In Vitro Cell.Dev.Biol.

Animal (2016) 52 (Suppl 1):S52S73

DOI 10.1007/s11626-016-0035-z

2016 WORLD CONGRESS ON IN VITRO BIOLOGY ABSTRACT ISSUE

Plant Posters

P-2000
In Vitro Conservation of Elite Germplasm, Isolation and UpRegulation of Some Important Biomolecules.VEENA
AGRAWAL. Department of Botany, University of Delhi110007, INDIA. Email: drveena_du@yahoo.co.in
Medicinal plants have been reckoned as valuable therapeutic aids
for alleviating various ailments of human-kinds. There has been
an exponential growth of herbal products both in developed and
developing countries. The medicinal plants are required globally
for several important industries. It is the exigency to develop
strategies not only for conservation of the valuable germplasm
but also for their recurrent production at large scale at the commercial level. Lab to land technologies have been developed for
mass production of at least 20 medicinal plant taxa like Arnica
Montana, Artemisia annua, Cassia aungustifolia, Calliandra
tweedii, Centella asiatica, Erythrina indica, Hollarhena
antidysenterica,Populus x euramericana, Psoralea corylifolia,
Simmondsia chinensis, Spilanthes sp., Stevia rebaundiana,
Terminalia bellerica, Vernonia anthelmintica, etc. through organogenesis and embryogenesis or both. Some herbal-based products such as anticancerous,larvicidal, insecticidal, and antimicrobial compounds have been developed employing in vitro technologies from Artemisia annua (artemisinin), Psoralea
corylifolia (psoralen, diadzein and genistein), Cassia
aungustifolia (sennosides A & B), Spilanthes sp. (spilanthol),
Centella asiatica (Asiatic acid and asiaticoside) and Stevia
rebaundiana (stevioside) in my laboratory. Bioactive compounds
like artemisinin, psoralen, spilanthol, sennosides A & B,
asiaticoside have been isolated and characterized. Their optimization has been done using various organic and inorganic elicitors. Key enzyme gene of psoralen synthesis pathway has been
cloned and characterized using molecular tools. A summary of
the work pertaining to germplasm conservation, extraction,

isolation and up-regulation of different herbal compounds including their bioefficacy shall be presented.
P-2001
Development of an Efficient In Vitro Regeneration System
Amenable to Agrobacterium-mediated Transformation of
Enset. J. M. MATHEKA1, J. Tripathi1, E. Gabre2 and L.
Trpathi1.1International Institute of Tropical Agriculture, C/o
ILRI, P. O. Box 30709-00100, Nairobi, KENYA and
2
Ethiopian Institute of Agricultural Research (EIAR), Addis
Ababa, ETHIOPIA. Email: j.matheka@cgiar.org
Ensete ventricosum is a clonally propagated perennial plant
under the banana family Musaceae. It is indigenous to
Ethiopia where about 20 million people depend on it for food,
animal feed and fiber (Bezuneh et al., 1967). Enset tolerates
persistent droughts, heavy rains, and flooding making it an
important food security crop. However, enset bacterial wilt
disease is the main constraint to enset production. Work is
under way to engineer resistance to bacterial wilt disease in
enset through a joint collaboration between International
Institute of Tropical Agriculture (IITA) and Ethiopian
Institute of Agricultural Research (EIAR). A tissue culture system for regeneration of enset through somatic embryogenesis
and multiple meristems was developed at IITA and is being
adapted for enset transformation. Multiple meristems of enset
were cultured on different media designed to induce somatic
embryos. The induction efficiency from multiple meristems
was highest (28%) on media with 1.5 mg/l 2,4-D and 0.22
mg/l zeatin. Various media were tested for generation of friable
embryogenic callus and shoot regeneration. Agrobacteriummediated transformation was performed on multiple meristems
with fluorescent protein and gusmarker genes. Plants were
confirmed transgenic by PCR and southern analysis.

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P-2002
Implementation of Precision Breeding for Grapevine
Improvement A Technical Perspective. ZHIJIAN T. LI and
Dennis J. Gray. Mid-Florida Research & Education Center,
University of Florida/IFAS, 2725 S. Binion Road, Apopka,
FL 32703. Email: zjli@ufl.edu
Over the last decade, many candidate genes of grapevine were
investigated in order to gain understanding of their functions and
usefulness in precision breeding (PB). Only PB will allow genetic improvement of elite grape varieties which must retain
their unique characteristics to remain useful. Among disease
resistance genes, eight unique basic-typeVvPR1b1-like genes
isolated from a disease resistant Florida hybrid bunch grape
(FHBG) (Vitis subg. Euvitis) selection BN5-4 and two Vitis
vinifera-derived genes - a seed-specific 2S albumin protein
gene, VvAlb, and a fruit-specific thaumatin-like protein gene,
VvTL1, were tested. All genes were placed under control of
strong constitutive viral promoters and introduced into a disease
susceptible Vitis subg. Euvitis hybrid Seyval Blanc. Modified
plants were first grown in the field for fitness testing and surviving lines were multiplied and used in subsequent greenhouse
tests for resistance to powdery mildew (Uncinula necator) using
both manual and natural inoculations. Several candidate genes
were determined to confer high levels of sustainable disease
resistance to field grown and greenhouse-maintained modified
plants. In addition, a Vitis-derived selectable marker using an
herbicide-tolerant acetolactate synthase (ALS) gene, VvALS2f,
was developed based on the combination of eight targeted amino acid alterations. In vitro tolerance to one sulfonylurea and
two imidazolinone herbicides was detected in both modified
grapevine and tobacco, thus allowing identification of gene insertion events based on host-derived DNA. However, packaging
all of these functionally defined multiple genes and other genetic
elements into a deliverable form without any foreign nucleotides
for insertion into recipient plants represents a great challenge
with the currently available DNA manipulation techniques.
Strategies to overcome this final obstacle will be discussed.
P-2003
Development of a Robotic Platform for Automated Protoplast
Isolation, Transformation, and Screening of Plant Suspension
Cultures. ELIZABETH M. DLUGOSZ1, Taniya Dhillon1,
Jason N. Burris1, Scott C. Lenaghan2,3, and C. Neal Stewart,
Jr.1. 1Department of Plant Sciences, University of Tennessee,
Knoxville, TN 37996; 2 Center for Renewable Carbon,
University of Tennessee, Knoxville, TN 37996; and
3
Department of Mechanical, Aerospace and Biomedical
Engineering, University of Tennessee, Knoxville, TN 37996.
Email: edlugosz@utk.edu

S53

Recently, plant protoplasts have been used for analysis

of gene expression, genome-editing, and gene-silencing.
Additionally, regeneration of plants from protoplasts has
further increased interest in the use of these systems for
plant genomics. In this work, a protocol has been developed for automation of high-throughput protoplast
isolation, transformation, and screening using switchgrass (Panicum virgatum) and tobacco (Nicotiana
tabacum) Bright Yellow 2 (BY-2) suspension cultures.
The robotic platform includes 2 liquid handlers for precise pipetting and washing of 96-well plates, a plate
incubator and shaker, a monochromator-based microplate reader, and a robotic arm, all housed within a
HEPA-filtered biosafety cabinet. Low-cost food-grade
enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that
are cost-prohibitive in high-throughput automated protoplast isolation and analysis. The complete procedure
from protoplast isolation to transformation can be conducted in under 4 hours. After PEG-mediated transformation, protoplasts were analyzed for transformation efficiency by monitoring expression of GFP and orange
fluorescent protein (OFP) reporter genes at 1-hour intervals over the course of 24 hours. This automated transformation and screening have proven to be useful for
high-throughput promoter screening as well as rapid
identification of target transgenic modifications. These
methods should be translatable to any plant suspension
culture/protoplast system, which is expected to enable
acceleration of crop genomics research.
P-2004
Use of Low Inoculum/Long Co-culture Treatments Lead to
Tremendous Enhancements in Agrobacterium-mediated
Transformation of Sunflower. Zhifen Zhang and JOHN J.
FINER. The Ohio State University, Department of
Horticulture and Crop Science, OARDC, 1680 Madison
Ave., Wooster, OH 44691. Email: finer.1@osu.edu
For successful transformation, plant tissues are typically excised and exposed to large numbers of Agrobacterium cells for
2-3 days. During this co-culture, the bacterial T-DNA is processed and delivered to the plant cell and nucleus, where it
eventually becomes integrated into the genome. Extending
the co-culture period could provide a greater opportunity for
Agrobacterium cells to find, bind to, and transform plant cells.
Since long co-culture periods result in bacterial overgrowth of
target tissues and induction of plant immunity, use of low
Agrobacterium inoculum levels was explored. We found that
use of very low inoculum levels (6 log dilution), in combination with long co-culture times (2 wk) enhanced

