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 morphology of plant shoots. We have shown that the house at ambient temperature and approximate humidity of mist reactor can be used for 1-step propagation of carrot cells 70%. These are epiphytes, leaves have smooth margins. Two into embryos and then fully rooted plantlets using poly-L-lysine appendages are present at the base of the petals. Nectary is (PLL) coated polypropylene or nylon strips to which cells were septal, interlocular, at the base of the ovary. Ovules present a attached prior to growth while hanging in the nutrient mist. It is acuminate chalazal appendage. Stigma in V. ensiformis is of also possible to propagate small attached explants of leaves the convoluted-blade type, stamens are exserted, while in V. since Artemisia annua leaf explants, for example, attached by saundersii presents included stamens. In vitro seed PLANT POSTERS germination in both species occurred in a higher percentage Camelina sativa, the oilseed crop of the Brassiaceae family, than in substrate, with differences among the three tempera- also known as false flax and gold of pleasure, is an herba- tures. Post-seminal development was classified in five stages. ceous annual dicotal C3 crop. It is now becoming popular to The results presented here are part of a research line that aims cultivate again due to its potential as a low cost biofuel crop, to characterize and study reproductive aspects of bromeliad or as a rich source of omega-3 fatty acids. Salinity is one of species from the Atlantic Forest, as well as their the most deleterious environmental factors limiting crop micropropagation to provide methodologies for large scale growth and yield. Because salinity is steadily affecting more production of plants. (Acknowledgements: CNPq, FAPESP, of the world’s land area each day, it is necessary to research NAP/MEPA-ESALQ/USP) ways to conserve lands for cultivation, or be able to breed plants that can grow and thrive in saline conditions. Salt P-2006 stress causes an initial water deficit and ion specific toxicity resulting in changes of K+and Na+ concentrations. Increased Identification and Characterization of the Genes Involved in the levels of Na+ and Cl− in cells inhibit plant growth by Fruit Color Development of European Plums (Prunus disrupting physiological processes, especially photosynthe- domestica L.). S. DINESH KUMAR1,S.Sherif1,K.S.Tiwari1, sis. Also, salinity causes changes in proline synthesis and G. Paliyath1, and S. Jayasankar1,2. 1Department of accumulation including induction of osmotic stress on Plant Agriculture, University of Guelph, Guelph, ON, plants and these stresses can be evaluated using different N1G2W1, CANADA and 2Department of Plant Agriculture, physiological parameters. It is important to examine salt University of Guelph, 4890 Victoria Ave. N., PO Box 7000, induced irregularities in plants’ cell cycle because in addi- Vineland Station, ON, L0R 2E0, CANADA. Email: tion to enable the plant to survive, the plant cell cycle may [email protected] also contribute to the mechanism by which the plant can survive. Another key area of research into salt stressed European plums (Prunus domestica L.) are normally purple plants is associated with post-translational modifications of black as the fruit ripens. The pigments that impart characteristic proteins related to cell cycle. Cell length measurements and purple color to the plums are primarily anthocyanins. However, cell death was estimated in the roots. Our work also includ- there are genotypes that exhibit lack of this dark color and remain ed the analyses of mRNA and proteins related to cell cycle. green or yellow even after ripening. It is hypothesized that in In summary all the physiological and molecular data will be such genotypes one or more genes responsible for biosynthesis discussed. of anthocyanins are not functional possibly due to mutation or genetic regulation. In this study, six varieties of European plums P-2008 that vary in color from dark purple to green are investigated for expression of anthocyanin biosynthetic pathway genes. We Ectopic Expression of an Osmotin in Indian Soybean Con- chose five genes namely phenylalanine ammonia lyase (PAL), fers Resistance Against Drought Stress. KONDETI chalcone synthetase (CHS), dihydroflavonol 4-reductase (DFR), SUBRAMANYAM, Andy Ganapathi, and Markandan anthocyanin synthetase (ANS), UDP-glucose:flavonoid 3-O- Manickavasagam. Plant Molecular Biology Laboratory, De- glucosyltransferase (UFGT). Our preliminary results indicate partment of Biotechnology & Genetic Engineering, down-regulation of CHS may lead to the loss of normal purple Bharathidasan University, Tiruchirappalli–620024, Tamil color resulting in green or yellow genotypes. Nadu, INDIA. Email: [email protected]

P-2007 Soybean [Glycine max (L.) Merrill] is an important legumi- nous seed crop that provides the largest source of vegetable Physiological and Molecular Studies of Salt Stress on protein to the human diet and the second-largest source of Camelina sativa.D.MORALES1, D. MILLER2,A.Jones3, vegetable oil. Soybeans contain 40% of protein and 20% of E. Kovak4, E. Lowry5,F.Ansary2,R.Anand6,N.Karelia2,A. oil, which is comparatively higher than any other legumi- Shuler2, S. Rudrabhatla2, and S. Potlakayla2. 1Shippensburg nous crops. USA leads the world in soybean production University of Pennsylvania, 1871 Old Main Drive with an annual output of about 80.7 million metric tons Shippensburg, PA 17257; 2Penn State Harrisburg, 777 West followed by Brazil, Argentina, China and India. The annual Harrisburg Pike, Middletown, PA 17057; 3University of Wy- production of soybean in India is 10.12 million metric tons. oming, 1000 East University Avenue Laramie, WY 82071; Globally soybean production is hampered by several abiotic 4St. Olaf College, 1520 Saint Olaf Avenue Northfield, MN factors. Drought is one among the several abiotic factors 55057; 5Kansas State University,116, Ackert Hall Manhattan, which limits the soybean production. In order to overcome KS 66506; and 6University of Pittsburgh, 4200 Fifth Ave the consequences of drought stress in soybean, we have Pittsburgh, PA 15213. Email: [email protected] transformed soybean cv. Pusa 16 via somatic embryogenesis PLANT POSTERS with stress induced and apoplastically secreted tobacco producer of high quality biodiesel. Apart from biodiesel, the osmotin (Tbosm) gene using Agrobacterium-mediated ge- plants were been widely cultivated as live fence and also in netic transformation. Integration of Tbosm in randomly se- production of anti-bacterial soaps. Jatropha is also practiced lected GUS assay-positive independently transformed in various traditional medicines as anti-cancer agent, anti- soybean plants was confirmed by polymerase chain reaction dote to snake bite, and also as a remedy in treating skin (PCR) and Southern hybridization. Reverse transcriptase diseases, rheumatism etc. The plant has a very low produc- PCR (RT-PCR) and Western blotting confirmed that the tivity rate and also susceptible to various abiotic and biotic Tbosm was expressed in transformed soybean plants. The factors which directly affect the growth and productivity of transformed soybean plants were challenged against poly- the plant. Being a perennial plant conventional breeding is ethylene glycol 6000 (PEG) and water deficit. The not much fruitful and hence genetic transformation is essen- transformed (T1) soybean plants were survived when the tial to counter the above said factors and improve the pro- plants were watered with up to 15% PEG 6000 and whereas, ductivity. Various tissue culture based protocols have been non-transformed plants failed to survive. In another experi- developed to obtain Jatropha transgenics but still the trans- ment,thetransformedsoybeanplantswereassessedfor formation efficiency is considerably low. Hence the neces- tolerance to water deficit stress by withholding irrigation sity of developing an alternate method to produce Jatropha for 7 days followed by rewatering. The transgenic plants transgenics with higher transformation efficiency is of prior survived and performed better under water deficit condi- interest. Consequently a pricking based In-Planta transfor- tions, while non-transformed plants failed to withstand mation method was developed to minimize the process cost, water deficit. When the plants were irrigated with water, time and man power with higher transformation efficiency. the transgenic plants recovered fully but the non- Various influential parameters like type of infection, transformed plants did not survive. The biochemical Agrobacterium strain, acetosyringone concentration and analysis revealed the T1 stress induced soybean plants vacuum infiltration have been evaluated to attain maximum accumulated higher amount of proline, chlorophyll, transformation efficiency. Among a variety of parameters APX, CAT, SOD, DHAR, MDHAR, and RWC than tested, the decapitated plants coupled with pricking and NT stressed plants. Leaf gas exchange measurements re- vacuuming in Agrobacterium tumefaciens EHA105 harbor- vealed that T1 stress induced soybean plants maintained ing pME 524 suspension at a pressure of 250 mm of Hg for higher net photosynthetic rate, CO assimilation, and stomatal 3 min recorded a highest transformation efficiency of 62%. conductance than NT stressed plants. The T1 stress induced Herbicide resistance trails and GUS gene expression initial- soybean plants produced 20–23 soybean pods/plant ly showed the incorporation of bar gene into the Jatropha containing 8.3–10.5 g of seeds at 15% PEG 6000 and non- genome. In addition, the integration of bar gene with the transformed soybean plant produced 18–20 soybean plant genome was confirmed with PCR and Southern hy- pods/plant containing 7.6–8.2 g of seeds at 5% PEG 6000. bridization and thereby reported a transformation efficiency Under water deficit for 7 days, the transformed soybean plants of 62%. Putatively transformed plants (Scion) of 2 months produced 17–22 soybean pods/plant containing 7.6–9.8 g of of age were then grafted with 3 months old of wild plants seeds and non-transformed plants failed to survive. The pres- (Stock or Root stock). A complete union was observed after ent investigation clearly shows that expression of Tbosm 4–5 weeks of grafting with a 100% success rate. Herbicide enhances drought tolerance in transformed soybean trails, GUS assay and molecular confirmations were plants. performed with the grafted plants as like the mother plants. In addition, genetic fidelity of the grafted plants was evalu- P-2009 ated with the aid of RAPD markers which proved the genetic stability of the grafted plants. In-Planta Transformation of Jatropha curcas (L.) and ex vitro Multiplication of Transgenics Using Grafting. P-2010 BALUSAMY JAGANATH, Andy Ganapathi, and Markandan Manickavasagam. Plant Molecular Biology Genetic Engineering of Fruit Rot Disease Resistance in Laboratory, Department of Biotechnology & Genetic Engi- Capsicum annuum. L Using Defensin gene (TvD1) Through neering, Bharathidasan University, Tiruchirappalli–620024, Stable Plastid Transformation. A. V. RAO, N. Murali Tamil Nadu, INDIA. Email: [email protected] Krishna, E. Raghu, and A. Sadanandam. Department of Biotechnology, Kakatiya University, Warangal-506 009, AP, Jatropha curcas (L.) is a perennial plant belonging to the INDIA. Email:[email protected] family Euphorbiaceae being cultivated around the globe for the production of biodiesel. The seed contains approximate- Chilli pepper is a spice and vegetable crop and its green ly 40% of oil (non-edible) which makes the plant a potent fruits are rich source of vitamins, especially C, while dry PLANT POSTERS chilli peppers impart hotness and pungency to foods. India acclimatized under culture room conditions, transferred to is the world’s largest producer of C.annuum L. pots, grown in the glass house, and confirmed through South- Colletotrichum capsici and C. gloeosporioides, are reported ern blotting. to cause anthracnose fruit rot diseases and responsible for major crop loss. A defensin that could fight the P-2011 Colletotrichum infection in chilli pepper is highly pertinent in plant biotechnology. Plant defensins are small, highly Tissue Culture, Transformation, and Genetic Diversity Stud- stable, cysteine rich antimicrobial peptides produced by ies of Jatropha curcas, a Second Generation Biofuel Crop. the plants for inhibiting a broad-spectrum of microbial path- BEHNAM TABATABAI1,2, Mohsen Zaidi1,Matthew ogens. Defensins with antimicrobial activity are potent can- Reitzel1, Shobha Potlakayala1,LaurenKong1,3, Camille didate genes for deployment in transgenic crops for Gonzalez-Morales1,4, Alison Shuler1,andSairam protecting them against pathogens. TvD1 appears to be a Rudrabhatla1. 1Penn State Harrisburg, 777 West Harrisburg potent defensin gene for fungal disease resistance in trans- Pike, Middletown, PA 17057; 2Morgan State University, genic crops. Plastids of higher plants are semi-autonomous 1700 East Cold Spring Lane, Baltimore MD 21251; 3NSF- organelles with a small, highly polyploid genome and their REU Student, Mills College, 5000 MacArthur Blvd., Oak- own transcription-translation machinery. The high ploidy land, CA 94613; and 4NSF-REU Student, University of level of the plastome enables a high degree of gene expres- Puerto Rico - Rio Piedras, Ponce de León Ave., San Juan, sion, and moreover the chloroplast transformation has also PR 00925. Email: [email protected] been proposed to remedy the problems associated with transgene dispersal, Gene silencing and position effect often Jatropha curcas L. (Euphorbiaceae) is an important second observed in nuclear transgenic plants has not been observed generation biofuel crop. In a time when energy needs are in genetically engineered chloroplasts. pRB94 is a plastid coming to the forefront of our nation’s concerns, develop- specific Vector that is suitable for organellar transforma- ment and improvement of alternate, sustainable biofuel tions. pRB94 vector and pTZ57R harboring defensin crops are critical. J. curcas is especially useful because it gene(TvD1) were transformed and maintained in E.Coli is a drought-tolerant, non-food crop that can be grown on (DHα)cultures. Both the vectors were double digested with marginal lands, thus not competing for the fertile agriculture restriction enzymes Kpn1 and BamH1. Digested pRB94 land used for food production. J. curcas seeds contain vector has resulted in a linear plasmid of 7.6 Kb, where as approximately 40% oil that can be used as a feedstock for a 228 bp fragment of defensin gene(Insert) was released biodiesel production. However, one of the limitations of J. from pTZ57R vector .Digested plasmids were separated by curcas is that it is adapted to warm, tropical and subtropical electrophoresis and the desired fragments were eluted climate and it is susceptible to cold stress. Combining the (Genetix). Linear pRB 94 vector is mixed with defensin tools of biotechnology such as tissue culture and genetic gene (insert) at the ratio of 1:3 and ligated with suitable engineering, value added traits (e. g. tolerance to biotic and buffers and at optimal conditions. Ligated vector is trans- abiotic stress) can incorporated to J. curcas leading to ac- ferred into competent E.Coli (DHα strain) Cells and the celerated production of superior cultivars. We report here colonies were screened for the presence of defensin gene the development of an efficient protocol for in vitro regen- by PCR and Restriction digestion. The indigenous built eration of J. curcas using various genotypes and explants particle delivery system (PDS-1000/He, Biorad) was used (leaf, nodal segment, and embryo). Likewise, a protocol for for bombardment using gold for propelling by acceleration genetic engineering of J. curcas via Agrobacterium-mediat- provided by helium gas. Defensin cloned vector is coated ed transformation was also optimized to improve its qualities with microcarriers (60 mg/ml) for transformation with 2.5 M such as tolerance to abiotic stress and oil yield. Lastly, Ran- calcium chloride and 0.1 M spermidine that allows DNA dom Amplification Polymorphic DNA (RAPD) was coated micro carriers to integrate into plastid. Standard performed on 32 J. curcas plants to test for genetic diversity. bombardment parameters were set by performing bombard- ment with different flight distances, pressure and vacuum P-2012 using GUS as reporter gene. The standardized protocol trails is adapted in Capsicum annuum cv-G4 for transformation of Development of Brassica juncea Suspension Culture and Its cloned defensin into plastids at 900/6 flight distance both for Transformation Using Particle Bombardment. LIU Y. SHEN. Capsicum with 25 mm of Hg pressure and putative Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN transformants were on selection for spectinomycin with a 46268. Email: [email protected] optimized lethal dose concentration at 250 mg/L. Molecular characterization was carried out for T0 transplastomic lines Embryogenic suspension