropropagation. Orangeburg, SC 29115. Email: [email protected] P-2005 Camelina sativa is a renewable biofuel crop with a high seed oil content. C. sativa grows well in semi-arid conditions, is cold- Morphological Characterization, Seed Germination and tolerant, and requires few inputs. Unfortunately, the high per- Micropropagation of Vriesea ensiformis and Vriesea centage of polyunsaturated fatty acids in Camelina oil causes saundersii, Ornamental Bromeliads from the Atlantic Forest oxidative susceptibility, making it unattractive for use in large inBrazil.S.SANTA-ROSA,L.K.Manarim,M.L.Rossi,and scale production. This disadvantage can be overcome through A. P. Martinelli. University of São Paulo, CENA, Av. genetic engineering via protoplast transformation. Successful Centenario, 303, Piracicaba, SP, 13416–930, BRAZIL. Email: transformation requires optimized protoplast isolation and puri- [email protected] fication protocols. We tested the effect of enzyme concentration, digestion temperature, purification methods, and C. sativa vari- Vriesea ensiformis and V. saundersii are bromeliads native to ety on protoplast yield. We achieved the highest viable proto- the Atlantic Forest in Brazil, targets of extractivism due to their plast yield by placing the Blaine Creek variety in a 250C ornamental characteristics, with V. saundersii currently a critical- incubation of 1% cellulose 1% macerozyme+mannitol solution ly endangered species. This study aimed to characterize both and using a washing solution isolation method. species and evaluate seed germination, post-seminal develop- ment and micropropagation rates, aiming to contribute to the P-2004 availability of these species and their preservation. Plants were characterized by observation of the material in the greenhouse Heading: Towards Automation of Micropropagation Using a and herbarium specimens. For germination studies, seeds Mist Bioreactor. L. FEI and P. J. Weathers. Worcester Polytech- obtained from plants grown in the greenhouse were rinsed and nic Institute, Dept. of Biology & Biotechnology, 100 Institute the seed appendage was removed prior to assepsy. Introduction Road, MA 01609. Email: [email protected]; [email protected] in vitro was in ½ MS supplemented with 30 gL-1 sucrose, 8 g L-1 agar and cultivated at 22, 27 and 32°C, under 16 h photoperiod. Micropropagation is challenging to automate mainly because of Additionally, seeds were sown in trays and kept in the green- the diverse