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PLANT POSTERS

Agrobacterium-mediated transformation of sunflower cotyledonary tissues by 10-1000 fold or more. The low inoculum/
long co-culture (LILC) method required both low inoculum
levels and long co-culture periods. No or low transformation
was obtained when using standard high inoculum with long coculture or low inoculum with standard short co-culture. Since
dipping plant tissues in a dilute bacterial suspension led to inconsistent inoculation loads, diluted bacterial suspensions were
drop-inoculated (10 l) onto target tissue to more precisely
control inoculum levels. qRT-PCR analysis of sunflower cotyledon tissues exposed to LILC revealed reduced or delayed
induction of selected defense response genes, suggesting that
use of LILC did not lead to defense or hypersensitive responses,
which would have resulted in inhibition of bacterial and/or plant
growth. LILC holds much promise for improving transformation rates of recalcitrant plants, and expanding the suitable targets for Agrobacterium-mediated transformation.

exhibited faster growth in the greenhouse. IVE is an engineered

matrix allowing both liquid culture and semisolid culture.
Improved air/water ratio in IVE allowed for superior rooting
success. The proposed system, in comparison with rooting on
agar brings certain number of advantages. The transport of this
type of culture, plant plus IVE plus Microbox, is resistant to
physical constraints. Elements plug plus plantation are easier to
handle and allow for mechanization for acclimatization. Indeed,
this protocol of in vitro rooting of walnut tree gives us a new
interest for mass production; rooting, transport and acclimatization linked with exportation business. This way, we can offer an
efficient plug and growth system easy to handle and directly
usable by nurseries.
P-2006

In Vitro Rooting Induction and Expression in Foam-based

Matrix Resulted in Improving Performance and Greenhouse
Acclimatization in Walnut Cultivars Fernor and Chandler.
L. JOUVE1, F. Van de Heyning2, and V. Rapaka3.1L & J BioTech,
R u e d u P on d a i l l a n , 4 6 20 0 S ou i l l a c , F R A N C E ;
2
Microbox Combiness, 1177 Avenue of the Americas,
7th Floor, New York, NY 10036; and 3Smithers-Oasis
Company, 919 Marvin Street, Kent, OH 44240. Email:
ljouve@lj-biotech.com

Improved Rooting in Cavendish Banana cv. Grande Naine Using

Coconut Shell Activated Charcoal and Naphthaleneacetic Acid
Supplements. R. SISON- FLAMIANO1, J. B. Madas1, L. G. C.
Itang 1, R. B. Gubalani1, V. R. S. Flamiano2, R. S. Diamonon 3,
E. S. Bautista3, L. D. V. Ligan3 and R. S. Casido3. 1Mindanao
State University, Science Department, College of Natural
Sciences and Mathematics, Barangay Fatima-General Santos
City 9500, PHILIPPINES; 2Greenlands Agritech Ventures,
Research and Development Division, General Santos City,
9500, PHILIPPINES; and 3Technical Education and Skills
Development Authority-Surallah National Agricultural School,
Surallah, South Cotabato 9512, PHILIPPINES. Email:
rflamiano@yahoo.com

It is well known that walnuts are hard to root and acclimatize.

Of the two cultivars tested, Fernor is more difficult to propagate
than Chandler. Fernor and Chandler were propagated using
DKW medium solidified by agar. Elongated stems were used
for induction and expression rooting processes. They were
placed on Oasis Foam (IVE) infused with agar induction medium and then placed in breathable Microbox containers from
Combiness. Following one-week induction in darkness and
four weeks under light for root expression, the comparison
between rooting rate and survival/shape after 2 subsequent
months of acclimatization under classical agar rooting condition and with foam was done. The experiments have been done
with a production significant aim. The assay must show a reality with an industrial aim. Three independent assays were done.
In each assay 240 plants were used. In this respect, the experiment involved 720 plants per cultivars. Results showed clear
benefits. In the case of Fernor 67% rooting was observed in
IVE and only 48% with agar alone. In the case of Chandler the
results showed the same tendency with 97% rooted in IVE and
90% rooted in agar. Rooted plants were then transferred into
Jiffy pots filled with peat-perlite based medium. In case of both
cultivars, the plant rooted in IVE acclimatized faster and

The present study was done to evaluate the efficacy of using

coconut shell activated charcoal in combination with
Naphthaleneacetic Acid (NAA) as rooting amendments to
full-strength Murashige and Skoog (MS) medium for
Cavendish cv. Grande Naine. Concentrations of NAA from
1-5 ppm, and coconut shell- acivated charcoal from 1.0% to
3.0% were added to full strength MS medium. Regenerated
microshoots of Cavendish banana were allowed to root for 21
days in the different MS amended media. Thereafter, the number of roots and root length were measured. Results of this
study revealed that rooting of Cavendish banana cv. Grande
Naine was best in full-strength MS medium amended with
3.0% coconut shell-activated charcoal and 2 ppm NAA combination. In this MS amended medium, each plantlet developed an average of 26 roots with the longest average roots of
92.22 cm. Higher concentrations of NAA at 4 ppm and 5 ppm
combined with 3.0% activated charcoal added to full strength
MS medium were shown to have inhibitory effects on the
number and length of roots. The addition of 3.0% coconut shell
activated charcoal to full strength MS Medium increased the
number of microshoot roots although the increase was not significant from the hormone-free, full-strength MS medium. Full

P-2005

PLANT POSTERS

strength, hormone-free MS medium had also been shown to

support rooting as with the different coconut shell activated
charcoal-amended MS medium with comparable number of
roots and root length per plantlet.
P-2007
Micropropagation of Lycium barbarum (Muragli) of Indian
Desert. SMITA SHEKHAWAT1, Narpat S Shekhawat2, and
Veena Agrawal1. 1Department of Botany, University of Delhi,
Delhi-110007, INDIA, and 2 J. N. V. University, Jodhpur342005, Rajasthan, INDIA. Email: smita.shekhawat@gmail.
com
Lycium barbarum, belonging to family Solanaceae, is
drought, heat and frost tolerant hardy shrub. Its fruits are used
in herbal medicine for treatment of chronic diseases such as
diabetes, cancer, hepatitis and male infertility. We have
attempted to characterize germplasm and develop tissue culture methods for micropropagation of L. barbarum of the
Indian Desert. Nodal shoot segments harvested from adult
plants were cultured on MS + 2.0 mg L-1 of BAP. From each
node 4.30 0.90 shoots differentiated. These were multiplied
by repeated transfer of mother explants. Culture conditions
were defined for production of 29-30 shoots per culture vessel. The maximum number (29.55 4.28) of shoots were
produced on 0.75 mg L-1 of BAP + 0.25 mg L-1 of Kinetin
+ 0.1 mg L-1 of IAA with average shoot length of 7.1
0.32 cm within 25-30 days. The highest number of shoots
(72%) rooted on one fourth strength of MS salts with
5.0 mg L-1 of IBA + 0.5 mg L-1 of NOA with 7.750.95
average number of roots. Sixty percent of the in vitro produced shoots could be rooted under ex vitro conditions
when treated with 500.00 mg L-1 of IBA for 3 minutes
on soilrite in green house with 5.800.82 roots from the
base (average length of 5.530.89 cm) of each shoot.
About 80% plantlets were hardened after 30- 40 days of
rooting. The hardened plantlets were transferred to polybags
containing garden soil, sand and organic manure. After proper
hardening in the green house plants were transferred to nursery and finally exposed to natural conditions.
P-2008
Engineering Low Cd Rice Through Expression of OXS3-like
Gene Fragments. DAVID W. OW and Changhu Wang. Plant
Gene Engineering Center, South China Botanical Garden,
Chinese Academy of Science, 723 Xingke Road,
Guangzhou 510650, CHINA. Email: dow@scbg.ac.cn
Cadmium is a toxic heavy metal and carcinogen and its entry
into the food chain has serious effects on food security and
public health. As rice is a staple food crop for half of the world