cultures have been utilized for the obtained from leaf disc transformation. These plants were study of plant metabolism and gene regulation due to their PLANT POSTERS faster cycle time and relative ease of manipulation. Devel- studies will focus on the in depth analysis of the differential opment of an embryogenic Brassica suspension cultures bands at the molecular level. In addition, we plan to present the that are capable of forming somatic embryos, are regenera- on-going 1D and 2D and gel analyses comparing the proteins ble, and amenable for genetic transformation would be very of American and Chinese chestnut species. useful for many applications. In the present paper we de- scribe an efficient system for Brassica juncea suspension P-2014 culture initiation, proliferation, somatic embryo induction, and regeneration. Different tissues including seedling coty- Callus Induction and Assessment of Genetic Diversity in ledons and epicotyl tissue from two different varieties of Simarouba glauca. J. KAKUTURA, M. Zaidi, M. Reitzel, Brassica juncea Zem-1 and Vniimk 405, as well as various A. Shuler, S. Potlakayala, P. Josekutty , and S. Rudrabhatla. growth regulators were evaluated for their effect on the Penn State Harrisburg, 777 West Harrisburg Pike, Middle- induction of embryogenic calli. Concentrations of 2, 4-D town, PA 17057. Email: [email protected] at 4-5 mg/l was found to be most effective for inducing embryogenic calli. Embryogenic suspension culture were Biofuels are renewable sources of energy produced from initiated from the calli with a medium containing 2,4-D plants and have lower greenhouse gas emissions compared (2 mg/l), TDZ (0.05 mg/l) and picloram (6.04 mg/l), or an to petroleum-based fuels. Simarouba glauca is an excellent alternate medium containing 4 mg/l 2,4-D and 1 mg/l kine- biofuel crop that bears seeds with an oil content of 65%. tin. Induction of somatic embryos was induced on a medium Since S.glauca has a long generation time and bears seeds containing 3 mg/l BAP and 5 mg/l AgNO3 under light after 5–6 years, it can be regenerated faster in sterile labo- condition. The embryos matured and regenerated into nor- ratory conditions through plant tissue culturing techniques. mal fertile plants. Suspension developed from juncea Callus was generated through indirect organogenesis in S. Vniimk 405 was transformed with plasmid containing YFP glauca when leaf explants were cultured on Murashige and reporter gene using particle bombardment. Transient expres- Skoog (MS) medium, SGC 1, supplemented with 100 mg/L sion was seen 7–24 hours after bombardment. The Ascorbic acid, 0.5 mg/l 6-benzylaminopurine (BA) and 0.5 - transformed callus events isolated on high herbicidal selec- 5.0 mg/l NAA (1-napthaleneacetic acid) (SGC 1) and on tion have shown Yellow Fluorescent Protein (YFP) expres- SGC 2 with 0.5 - 5.0 mg/l NAA and 0.5 – 5.0 mg/L 2,4-D sion. (2,4-dichlorophenoxyacetic acid) and 3% sucrose. MS me- dium containing 2.5 mg/L NAA (SGC 1) and 4.5 mg/L P-2013 NAA with 4.5 mg/L 2,4-D (SGC 2) was noted to be the most effective in inducing friable embryogenic callus (FEC) Genetic Comparison of Castanea dentata and Castanea in S.glauca. SGC 2 showed better responses compared to mollissima Using RAPD Analysis. N. BRILEY1,S. SGC 1 displaying the importance of 2,4-D in combination Potlakayala2, M. Reitzel2, K. Vescio1, M. Zaidi2, A. Shuler2, with NAA for callus generation. A random amplified poly- and S. Rudrabhatla2. 1Undergraduate; Penn State Harrisburg morphic DNA analysis (RAPD) analysis was also done to 777 West Harrisburg Pike, Middletown, PA 17057 and see the genetic differences between the S. glauca genotypes 2Penn State Harrisburg 777 West Harrisburg Pike, Middle- used in this study. Plant 5’s high callus frequency was the town, PA 17057. Email: [email protected] genetopic difference analyzed by the absence and presence of bands compared to plant 19 and 21 analyzed by RAPD. Castanea dentata, the American chestnut, is all but extinct thanks to a fungal predator brought to America by the impor- P-2015 tation of Castanea mollissima (Chinese chestnut). Cryphonectria parasitica ravaged the C. dentata population; Assesment of Salinity Tolerance in Sugarcane Through As- yet, somehow had no effect on the C. mollissima plants. In an sociation Mapping and Functional Genomic Analysis. effort to track down the genetic source of resistance in the KALPANA SENGAR and R. S. Sengar. Tissue Culture Chinese species, our study was designed to see the similarities Lab, College of Biotechnology, Sardar Vallabh Bhai Patel and differences between the American and Chinese chestnut University of Agriculture & Technology, Meerut-250110, plants at a molecular level. A random amplified polymorphic INDIA. Email: [email protected] DNA (RAPD) analysis was performed using polymerase chain reaction (PCR) involving 15 different primers. The resulting Salinity has significant importance in the production of PCR was separated using a 1% agarose gel and analyzed for sugarcane because it affects the growth rate and sucrose similarities and differences based on common band sizes. The yield of sugarcane crop. In the past decade considerable results showed some distinct bands mainly around 700 and progress has been made in understanding and manipulating 1200 base pairs that existed only in the Chinese species. Future the sugarcane genome using various biotechnological and PLANT POSTERS cell biological approaches. Advances in genomics and mo- understand the underlying molecular mechanisms of non- lecular markers, and progress in understanding the molecu- neuronal acetylcholine expression and its effect on cellular lar aspects of sucrose transport, accumulation and selection and developmental processes. Specifically, the bamboo trans- for biotic and abiotic stresses. Salinity stress response in formation system is being utilized to genetically alter the multigenic, as a number of processes involved in the toler- expression of cloned genes of the acetylcholine pathway ance mechanism are affected, such as various compatible coding for choline acetyl transferase (ChAT) and acetyl cho- solutes/osmolytes, polyamines, reactive oxygen species and line esterase (AChE). The presence of non-neuronal acetyl- antioxidant defense mechanism, ion transport and compart- choline in plants and animals has been implicated in the mentalization of injurious ions. Assesment of various as- regulation of cell differentiation, phyotochrome-mediated pro- pects of the physiology of sugarcane affected by increasing cesses, cytoskeletal organization, immune function, and ion levels of soil salinity during the growing season is very transport. The presence of acetylcholine in bamboo shoots at a important, whether or not there are differences in the phys- significant concentration of 2.9 μmol/g presents a unique iological responses of different varieties to saline conditions. potential to examine bamboo as a non-human model system The trial focused solely on salinity, with the effects of Na + to decipher the molecular events of the non-neuronal acetyl- and sodicity as a variable, when assessing the effect of high choline pathway. Altering the expression of the ChAT and electrical . To assess leaf water potential, stomatal conduc- AChE genes in transgenic bamboo will provide valuable in- tance and light-saturated rate of photosynthesis of these sights in the role of acetylcholine in non-neuronal phenomena varieties investigations were carried out under the experi- involving cell gene expression, proliferation, differentiation mental conditions provided. Throughout the trial it was found and organization. This will in turn illuminate our understand- that the high levels of salt adversely affected growth for all ing of the non-neuronal acetylcholine’s role in pathogenesis of varieties; with increasing salinity total above-ground biomass diseases such as acute and chronic inflammation, local and decreased significantly. Although sucrose yields (tons/ha) of systemic infection, dementia, atherosclerosis and cancer. An all varieties showed a trend of decline with increasing salinity, Agrobacterium-mediated bamboo genetic transformation sys- some showed significant treatment effects. Leaf water poten- tem was developed with a GFP construct along with an tials at both pre-dawn and midday were found to be lower as environmental-friendly non-antibiotic phosphomannose EC increased, implying a mild water stress. Some showed isomerase (pmi) selection system. The bamboo transformation moderate sensitivity to salt because their responses to salinity, system will be utilized to over express the ChAT gene and although variable depending on the parameter being assessed, partially silence the AChE gene. The transgenic plants will be were never as severe as the response of the variety which analyzed for the altered concentrations of acetylcholine to showed significant treatment effects, which displayed more establish the effectiveness of the bamboo genetic transforma- pronounced reactions at higher levels of salinity. Various tion model to characterize the molecular phenomena behind genes/cDNAs encoding proteins involved in the above men- the non-neuronal acetylcholine system that may suggest mo- tioned processes have been identified and isolated. The role of lecular therapeutic targets. genes/cDNAs encoding proteins involved in regulating other genes/proteins, signal transduction process involving hor- P-2017 mones like ABA and strategies to improve salinity stress tolerance have also been discussed. This information have The Complete Chloroplast DNA Sequence from Cowpea opened up possibilities for its application to overcome various (Vigna unguiculata L. Walp). E. O. P. L. NOGUEIRA1,A.P. biotic and abiotic stresses, which are serious threat to Z. Mota1,2, and F. J. L. Aragão1. 1Embrapa Recursos Genéticos sustained sugarcane production to fulfill the sugar demand e Biotecnologia, PqEB W5 Norte, 70.770-917, Brasília, DF, of ever increasing population. BRAZIL and 2Universidade de Brasília, Departamento de Botânica, Campus Universitário Darcy Ribeiro, 70.910-900, P-2016 Brasília, DF, BRAZIL. Email: [email protected]

Transgenic Bamboo as a Non-human Model to Understand the Chloroplasts possess their own genome, known as Molecular Basis of the Non-neuronal Acetylcholine Expression. plastosome, which is circular with highly conserved four CHANDRAKANTH EMANI. Department of Biology, Western regions: two inverted repeats (IR) designated IRa and IRb; a Kentucky University-Owensboro, 4821 New Hartford Road, large single copy (LSC) and a small single copy (SSC) Owensboro, KY 42303. Email: [email protected] regions. Although analyses of different chloroplast genomes demonstrate that they are highly conserved among plants The principal goal of this project is to develop a genetic with little or no differences within species, more than 40 transformationsysteminPhyllostachys bambusoides different plastosomes of higher plants have been sequenced (bamboo) and utilize it as a non-human model to better and are available in different databases, being important PLANT POSTERS tools for comparative genome analysis. Cowpea is a very clonal propagation of physic nut, allowing material homo- important leguminous crop grown in different parts of the geneity, as well as the production of seedling on commercial world, in addition, different research groups are seeking to scale and better quality. Thus, the objective of this study was improve different varieties of this crop. The availability of to evaluated the effects of growth regulators on the hypo- specific and precise information regarding cowpea’s evolu- cotyls of physic nut grown in vitro and detect a possible tion and domestication will help in transgenic and breeding establishment of oxidative stress induced by these growth programs which will ensure the development of elite varie- regulators, using analysis of MDA (malondialdehyde) pro- ties. In this work, the complete chloroplast genome of V. duction and the activities of the enzymes CAT (catalase) and unguiculata was generated using Platinum FLX method, SOD (superoxide dismutase). The plant materials were which gave a total of 9,909 reads. When analyzed using obtained from the germplasm collection of EMBRAPA MIRA 3.0, the reads led to the identification of a final contig Agroenergia, from Rio Verde/GO and Xambrê/PR, both with 152,415 bp. The IRs of the plastosome comprise Brazilian cities. The culture medium used was WPM 25,883 bp each, while LSC contains 81,468 bp and SSC (Woody Plant Medium) with 3% sucrose, pH 5.8, solidified contains 19,181 bp. Chloroplast genome annotation showed with 0.5% agar and supplemented with growth regulators, that V. unguiculata chloroplast contains 120 genes, 25 of resulting in five treatments: T1 (control), T2 (4.5 μMof which are located in the repetitive region. Comparative TDZ), T3 (4.5 μM of TDZ + 2.8 μM of IAA), T4 (4.4 μM genome analyses available from other leguminous species of BAP) and T5 (4.4 μM of BAP + 2.8 μM of IAA). The showed high degree of conservation in chloroplast genome morphophysiological analysis highlighted the success of regions. In parallel and based on the complete genome of V. BAP in inducing adventitious buds. The results from the unguiculata, nine (9) pairs of primers were used to analyze MDA production and the activities of antioxidant enzymes 36 different cultivars from Brazil and Nigeria. Primer 7 was showed that the physic nut hypocotyls cultivated in vitro selected for polymorphism analysis which led to the identi- were under oxidative stress, specially the control group fication of two distinct groups, designated haplotype 0 and (T1). It confirmed that the major factor in causing stress haplotype 1. This divergence leads us to hypothesize that and consequently the lower induction of adventitious buds Brazilian cultivars may have originated from other regions in T1, was the absence of the growth regulator. of the world. Acknowledgements: EMBRAPA and FINEP. P-2019 P-2018 Nicotiana tabacum In Vitro Cultures: a Promising Platform Growth Regulators Effects in the Micropropagation of for Producing Recombinant Human Prethrombin. M. A. 1 1 2 1 Physic Nut (Jatropha curcas) and in the Promotion of ALVAREZ , M. Laguía Becher , Z. Zaldúa , E. Marzol , Oxidative Stress. F. A. ARTIOLI-COELHO1,F.A. P. L. Marconi1, and W. Velander2. 1Instituto de Ciencia y Piotto2,andM.Almeida1. 1Pádua Dias St., No 11, Tecnología Dr. César Milstein, CONICET/Fundación Pablo Piracicaba, SP, Dept. of Biology, School of Agriculture Cassará, Saladillo 2468 (1440), Ciudad de Buenos Aires, “Luiz de Queiroz”–USP (University of São Paulo), Argentina, ARGENTINA and 2Dpt. of Biochemical Engi- 13418–900, BRAZIL and 2Pádua Dias St., No 11, neering, University of Nebraska, Lincoln, NE 68588–0431. Piracicaba, SP, Dept. of Genetics, School of Agriculture Email: [email protected] “Luiz de Queiroz”–USP (University of São Paulo), 13418–900, BRAZIL. Email: [email protected] Active human prothrombin, a glycoprotein involved in blood coagulation, has been expressed in CHO and BHK Physic nut (Jatropha curcas), belonging to family cells. Plants (including in vitro cultures) are gaining atten- Euphorbiaceae, is preferably used as feedstock for biodiesel, tion for the production of recombinant proteins. Its advan- representing an alternative to the fossil fuels use, which due tages include that they can rapidly be bulked to large to the intensive use, have promoted gas emissions green- biomass, its relatively inexpensive maintenance, and that house, with harmful environmental impacts. Facing the they do not harbor mammalian proteins or pathogens. We most important characteristics, physic nut stands out for have expressed prethrombin-2 (FII), the smallest single- being one of the unique oilseeds that does not compete chain precursor to thrombin, in Nicotiana tabacum. We have directly with the food production. However, being an designed two constructs bearing the human FII coding se- alogamous plant, the propagation of the species by seeds quence (864 bp), under the control of the CaMV35S pro- results in non-uniform crops, making itself an obstacle for moter, fused to the signal peptide 2S2 from A. thaliana to the production of this culture. Moreover, it also does not deliver the protein to the secretory pathway, the Kozak guarantee the quality of the oil. Therefore, the sequence, and with or without adding the C-terminal KDEL micropropagation represents an excellent alternative to sequence for retrieving the protein to the endoplasmic PLANT POSTERS reticulum (pFII/-A/RE). Those constructs were introduced only observed in pollen grains, starting when the flower into the plant expression binary vectors pK7WG2 and opened and initiating the senescence process; at this point, p35SGAT (pK7WG2:pFII-A/RE and p35GAT:pFII-A/RE). desiccated mature pollen grains are released from anthers. In Then, they were introduced by electroporation into addition, dehydrated developing pollen grains also Agrobaterium tumefaciens that was used to agroinfiltrate expressed the gus gene. This is the first study on a castor N. tabacum young plants. To analyze if post- bean DREB gene and presenting pollen-specific expression. transcriptional gene silencing (PTGS) could interfere with Acknowledgements: EMBRAPA, CNPq and FUNARBE. FII expression a co-agroinfiltration in the presence of Turnip crinkle virus capside protein (CP-TCV) was performed. The P-2021 Western blot analysis performed in plant extracts confirmed the protein integrity, showing a specific band of approxi- Optimization of the In Vitro Propagation Method for a Seed- mately 35 kDa in the two ER versions but, surprisingly only less Cultivar of Ziziphus spinachristi. J. ASHKANANI and C. in the presence of the PTGS CP-TCV suppresor in the Sudhersan. Environment and Life Science Crnter, Kuwait apoplastic versions. Our preliminary assessment is that the Institute for Scientific Research, KUWAIT. Email: recombinant pre-FII here expressed is in the proper molec- [email protected] ular weight range and structure needed for coagulation function. A seedless cultivar of Ziziphus spinachristi was identified among the seedling populations grown in Kuwait. Ziziphus P-2020 fruits used to have 1–2 seeds enclosed by a hardy stone. This particular cultivar produced seeds but hardy stone formation Characterization of a Pollen-specific and Desiccation- around the seeds was arrested and finally the seeds aborted associated AP2/ERF Type Transcription Factor Gene from before the fruit ripening stage. This character was observed for Castor Bean (Ricinus communis L.). F. J. L. ARAGÃO1,T. 3 years and confirmed about the seedless character. The fruits M. Cipriano1,2,andA.T.Morais1,2. 1Embrapa Recursos were tasty and more palatable than the normal stony fruits of Genéticos e Biotecnologia, PqEB W5 Norte, 70770–900 other Ziziphus species. Clonal propagation of this seedless Brasília, DF, BRAZIL and 2Universidade de Brasília, cultivar in large numbers for its palatable tasty fruits required Departamento de Botânica, Campus Universitário Darcy micropropagation. Therefore, this study was undertaken. The Ribeiro, 70910–900 Brasília, DF, BRAZIL. Email: objective of the present study was to develop a suitable [email protected] method of clonal plantlet regeneration under in vitro condi- tion. Shoot buds were collected from the seedless cultivar DREB transcription factors (TF) belong to the superfamily maintained at the KISR campus and surface sterilized using of AP2/ERF and their involvement in protein-protein in- 50% Chlorox® with a drop of soap solution for 15 min. Leaf teractions and DNA binding has been proposed. AP2/ERF disc of 5 mm2 with central vein, stem nodal segments and proteins have important functions in the transcriptional reg- shoot tip explants isolated under the laminar hood were used ulation of a variety of biological processes related to growth for the regeneration experiments. MS culture media with and development, as well as various responses to environ- different plant growth regulators (PGRs) BA and 2,4-D alone mental stimuli, regulating expression of plant biotic and or in combination were used for the experimental study. abiotic stress-responsive genes. In this study an AP2/ERF Cultures were maintained under 16 h photoperiod of 1000 TF gene was isolated from castor bean (Ricinus communis lux light intensity at 25±2˚ C. All cultures were subcultured L.) and its expression was characterized under abiotic stress. once per week 4 times initially to control the browning and The RcDREB1 deduced amino acid sequence was com- later on explants were subcultured once in 20 days. PGR- pared to other AP2/ERF TF proteins and presented 38– free MS medium was used as experimental control. Three 78% identity. Phylogenetic analysis classified it as a mem- types of regenerations: 1. adventitious shoot regeneration ber of the CBF/DREB subfamily, rooting with the subgroup from stem nodal segments, 2. axillary shoot multiplication A-5. The RcDREB1 promoter was fused to the gus reporter from shoot tip explants and 3. somatic embryogenesis from gene and used to transform tobacco. Transgenic plants were leaf disc explants were obtained. Axillary shoot multiplica- exposed to various abiotic stress treatments (low and high tion through stem nodal cuttings from in vitro plantlets temperatures, drought, salinity and exogenous ABA) and no obtained via shoot tip elongation or adventitious shooting detectable GUS expression was observed, suggesting that or somatic embryogenesis method and in vitro rooting of the RcDREB1 promoter is not active under tested condi- shoots using NAA was standardized for large scale plant tions. In silico analyses revealed the presence of three copies production. Few plants were grown in the field and con- of the regulatory late pollen-specific element (AGAAA) in firmed the clonal nature through the similar seedless fruit the RcDREB1 5´-region. Interestingly, GUS expression was producing character of the mother plant. PLANT POSTERS

P-2022 conventional methods is difficult due to the non-persistent virus transmission by aphid vectors and the unavailability of Micropropagation of Emblica officinalis for Large-Scale resistant/tolerant varieties. An alternative for controlling viral Plant Production. L. AL-SABAH, S. Jibi Manuel, C. diseases is the development of transgenic plants expressing Sudhersan, A. Al-Ajeel, and S. Al-Melhem. Environment gene fragments derived from the pathogen genome. Plants of and Life Science Centre, Kuwait Institute for Scientific P. edulis f. flavicarpa were obtained via Agrobacterium Research, KUWAIT. Email: [email protected] tumefaciens with two different gene constructs, both with frag- ments of the CABMV coat protein (CP) gene and the nptII as a Emblica officinalis locally known as amla is a multipurpose selection gene. The pCABMV-asCP contains a 610 bp frag- tree species. The fruits of this species are traditionally used in ment of the CP gene in an antisense orientation and the herbal preparations for curing several diseases and believed to pCABMV-dsCP contains a 208 bp fragment of the CP gene increase the defense against many diseases. It is particularly in a hairpin design. The transgenic plants were identified by used for the treatment of cancer, diabetes, liver disorders, heart PCR with primers for detection of the nptII gene and the CP diseases, ulcer, hemorrhage, diarrhea, dysentery, anemia and gene fragment. For the pCABMV-asCP and pCABMV-dsCP ophthalmic disorders. Due to its medicinal value and tolerance constructions respectively, 15 and 13 transgenic lines were to arid climatic conditions, this species was recently introduced identified. These plants were propagated and inoculated me- to Kuwait for fruit production and greenery purpose. Few chanically with the CABMV-SP isolate. The plants were ana- plants were highly acclimatized to the local harsh climatic lyzed by ELISA for virus detection. The virus replication was conditions and showed vigorous vegetative growth. In order not detected in 8 lines containing the pCABMV-asCP and to multiply these well-acclimatized plants in large numbers for 8 lines containing pCABMV-dsCP gene constructs. Some lines the greenery purpose and fruit production in Kuwait, showed partial resistance, i.e., only in some propagated clones micropropagation study was undertaken in our laboratory. Pre- the virus replication was not detected. The plants will be re- vious published reports on micropropagation of this species did inoculated by viruliferous aphids (Myzus persicae) and virus not help much for the large-scale plant production. Therefore, titer will be quantified by RT-qPCR. Experiments were conducted to develop and standardize micropropagation technique for large-scale plantlet production P-2024 using different tissue explants and MS basal medium with different additives. Both somatic embryogenesis and adventi- Amaranthus hypochondriacus In Vitro Culture for the Pro- tious shoot proliferation methods for large-scale plant produc- duction of Bioactive Sustances. B. GUADARRAMA1,M. tion was optimized. Technique for the control of severe tissue Rodriguez2, C. Pérez3,J.Vernon1, and F. Cruz1. browning during culture initiation using explants from field 1Universidad Autónoma Metropolitana-Iztapalapa, Av. San grown trees was standardized. Rooting was achieved using half Rafael Atlixco 186, Col. Vicentina, México D.F. 09340, strength MS with 20 g sucrose and 3 mg/l IBA. Rooted plant- MEXICO; 2Centro de Desarrollo de Productos Bióticos, lets were hardened with 100% success using in vitro photoau- Instituto Politécnico Nacional, Apdo. Postal 24, Yautepec, totrophic culture phase prior to the greenhouse hardening. Morelos, 62731, MEXICO; and 3Universidad Autónoma Thousands of plants were produced and acclimatized. Few del Estado de México, Paseo Colón esq. Paseo Tollocan plants were planted in the field for evaluation. S/N, Col. Residencial Colón, Toluca, México. 50120. MEX- ICO. Email: [email protected] P-2023 It is necessary to look for alternative sources of natural dyes Progress Towards Genetic Transformation of Passiflora due to rising environmental costs and adverse health effects of edulis f. flavicarpa for Resistance to Cowpea-aphid Borne colorful synthetic additives applied in foods, beverages, phar- Mosaic Virus (CABMV). M. F. CORREA1,A.C.B.A. maceuticals, and cosmetics. Amaranthus hypochondriacus is Monteiro-Hara1,A.P.O.AmaralMello2,R.Harakava3,J. a specie endemic of Mexico and may be a viable option to A. M. Rezende2, and B. M. J. Mendes1. 1University of São obtain betalains: betaxanthins (yellow) and betacyanins (red). Paulo, CENA, Av. Centenário, 303, Piracicaba, SP, 13416– These water-soluble nitrogen compounds have antioxidant 903 BRAZIL; 2University of São Paulo, ESALQ, Piracicaba, and anticarcinogenic properties. Betalins content in plant is SP, BRAZIL; and 3Instituto Biológico, São Paulo, SP, BRA- less to 1%, in addition to the agronomic management of the ZIL. Email: [email protected] crop of amaranth is poor and only intended for the production of grain, but not to obtain natural dyes. The objective of this Brazil is the largest producer of passionfruit and the passionfruit work was to obtain betalain production in plant cell culture of woodiness disease (PWD), caused by CABMV, is the most A. hypochondriacus in vitro (callus and suspension cells) and important disease of the culture. The control of the disease by made a partial physicochemical characterization. Seeds of the PLANT POSTERS plant (INIFAP-2011) were sterilized under aseptic conditions Pistacia lentiscus L. is an economically important member and planted on MS medium 100%, leaves of seedling (3 to of Pistacia genus which has valuable secondary metabolites. 5 cm) were used as explants placed on MS medium to 50%. Among them, mastic resin (gum) is harvested as spice from 2,4-D (0.0 - 3.0 mg / L) and BAP (0.0 - 2.0 mg / L) were the cultivated trees grown especially in Cesme, Turkey and essayed as phytohormons. Treatment with 2,4-D (1.5 mg / L) Chios Island, Greece. Mastic gum from lentisk (mastic tree) and BAP (1.5 mg / L) was selected to establish the cell line to is evaulated for several applications such as perfumery, have a high percentage of friable callus induction (96%) and lacquers, varnishes and alcolic beverages. However, as yellowing after 30 days culture. Cell cultures were established lentisk germplasm started to face genetic erosion in vitro in suspension (inoculum: 5% fresh weight). Thin layer chro- storage techniques became important for ex situ conserva- matography, UV–VIS absorption spectra, color properties, tion of the species. Besides micropropagation, slow growth and auto fluorescence of cells exhibiting yellow color, it was storage technique is also used and preferred for medium- determined that the cell line amaranth produces betaxanthins. term conservation of plant germplasm due to the reduction Suspension cultures of A. hypochondriacus are spreading to of periodic subculture requirement by decreasing nutrient evaluate betaxanthins production in bioreactor. and/or light intensity and/or culturing in dark. Thus, slow growth storage technique is used in this study for optimiza- P-2025 tion of medium-term conservation of lentisk. To obtain optimal storage period, P. lentiscus shoots were transferred Optimization of Grapevine Transient Transformation Via to standard proliferation medium (MS medium containing Biolistic Bombardment of Somatic Embryos. K.-H. KIM, 1 mg/L BA) and maintained at 4°C in the dark for 2, 4, Z. T. Li, J. R. Jasinski, and D. J. Gray. Mid-Florida Research 6 month. After appropriate storage period shoot apices were & Education Center, University of Florida/IFAS, 2725 excised from conserved microshoots and transferred to stan- Binion Road, Apopka, FL 32703–8504. Email: dard proliferation medium and standard culture conditions [email protected], [email protected] (25°C under a 16 h photoperiod of 36 μmol m−2 s−1 photo- synthetic photon flux provided by cool-white fluorescent Somatic embryos (SE) of Vitis vinifera ‘Thompson Seedless’ lamps). Highest proliferation rate (100%) was determined were pre-cultured on DM medium for 1, 3, and 5 days in the after 6 month of storage. Moreover, genetic stability of the dark before bombardment. The embryos were bombarded with 6 month-stored plantlets was assessed by amplified frag- 0.6 μmand1μm gold particles coated with pE2113LGKH ment length polymorphism (AFLP) analysis and high levels plasmid DNA containing an enhanced green fluorescence pro- of genetic stability between the stored planlets and donor tein (EGFP)/ neomycin phosphotransferase II (NPTII) fusion plants were obtained after they were stored 6 month. Thus, gene. The embryo pre-culture conditioning affected the frequen- optimized slow growth protocol could be applied to not only cy of GFP expression after biolistic transformation. Bombarded other Pistacia species but also other woody plants to safe- embryos derived from pre-culture for 1 and 3 days yielded guard their germplasm. higher rates transient GFP expression than 5 days. Gold particles in 1 μm diameter resulted in more GFP expression than in P-2027 0.6 μm diameter. The distance between the particle launch point and target cells significantly affected the frequency of GFP Cryopreservation of Pistacia lentiscus L. (Mastic Tree). I. expression. The highest number of GFP spots per explant was KOC1, Y. Çiftçi Özden1, and A. Onay2. 1Gebze Institute of achieved with a distance of 6 cm as compared to 3, 9, and 12 cm. Technology, Department of Molecular Biology and Genet- These results are useful for optimizing transformation efficiency ics, 41400, Kocaeli, TURKEY and 2Dicle University, De- using biolistic bombardment and SE as explants. partment of Biology, 21280, Diyarbakir, TURKEY. Email: [email protected], [email protected] P-2026 Pistacia lentiscus L. is an economically important member Determination of Genetic Stability of Medium-term Stored of Pistacia genus together with pistachio (Pistacia vera L.) Lentisk Plantlets by AFLP Markers. I. KOC1, H. Akdemir1, and widespread throughout the Mediterranean region. It A. Onay2, A. Altinkut Uncuoglu3, and Y. Çiftçi Özden1. produces a valuable resin called mastic which is used in 1Gebze Institute of Technology, Department of Molecular toothpaste, gum, perfume and medicine industry. As genetic Biology and Genetics, 41400, Kocaeli, TURKEY; 2Dicle erosion of Pistacia lentiscus has been occurring due to the University, Department of Biology, 21280, Diyarbakir, problems faced in its generative and vegetative propagation TURKEY; and 3 Marmara University, Department of and rapid urbanization, in vitro conservation strategies are Bioengineering, 34722, Istanbul, TURKEY. Email: promising. Besides in vitro propagation and conservation [email protected], [email protected] techniques, germplasm can be stored with the application of PLANT POSTERS cryopreservation techniques. Thus, in this study droplet vitri- floral development. To efficiently study this system, an fication and one-step freezing technique was assessed for long optimised protocol for the successful extraction of proteins term conservation of mastic tree. Stock plants were cold from florets is crucial and will be discussed here. hardened for 2 weeks in dark at 4°C. Explants excised from cold hardened plants were then pre-cultured in Murashige and P-2029 Skoog’s (MS) medium containing 0.5 M sucrose prior to LN immersion. Among the methods assessed, 17.6% of regener- Establishment of Cell Suspension Culture of Bursera linanoe ation was achieved with incubation of basal shoots in the (La Llave) for the Production of Secondary Metabolites. L. droplets of PVS2 (Plant vitrification solution) for 45 min at PAVÓN1,S.Evangelista1,G.Sepúlveda1,V.Chávez2,andM. 0°C followed by direct immersion in liquid nitrogen. After Rodríguez1. 1Centro de Desarrollo de Productos Bióticos, that, basal buds were rapidly thawed and cultured in MS Instituto Politécnico Nacional, Apdo. Postal 24, Yautepec, medium containing 1 mg/L BA. Morelos, 62731, MEXICO and 2Laboratorio de Cultivo de Tejidos Vegetales, Jardín Botánico del Instituto de Biología, P-2028 Universidad Nacional Autónoma de México (UNAM), Apdo. Postal. 