S55

population, Cd in rice grain has become a major source of

dietary Cd for some parts of the world. Previous gene discovery efforts for the development of low Cd rice have been
linked to cation transport; and for the two most effective
genes, OsHMA3 and OsNRAMP5, expression in yeast can
cause a Cd sensitive phenotype. Rather than limiting the focus
to these leadscation transport and Cd hypersensitivitywe
decided to broaden the consideration to include Cd
hypertolerance. Since there are numerous cellular targets for
this metal, enhanced tolerance to Cd would not likely be due
to target insensitivity. Instead, we reasoned that it would be
due to one or more of the following: enhanced repair of damage, intracellular sequestration, organ-specific sequestration,
hyper-export, or hypo-uptake of the metal; and any of the
latter three possibilities could potentially lead to low grain
Cd. Therefore, DNA fragments that enhance Cd tolerance in
the fission yeast were tested in transgenic plants. We report
that fragments derived from two rice OXS3(OXIDATIVE
STRESS 3) homologs can reduce Cd accumulation in rice
grain as well as throughout the plant, and without affecting
grain yield or content of Mn, Fe, Zn and Cu.
P-2009
Robust Genetic Transformation of Sorghum (Sorghum
bicolor) Using Green Regenerative Tissue. SRINIVAS
BELIDE, Thomas Vanhercke, James Robertson Petrie, and
Surinder Pal Singh. CSIRO Agriculture, Canberra, ACT,
AUSTRALIA, 2601. Email: Srinivas.belide@csiro.au
Sorghum is one of the worlds most important cereal crops and
is grown for multiple applications. A new generation of researchers and entrepreneurs have re-discovered sorghum and
are now investigating its potential as a promising biofuel crop.
Metabolic engineering can enhance the value of the traits such
as the energy dense, high oil leaves we have recently reported.
Highly efficient and fast transformation methods, however,
are prerequisite for metabolic engineering projects that manipulate complex pathways. Though significant improvements in
sorghum transformation have been reported recently, the efficiency is lagging behind many other monocot species. The
present study reports a robust microprojectile mediated transformation system for sorghum (TX430) that is independent of
environmental factors. Immature embryos (12-15 DAP) were
used to induce green regenerative callus and maintained for
more than one year without losing potential for regeneration
and transformation on modified MS media. More than 650
potential transgenic shoots were obtained from six different transgenic constructs using a mix and match combination of co-transformation over a six month period. The
average transformation efficiency (total number of independent transgenic events divided by the total number of
bombarded green calli) was 46.6% (single construct).

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PLANT POSTERS

Around 93% of putative transgenic shoots were positive

for NPTII by PCR and a co-transformation frequency of
the non-selected construct was 59%. The transformation
procedure, from bombardment to establishing a transgenic
plant in the glasshouse, took 12-14 weeks. Efficiencies are
up to two-fold higher than published reports of TX430
sorghum transformation. This new transformation system
has proved to be reliable and reproducible in our lab and
can likely be extended to sweet sorghum. Preliminary results from a series of stably-transformed constructs aimed
at increasing the oil content in vegetative sorghum biomass
will be presented.

drive greater attention towards Spilanthes sps to explore

new therapeutic drugs.

P-2010

The value of doubled haploids in genetic analysis and modern

plant breeding has been known for a long time. Goals of the
WICT (Wheat Improvement through Cell Technologies) project of the Canadian Wheat Alliance include the development
of an efficient isolated microspore culture system (IMC) for
spring and winter wheat that is effective with a broad range of
wheat cultivars, as well as the establishment of an effective
and efficient microspore transfection protocol using
nanocarriers. Both activities involve the testing of a wide
range of variables, including different culture and media conditions, cultivars and nanocarrier: DNA complexes.
Unfortunately, the regular large scale wheat microspore extraction protocol requires a minimum of 10-12 spikes to be
processed in one extraction, with a maximum of 2 treatments
processed in one batch. We carried out experiments to develop
a small scale culture technique for wheat microspores. With
this method, up to twenty different treatments can be processed in one batch using as few as 10 anthers per treatment.
This has significantly reduced the time and labor required for
the testing of multiple treatments for the production of doubled haploid plants.

Development of Substantial Biotechnological Methods for

Production of Pharmaceutically Active Biometabolites from
the Genus Spilanthes. RAKHI CHATURVEDI and R.
Radhika. Department of Biosciences and Bioengineering,
Indian Institute of Technology Guwahati, Guwahati 781039, Assam, INDIA. Email: rakhi_chaturvedi@yahoo.
co.uk, rakhi_chaturvedi@iitg.ernet.in
The genus Spilanthes is from the paramount family
Compositae (Asteraceae) of the order Asterales. It is rich in
N-alkylamides, which are recently classified as imperative and
large group of bioactive secondary metabolites. To attest their
traditional applications, insight scientific investigations are
highly crucial to find out the biochemical capacity. In vitro
tissue culture methods, unlike the conventional methods, favor consistent and uniform availability of plant biomass,
throughout the year. Therefore, the methods have been implemented in the genus Spilanthes for the development of cell
biomass from two different explants, such as leaves and flower heads, which are considered to be the main repositories of
these alkylamides. The production of N-alkylamides are
screened and quantified by following traditional method,
like Thin Layer Chromatography (TLC) followed by highthrough put techniques, like High Performance Liquid
Chromatography (HPLC), Mass Spectrometry (MS) and
Infrared Spectroscopy (IR). The study clearly revealed that
the cell biomass from leaf-origin showed the presence of
predominant N-alkylamide (2E,6Z,8E)-N-Isobutyl-2,6,8decatrienamide (spilanthol), whereas, the cell biomass
from the flower-head source contained two alkylamides,
spilanthol and (2E,4Z)-N-isobutyl-2,4-undeca-8,10diynamide (UDA). The quantity of spilanthol in cell biomass from flower-head was comparatively greater (2.23
0.04 mg/g DW) than those present in cell biomass from
leaf-origin (1.75 0.03 mg/g DW) and flower-head from
field grown parent plant (control; 0.83 0.12 mg/g DW).
The UDA content in the cell biomass from flower-head
was (4.30 0.22 mg/g DW). This nutshell of work will

P-2011
A Rapid Small-scale Wheat Microspore Culture Technique
for Screening Multiple Variables. P. L. POLOWICK, P. K.
Bhowmik, G. Nowak, J. L. Enns, and A. M. R. Ferrie.
National Research Council Canada, 110 Gymnasium Place,
Saskatoon, SK, S7N 0W9, CANADA. Email:
Patricia.Polowick@nrc-cnrc.gc.ca, Pankaj.Bhowmik@nrccnrc.gc.ca

P-2012
Isolating and Identifying the Proteins Contributing to
Nanoparticle Formation in English Ivy (Hedera helix). JASON
N. BURRIS1, Scott C. Lenaghan2,3, C. Neal Stewart, Jr.1.
1
Department of Plants Sciences, University of Tennessee,
Knoxville, TN 37996; 2Center for Renewable Carbon,
University of Tennessee, Knoxville, TN 37996; and
3
Department of Mechanical, Aerospace and Biomedical
Engineering, University of Tennessee, Knoxville, TN 37996.
Email: jburris1@utk.edu
Nanoparticles isolated from English ivy have previously
shown promise in both the biomedical field and cosmetics
industry. However, to truly demonstrate the feasibility of using
these nanoparticles in these fields, it is necessary to understand
the makeup of the nanoparticles. Previous studies have demonstrated that the nanoparticles are proteinaceous