70–614, CP 04510 Distrito Federal, MEXICO. Email: The Mantled Phenotype in Oil Palm and an Optimised [email protected] Protein Extraction Protocol from Inflorescence Tissues of Oil Palm to Study This. H. Y. LOONG1, C. N. Choo2,C.K. Bursera linanoe is a tree used as a renewable natural source Wong3, and W. C. Wong1. 1Advanced Agriecological Re- to obtain essential oils, mainly the monoterpenes linalool search Sdn. Bhd., Biotechnology Section, Crop Improve- and linalyl acetate are the compounds responsible for the ment, AAR-UNMC Biotechnology Research Centre, Jalan characteristic aroma of this specie. Due to the high demand Broga, 43500 Semenyih, Selangor, MALAYSIA; 2Ad- of the use of these monoterpenes in cosmetic and pharma- vanced Agriecological Research Sdn. Bhd., Tissue Culture ceutical industries, there is overexploitation of the species. Section, Crop Improvement, Tissue Culture Laboratory c/o Alternatively, essential oils of B. linanoe could be produced Ladang Tuan Mee, KM29, Jalan Sungai Buloh/Kuala Se- by cell culture; but, there is no information on the in vitro langor, 47000 Sungai Buloh, Selangor, MALAYSIA; and establishment of Bursera cultures. The objective of this 3Advanced Agriecological Research Sdn. Bhd., Plant study was to establish a cell line in suspension of Bursera Breeding Section, Crop Improvement, Locked Bag 111, linanoe and characterize its growth in Erlenmeyer flask. Paloh Post Office, 86009 Paloh, Johor, MALAYSIA. Email: Leaf, internode and axillary bud were used as explants to [email protected] generate callus. The explants were placed on Murashige– Skoog medium with two different plant growth regulators: Reproductive development is a crucial phase in a plant’slife Naphthaleneacetic acid (NAA) concentrations of 0, 0.5, 1.0, cycle. While much work has been done to elucidate this 2.0 yr 3.0 mg L-1 and 6-Benzylaminopurine (BAP) concen- process in model plants, much more needs to be done to trations of 0, 0.1, 0.5 yr 1.0 mg L-1. Explants were cultivated translate this knowledge and reach the same level of under- at 25°C, under complete dark conditions. The treatment of standing in non-model crops like oil palm. In oil palm, one of NAA (3.0 mg L-1)andBAP(0.5mgL-1) using axillary bud the major impediments to large-scale clonal production is the and internode were the most efficient to induce callus (85 and occurrence of a somaclonal variant known as ‘mantling’.This 95%, respectively). It was not response in callus formation variant is actually an upset in individual flower sex determi- using the leaves as explant. Cell suspension culture was nation. In the female flower, the stamen primordia undergo a initiated by transferring 3–5 g fresh weight of friable callus homeotic transformation into supplementary carpels. This in Erlenmeyer flask (250 mL), containing 50 mL of the same leads to an aborted oil palm fruit bunch and is an industrial medium used for callus induction. The culture was incubated concern as it only manifests in the field a few years after the on a gyratory shaker, at 90 rpm and 25°C, under condi- plantlets have left the laboratory and is a waste of resources. tions of 16 h light /8 h dark. Cell growth reached a Solving this problem requires a deep understanding of the maximum biomass production of 10.34 g DW L-1 with a biology and processes involved in its formation. To achieve μ of 0.10 days-1. During the growth kinetic, the viability this, the phenotypic data of the fruits and inflorescence in was of 60% and pH had values between 4 and 5. Essays mantled palms was characterised to identify the developmen- of thin layer chromatography indicated that cell suspension tal stage where these changes are occurring. Based on the culture accumulated linalool and linalyl acetate. In conclu- phenotypic data obtained, the mantled phenotype of supernu- sion, the cell suspension culture of B. linanoe was merary carpellate tissue could be observed in florets very early established and obtained the growth parameters. These cell during inflorescence development, showing that cells become suspensions can be used to culture in bioreactor and determined in this developmental pathway at this stage of obtaining the essential oil of B. linanoe. PLANT POSTERS

P-2030 Wheat streak mosaic virus (WSMV) and is one of three viruses of the wheat mosaic complex affecting wheat in Production of Anti-inflammatory Metabolites by Transformed the Great Plains of the United States. The current Hairy Root Cultures of Lopezia racemosa Cav. IRENE disease management strategy incorporates the deploy- PEREA-ARANGO1, Norma Moreno-Anzúrez1,Jesus ment of resistant varieties, mite vector control and Arellano1, Laura Alvarez-Berber2, and Patricia Castillo- various cultural practices, however, it is not fully ef- España2. 1Centro de Investigación en Biotecnología, fective. As an alternative strategy, we evaluated the use Universidad Autónoma del Estado de Morelos, Avenida of interference RNA to generate resistance to these Universidad 1001 Colonia, Chamilpa CP 2009 Cuernavaca, wheat viruses. RNAi expression vector was created Morelos, MEXICO and 2Centro de Investigaciones Químicas, from the sequence of the 6 K2-NIa protein of WSMV Universidad Autónoma del Estado de Morelos, Avenida and immature embryos of the wheat cultivar ‘Bobwhite’ Universidad 1001, Colonia, Chamilpa CP 2009 Cuernavaca, were ico-transformed by biolistic particle delivery sys- Morelos. MEXICO. Email: [email protected] tem with the RNAi expression vector and pAHC20, which contains the bar gene for glufosinate selection. Plants remain as an alternative source of new active ingre- After tissue culture and plant recovery, putative dients to cope with symptoms of many diseases. Chronic transformed plants were analyzed through PCR for the inflammation has been associated with benign and malig- presence of the appropriate RNAi 6 K2-NIa gene. nant neoplasms. Among compounds derived from plants Transgenic T1 seeds were collected and each line was currently used as anticancer drugs we have taxol, docetaxel, tested for transgene expression via RT-PCR. To deter- and camptothecin. However, these substances are chemical- mine viral resistance, T1 progeny were mechanically ly complex and despite advances in synthetic chemistry, its inoculated with the corresponding virus. Viral presence obtaining still depends from biological sources. Lopezia was established by ELISA. In the T1 generation, resis- racemosa Cav, commonly known as "stroke herb" or "can- tance was seen in up to 80% of the plants evaluated for cer herb" is a plant used in Mexican traditional medicine to the 6 K2-NIa construct. These plants have undergone heal inflammatory diseases. This plant produces the com- single plant selection up to T4 and continue to show pound β–sitosterol–6–O–palmitoyl–3–Oglycoside (LR1) high level of resistance when challenged with the virus. which had been isolated in low yield from the dichloromethane extract of whole wild plants of L. P-2032 racemosa. LR1 presents cytotoxic activity in cell lines of breast, prostate, T–cell leukemia and Burkitt lymphoma. The In Vitro Manipulations of Wheat for Genotype-independent aim of our work is the establishment of transformed hairy Genetic Engineering Via Agrobacterium tumefaciens.M.M. root cultures of L. racemosa to produce LR1 and/or other SHIRKE,S.Rohila,K.Buehner,M.EldakakandJ.S. metabolites with anti-inflammatory activity at highest or Rohila. Department of Biology and Microbiology, South similar concentration than wild plants. Using the Dakota State University, Brookings, SD 57007. Email: Agrobacterium rhizogens strain ATTC 15834 carrying a [email protected], [email protected] binary vector containing the reporter gen of a red fluorescent protein, we carried out transformation experiments. Infected Wheat is a commercially important crop of the USA leaves of L. racemosa produced hairy roots 16 days after and is consumed worldwide. Climatic changes, dis- infection and were expressing the reporter gen as we ob- eases, and pests cause fluctuations in its yield from served by epi-fluorescence. These roots were selected by its year to year. In order to sustain with the increasing rate of growth and fluorescence. Results on the production of demand for wheat and to avert the risk for grain anti-inflammatory metabolites are presented. shortages in near future, FAO has projected to double the crop productions by the year 2040. In 20th century, P-2031 the conventional wheat breeding has played crucial role in green revolution, but in 21st century the use RNAi Mediated Viral Resistance in Transgenic Wheat. of in vitro tools including biotechnology has become JESSICA L. RUPP1, Luisa Cruz1,2, John P. Fellers3, available to assist modern wheat breeding programs. and Harold N. Trick1. 1Department of Plant Pathology, While there are successful stories of using transgenics Kansas State University, Manhattan, KS 66506; 2Department in many commercial crops such as rice, maize, barley, of Entomology and Plant Pathology Auburn University, AL sorghum, soybean, cotton and canola, the use of ge- 36849; and 3USDA-ARS Hard Winter Wheat Genetics netic engineering in wheat improvement is negligible. Research Unit, Manhattan, KS 66506. Email: The genetic engineering approach could bring a speed [email protected] to the wheat breeding programs by transferring the PLANT POSTERS desired genetic traits into the wheat genome at a faster PCR analyses revealed that ectopic expression of the pace and with no boundaries. Our current study 35S-VaFT caused upregulation of two tobacco floral focusses on exploiting the in vitro manipulations of activator genes, APETALA1 (AP1)-like (AF385746) and wheat for the use of genetic engineering in future. LEAFY (LFY)-like (U16174). Expression of the 35S- For this we show that a successful integration and VaFT in blueberry cultivar Aurora resulted in an ex- expression a foreign gene (GUS) in the wheat genome tremely early flowering phenotype, which flowered dur- is achievable. Biolistics and Agrobacterium-mediated ing in-vitro-culture, a growth stage when nontransgenic genetic transformation (AMGT) are the two most prom- shoots had not yet flowered. These results demonstrate ising tools to achieve these goals but AMGT has some advan- that the VaFT is a functional floral activator with po- tages over the biolistics (such as a low copy number); thus tential for shortening juvenility and reducing chilling AMGT was a method of choice for us. In this study, we have requirements in blueberry and other woody plants. developed a robust protocol for in vitro manipulations that is found suitable with 20 genotypes tested. During the study P-2034 these 20 different wheat cultivars along with four different A. tumefaciens strains were evaluated. pCambia1305.2 carrying Setaria viridis Transformation Provides a Useful Tool for GUSplus as a reporter gene and Hygromycin as plant selection Maize Functional Genomics. J. VAN ECK1,K.Swartwood1, marker was used for wheat transformations. Further, the pro- C. Azodi1, E. Vollbrecht2, E. Unger-Wallace2,J.Strable2,D. tocol was optimized using three cultivars of spring and winter Stern1, L. Feiz1, and T. Brutnell3. 1The Boyce Thompson wheat each. Stable integration and expression of GUS gene in Institute, 1 Tower Road, Ithaca, NY 14853; 2Department of transgenic plants was confirmed by PCR analysis. Transfor- Genetics, Development, and Cell Biology, Iowa State Univer- mation efficiency of wheat embryogenic calli derived from sity, Ames, IA 50011; and 3Donald Danforth Plant Science immature embryos was recorded to be 0.5%. Center, 975 North Warson Road, St. Louis, MO 63132. Email: [email protected] P-2033 Setaria viridis (green millet), a relative of the cultivated The Vaccinium corymbosum FLOWERING LOCUS T-like Setaria italica, has the potential to serve as a model Gene (VaFT) is an Effective Flowering Activator for Short- species for C4 photosynthesis studies. Resources in- ening Juvenility and Reducing Chilling Requirements in cluding genome sequence, RNA-seq data, and transfor- Blueberry. GUO-QING SONG, Aaron Walworth, Dongyan mation are being developed to advance S. viridis as a Zhao, Ning Jiang, and James F. Hancock. Plant Biotechnol- model. Advantages of using S. viridis include its small ogy Resource and Outreach Center, Department of Horti- size, a seed-to-seed generation time of 6 – 8weeks, culture, Michigan State University, East Lansing, MI 48824. and its simple growth requirements. An Agrobacterium Email: [email protected] tumefaciens transformation method has been developed that utilizes seed-derived callus. Mature seeds from Blueberry is a woody perennial bush with a longer juvenile which the seed coats have been removed are cultured period than many annual crops and requiring vernalization on an MS-based callus induction medium (CIM) that to flower normally. Few studies have been reported on the contains 2,4-D and kinetin. Following infection of molecular mechanism of flowering in blueberry or other 6-week-old callus, there is a cocultivation period of woody plants. Because FLOWERING LOCUS T (FT) from 3 days after which the callus is transferred to selective Arabidopsis thaliana plays a multifaceted role in generating CIM. The vectors we use contain the hygromycin mobile molecular signals to regulate plant flowering time, phosphotransferase II gene as the plant selectable isolation and functional analysis of the blueberry FT-like marker and hygromycin is used as the selection agent. gene (VaFT) will facilitate the elucidation of molecular The current transformation efficiency ranges from 5 – mechanisms of flowering in woody plants. Based on 10%, however, efforts are underway to improve the EST sequences, a 525-bpVaFT was identified and cloned efficiency to provide a more high-throughput applica- from the cDNA library of a tetraploid, northern high- tion. Overexpression of maize ramosa1 (ra1), which is bush blueberry (Vaccinium corymbosum L.) cultivar, hypothesized to be associated with inflorescence diver- Bluecrop. Ectopic expression of 35S-VaFT in tobacco sity in the grasses, resulted in altered inflorescence induced flowering an average of 28 days earlier than phenotypes (spikelet and bristle density, and bristle wild-type plants. Real-time reverse-transcription (RT)- length) in S. viridis transgenic lines. Gene-knockout PLANT POSTERS

(RNAi-mediated) experiments with a putative maize Rubisco used to determine the copy number of the marker gene, assembly factor gene resulted in S. viridis transgenic lines neomycin phosphotransferase (nptII) in each independent with the expected phenotype of pale green leaves and reduced transgenic line. To improve the rooting efficiency of trans- overall plant growth. Based on the results from these early genic black cherry shoots, half-strength Murashige and experiments, it appears that S. viridis holds promise as a model Skoog medium supplemented with 2% (v/v) sucrose, for studies on the function of maize genes. 5.0 μM napthaleneacetic acid, 0.1 μMkinetin,and 0.793 mM phloroglucinol was tested. P-2035 P-2036 Improvement of Agrobacterium-mediated Transformation of Mature Black Cherry (Prunus serotina Ehrh.) and Induced Mutations for Enhancing Variability of Giant Rooting of Transgenic Shoots. Y. WANG1 and P. M. Pijut2. Miscanthus (Miscanthus x giganteus). DINUM PERERA1, 1Purdue University, Dept. of Forestry and Natural Re- Brian S. Baldwin1, and Nancy A. Reichert2. 1Dept. of Plant sources, Hardwood Tree Improvement and Regeneration & Soil Sciences, Mississippi State University, Mississippi Center (HTIRC), 715 West State St., West Lafayette, IN State, MS 39762 and 2Dept. of Biological Sciences, Missis- 47907 and 2USDA Forest Service, Northern Research Sta- sippi State University, Mississippi State, MS 39762. Email: tion, HTIRC, 715 West State St., West Lafayette, IN 47907. [email protected] Email: [email protected], [email protected] Giant miscanthus (Miscanthus x giganteus; Mxg) is a key An efficient Agrobacterium-mediated transformation system candidate energy crop for use in biomass to liquid fuel for an elite mature genotype of black cherry was developed production. Mxg is a natural sterile triploid lacking genetic basedonanexistingprotocoldevelopedinourlab.To variation, the basis for selection. Genetically uniform mono- increase transformation efficiency, vacuum infiltration, son- cultures of Mxg are inherently vulnerable to pathogenic ication, and the combination of the two treatments were epidemics. Thus, induced mutation techniques are particu- applied during the co-cultivation of leaf explants and larly important in Mxg. The aim of this study was to induce Agrobacterium tumefaciens strain EHA105 harboring a variation in Mxg through in vitro chemical mutagenesis. A PsAGAMOUS (PsAG) RNAi plasmid. The effects of differ- previously developed in vitro propagation protocol for Mxg ent concentrations of Agrobacterium suspension including was utilized during the in vitro mutagenesis procedure.