PLANT POSTERS

biomolecules devoid of metal ions. In this work, we utilized a

proteomic- and transcriptomic-based approach to identify the
specific proteins involved in the formation of the nanoparticle
adhesive complex. It was found that the use of dithiothreitol
(DTT) during extraction permitted the separation of a single
high molecular weight band observed in other studies, into
nine distinct protein bands ranging in molecular weight from
10 kD to 175 kD. In addition, DTT was found to have no
effect on the size or stability of the nanoparticles, as assessed
by dynamic light scattering (DLS) and Zeta potential analysis.
Further analysis of the protein bands using LC/MS/MS
allowed for sequencing of the top 13 protein candidates involved in nanoparticle formation. To identify the ivy specific
sequence of these genes, the sequencing data was blasted
against transcriptomic data obtained for the adventitious roots
of English ivy. PCR was conducted using primers designed
from conversion of the protein sequences to validate the transcriptomics data, and generate English ivy specific sequence
for the genes of interest. Based on these results, candidate
proteins were identified that contribute to the formation of
the English ivy nanoparticles, providing further data on the
structure and creation of these important nanostructures.
P-2013
Lincosamide Nucleotidyltransferase Genes Are Highly
Efficient Selectable Markers for the Recovery of Transgenic
Events in Higher Plants. MASSIMO BOSACCHI, Kliment
Todosov, Daniel Ilg, and Pal Maliga. Rutgers, The State
University of New Jersey. Email: massimo.bosacchi@
rutgers.edu
Transformation, the technology that moves genes into
plants, is the foundation of agricultural biotechnology
and a major tool in plant genetic research. A key element
of transformation technology is a selectable marker gene
to enable identification of transgenic events. However,
there are a limited number of selectable marker genes
efficacious enough for routine use. As academic and industry research increasingly focuses on complex polygenic traits, the discovery and development of new efficient
marker genes will facilitate the stacking of multiple genes
in plants. We designed a novel selection scheme based on
the toxic effects of lincosamide antibiotics on plant cells
in tissue culture. We established that a combination of
clindamycin and lincomycin, to respectively block callus
proliferation and plantlet formation, is the optimal selection regiment. We identified seven candidate genes from
microbial studies and tested their performance as selectable markers. In tobacco transformation experiments, four
genes yielded resistant plants with high efficiency:
lnuAN2, lnuB, lnuD and lnuF. We analyzed putative transgenic events with western blotting, confocal microscopy

S57

and segregation analysis of F1 progeny. The lnuBgene

yielded transgenic plants with a high transformation frequency and low escape rate. In tissue culture, resistant
lnuB shoots develop more slowly, but arise as distinct
plantlets with resistant proliferating callus largely absent.
This characteristic makes event recovery quick and efficient and is probably a factor in the low escape rate. The
larger size of lnuB may be a reason for this phenomenon.
Codon-optimization of the three ineffective genes did not
improve their performance.
P-2014
Micropropagation of Stevia rebaudiana Bertoni in a
Temporary Immersion Bioreactor; Phytochemicals
Analysis of Plants. D. VILLAMARN-GALLEGOS1, D.
Oviedo-Pereira1, G. Seplveda-Jimnez1, S. EvangelistaLozano1, J. Molina-Torres2, and M. Rodrguez-Monroy1.
1
Instituto Politcnico Nacional, Centro de Desarrollo de
Productos Biticos, Departamento de Biotecnologa, Calle
CEPROBI 8, Col. San Isidro, Yautepec, MorelosMXICO, CP. 62730 and 2 Instituto Politcnico
Nacional, Centro de Investigacin y de Estudios
Av a n z a d o s , D e p a r t a m e n t o d e B i o t e c n o l o g a y
Bioqumica, Km. 9.6 Libramiento Norte Carr, IrapuatoLen Irapuato G to-M XI CO, CP.36821. E m a i l :
dvillamaring1400@alumno.ipn.mx
S. rebaudiana produces edulcorant (steviol glycosides) and
antioxidant compound (phenols and flavonoids).
Micropropagation of S. rebaudiana in semisolid media causes
hyperhydricity in tissues and a low rate of shoot multiplication. Temporary Immersion Bioreactors (TIB) is an alternative system to generate micropropagated plants with better characteristics of develop and physiological. The aim of
this study was to implement the S. rebaudiana micropropagation in TIB and to know the phytochemical profile
of micropropagated plants. Nodal segments (3 cm) were
used as explants and planted in a glass container (1 L) with
MS medium (300 mL) and was evaluated the effect of
phytohormones (mg L-1): IBA (0.37), AIA (0.25), IBA +
BAP (0.1 + 0.25), IBA + AIA (0.1 + 0.25), IBA (2.0) and
BAP (2.0). The content of steviol glycosides, phenols, and
flavonoids were evaluated by HPLC. The best condition to
obtain plants was adding only IBA in the culture media; in
the which, was generated 10.7 shoots per explant, with root
of 1.4 cm and 44 leaves per plant. The profiles of steviol
glycosides of plants acclimated and the plant mother were
similar. The plants acclimated in greenhouse showed a
content of stevioside, rebaudioside A and dulcoside A of
59.6, 3.5, and 4.9 mg g-1 dw, respectively; and the content
of stevioside, rebaudioside A and dulcoside A in the mother plant was 45.6, 3.2 and 4.1 mg g-1 dw, respectively. The

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mother plant, TIB shoots, and acclimated plants produce a

similar profile of phenols and flavonoids. We conclude that
the TIB system is an alternative for the massive propagation of S. rebaudiana plants with the same phytochemical
profile that the mother plants.
P-2015
Establishment of In Vitro Culture of Bougainvillea
glabra Choise Variety Sorpresa Mexicana. C. M.
RODRIGUEZ-SALAZAR, J. Romn-Reynosa, S.
Evangelista-Lozano, and A. R. Jimnez-Aparicio.
Instituto Politcnico Nacional, Centro de Desarrollo de
Productos Biticos, Departamento de Biotenologa,
Carretera Yautepec-Jojutla, Km. 6, calle CEPROBI No. 8, Col.
San Isidro, Yautepec, Morelos, MXICO, C. P. 6273. Email:
crodriguezs1405@alumno.ipn.mx
Bougainvillea glabra Choise (buganvilia), belonging of
Nyctaginaceae family, is an ornamental woody shrub
with a wide ranges varieties. Studies have been shown
that bracts have pigments as betacyanins, which are interest in the food industry. So the aim of this study was
establishment of in vitro culture of the variety Sorpresa
in semisolid medium. To callus induction, was started
from explants of young leaves, were disinfected and
seeded on MS to 50% basal medium, for isolation.
Later were transferred to culture medium MS to 100%,
added with plant growth regulators (PGR); cytokinin
KIN and the auxin 2, 4-D (2, 4-D dichorophenoxyacetic
acid), in different concentrations. Culture was incubated
at 25C in total darkness. Observations were realized
every 3 days under darkness. The callus tissue formation
was at 26 days after seedtime; the best concentration of
PGR was 1.0 mgL-1 to both regulators. Were realized
two subcultures with the same treatment, under photoperiodic 16 h light and 8 h darkness and 3 mol m-2s-1
light intensity. Established mass culture callus will be
possible implement another system culture, to evaluate
the cell growth kinetics and pigments content.
P-2016
Stacking of Two Intragenic Herbicide Resistance Genes into
Sugarcane for Improved Weed Control. T. N. SILVA, T. Oz,
and F. Altpeter. Agronomy Department, Plant Molecular and
Cellular Biology Program and Genetics Institute, University
of Florida - IFAS, Gainesville, FL. Email: tallyta@ufl.edu,
altpeter@ufl.edu
Herbicide resistant traits are used on more than 80% of the
estimated 134 million hectares of transgenic crops grown
worldwide annually, with 5-enolpyruvoylshikimate-3-

phosphate synthase (epsps) being by far the most utilized

(Green and Owen, 2011). EPSPS is a key enzyme in the aromatic amino acid synthesis. It is the target of the broad spectrum systemic herbicide glyphosate. The wide spread use of
glyphosate triggers natural response to the selection pressure
imposed, causing resistant weed populations to evolve rapidly.
An alternative to this issue is the stacking of herbicide resistant
genes, which will allow for herbicide rotation. As epsps gene
has great potential as a selectable marker as well, this study
aimed to develop intragenic selection protocol for sugarcane
using a mutant sorghum epsps as a selectable marker and to
stack a mutated acetolactate synthase (als) gene with epsps.
This will allow herbicide rotation and broad spectrum weed
control. Als is involved in the biosynthetic pathway of
branched-chain amino acids. It is the target of highly selective
ALS-inhibiting herbicides including sulfonylureas and
imidazolinones. Two vectors encoding mutant als and epsps
genes from Sorghum bicolor were constructed using a constitutive ubiquitin promoter and the HSP 3UTR from S. bicolor.
The minimal expression cassettes of mutant als and epsps
were co-introduced into sugarcane by biolistic gene transfer.
A selection protocol using glyphosate was developed by identifying suitable concentration for recovery of transformed
events. Selection protocol, regeneration of events and quantification of the transcript levels of als and epsps will be presented at the conference.
P-2017
Plant Regeneration in Atlantic White Cedar (Chamaecyparis
thyoides) Via Somatic Embryogenesis. CHANGHO AHN,
Paul M. Montello, Ryan Tull, and Scott A. Merkle. Warnell
School of Forestry and Natural Resources, University of
Georgia, Athens, GA 30602. Email: dosanahn@uga.edu
Atlantic white cedar (AWC; Chamaecyparis thyoides) is
an evergreen conifer and has been important for timber
production due to its wood quality. However, its populations in the eastern U.S. have declined over the past two
centuries. In vitro mass propagation technologies offer
opportunities to expedite mass clonal propagation of elite
trees. In particular, somatic embryogenesis (SE) has
shown promise for improvement of important coniferous
species and could aid greatly with germplasm conservation and restoration efforts. The objectives of the study
reported here were to: 1) determine the effect of plant
growth regulators (PGRs) and collection date on induction
of SE in AWC, 2) test the effect of the combination of
abscisic acid (ABA) and activated carbon (AC) on AWC
somatic embryo production, and 3) optimize the range of
polyethylene glycol (PEG) concentrations and molecular
weight for their potential to promote somatic embryo production. Immature AWC cones were collected from three

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source trees during July and August. Whole megagametophytes

with zygotic embryos were cultured on a modified half-strength
EM with three different PGR treatments. Both PGR treatment
and cone collection date had significant effects embryogenesis
induction (P < 0.001 and P = 0.002, respectively), with EM
with no PGRs giving the highest embryogenesis induction.
While ABA concentration did not affect somatic embryo production (P = 0.547), AC had a significant effect (P < 0.001),
with 2 g l-1 producing more embryos than 0 g l-1. We also found
that PEG concentration significantly affected somatic embryo
production (P < 0.001), although molecular weight of the PEG
did not (P = 0.586). Five g l-1 PEG (4000) produced the highest
numbers of cotyledonary-stage somatic embryos. Our study
can help improve AWC somatic embryo production efficiency.
This system may also be useful for somatic embryo development and maturation in other Chamaecyparis species.

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was 26 1.4 , whereas only 19 0.6 flower buds were formed

on the untransformed plants. Furthermore, we didnt observe
any morphological differences in the flower formation and fruit
development between all heat shock treated and untreated
plants. This study demonstrated that the FT gene expression
identified at mRNA level could be successfully induced after 2
to 4 hours of the heat treatments and could lead to the increased
number of flowers but not a shorter flowering cycle in cotton.
P-2019
Effects of Transgenically Expressed Antimicrobial Peptides in
Wheat on the Pathogenic Fungi and Bacteria. J.
BRUNGARDT, K. Neugebauer, B. Tian, N. Tyagi, C. R.
Little, and H. N. Trick. Department of Plant Pathology,
Kansas State University, 4024 Throckmorton Plant Sciences
Center, Manhattan, KS 66506. Email: jxbrungardt@ksu.edu

P-2018
Effect of Heat Inducible Flowering Locus T (FT) Gene on the
Flowering in Cotton (Gossypium hirsutum L.). S.
BARAMPURAM, G. Allen, and S. Krasnyanski. North
Carolina State University, Department of Horticultural
Science, Campus Box 7550, Partners II Bldg, Room 1200,
Raleigh, NC 27695-7550. Email: sbaramp@ncsu.edu
It was shown that the expression of Flowering Locus T (FT)
was effective in promoting early flowering in various plant
species. A cotton plant with a shorter flowering cycle could
accelerate breeding efforts and speed up the development of
elite cotton germplasm. In this study we demonstrate the effect
of the Arabidopsis FT gene expression under control of heat
shock promoter in transgenic cotton plants. One month old T2
transgenic plants transformed with Arabidopsis FT (flowering
locus T) gene under control of the soybean heat shock inducible promoter (HSP) were used for heat shock treatments to
induce the FT gene expression. Transgenic HSP:FT positive
and untransformed plants were subjected to the heat treatments for 1 h daily at 42C for 2, 4, 6 and 8 days, respectively.
All treated plants including transgenic, untransformed and untreated HSP:FT plants were moved to the green house for
further plant development. RT-PCR analysis of the leaf samples collected from the heat shock treated HSP:FT plants
showed the expression of FT gene at mRNA level and the
absence in untransformed and untreated HSP:FT plants. All
plants were scored for the time of flowering, number of flower
buds and fruits developed on the plants. No significant difference was recorded in the flowering time of transgenic and
untransformed cotton plants. However, the number of the flower buds formed on HSP:FT plants treated with the heat shock
for 2 and 4 days is higher in comparison to the 6 and 8 days
treated and untreated plants. The average number of the flower
buds formed on HSP:FT plants after 2 days of heat treatments

Antimicrobial peptides (AMPs) are produced in almost all

living systems and are an important line of defense against
bacterial and fungal pathogens. Several classes of AMPs have
been transformed into plants to successfully confer disease
resistance to plant pathogens. We have selected three AMP
genes that are highly efficient in their native systems and
expressed them in wheat (Triticum aestivum) under the control
of the maize ubiquitin promoter. Using biolistic transformation, we have independently co-bombarded immature wheat
embryos with these constructs and pACH20, containing the
gulfosinate resistance gene (bar), and generated several independent events. These events were challenged with multiple
important wheat pathogens. Preliminary results indicated that
the transgenic lines expressing one AMP (DONAR32)
inhibited the growth of Xanthomonas translucens, the causal
agent of bacterial leaf streak. We will also report the results of
transgenic lines challenged with an array of fungal wheat
pathogens including Fusarium graminearum, the foliar infecting Puccinia triticina and the seed borne pathogen Tilletia
laevis.
P-2020
Use of Automation for Explant Preparation, Handling
Agrobacterium Infection and Plating of Explants on Cocultivation Media for Soybean Transformation. SIVA
CHENNAREDDY1, Lyle McCarty2, Toby Cicak1, Geny
Anthony 1 Tom Parsons 2 , Eddie Gee 2 , K atherine
Armstrong3, Paul Morabito2, and Rodrigo Sarria3. 1Dow
AgroSciences, West Lafayette, IN; 2 Dow Chemical,
Midland, MI; and 3Dow AgroSciences, Indianapolis, IN.
Email: schennareddy@dow.com
At Dow AgroSciences, efficient and robust transformation
protocols have been developed for high throughput transgenic