OD600 of 0.5, 1.0, 1.5, and 1.8, as well as co-cultivation Mutagenic dosage was optimized using various concentra- duration of 3 days or 4 days on transformation efficiency tions (0.01%, 0.1%, 0.5%, 1%, and 3%) of ethyl were examined using the same strain harboring pBI121- methanesulfonate (EMS) and time periods (45 and 90 min) mdl4 and pBI121-PsTFL1, respectively. Among all the with 2% dimethyl sulfoxide (DMSO) incorporated into the treatments, 15 min vacuum infiltration was found to be mutagen solution. Mxg immature inflorescence explants optimal and produced the highest transformation efficiency (1–2 mm), embryogenic and shoot-forming calli (1– (21.7%) using the RNAi plasmid pART27 as the binary 2 mm3) were treated with ten mutagenic dose treatments vector containing a PsAGRNAi cassette. The OD600 of 1.0 in order to determine the optimum mutagen dosage. and 1.5 resulted in higher transformation efficiency (5.0%) Depending on the EMS concentration used, the mean than OD600 of 0.5 and 1.8 when the construct pBI121-mdl4 survival rate of callus cultures decreased from 85% was used for transformation. Transformation efficiency of (0.1% EMS) to 32% (3% EMS); whereas with inflores- 5.0% was also obtained from the 3 days co-cultivation using cence explants, the mean survival rate decreased from construct pBI121-PsTFL1, whereas no shoots were able to 80% (0.1% EMS) to 29% (3% EMS) after treated with regenerate after the 4 days co-cultivation. The results indi- 3% EMS for 90 min. The dose at which 50% of the cated that the binary vectors had an impact on transforma- callus/explants died (LD50) was used as the optimum tion efficiency when the exact same conditions of EMS dose. Mxg calli and inflorescence explants were transformation were applied. Polymerase chain reaction treated with LD50, 2 × LD50, and 3 × LD50 EMS (PCR) analysis of individual putative transgenic shoots dosages and the regenerants were transferred to soil. Inter was conducted to verify the integration of transgenes and simple sequence repeat (ISSR) marker has been utilized transformation efficiency was calculated as the number of to identify variants in the regenerated plants. Putative callus that formed transgenic shoots relative to the initial mutant population will be screened and evaluated in the number of leaf explants per treatment. QPCR analysis was field during 2013 for future selection. PLANT POSTERS

P-2037 Al. The parameters analyzed were fresh and dry weight of roots and shoots of plants after 25 days in the greenhouse for Gene Expression Differences in Wheat Induced by Six rooting. The results show the superiority development of Puccinia triticina Races. K. NEUGEBAUER1, M. Bruce2, clone A, and it is evident that the best treatment is the one J. Glasscock3, and J. Fellers2. 1Kansas State University, which was added 1.0 mg.L-1 of aluminum in the culture Department of Plant Pathology, 4024 Throckmorton Hall, medium, because this treatment was the best result in bio- Manhattan, KS 66506; 2USDA-ARS Hard Winter Wheat mass gain shown by parameter dry weight, furthermore, was Genetics Research Unit, 4008 Throckmorton Hall, Manhat- observed in this dose a better rooting during in vitro prop- tan, KS 66506; and 3Cofactor Genomics, 3141 Olive St., St. agation, thus better development of the part area, concluding Louis, MO 63103. Email: [email protected] that the aluminum in low concentration in the culture medi- um has a beneficial effect on plant ex vitro rooting. Puccinia triticina, the casual agent of wheat leaf rust, is a devastating disease that can cause up to 20% yield loss. P-2039 During fungal infection the host plant recognizes proteins, secreted effectors, and other molecules, which trigger a host Protocol for Somatic Embryogenesis from Ovaries of defense response. Changes in the pathogen effectors and Seedless Sweet Orange Cultivar. A. P. MARTINELLI 1;M. strong varietal selection pressure are responsible for the rapid Curtolo 2;R.R.Latado2 and J. C. Cardoso1,3. 1University of development of new rust races. The question arises whether São Paulo, CENA, Av. Centenario, 303, Piracicaba; 2Centro different leaf rust races utilize different pathways in wheat. Six APTA Sylvio Moreira, IAC, C.P. 04, Cordeirópolis, SP; and races of leaf rust were evaluated on a single variety of wheat in 3UFSCAR, Centro Ciências Agrárias, Araras, SP, BRAZIL. a time course study and the RNA was sequenced. Thirty-four Email: [email protected] [email protected] wheat genes with expression variation induced by different races were initially identified. Sixteen of those genes are being The use of adult tissues, instead of seeds, can provide new characterized for specific timing of expression and whether applications and tools in transformation protocols for seedless their expression is necessary for the rust to infect, (i.e. are they citrus breeding. Navel oranges, seedless cultivar(s), can benefit susceptibility genes?). from these studies. The aim of this work was to obtain a somatic embryogenesis protocol and plant regeneration from ovary P-2038 tissues of the sweet orange Washington Navel. Isolated ½ pistils were used as explants, obtained from bud flowers in pre- Effect of Aluminum on Ex Vitro Rooting of Different anthesis. The explants were grown in Wood Plant Medium Clones of Tectona grandis Lf Propagated In Vitro. R. S. (WPM) for 40 d and ovary-derived-callus were transferred to BARBOSA and A. N. Gonçalves. Pádua Dias Av., No 11, N6 medium. Both media were supplemented as described by Dept. of Forest Science, University of São Paulo – Luiz de Cardoso et al. 2012 (Plant Cell Tiss Organ Cult 109:171–177). Queiroz College of Agriculture,13418900, BRAZIL. Email: Callus from ovaries were subsequently transferred to MS me- [email protected] dium for somatic embryo growth, germination and plant devel- opment. Fifty shoots from germinated embryos were Tectona grandis L.f. is popularly known as teak, being a micrografted into citrange ‘Carrizo’ and cultivated for 45 d in tropical tree species, native from India, Laos and Thailand, liquid MS medium. Ploidy was assessed by flow cytometry, with high market value due to its characteristics of fine using 25 plants, and the origin of the embryos was confirmed wood and sturdy. In general, the forest activity is aimed at by TRAPs (Targeted Region Amplified Polymorphism) sandy soils with low fertility, having many levels of ele- markers. Part of the explants previously established did not ments considered toxic to plants. Among these elements, develop callus (27.5%), 7.5% presented swollen ovary tissues aluminum stands, collaborating with decreasing soil pH and and 65% showed callus formation in the WPM medium. The impairing the availability of some key elements in the min- first multiplication of callus in N6 medium resulted in eral nutrition of plants. Even in small amounts Al3+ can 0.21 g/callus and a multiplication factor of 1.55x. Embryogenic inhibit root growth in a few minutes or hours, in various callus were obtained only in 10% of ovary-derived-callus in N6 plant species. Therefore, this study aimed to evaluate the medium. In average 47 embryos (85.1%) were obtained per effect of aluminum on ex vitro rooting of shoots obtained callus, resulting in 40 plants. Around 90% of micrografted from in vitro propagation of internodal sections of clones A, plantlets were successfully acclimatized in the greenhouse. B and C of Tectona grandis from L.f Bioteca .company The cytometric analysis showed that 100% of recovered plants Ltda. The experiment was designed entirely randomized, were diploid (2n) and the molecular analysis by TRAPs with 4 doses of AlCl3 and 5 replicates per treatment. Each showed the similar profile between regenerated plants and treatment corresponded to 0, 0.1, 1.00 and 10.00 mgL-1 of donor plants. PLANT POSTERS

P-2040 An efficient plant propagation system was developed by asymbiotic germination of seeds of Stanhopea hernandezii Micropropagation and Cryopreservation of the Critically (Kunth) Schltr. Seeds of the capsules was germinated Imperiled Mespilus canescens (Stern’s medlar). VALERIE asymbiotically (90%) on half strength Murashige and C. PENCE and Kristine Lindsey. Center for Conservation Skoog’s (MS) basal medium with 3% sucrose, activated and Research of Endangered Wildlife, Cincinnati Zoo & charcoal and solidified with 0.18% Gelzan™. Multiple Botanical Garden, 3400 Vine Street, Cincinnati, OH shoot buds and protocorm-like body (PLB) were induced 45220. Email: [email protected] on full strength MS basal medium containing 3% sucrose, 0.18% Gelzan™ and different concentrations and combina- Mespilus canescens (Stern’s medlar) is a small tree in the tions of auxins [α-naphthalene acetic acid (NAA) and Rosaceae found in one population of about 25 individuals in dichlorophenoxyacetic acid (2,4-D)] and cytokinins [6- Arkansas. It was first discovered in 1990, and, although it pro- benzylaminopurine (BAP) and thiadiazuron (TDZ)], after duces flowers, it does not currently produce fruit or seeds. It has 4 weeks of culture in photoperiod, a higher response of the global ranking of G1, or critically imperiled. As a species for PLBs formation was induced by TDZ (5.0 mg l-1). PLBs which seeds are not available for traditional seed banking, it falls developed into intact plantlets about 5.7 cm in height when into the category of an “exceptional species,” or one for which on basal medium with 5.0 mg l-1 BAP or combined with alternative methods of ex situ conservation are needed. Research 0.5 mg l-1 of NAA. Healthy plantlets, transferred to plastic at CREW has been directed at developing in vitro methods to trays containing pine bark, wood charcoal and clay grains propagate the plant, as well as to provide tissue for cryopreser- (3:1:1), more than 95% of the rooted plantlets were success- vation and long-term tissue banking. Several attempts were fully acclimatized within 60 days. made, with material received from collaborators in successive years, to establish in vitro propagating cultures without success. P-2042 Successful establishment of shoot cultures was eventually achieved by culturing young, elongating buds on MS medium Total Medium Nitrogen and Ammonium to Nitrate Ratio plus 1 mg/L BAP and 0.5 mg/L IBAwith 100 mg/L Benlate and Vary for Optimal Growth of Four Pyrus Species In Vitro. 0.25% gellan gum. Cultures were then transferred to and Sugae Wada1, Randall P. Niedz2 and BARBARA M. maintained on MS medium plus 1 mg/L BAP on 0.8% agar. REED3. 1Dept of Horticulture, Oregon State University, Rooting has been achieved on WP medium with 1 and 2 mg/L Corvallis, OR 97331–7304; 2USDA-ARS U.S. Horticultural IBA. Rooted plants have been acclimatized and planted in raised Research Laboratory, 2001 South Rock Road, Ft. Pierce, FL beds at CREW, as well as returned to collaborators at the 34945–3030; and 3USDA-ARS National Clonal Germplasm Missouri Botanical Garden and the Morton Arboretum. Shoot Repository, 33447 Peoria Rd., Corvallis, OR 97333–2521. tips from propagating cultures that have been cold treated for one Email: [email protected] week have been isolated, and cryopreserved using the encapsu- lation dehydration method. Survival of 50-70% of shoot tips has Nitrogen (N) is a key nutrient in plant growth that controls been observed after exposure to LN. Some shoot tips have also metabolic and physiological development. Nitrate (NO3)and been banked for long-term storage in CREW’s CryoBioBank. ammonium (NH4) are the two major forms of N typically used The demonstration of these methods provides a protocol that in tissue culture growth media. Total N and ratio of the two could be used for other genotypes from the wild population to forms influence growth and differentiation and the results vary preserve them ex situ in long-term cryostorage as a resource for with the plant type. This study modeled the optimum NH4 and the future. (Protocol development was supported in part by a potassium (K) ratios and total NO3 required for growth of pear grant from the Institute of Museum and Library Services in shoot cultures. Pyrus communis (‘Horner 51’ and ‘OHxF 87’), collaboration with the Center for Plant Conservation.) P. cordata, P. pyrifolia ‘Sion Szu Mi’,andP. ussuriensis ‘Hang Pa Li’ cultures from the National Clonal Germplasm Repository P-2041 collections were evaluated for N requirements using a surface response methodology. Data was collected on overall plant In Vitro Seed Germination and Seedling Development of quality, shoot length, multiplication, leaf color/size, leaf Stanhopea hernandezii (Kunth) Schltr. PATRICIA spotting/necrosis and callus. The design model was significant CASTILLO-ESPAÑA, Oswaldo Enciso Diaz, Jesus at P <0.0001(α = 0.05). Significant improvement was seen in Arellano Garcia, and Irene Perea-Arango. Centro de overall quality, shoot multiplication, shoot length, and reduction Investigación en Biotecnología, Universidad Autónoma del of callus for all five genotypes. Higher total N was the critical Estado de Morelos, Avenida Universidad 1001, Colonia factor for quality improvement in the P. communis cultivars

Chamilpa CP 2009, Cuernavaca, Morelos, MEXICO. Email: while decreased NH4/NO3 ratios were best for increasing shoot [email protected] production. NH4/NO3 ratios and total N varied for the other PLANT POSTERS three species as well. Improved quality for P. cordata required Somatic embryogenesis is an in vitro induced process, by more total N and increased NH4. ‘Hang Pa Li’ and ‘Sion Szu which somatic cells divide and differentiate into an embryo Mi’ both required lower total N and less NH4 for improved through characteristic embryological stages. Since somatic em- quality and better shoot multiplication. Improved leaf color and bryos resemble zygotic embryos in many aspects, somatic reduced leaf necrosis were seen with ‘Sion Szu Mi’ and ‘Hang embryogenesis become a model for studying the morphologi- Pa Li on the optimum N ratios. cal, physiological, biochemical and molecular events occurring during embryogenesis in plants, besides the possibility of an P-2043 efficient protocol for clonal propagation in reduced area. There are reports on somatic embryogenesis of Passiflora spp, such as Minor Nutrients are Critical for the Improved Growth of P. giberti, P. cincinnata and the economically important Corylus avellana Shoot Cultures. Charles Hand1 and P. edulis, the yellow or sour passionfruit. However, somatic BARBARA M. REED2. 