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soybean production. Split seed soybean explants are prepared

from surface-sterilized and water imbibed seeds. To prepare a
soybean explant manually, a researcher uses forceps and scalpel to trim the embryonic axis, split the seed into two halves,
and removes the seed coat. The explants are treated with
Agrobacterium solution and placed on solid co-cultivation
media. Dow AgroSciences has successfully automated split
seed explant preparation, Agrobacterium infection of explants, and placement of the explant onto co-cultivation media. This automation improves efficiency in transgenic plant
production, increases capacity, improves reliability and improves safety for the researchers. Three steps of the transformation procedure have been automated using this system and
will be discussed: 1) Split-seed explant preparation, 2)
Agrobacterium infection of explants and 3) Placement of
Agrobacterium treated explants onto co-cultivation medium.
Automation of these steps creates cost savings by reducing
man-hours required to perform those activities. Safety is significantly improved for the individual researchers by removing the need to use scalpels, reducing the risk of ergonomic
problems, and reducing the time spent handling
Agrobacterium. Finally, this automation tool results in increased quality and consistency by reducing experimental variability.
P-2021
Isolation and Characterization of Four Rubulose-1,5bisphosphate Carboxylase/Oxygenase Small-subunit
Promoters from Soybean. ERIC DEAN and John J. Finer.
Department of Horticulture and Crop Science, OARDC/The
Ohio State University, 1680 Madison Ave. Wooster, OH
44691. Email: dean.1063@buckeyemail.osu.edu,
finer.1@osu.edu
Promoter regions located upstream of coding sequences contain regulatory elements and motifs that are primarily responsible for transcriptional regulation. Genes can be regulated in
response to developmental stage, tissue type, or various environmental stimuli. Many plant transgenes are regulated by
constitutive promoters such as the Cauliflower Mosaic Virus
35S (35S) promoter. While constitutive transgene expression
has been valuable for basic and applied research, a more
targeted approach to transgene regulation is desirable for more
precise control of transgene expression. The discovery of novel promoters and promoter elements adds to the available tools
for transgene regulation, and provides a deeper understanding
of the complexity of transcriptional regulation. Four soybean
RuBisCO small-subunit gene promoters (GmRbcs) were
selected based on sequence homology of the gene coding
regions. Three of the predicted coding regions (GmRbcs13) were very similar to each other while the fourth coding
region (GmRbcs4) only showed about 50% amino acid

homology. RNAseq analysis of the native Rbcs genes

showed high expression for GmRbcs1-3 in young leaf tissue while highest expression of the GmRbcs4 gene was in
root and nodule tissue. All promoters were then isolated
and cloned upstream of the green fluorescent protein (gfp)
gene and introduced into lima bean cotyledons via particle
bombardment. Images were collected every hour for
100 hours and image analysis was used to quantify promoter strength. The Rbcs1 promoter showed the highest
transient expression followed by Rbcs4. All of the Rbcs
promoters showed relatively high transient expression
levels when compared to the 35S promoter. Following introduction of these promoters into soybean hairy roots
using Agrobacterium rhizogenes, the Rbcs1 promoter
again showed high expression. Characterization of these
promoters will be useful in developing more targeted and
efficient transgene regulation, as well as contribute to our
understanding of transcriptional regulation.
P-2022
Development of TA-cloning Vectors for Promoter Screening
in Monocots and Dicots. TANIYA DHILLON 1 , E. M.
Dlugosz1, J. N. Burris1, S. C. Lenaghan2, 3, and C. N.
Stewart Jr. 1 . 1 Department of Plant Sciences, 2431 Joe
Johnson Drive, 252 Ellington Plant Sciences Building,
Knoxville, TN 37996; 2 Center for Renewable Carbon,
University of Tennessee, Knoxville, TN 37996; and
3
Department of Mechanical, Aerospace and Biomedical
Engineering, University of Tennessee, Knoxville, TN 37996.
Email: tdhillon@utk.edu
Promoter-reporter assays are commonly used to study
the strength and tissue-specificity of a promoter in a
given system. To facilitate efficient screening of plant
promoters, we have designed TA-cloning vectors for
analyses in monocot and dicot plants. These vectors
contain two reporter genes an endoplasmic reticulum
(ER) targeted Green Fluorescent Protein (mGFP-5ER)
under the control of either the switchgrass (Panicum
virgatum L.) ubiquitin promoter (PvUbi1+3) for use in
monocots (Monocot Vector) or the Cauliflower Mosaic
Virus (CaMV) 35S promoter for use in dicots (Dicot
Vector), and a variant of the Red Fluorescent Protein
(pporRFP) under the control of a test promoter. The test
promoter can be cloned-in using TA-cloning, which is a
convenient and economical method compared to other
cloning approaches. The vectors use Kanamycin resistance as the bacterial selection marker and are best suited for transient expression studies using particle bombardment or PEG-mediated transformation. As a proof
of concept, we clonedPvUbi1+3, PvUbi2, 35S, and NOS
promoters in both the monocot and dicot vectors and

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tested their strength in switchgrass and tobacco BY-2

protoplasts using PEG-mediated transformation followed
by microscopy and protein quantification assays.
Consistent with previously published data, our results
showed that PvUbi1+3 and PvUbi2 are strong monocot
promoters, whereas 35S and NOS effect stronger gene
expression in the dicot system. These vectors can also
be used to identify promoter regions or motifs essential
for downstream gene expression using promoter deletion
mutants and subsequent cloning. To this effect, we have
tested deletion mutants of the PvUbi1+3 promoter using
the monocot vector to identify 5 cis-regulatory regions
essential for its activity.
P-2023
E c t op i c M a n i p u l a t i o n o f t h e F l o r a l / F r u i t O rg a n
Developmental Pathway by Grapevine Phylloxera
(Daktulosphaira vitifolia) as a Mechanism for Gall
Formation on Leaves of Vitis. TRUDI N. L. GRANT1,
Zhijian T. Li1, Deborah A. Dean1, Jack C. Schultz2, and
Dennis J. Gray1. 1University of Florida/IFAS, Mid-Florida
Research & Education Center, Apopka, FL 32703 and
2
University of Missouri, 105 Bond Life Sciences Center,
Columbia, MO, 65211. Email: trudigrant@ufl.edu
The highly specialized galls induced by grape phylloxera
(Daktulosphaira vitifolia Fitch) on leaves of grapevine are
structurally similar to floral and fruit structures. It is not
understood as to how the insect directs the hosts natural
resources to be diverted for its own shelter and food but it
is proposed that phytohormones are responsible for activation and redirection of flower/fruit developmental pathways. Molecular insights into the mechanisms underlying
this complex insect-plant interaction generated through
RNA sequencing data and RT-PCR revealed several key
genes involved in floral development and hormone signaling. Expression profiles of the following endogenous
genes were determined to vary during gall development
compared to non-galled controls: LEAFY, the Vitis homologs that regulate the transition to flowering, WUSCHEL,
which maintains the pluripotent stem cell pool in the shoot
apical meristem and AGAMOUS, a carpel-identity gene.
Transgenic plants with targeted silencing of these genes
were created to determine the correlation between changes
in their expression and successful galling. Evaluation of
transgenic selections of Seyval Blanc (Vitis spp.), a susceptible hybrid cultivar, by direct challenge with
D. vitifolia revealed plants with increased resistance to
phylloxera, with some exhibiting limited-to-no gall formation at all. This is an important step in understanding the
insects ability to manipulate plant morphogenesis in order
to create such specialized structures. Controlling gene(s)

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identified via this knock-out approach can be modified to

create phylloxera resistant cultivars.
P-2024
Presence and Effects of Melatonin in Transgenic Switchgrass
Overexpressing Ovine AANAT and HIOMT Genes. YANHUA HUANG1, SIJIA LIU1, Shan Yuan1, Jiu-Chang He2,
and Yun-Wei Zhang1. 1Department of Grassland Science,
College of Animal Science and Technology, China
Agricultural University, Beijing, 100193, CHINA and 2Key
Laboratory of Animal Genetics and Breeding of the Ministry
of Agriculture, National Engineering Laboratory for Animal
Breeding, College of Animal Science and Technology, China
Agricultural University, Beijing, 100193, CHINA. Email:
zywei@cau.edu.cn; huangyanhua666@163.com
Switchgrass (Panicum virgatum L.) has been increasingly
recongnized as an important dedicated penennial bioenergy
crop. A major goal of the plant is to improve the biomass
production through metabolic engineering. Melatonin is a
well-known bioactive molecule with an array of healthpromoting properties, e.g. potent antioxidant capability. Its
valuable to detect the physiological function of melatonin in
transgenic switchgrass overexpressing the homologous sheep
AANAT and HIOMT genes , which are responsible for the last
two steps of melatonin synthesis. We generated transgenic
switchgrass using Agrobacterium-mediated gene transformation under the constitutive ubiquitin promoter. The integration
and expression of the transgenes were confirmed in transgenic
seedlings by PCR, RT-PCR and Southern analyses. High expression levels of transgenes were observed by quantitative
real-time PCR. Compared with wild-type and transgenic control (EV; expressing the empty vector only), the transgenic
switchgrass showed enhanced melatonin levels. Moreover,
melatonin concentrations varied among switchgrass tissues
(roots, stems, leaves and spikes) and different treatments (light
and dark). Relatively high concentrations of melatonin was
discovered in roots while other tissues decreased gradually
from the stems and leaves to spikes. In addition, the levels
of melatonin in switchgrass had been found to be
photoresponsive, with higher content in dark periods but lower content in light. Finally, the transgenic switchgrass with
high levels of melatonin exhibited increased heights, suggesting the significant potential for melatonin to act as a regulator
in plant growth. These findings would laid foundation for
further exploring the molecular mechanism of melatonins
effect on switchgrass.
P-2025
Resistance to Fusarium oxysporum f. sp. gladioli in
Transgenic Gladiolus Plants Expressing Either a Bacterial

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Chloroperoxidase or Fungal Chitinase Genes. K. KAMO1, D.