1Dept of Horticulture, Oregon embryogenesis has yet to be characterized in P. alata,thesweet State University, ALS 4007 Corvallis, OR 97331 and passionfruit, of commercial importance for the production of 2USDA-ARS National Clonal Germplasm Repository, fruits, as an ornamental vine, that produces large, colorful 33447 Peoria Rd., Corvallis, OR 97333–2521. Email: flowers, and for its potential for rootstock use for P. edulis. [email protected] The aim of this work was to induce somatic embryogenesis in P. alata associated with histological analysis to describe the Hazelnut (Corylus avellana) is typically difficult to embryogenic process in vitro. Zygotic embryos of P. alata were micropropagateandmanyfailtothriveonstandardgrowth excised from mature seeds, incubated on MS medium media. Our initial study of mineral nutrients in C. avellana supplemented with 72.4 μM 2,4-diclorophenoxyacetic acid showed that five hazelnut cultivars had improved growth and (2,4-D) and 4.4 μM 6-benzyladenine (BA) in darkness at 27± shoot quality with changes in mineral nutrients, including dou- 2°C. Explants with embryogenic callus were collected at regu- bling the Driver and Kuniyuki medium (DKW) minor elements. lar intervals for microscopical analysis. Calli formation oc- This study was designed to optimize the individual minor min- curred predominantly at the adaxial surfaces of cotyledons eral nutrients from DKW medium and to determine if Ni is after 12 days of culture, proliferating throughout the explant . required for optimal growth. Six factors: H3BO3,CuSO4 5H2O, after 24 days, revealing distinct cell types and globular struc- . . . MnSO4 H2O, Na2MoO4 2H2O, Zn(NO3)2 6H20, and tures. The present work contributes to an ongoing project on . NiSO4 6H2O were tested in a surface response design with 39 embryogenesis in Passiflora spp. and the following develop- treatment combinations. The treatment concentrations varied ment of the aril, which is of high economic importance. from 0.5x to 4.0x DKW concentrations; Ni, not present in (Acknowledgements: FAPESP, CNPq). DKW, ranged from 0 to 6 μM. There was wide diversity of response, but in general, higher concentrations (4x) of B and Mo P-2045 increased overall shoot quality, length and multiplication for the three C. avellana cultivars. There were many significant inter- Performance of Tissue-cultured and Seed-grown Plants of actions of minor nutrients that affected shoot growth and quality. Heirloom Tomatoes Under Greenhouse Conditions. O. High B concentrations significantly improved quality for VÁZQUEZ MARTÍNEZ1, Balch E. M. Pérez-Molphe2, ‘Jefferson’, shoot quality, length and number for ‘Dorris’,and Castañeda O. E. Medina1, Fuentes Y. M. Ochoa3,GourcyF. shoot length and number for ‘Sacajawea’.HighMoimproved Ramos1, and Gavilán M. Urrestarazu4. 1Av. Universidad #940 some responses for all cultivars. Ni interacted significantly with col. Ciudad Universitaria, Department of Plant Breeding, several other minor nutrients and affected responses of all three Universidad Autónoma de Aguascalientes, MÉXICO; 2Av. cultivars. The addition of 6 μM Ni, interacting with high con- Universidad #940 col. Ciudad Universitaria, Department of centrations of Mn, greatly improved shoot quality and length in Chemistry, Universidad Autónoma de Aguascalientes, MÉX- ‘Sacajawea’. Interactions of Ni were significant for the other ICO; 3Calzada Antonio Narro #1923 col. Buenavista, cultivars as well and altered the requirements for other nutrients, Department of Agricultural Parasitology, Universidad but did not necessarily improve the overall shoot response. Autónoma Agraria Antonio Narro, MÉXICO; and 4Carretera a Sacramento s/n. la Cañada, Department of P-2044 Plant Production, Universidad de Almería, ESPAÑA. Email: [email protected] Somatic Embryogenesis from Mature Zygotic Embryos of Passiflora alata.S.R.SILVEIRAandA.P. Heirloom varieties of tomato may offer greater culinary appre- MARTINELLI. University of São Paulo, CENA, Av. ciation and they are well suited to meet the requirements of the Centenario, 303, Piracicaba, SP, 13416–903 BRAZIL. fresh food and organic food markets. Intensive production may Email: [email protected] increase productivity trough tissue culture. Although reports are PLANT POSTERS available for propagation of tomato via nodal tissue culture, study are to develop lineages of papaya resistant to relatively few results are available on the performance of tissue- both viruses (PRSV and PSV) using the technique of cultured tomatoes in the field. The objective of this study was to gene silencing by RNA interference. For this, efficient evaluate the overall performance of tomato micropropagated somatic embryogenesis and organogenesis systems for plants under semi-hydroponic greenhouse conditions. Multipli- Sunrise and Formosa cultivars using immature zygotic cation of various heirloom and some commercial varieties have embryos, hypocotyls and cotyledons from in vitro ger- been made through a simple method when nodal segments minated seedlings were obtained. A RNAi cassete were cultivated on MS medium (Single-nodal method). Seeds containing the conserved region of Papaya ringspot of 11 different Heirloom varieties were sown at the same time virus and meleira virus driven by CaMV35S promoter as the tissue-cultured plants were transplanted. Tissue-cultured was constructed. Agrobacterium tumefaciens- (strains plants were transplanted and rooted directly in the pot mix LBA4404 or EHA105) and biolistics-mediated transfor- substrate. Plantlets were acclimatized for less than 30 days, mation protocols are under development and have transplanted and grown on coco-peat slabs and cultured in a shown to be very successful in transforming the gus semi-hydroponic greenhouse. All plants were fertilized with a gene into papaya. After transformation the explants are nutrient solution at four reproductive stages. Unpaired t-tests being screened for nptII or bar activity on either kana- were used to compare means of seeds and tissue cultured plants. mycin or glufosinate selection media. Putative No qualitative abnormalities were observed among varieties for transformants are then being assayed for the presence vegetative traits reflecting absence of epigenetic effects. No of the gus gene via histochemical assay and PCR. significant differences in main shoot length and diameter were Acknowledgements: EMBRAPA, CNPq and observed between tissue-cultured and seed-grown plants of all FUNARBE. varieties. Nevertheless, the diameter of most tissue-cultured varieties was significant larger and produced more fresh bio- P-2047 mass than the seed-grown varieties (P 0.05). This response has not been widely reported for tomato. No significant differences Development of Transgenic Imazapyr-tolerant Cowpea were observed on fruit quality (pH, firmness, total soluble (Vigna unguiculata). C. T. CITADIN1,2; A. R. R. Cruz1,2 solids and antioxidant capacity) between tissue-cultured and andF.J.L.Aragão1. 1Embrapa Recursos Genéticos e seed-grown plants. Biotecnologia, PqEB W5 Norte, 70.770-917 Brasília, DF, BRAZIL and 2Universidade de Brasília, Campus P-2046 Universitário Darcy Ribeiro, 70910–900 Brasília, DF, BRA- ZIL. Email: [email protected] (presenting au- Progress Toward Genetic Transformation and Gene Silencing thor), [email protected] (corresponding author) for Carica papaya L. Resistant Plants to Ringspot Virus (PRSV) and Sticky Virus (PSV). G. B. CABRAL1,C.S. Cowpea is one of the most important and widely cultivated Ramalho2,M.L.P.Tinoco1,F.Boff2,E.F.M.Abreu3,H.C. legumes in many parts of the world. Its cultivation is dras- Oliveira2,G.R.Vianna1, and F. J. L. Aragão1. 1EMBRAPA tically affected by weeds, causing damages during growth Genetic Resources and Biotechnology, PO Box 02372, and development of plants, competing for light, nutrients Brasília, DF, 70770–900, BRAZIL; 2University of Brasília, and water. Consequently, weed control is critical, especially Campus Universitário Darcy Ribeiro, S/N - Asa Norte, Brasília, using no-tillage farming systems. In tropical regions, no-till DF, 70910–900 BRAZIL; and 3EMBRAPA Cassava & Fruits farming is much easier with the use of herbicides to control Research Center PO Box 007 Cruz das Almas, BA, 44380– weeds. This study was conducted to evaluate the possibility 000, BRAZIL. Email: [email protected] of obtaining transgenic cowpea plants resistant to imidazolinone, which would facilitate weed control during Brazil is a leading producer of Carica papaya L., account- the summer season. The biolistic process was used to insert ing for 10% of worldwide acreage and 25% of production in a mutated acetohydroxyacid synthase coding gene (Atahas) 2010. The development of papaya crop has been limited which confers tolerance to imazapyr. The transgene integra- by several factors such as its dioecious nature, tion was confirmed by Southern blot analysis. Out of ten heterozygousity, lack of commercial vegetative propa- lines tested for tolerance to 100 g ha-1 imazapyr, eight gation methods and susceptibility to diseases, especially presented some tolerance. One line (named 59) revealed viruses. The main viruses of this crop in Brazil are high herbicide tolerance and developmental growth compa- ringspot virus (Papaya ringspot virus,PRSV)and rable to non-transgenic plants. This line was further tested sticky virus (PSV). Using the gene silencing by RNA for tolerance to higher herbicide concentrations and interference (RNAi) is a viable strategy for obtaining presented tolerance to 400 g ha-1 imazapyr (4-fold the com- papaya resistant to multiple viruses. The goals of this mercial recommended dose) with no visible symptoms. Line PLANT POSTERS

59 will be the foundation for generating imidazolinone- Endive (Cichorium endivia) is a self-compatible leafy veg- tolerant cowpea varieties, which will facilitate cultivation etable crop. The origin of domestication and cultivation of of this crop in large areas. endive is believed to be in the Mediterranean. Morpholog- Acknowledgements: EMBRAPA, CNPq and FINEP. ically it can be distinguished between two major groups of cultivars: var. latifolium (broad-leaved) and var. crispum P-2048 (curly-leaved). As a relatively minor crop, selfing endive cultivars may be founded on a narrow genetic base, despite A Comprehensive Genetic Analysis of the RPA70 Gene the substantial morphological diversity. It was suggested Family in Arabidopsis thaliana Reveals Unique and that Cichorium pumilum or Cichorium calvum might be Overlapping Roles in DNA Repair, Replication, and Meio- endive’s wild progenitor. A measure of the allelic diversity sis. B. B. AKLILU and K. M. Culligan. Department of in the compared populations of domesticated endives (land- Molecular, Cellular and Biomedical Sciences, University of race and cultivar origin), C. calvum, and C. pumilum would New Hampshire, Durham, NH 03824. Email: provide an insight into the role these wild species played in [email protected] the domestication of endive. Knowledge of C. calvum and C. pumilum genetic variability would be helpful for Replication protein A (RPA) is a heterotrimeric, single- expanding the endive breeding stock. We screened 138 stranded DNA-binding protein complex that is required for plants – two wild C. calvum accessions (15 plants/ popula- multiple processes in eukaryotic DNA metabolism, includ- tion), two wild C. pumilum accessions (15 plants/ popula- ing replication, repair and recombination of DNA. RPA tion) and 13 accessions of landrace endives and cultivars (6 homologues have been identified in all eukaryotic organ- plants/ per population). Twelve polymorphic microsatellite isms examined and are composed of subunits that are ap- markers were developed, and they cross-amplified on all proximately 70, 34, and 14 kDa. In contrast to single RPA70 three species. Allelic profiles of all individuals were ana- subunit encoding genes found in yeasts and most animals, lyzed using Arlequin, HP-Rare. The genetic relationships Arabidopsis encodes five phylogenetically distinct RPA70 based on marker similarities were visualized in Structure. C. subunits. DNA sequence analysis, DNA damage sensitivity, pumilum was confirmed to be endive’s wild progenitor DNA replication assays and fertility analysis revealed that based on Fst values and sharing the same genetic cluster each of the five Arabidopsis RPA70 gene family members with landrace endives. The highest allelic richness 3.48 was play unique roles in repair, replication, and/or recombina- observed in a wild C. pumilum population. Surprisingly, tion of DNA. Genetic analysis of the respective mutants endive cultivars showed heterozygosity in several loci, suggest that RPA70C and RPA70E each play unique roles suggesting a high rate of outcrossing in endive’s selfing in the repair of DNA damage, with RPA70C playing a breeding system. leading role in response to DNA double strand breaks and damage caused by replication blocking agents. In contrast, P-2050 the rpa70b rpa70d double mutant has a severe replication defect, revealed by a slower rate of DNA replication, Effect of the PARP Inhibitor 3-Methoxy Benzamide on inhibited growth and developmental phenotypes. This sug- Shoot Regeneration and Microtuber Formation of the Blue gests that RPA70B and RPA70D play a leading, and perhaps Potato Solanum andigenum. KHALID AKARI, REEMA overlapping role in the initiation and/or maintenance of SALEH, Venkarteswari, J. Chetty, Dora Garcia, and Martha DNA replication. Lastly, the single mutant rpa70a displays L. Orozco-Cárdenas. Plant Transformation Research Center, a partially sterile phenotype (reduced seed set; Osman et al., University of California, Riverside, CA 92521. Email: 2009), while the rpa70a rpa70c double mutant displays [email protected] complete sterility, suggesting both RPA70A and RPA70C play unique roles in meiotic progression. In summary, we Potato (Solanum tuberosum) is the third largest global food present evidence that the RPA70 gene family in Arabidopsis crop after wheat and rice, with an annual production represents a diversification of gene functions within this approaching 325 million tons. In addition to human consump- unique gene family in plants. tion, potato is also another possible source of feedstock for bio- energy production. The blue potato (Solanum andigenum) P-2049 originating from the Andean Valleys of Peru has an additional benefit from other potato species in that they contain higher Genetic Ancestry and Diversity in Endive (Cichorium levels of antioxidant rich flavonoids which prove to be good endivia). T. ZAVADA and R. Kesseli. Biology Department, for fighting against chronic diseases. An efficient regeneration University of Massachusetts Boston, 100 Morrissey Blvd., protocol for S. andigenum was developed using internode Boston, MA 02125. Email: [email protected] explants obtained from plants micropropagated in vitro. Eighty PLANT POSTERS percent of the explants cultured on Murashige and Skoog (MS) results show that, of the fifteen treatments consisting of medium supplemented with B5 Vitamins, Myo-inositol different cytokinin types and concentrations, only one (100 mg/l), Sucrose (30 g/l), Zeatin (1 mg/l), Indole Acetic treatment (2 μM BA) resulted in stable multiplication Acid (0.5 mg/l), and Agar (0.8%) were able to regenerate rates over repeated subcultures. These results prove that shoots. Furthermore, the effect of 3-Methoxy Benzamide tissue culture protocols should not be constructed based (3-MB), an inhibitor of the enzyme Poly (adenosine 5’-diphos- on single culture medium optimization experiments. phate (ADP)-Ribose) Polymerase (PARP) on shoot regenera- tion, clonal propagation, microtuber formation and P-2052 Agrobacterium-mediated transformation was also evaluated. Addition of 3-MB significantly increased growth rate of shoots Comparative Study of Different Cryopreservation Tech- by about 72%, and the formation of microtubers in over 21% niques for the Long-term Conservation of Two Orchid of the micropropagated plants. In addition, the plants grown in Species. M. MATA-ROSAS and E. C. Lastre-Puertos. the presence of 3-MB had a higher content of anthocianin Instituto de Ecología, A.C. Carretera Antigua a pigments. Preliminary results also indicated higher shoot re- Coatepec 351, El Haya, Xalapa, Ver. 91070, MEXICO. generation and transformation rates of explants cultured in the Email: [email protected] presence of 3-MB. Altogether, these results indicate the poten- tial use of PARP inhibition on the clonal propagation and the Many species and wild populations of orchids are become genetic improvement of S. andigenum. rare or endangered as direct or indirect results of two human activities: habitat alteration and over-collection; P-2051 tissue culture represents an excellent tool for helping achieve conservation and sustainable use goals for this Micropropagation of Sagittaria latifolia, an Illustration of a resource, but most of the orchids are in vitro fast- Cytokinin Carry-over Effect Following Repeated growing, and conservation of their germplasm through Subcuture. N. H. HOANG and M. E. Kane. Environ- tissue culture requires frequent maintenance, for this rea- mental Horticulture Department, University of Florida, son, the cryopreservation techniques are an excellent al- Bldg. 68, PO Box 110675, Gainesville FL 32611–0675. ternative the long-term conservation of valuable Email: [email protected] germplasm. Several methods have been developed, but their uses in orchids are limited. Hence, in the present Cytokinin carry-over effects on shoot multiplication upon paper a comparative study of the long-term conservation repeated subculture, rooting, and ex vitro survival have of protocorms of two orchids species, based on variants of been practical issues facing large-scale in vitro plant four different cryopreservation methods: encapsulation- production. Many micropropagation studies are published dehydration, encapsulation-vitrification, encapsulation- using data from one Stage II culture cycle with the dehydration-vitrification and vitrification, was carried out. assumption that the procedure is also optimized for mul- Different variables were essayed: percentage of dehydra- tiple subculture cycles which is not always true. Conse- tion of alginate beads, concentration and incubation period quently, attempts by commercial tissue culture labs to on cryopreservation solution, preculture of protocormos in apply these published protocols are sometimes met with different abscisic acid concentration. The survival rate failure. Clearly, there exists a gap between science and ranged from 16% to 100%. The preculture of protocormos application that is large and needs to be addressed. In the in culture media added with abscisic acid was essential to current study Sagittaria latifolia, a native wetland spe- achieved high percentages of protocorms survival. This cies, was used as a model species to investigate cytoki- study is important, since it helps to extend the knowledge nin carry-over effects during in vitro propagation. In vitro about the cryopreservation of orchids and establish the shoots were multiplied on basal half-strength MS medi- bases to conduct more studies with different orchid spe- um supplemented with two cytokinins (benzyladenine cies, and allow the long-term conservation of this valuable [BA] or meta-Topolin [mT]) at different concentrations plant genetic resource. (0-20 μM). Rhizome tip explants were repeatedly trans- ferred to the same media for four 4-week culture cycles. P-2053 Each time explants were transferred, the numbers of leafy rhizome tips, corms, and roots were also recorded. To Optimization of Cryopreservation Protocols for Shoot Mer- investigate the carry-over effect on acclimatization, leafy istem Tips of Sea Oats (Uniola paniculata). J. J. SADLER rhizome tips from four selected treatments (2 μM BA, and M. E. Kane. Environmental Horticulture Department, 4 μM BA, 1 μMmT,3μM mT) were harvested every PO Box 110675, University of Florida, Gainesville, FL subculture and outplanted into the greenhouse. Our 32611–0675. Email: [email protected] PLANT POSTERS

Sea oats (Uniola paniculata) plantings serve as a stabilizer had an impact on transformation frequencies. Bulb scales and builder of coastal dunes, which decrease the destructive cultured overnight on Murashige and Skoog’smedium forces of hurricanes and storms. Micropropagation of di- containing 1.0 mg/L picloram before bombardment with verse sea oats genotypes from multiple Florida populations 0.6 μmgoldresultedinatransformationfrequencyof is limited due to labor costs and culture space requirements 0.9 %. A one week or one month preculture time on compounded by the loss of specific donor populations. Murashige and Skoog’s medium containing 1.0 mg/L piclo- Cryopreservation protocols were developed to reduce these ram yielded higher, 4.2% and 5%, transformation frequencies, limitations but still maintain genetic diversity. Cryopreserva- respectively. When bulb scales were cultured on Murashige tion, the ultra-low storage of plant tissues in liquid nitrogen and Skoog’s medium with either 2.0 mg/L dicamba or (−196°C) is an effective technique for long-term plant germ- 0.5 mg/L picloram, lower frequencies of transformation, plasm storage. Protocols employing vitrification and 1.2% and 0.8%, respectively, were observed. Direct bombard- encapsulation-dehydration of shoot meristem tips were opti- ment of bulb scales eliminates the time needed for callus mized in this study. Shoot meristem tips could be induction and multiplication when embryogenic callus is cryopreserved using either method. Multiple studies were bombarded. conducted to optimize significant cryopreservation steps. The vitrification protocol was optimized by inclusion of a P-2055 2 M glycerol and 0.4 M sucrose loading solution pretreatment, 40 minutes exposure to either PVS2 or A9, a modified PVS2 Comparison of Larvicidal Effect of Rhizomes of Tissue solution, and one rehydration step with 1.2 M sucrose. The Cultured and Naturally Grown Plants of Kaempferia galan- different vitrification steps screened resulted in regrowth rang- ga (Zengiberaceae). R. M. U. S. SENARATH, Jimmy G. ing between 13% and 30% and averaged about 25%. Catanes, and S. Senarath. Faculty of Arts and Sciences, Encapsulation-dehydration resulted in higher average Virgen Milagrosa University Foundation, PHILIPPINES. regrowth. Meristem tips were encapsulated in a 0.75 M su- Email: [email protected] crose and 3% alginic acid gel and polymerized into a bead using 100 mM CaCl2. Beads were placed in 0.75 M sucrose Kaempferia galanga (Zengiberaceae) commonly named as for 20 hours before drying in a laminar flow hood for 0, 2, 4, 8, aromatic ginger or galangal is a medicinal plant used in folk or 12 hours. Regrowth (28.3%) occurred only after 4 hours of medicine as well as in culinary purposes in South East Asia. drying when a moisture content (MC) of 27.2% was obtained. It has been reported that the rhizomes of K. galanga con- Regrowth did not significantly decrease with subsequent dry- tains chemicals that are potent insecticides and have poten- ing to 19.6% MC. These studies show that cryostorage of sea tial in mosquito control. The plant has been overexploited oats shoot meristem tips can be successfully achieved. and listed under threatened category in Sri Lanka and India. Therefore developing tissue culture techniques for plant P-2054 regeneration of K. galanga would be beneficial. Evaluation of extracts of tissue cultured plants against larvae of Aedes Optimized Conditions for Biolistic-mediated Transfor- aegypti , also has been studied which could give a prelim- mation of Lilium longiflorum ‘Nellie White’.K. inary idea about the presence of Kaempferol in tissue cul- KAMO. Floral & Nursery Plants Research Unit, U.S. tured plants and the possibility of tissue cultured plants in National Arboretum, USDA, Beltsville, MD. Email: herbal industry. Objectives of the study were, to develop a [email protected] protocol for tissue culture of Kaempferia galanga from axillary buds obtained from rhizome cuttings and to evalu- Lilies are currently the number one cut flower in the US ate the insecticidal activity of extracts of tissue cultured with a wholesale value of $6.1 million. They are also plants comparing to natural plants. Rhyzomes of K. galan- marketed as a pot plant and grown in gardens. A variety of gal has been wrapped in wet tissues and allowed the axillary tissues were used for biolistic-mediated transformation of buds to be elongated. Axillary buds were removed carefully Lilium longiflorum ‘Nellie White’. Transgenic plants were and surface sterilized. Explants were cultured on Murashige not recovered from five-month-old, “non-select” callus or and Skoog (MS) medium supplemented with BAP (Benzyl suspension cells that had been bombarded with pDM327 amino purine) and IAA (Indole-3-acetic acid) for shoot induc- that contains the bar-uidA fusion gene under control of the tion. For root induction elongated shoots were either continu- CaMV 35S promoter. In comparison, ten transgenic plants ously subcultured in the same medium or separated after were recovered from “select” callus that had been selected 3 weeks and cultured in MS medium supplemented with for its embryogenic-like appearance. Transgenic plants were lowered concentration of BAP and IAA. Rooted plantlets also obtained following direct bombardment of precultured were carefully removed from the medium washed in luke bulb scales. Both preculture time and preculture medium water to remove all traces of agar and acclimatized in a potting PLANT POSTERS mixture of 1: 1: 1 soil: sand : compost. Rhizomes were overexpression cassette. Transformants undergo genetic and collected from tissue cultured plants as well as naturally molecular characterization. Analysis of transgene stability and grown plants. Kaempferol present was extracted separately effectiveness will be further confirmed in T1 progeny by in hexane, methanol and water and different concentrations genetic testing, relative water content, and stress testing were tested against the forth instar larvae of A. aegypti.Mor- (drought/salt/cold). Transformation of soybeans in the aca- tality was observed hourly and LD50 was calculated after 24 h demic field is unpopular due to difficulties in tissue culturing incubation at room temperature. MS medium supplemented and explant preparation. In addition to implications in global with 2.0 mg/L BAP and 0.5 mg/L IAA found to be the best agriculture, this research will be a vital addition to the litera- medium for shoot elongation and multiplication. Increased ture describing these precise techniques. concentration of BAP affects negatively on shoot elongation and multiplication. Best root induction medium was MS me- P-2057 dium supplemented with 1.0 IAA with no added cytokinin. For acclimatization 1: 1: 1 Sand: soil and compost mixture Genetic Transformation of Citrus sinensis cv. ‘Natal’ for found to be the best. When consider about the larvicidal Resistance to Huanglongbing. C. R. OLIVEIRA1,E.C.R. activity although it is a preliminary study, it indicated that Tavano2, R. Harakava3, F. A. A. Mourão Filho1, and B. M. the tissue cultured plants have higher concentrations of sec- J. Mendes2. 1Universidade de São Paulo, ESALQ, ondary metabolites than natural plants. Although the hexane Piracicaba, SP, BRAZIL; 2Universidade de São Paulo, extracts showed the highest activity against mosquito larvae CENA, Av. Centenário, 303, Piracicaba, SP, 13416–903, water extracts also could be used for controlling mosquito BRAZIL; and 3Instituto Biológico de São Paulo, SP, BRA- larvae. As this is a preliminary study, improving the tech- ZIL. Email: [email protected] niques for chemical extraction and testing larvicidal effect with further modifications are recommended. Brazil is the largest producer of sweet orange and concen- trated frozen orange juice in the World. However, diseases P-2056 such as Huanglongbing (HLB) have caused great losses in the Brazilian citrus industry. HLB is associated with Engineering Drought and Salt Tolerant Soybeans by Candidatus Liberibacter spp., limited to the phloem and Overexpressing AVP1, the Vacuole Proton Pump. M. PE- causes generalized plant debility and reduces fruit yield. TERSON and P. Zeng. Department of Natural Sciences, The aim of this work was to obtain Citrus sinensis cv. College of Agriculture and Technology, State University of ‘Natal’ transgenic plants, via Agrobacterium tumefaciens, New York, Cobleskill, NY 12034. Email: with the gene that encodes attacin A antibacterial peptide [email protected], [email protected] (attA) driven by a phloem-specific protein 2 gene promoter (CsPP2) from C. sinensis and phloem-specific protein 2 Salinization and drought conditions are major constraints to gene promoter (AtPP2) from Arabidopsis thaliana.The global crop production. The increased rate of desertification, genetic transformation experiments were performed using irrigation with brackish water, and over use of fertilizers has epicotyl segments obtained from in vitro seedlings. Ten increased these stresses, especially in developing nations. experiments were conducted with approximately five hun- Development of resistant cultivars has become an important dred explants each and ten transgenic plants were identified agronomic issue worldwide. AVP1, a simple pyrophosphate- by PCR, being two transgenic plants containing the gene driven proton pump, transports H+ ions from the cytosol into construct pCAtPP2/attA and eight containing the gene con- the plant cell vacuole. Overexpression of the AVP1 gene in struct pCCsPP2/attA. Plants were acclimatized in green- several model plants was shown to create a unique system of house conditions and will be evaluated for resistance to ion gradients which confers drought and salt tolerance. The Candidatus Liberibacter spp. induced gradients increase cellular water absorption while also increasing auxin flux, which enhances root system devel- P-2058 opment. These observations suggest that crops overexpressing AVP1 would be useful in regions struggling with salinization Agrobacterium tumefaciens-mediated Transformation of and drought conditions. When considering commercial and Atropa belladonna. GUO-QING SONG and Aaron cultural impacts, a soybean cultivar that overexpresses AVP1 Walworth. Plant Biotechnology Resource and Outreach would have vast potential. Soybean crops are vulnerable to Center, Department of Horticulture, Michigan State Univer- environmental stresses but prove to be an ideal source of sity, East Lansing, MI 48824. Email: [email protected] protein, oils, and many of the nutrients and vitamins associat- ed with malnutrition in developing nations. Agrobacterium Belladonna or Deadly Nightshade (Atropa belladonna L.) is was used to transform soybeans with the AVP1 an important medicinal plant in the family Solanaceae. In PLANT POSTERS this study, we developed a reliable protocol for efficient transformation, improving the quality of transgene integra- transformation of Atropa belladonna using Agrobacterium tion and sufficient gene regulation to meet the needs of crop tumefaciens. Hypocotyl and cotyledon explants were co- improvement and functional genomics. Our specific interest cultivated with three opine-type Agrobacterium strains in soybean genetic engineering is to regulate several eco- (LBA4404: pBISN1, GV3101: pBISN1, and EHA105: nomically important genes conditioning soybean seed traits, pBISN1). Selection and regeneration of transformed cells abiotic stress, virus resistance, etc. Some of these studies are were conducted on two regeneration media (RM1 and conducted as collaborations with on- and off-campus re- RM2), each containing 100 mg l-1 kanamycin and searchers. More details of these activities will be presented 250 mg l-1 timentin. Regardless of the A. tumefaciens, 84- at the conference. 96% of hypocotyl and cotyledon explants on selection RM1 and 28%-61% of those on selection RM2 produced at least P-2061 one GUS- and PCR-positive transgenic event after 2-month selection. Most transformants exhibited a normal phenotype Optimization of the Heat Shock Induction Conditions for while growing in the greenhouse. Southern blot analyses Control of GFP and FT Transgene Expression in Cotton 1 confirmed stable integration of the gusA transgene in T1 (Gossypium hirsutum L.). S. BARAMPURAM ,C.H. plants. Histochemical GUS assays indicated the gusA ex- Haigler2, G. Allen1 and S. Krasnyanski1. 