Lakshman1, R. Pandey1, M. A. Guaragna1, P. Okubara2, K.
Rajasekaran3, J. Cary3, and R. Jordan1. 1Floral and Nursery
Plants Research Unit, USDA, National Arboretum, Beltsville,
MD; 2Root Disease and Biological Control Research Unit,
USDA, Pullman, WA; and 3Food and Feed Safety Research
Unit, Southern Regional Research Center, USDA, New
Orleans, LA. Email: Kathryn.Kamo@ars.usda.gov
Three antifungal genes, a non-heme chloroperoxidase from
Pseudomonas pyrrocinia, and an exochitinase and endochitinase
from Fusarium venetanum under regulation by the CaMV 35S
promoter, were used to transform Gladiolus for resistance to
Fusarium oxysporum f. sp. gladioli. Gladiolus plants were confirmed to be transgenic by Southern hybridization. Semiquantitative RT-PCR of RNA isolated from leaves and roots
demonstrated expression of the Fusarium exochitinase and
endochitinase genes in transgenic plants compared to controls.
All transgenic plants expressing the Fusarium exochitinase or
endochitinase gene had chitinase activity higher than that of the
control plants. Semi-quantitative RT-PCR verified that three of
the four plant lines with the chloroperoxidase gene expressed the
transgene in leaves and roots while no expression was detected in
control plants. Western hybridization confirmed the presence of
the chloroperoxidase protein in both leaves and roots of transgenic plants. Cell extracts from one endochitinase plant line
inhibited growth of germinated F. oxysporum spores more
consistently than extracts from the four chloroperoxidase
and three endochitinase plant lines. Three
chloroperoxidase, two exochitinase, and three
endochitinase transgenic plant lines sustained a significantly lower density of hyphae on roots compared to roots of
non-transformed Gladiolus plants three to four days following exposure of the roots to Fusarium. Shoots from two
plant lines, one containing a chloroperoxidase and the other
an endochitinase gene, had less necrosis when rated on a
scale of 1-3 and appeared visually to be healthier and without obvious Fusarium infection than non-transformed, regenerated Gladiolus plants 17-21 days following exposure
to Fusarium oxysporum.
P-2026
Molecular Genetic Evaluation and In Vitro Selection of Onion
(Allium cepa L.) Plants Resistant to White Rot Disease. A. A.
MOHAMED1, A. T. Abdel-Rahem2, K. Z. Ahmed2, and S. A.
Osman 2 . 1 Field Crops Research Institute, Agricultural
Research Centre, Giza, 12619, EGYPT and 2Department of
Genetics, Faculty of Agriculture, Minia University, El-Minia,
61517, EGYPT. Email: abdelraheemelhakeem@yahoo.com
In vitro selection is one of the most effective and efficient
methods for plant improvement. In the present work, we

established a suitable protocol for in vitro selection for

Alliumwhite rot disease (Sclerotium cepivorum) tolerance in
Egyptian onion varieties (Giza 20, Giza 6 and Beheri Red).
Oxalic acid (the phytotoxin produced by Sclerotium
cepivorum) was used as selective agent. The response of onion
explants to OA were studied. Cotyledon explants from the
varieties were cultured on toxic MSBDK medium supplemented with 0, 3, 6 and 12 mM OA. The mean percentage survival of calli on MSBDK free toxic medium was
70.7% for all tested onion genotypes, however, MSBDK
stressed medium with 3 mM OA reduced the percentage
of viable calli to 42.1%, and the highest OA concentration (12 mM) completely inhibited calli induction from
cotyledons explants. Toxicity of OA to onion calli was
calculated as an LD80 for OA where a medium supplement with 3 mM OA retarded 80% of calli growth which
delivered a suitable in vitro selection protocol. Among
156 tested calli of Beheri Red only 23 calli (14.7%)
survived on toxic medium for 45 days. Similarly there
was 15.6% survival for Giza 20 calli. However 40.1% of
the Giza 6 calli survived. Plantlets were regenerated from
surviving calli and transplanted to ex vitro and formed
bulb after acclimatization. DNA (RAPD) analysis was
applied, six arbitrary 10-base primers were used, the results of DNA fragments showed unique bands where
exclusively present in all regenerated plants (from putative resistant calli) and not found in donor plants, in
primer OPA-6 found band with size (689 bp) and in
OPA-7 primer band with size (962 bp). The results presented here offer a useful method for the production of
germplasm with potential resistance to white rot disease.
P-2027
A Streamlined Approach of Gene Stacking Based on Site
Specific Recombinases and Nucleases. B. P. PATHAK1,2, J.
Underwood2, S. Nandy2, S. Zhao2, and V. Srivastava2. 1Cell
and Molecular Biology Program, University of Arkansas,
Fayetteville, AR 72701 and 2Department of Crop, Soil &
Environmental Science, 115 Plant Science Building,
University of Arkansas, Fayetteville, AR 72701. Email:
bppathak@email.uark.edu
Multigene transformation for various trait deployments in
the transgenic crops pose the challenges of serial transformations, marker-removal, and random gene integration.
Here, we describe a streamlined strategy of recombinasemediated site-specific gene integration and engineered nucleases for gene stacking and marker gene removal. We
could successfully create a site specific integration locus
in rice using the Cre-lox system. The selection marker
flanked by I-SceI or ZFN sites was excised out at ~20
30% efficiency. Subsequently, in the retransformation, we

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integrated the second gene-of-interest adjacent to the first

one. We could also efficiently excise out the selection
marker using a heat inducible I-SceI. However, the efficiency of heat inducible ZFN for marker removal appeared to be much lower, and needs further investigation.
This study is a proof of concept while testing each components functional efficiency. A combination of these
components can drive integration of new genes into the
pre-engineered site leading to stacking of potentially unlimited number of genes.
P-2028
Characterization and RNAi Suppression of a Tonoplast
Monosaccharide Transporter (TMT) of Sugarcane. SEYED
ALI RAVANFAR1, Ratna Karan1, Hui Liu2, John Shanklin2,
and Fredy Altpeter 1 . 1 Agronomy Department, Plant
Molecular and Cellular Biology Program, Genetics
Institute, University of Florida - IFAS, Gainesville, FL
32611; and 2Biosciences Dept., Brookhaven National
L a b 4 6 3 , 5 0 B e l l Av e , U p t o n , N Y. E m a i l :
altpeter@ufl.edu; ravanfar@ufl.edu
Sugarcane is a prime feedstock for commercial bioethanol
production. Metabolic engineering might lead to advanced
biofuels with higher energy density. Therefore, we are exploring the prospects of diverting the carbon flux in sugarcane.
This strategy involves the analysis of combinations of gene
expression cassettes supporting biosynthesis of highly concentrated stores of metabolic energy. Here we evaluate the role
of alternative sugar transporters for carbon partitioning.
Quantitative RT-PCR data of genes, homologous to members
of the tonoplast monosaccharide transporter (TMT) family will
be described from different sugarcane tissues. Phenotypes following RNAi suppression of a candidate TMT gene will also
be presented.
P-2029
Chemical Mutagenesis of Sugarcane to Confer Herbicide
Resistance. SEYED ALI RAVANFAR, Calvin Howard, Tina
Strauss, and Fredy Altpeter. Agronomy Department, Plant
Molecular and Cellular Biology Program, Genetics Institute,
University of Florida - IFAS, Gainesville, FL. Email:
altpeter@ufl.edu; ravanfar@ufl.edu
Sugarcane is an important C4 crop for commercial production
of table sugar and bio-ethanol. Costs and delays associated
with regulatory approval for a transgenic event are substantial
impediments to the commercialization of transgenic crops.
Generation of randomly mutagenized events can result in crop
improvement and does not require regulatory approval.
Acetolactate synthase (ALS) is a key enzyme in the