1North Carolina pression in T1 seedlings. This work paves a way for im- State University, Department of Horticultural Science, Cam- provement of Belladonna through genetic engineering. pus Box 7550, Partners II Bldg, Room 1208, Raleigh, NC 27695-7550 and 2North Carolina State University, Depart- P-2059 ment of Crop Science, Williams Hall 4405, Campus Box 7620, Raleigh, NC 27695-4405. Email: [email protected], Fostering Plant Science Research at MU Plant Transformation [email protected] Core Facility. S. VALDES, H. Y. Lee, L. Zhou, N. Wan, X. Yin, S.Y.Park,H.B.Li,J.K.Liu,U.Baykal,S.Lu,D.Pham,M. Cotton is a natural source of textile fiber that is cultivated Folta, P. Do, C. Wu, and Z. Zhang. Plant Transformation Core worldwide, making it one of the most important economic Facility, Division of Plant Sciences, University of Missouri, crops. However, improvements by traditional breeding are Columbia, MO. Email: [email protected]; www. limited by the length of time required for flowering. A plantsci.missouri.edu/muptcf cotton plant with a shorter flowering cycle would accelerate breeding efforts and speed the development of elite cotton Since its establishment in 2000, the University of Missouri germplasm. Recently, it was reported that overexpression of (MU) Plant Transformation Core Facility has been provid- the Flowering locus (FT) gene reduced the time required for ing state-of-the-art research support services in genetic en- flowering. Initially we attempted to overexpress FT gene in gineering of several major crops species. Located inside the cotton controlled by constitutive CMW 35S promote, but new MU central campus greenhouse, the Sears Plant were unsuccessful because of developmental arrest in Growth Facility, the Plant Transformation Core Facility is regenerated FT-expressing transformants. Currently we aiming at promoting gene discovery, crop improvement, and are testing the use of a heat-shock inducible promoter from funding opportunities for the plant science research commu- soybean (HSP) to regulate FT expression. We are using two nity. Our staff is strongly dedicated to providing various constructs in our cotton transformation experiments, which types of transformation support services and conducting include HSP:FT and HSP:GFP (Green Fluorescent Protein). research in transgenic technology development with a focus Embryogenic callus induced from the hypocotyls has been on maize (Zea mays), soybean (Glycine max), switchgrass transformed by Agrobacterium carrying either HSP:FT or (Panicum virgatum), sorghum (Sorghum bicolor). The facil- HSP:GFP construct and maintained on kanamycin- ity service categories include both standard and customized containing selection media. Genomic DNA from putative transient as well as stable transformation for maize and transgenic plants has been screened by PCR to test for the soybean upon the user’s request. The transformation sys- presence of either the FT or the GFP transgene. The trans- tems for all the above crops utilize Agrobacterium-mediated genic HSP:GFP embryogenic cultures and regenerated cot- approaches and somatic embryogenesis processes except for ton plants are now being used to optimize the heat-shock soybean. We employ the Agrobacterium-mediated cot-node induction conditions using varying temperature treatments transformation system coupled with organogenesis regime and exposure. The use of GFP expression can be easily for soybean transformation. We are now establishing effi- visualized, which allows us to test a variety of heat shock cient transformation system and soon will be ready for conditions. Later, the optimal heat shock conditions will be wheat transformation services. Current research activities applied to FT transformed regenerated plants to induce the are geared towards developing high-throughput FT gene expression. PLANT POSTERS

P-2062 carcinoma cells, attributable to phenolic compounds (flavo- noids and tannins). It is usually harvested from wild Adventitious Root Cultures of Cuphea aequipetala Cav.: Ef- populations, thus, to develop techniques for in vitro conser- fect of Culture Medium and Auxins. B. P. Martínez-Bonfil, A. vation of C. aequipetala and to provide a source of culti- Cruz-Hernández, A. R. López-Laredo, J. L. Trejo-Espino, and vated plant material, in the present study, we investigated G. TREJO-TAPIA. Centro de Desarrollo de Productos the effects of BAP and two auxins (IBA and 2,4-D, 2.5 μM) Bióticos, Instituto Politécnico Nacional, Morelos, MÉXICO, on callus and shoot induction from different explants (nodal C.P. 62731, Email: [email protected] segments, internodes, and leaves). We found that BAP alone or combined with IBA induced shoot formation from nodal Cuphea aequipetala Cav. (Lythraceae) is a highly valuable segments with a high proliferation rate (70%). Shoots de- medicinal plant used to treat tumors and other diseases. It veloped roots in vitro in the same shoot induction medium grows in pine-oak woods in Mexico andits curative proper- (containing BAP) before being transferred to potting mix, ties have been associated to the accumulation of antioxi- and the resulting plants successfully acclimatized and dants such as phenolic compounds. To provide an reached maturity. Total phenolic compounds and flavonoids alternative source of material for production of bioactive concentrations varied depending on the organ type (leaf, compounds, we established adventitious root cultures of stem, or root). i.e. average flavonoids content was 21.59 Cuphea aequipetala Cav. and evaluated their content of μg quercetin equivalents/g DW, being significantly more phenolic compounds and antioxidant activities. Cultures abundant in leaves and roots than in stems. Similarly, leaf were initiated from root tips of C. aequipetala in vitro extracts showed the highest antioxidant activity (measured plantlets using B5 and SH culture media containing either as reducing power, 329.47±0.6 μmol trolox/g DW) and root indole butyric acid (IBA) or α-naphthalene acetic acid extracts the lowest (89.10±0.3 μmol trolox/g DW). C. (NAA) (0, 5 and 10 μM).The greatest growth index aequipetala plants were able to successfully acclimatize (16.6) was achieved at 14 days with B5 medium plus and grow to maturity, and they retained their ability to 5 μM IBA compared to SH culture medium plus 5 μM accumulate antioxidants, being an alternative to wild plants IBA (4.8). In B5 culture medium, roots remained green as a source of bioactive compounds. along culture period whereas under other treatments, the roots tended to oxidize. The highest content of total P-2064 phenolic compounds (9.1±0.1 μg GAE (gallic acid equivalents)/g dry weight (DW)) and flavonoids (37.5± Assessment of Salt Tolerance in Taro and Sweet Potato: In 0.7 μg quercetin equivalents (QE)/g DW) were observed Vitro Selection, Greenhouse and Field Evaluation. using B5+5 μM IBA at 14 days. Their antioxidant VIRENDRA M. VERMA. Micronesia Plant Propagation properties were compared using in vitro assays (scaveng- Research Center, Kosrae Agricultural Experiment Station, ing of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’- Cooperative Research and Extension, College of azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) Micronesia-FSM, Kosrae, MICRONESIA. Email: (ABTS) radicals, and reducing power using [email protected] phosphomolybdenum assay). Adventitious root cultures of Cuphea aequipetala are a promising system for further Salinity, an abiotic stress that combines elements of water studies on scale-up and antioxidant compounds biosyn- deficiency and sodium toxicity is among the most serious thesis. and widespread of agricultural problems on islands resulting in lost crop yield and arable land. Therefore, the efforts to P-2063 develop salt-tolerant plants are of immense importance to increase crop productivity. In recent years, tissue culture Exploring the Antioxidative Properties of Cuphea aequipetala based in vitro selection has emerged as a feasible and cost- Cav. Cultivated Plants. B. P. Martínez-Bonfil, M. Pineda- effective tool for developing salt tolerant plants. Sweet Montero, A. R. López-Laredo, G., G. Salcedo-Morales, S. potato and taro are most important staple food crops in the Evangelista-Lozano and G. TREJO-TAPIA. Centro de Pacific Region for local consumption as well as for export. Desarrollo de Productos Bióticos, Instituto Politécnico These crops contribute significantly to the socio-economics Nacional, Morelos, MÉXICO, C.P. 62731, Email: and provide livelihood to almost all island people and thus [email protected] are crucial for ensuring nutritional and economic security. Both sweet potato and taro are placed on high agricultural Cuphea aequipetala Cav., (Lythraceae, “hierba del cancer”) priority but limitations in availability of salt tolerant germ- is a medicinal plant native to Mexico with proven biological plasm, and disease-free and elite seedlings, is a major bot- activities such as cytotoxicity against larynx and prostate tleneck in production. Therefore, the study was undertaken PLANT POSTERS for assessment of salt tolerance in taro and sweet potato occured on MS medium with 10 μMBAP,1μM Indole-3- through in vitro selection, followed by greenhouse and field butyric acid (IBA), 0.5 μMGA3, 80 mg/l L-Glutamine and evaluation. To establish aseptic cultures for collected germ- 20 mg/L L-Serine. Cytological squash preparation from root- plasm of taro and sweet potato, various experiments were tips of in vitro plants revealed the haplid number of chromo- performed. Different concentrations of sodium chloride somes as 2n=x=15, while squash preaparatin from parent were used for in vitro selection of salt tolerant germplasm. plant (control) revealed the diploid number of chromosomes This in vitro selected germplasm was further evaluated for as 2n=2x=30. Further confirmation of haploid status of in salt tolerance in the greenhouse and finally was field eval- vitro plants was done by flowcytometry analysis of leaves of uated at eight sites in four replications. Some varieties of in vitro plants as well as parent plants. taro and sweet potato performed very well at coastal sites. Results based on various physiological and morphological P-2066 parameters collected during the research are presented in this paper. Micropropagation of Dyckia distachya in Different Culture Media Physical State and Differences in Plant Endogenous P-2065 Microorganisms.C.HEUSER,C.K.Salomão,E.H.Souza, F.S.Cannavan,S.M.Tsai,andA.P.Martinelli.Universityof An Efficient Protocol for Androgenic Haploid Production in São Paulo, CENA, Av. Centenario, 303, Piracicaba, SP, 13416– TV21 Cultivar of Tea (Camellia assamica ssp. assamica 903 BRAZIL. Email: [email protected] (Masters). V. K. MISHRA and R. Chaturvedi. Department of Biotechnology, Indian Institute of Technology- In vitro plant microbiota represents a rather unexplored Guwahati, Guwahati-781039, Assam, INDIA. Email: area of microbiology compared to in field plants. This [email protected], [email protected], association is a result of many factors and considering [email protected] that plants can benefit from this interaction, the aim of this study was to evaluate different culture conditions Camellia assamica ssp. assamica (Masters), commonly for the micropropagation of Dyckia distachya Hassler known as Tea, belongs to the family Theaceae. It is a and also explore, by using the culture-independent mo- socio-economic crop and plays an important role in lecular approach, the microbiota diversity in D. country’s economic development by uplifting foreign ex- distachya under diverse micropropagation environments. change. Tea is highly heterozygous and heterogenous in Explants were cultivated in MS medium supplemented nature due its cross pollinating behavior. In vitro production with sucrose (30 g L-1), NAA (1 μM), kinetin (2 μM) of haploids will act as a precursor to raise homozygous in three physical states of the medium: liquid static diploids of tea, in single step by treating the haploids with (LS), liquid under agitation (60 rpm) (LA) and solidi- mutagen like colchicine. The present study evaluates the fied with agar (8 gL-1) (S). Three subcultures were role of plant growth regulators, carbon source, amino acids performed every 30 d, with leaves collected on the and physical factors on in vitro androgenesis. Androgenic second subculture (60 d) and stored for further molec- haploids of TV21 cultivar of tea are produced by anthers ular analysis. After the third subculture cultures were cultured at early- to late-uninucleate stages of pollen grains. evaluated for number of shoots. The leaves collected Haploid development occurred via callusing. The maxi- were evaluated by using molecular techniques, based mum (96%) induction of calli from anther cultures on heterogeneous nucleic acids amplified internal 16S was obtained on Murashige and Skoog’s (MS; 1962) rRNA gene by PCR, followed by T-RFLP. The results basal medium with 6% glucose and supplemented with for micropropagation showed that LA was the best 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 μM6- treatment for average number of shoots, producing furfurylaminopurine (Kinetin), 800 mg/L L-Glutamine 10% more than LE and 30% more than to S. The and 200 mg/L L-Serine. Proliferation of callus occurred molecular results showed that although the numbers of when 6% Glucose was replaced with 3% Sucrose in OTU found were similar (13–14), their relative abun- callus induction medium. Regeneration via embryogenesis dance for cultures in LA was clearly different compared wasobtainedonMSmediumwith10μM6- to the other two treatments. Further research is needed benzylaminopurine (BAP), 3 μM Gibberellic acid (GA3), to increase the understanding about the associations 800 mg/L L-Glutamine and 200 mg/L L-Serine after three between bacteria and plants under micropropagation months. Ten times reduced concentrations of growth regula- and their potential role in favoring plant multiplication tors and amino acids of embryo induction medium were and/or development. (Acknowledgements: FAPESP, responsible for maturation of embryos. Embryo germination CNPq). PLANT POSTERS

P-2067 cial sodium hypochlorite solution (2.5% active chlorine, 30 min), followed by three rinses in autoclaved water In Vitro Germination and Post-seminal Development in and introduced in vitro in ½ MS medium, under a 16 h Dyckia distachya Hassler, an Endangered Bromeliad Spe- photoperiod and different culture temperatures (22°C, cies. K. SALOMÃO, E. H. Souza, M. L. Rossi, and A. P. 27°C e 32°C), for germination. Post-seminal develop- Martinelli. University of São Paulo, CENA, Av. Centenario ment was followed at 27°C. Seeds of D. distachya are 303, Piracicaba, SP, 13416–903, BRAZIL, Email: brown, small, ovate, with membranaceous persistent [email protected] wings. Best germination was observed at 22 and 27°C, around 97% and 93% at 32°C. No contamination or The Atlantic Forest biome is known for its wide bio- seedling mortality was observed after germination. logical diversity. The disturbance and non-sustainable Seeds were considered germinated when the radicle tip exploitation of this environment and ecosystems caused protrusion was visible, which occurred three days after considerable loss of biodiversity, as exemplified by in vitro introduction. Seedling establishment is epigeal Dyckia distachya Hassler, an endangered bromeliad. As and the first eophyl leaf was observed already after four part of a research line in bromeliad reproduction, that days in vitro. At 12 days the young plant already shows aims to contribute to taxonomical, ecological and con- a shortprimaryrootwithmanyroothairs.Germinationand servational studies in this family, this work aimed to seedling establishment proved efficient and viable for the evaluate in vitro germination and morpho-anatomy of production of large numbers of seedlings or for in vitro post-seminal development in D. distachya. Seeds multiplication and reintroduction in adequate environment or obtained from plants cultivated in the greenhouse were for commercial. (Acknowledgements: CNPq, CAPES, disinfested in ethanol (70%, 3 min) and 50% commer- FAPESP, NAP/MEPA - ESALQ/USP).