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biosynthetic pathway of branched-chain amino acids, and also

a target for ALS-inhibiting herbicides including sulfonylureas
and imidazolinones. Mutations in the ALS gene that confer
herbicide resistance are well known. In this study, embryogenic calli of two commercially important sugarcane cultivars
were treated with two different chemical mutagens, sodium
azide (NaN3) or ethyl methanesulphonate (EMS) in different
concentrations and for different exposure times. For selection
of putative herbicide resistant ALS-mutants the treated calli
were subcultured on media supplemented with the herbicide
bisparibac sodium. Response of the regenerated plantlets from
alternative mutagen treatments to different concentrations of
ALS-type herbicides will be presented
P-2030
Aeroponic: Hi-tech In-vitro Cultivation Technique for Disease
Free and Clean Leafy Vegetables Production. KALPANA
SENGAR. Tissue Culture Lab, College of Biotechnology,
Sardar Vallabh Bhai Patel University of Agriculture &
Te c h n o l o g y, M e e r u t - 2 5 0 1 1 0 , I N D I A . E m a i l :
kalpana.sengar19@gmail.com
Aeroponic technique allows a more efficient use of water and
nutrition in a mist form to apply in a system, as well as a better
control of climate and pest factors. The degraded arable land
and soil health due to unrestricted application of chemicals
and fertilizers results in poor food quality production.
Aeroponic production increases crop quality and productivity,
with higher competitiveness and economic incomes.
Aeroponic effectively prevent fungal disease due to water deposition on leaf surface and eliminates the possibilities of root
diseases by allowing sufficient porosity for drainage of excess
water as well as increases oxygen availability to the root zone.
We used three vegetables crops, Spinach, lettuce potatoes and
tomatoes, germination from seeds occurs within 10 days in all
vegetables varieties and full crop grows according to its crop
cycle which yielded double quantity disease free and residual
free clean hygienic vegetable produce. Aeroponic system
complemented by plant tissue culture promises a great potential to transform seed and explants to full crop production.
Considering the potential benefits of the system such as rapid
production of seed, spacious, good nutrient monitoring system, improvement of growth and survival rate of plantlets,
constant air circulation and ecologically friendly, this system
has a potential of revolutionizing vegetable production.
P-2031
Development of Reproducible Micropropagation Method and
Screening of Bioactive Metabolites from Tinospora cordifolia
(Willd.) Miers ex Hook. F. Thoms. VARTIKA SRIVASTAVA
and Rakhi Chaturvedi. Department of Biosciences and

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Bioengineering, Indian Institute of Technology Guwahati,

Assam-781039, INDIA. Email: rakhi_chaturvedi@iitg.
ernet.in, rakhi_chaturvedi@yahoo.co.uk
Tinospora cordifolia, of the family Menispermaceae, is one of
the ancient Ayurvedic rejuvenating plants, having promising
activity against diabetes, cancer, dengue and malaria. It contains plethora of metabolites like, alkaloids and terpenoids.
Utilization of the plant for the enormous benefits may lead
to the erosion of plant diversity and the metabolite profile
can be affected by the environmental fluctuations. Therefore,
plant tissue culture technology is followed for large scale
propagation of Tinospora using in vitro nodal segment culture, a significant way of obtaining true-to-type plantations.
Nodal segments bearing axillary buds were surface sterilized
with 0.4% Tween-20, 0.1% HgCl2 and 70% ethanol under
aseptic conditions and inoculated in Murashige and Skoog
medium supplemented with benzylaminopurine (5 M).
Fully elongated shoots having an average of 6 nodes per shoot
with an average height of 7 cm were obtained in four weeks.
Occasionally, multiple shoot proliferation was also observed
in some of the cultures in the same medium composition
which also imparts large scale propagation of shoots. The
in vitro leaves and stem could be utilized for chemical analysis
of bioactive compounds. Alkaloids, like berberine and
palmatine, have been identified and characterized from field
grown Tinospora. Extracts have been prepared from stem and
leaves in methanol and compounds were analyzed through
Thin Layer chromatography (TLC) using chloroform and
methanol. The extracts were further purified using High
Performance Liquid Chromatography (HPLC) using acetonitrile and 0.01M KH2PO4 (80:20) solvent system. Presence of
berberine and palmatine at 336 and 352 m/z values have been
confirmed by Mass Spectrometry of TLC and HPLC eluents.
Corresponding study have been performed in in vitro cultures
of Tinospora which can be used as a reproducible protocol for
the extraction of bioactive metabolites throughout the year,
irrespective of seasonal variations.
P-2032
Effect of Temporary Immersion Bioreactor on Haploid Embryo
Regeneration of Pepper (Capsicum annuum L.). YUENYAD
TEERAWATSAKUL1 and Darush Struss2. 1Department of
Biology, Faculty of Science, Chaing Mai University, 50002,
THAILAND and 2Hortigenetics Research (S. E. Asia) Ltd., 7
M. 9 Chaing Mai Praw Rd., Sansai, Chiang Mai 50270,
THAILAND. Email: yongyuenyad@gmail.com,
yuenyad.teerawatsakul@eastwestseed.com
Double haploid has been used in pepper breeding programs by
several techniques. Shed microspore culture is the new technique
to culture pepper microspore, however it has a limitation in

regeneration about 15% of 100% the induction stage

(Somsamai et al., 2014). The objective of this studies was to
apply temporary immersion bioreactor (TIB) in pepper haploid
plant regeneration to improve the regeneration efficiency by
using strength MS liquid medium containing with 0.1 M
BA. Transferred haploid embryo in globular stage to TIB and
using immersion time 2 minutes every 4 hours and 2 minutes
every 6 hours. The results shown that regeneration efficiency rate
increased 4 times in the quantity of haploid regenerant (avg. 30
plantlets), 2 times faster in haploid plantlet growth (avg. 3 cm.
height in 3 weeks). The different immersion times has no significant effect on plant growth. TIB could be benefitial for pepper
shed microspore culture to improve the regeneration stage.
P-2033
The Effects of Hexavalent Chromium on Vigna Radiata
Germination, Root System Architecture and Growth. R.
PRIYA SAHAI1, M. Varma2, and R. R Sahai3. 1Apple Valley
High School, 11837 Navajo Road, Apple Valley, CA 92308;
2
Trika Medical Inc., 18095 Hwy 18, Ste. B, Apple Valley, CA
92307; and 3Georgetown University, 37th and O St. NW,
Washington, DC 20057. Email: ritikapriyasahai@gmail.com
Hexavalent Chromium (CrVI) is a soil and water pollutant in
Hinkley California. Toxic effects manifest as disease & cancer
in local population. 10% of Hinkleys population has cancer
whereas the national level is 4%. I examined the effects of
CrVI on Vigna Radiata seeds, germination, plant growth, root
architecture and root system. V. Radiata seeds were germinated using water from Hinkley CrVI, tested at 3.1ugm/ l, Apple
Valley(AV) CrVI, 0.33 ugm/l, & Arrowhead bottled water
CrVI, 0. Experiment conducted in constant conditions of 720
F 12hr light/dark cycle and daily watering. Growth observed
twice daily for 2 wks. Germination rate, rate of sprout occurrence, root length, hair appearance noted. At 2 wks root samples were microscopically examined. Seeds watered with
Hinkley water germinated with initial sprout development .
At 1 wk sprouts were 2mm and about 1 cm at the end of
2wks. No root hairs or cotyledons appeared. Sprouts did not
grow to have leaves. 93% of the seeds germinated and 88%
sprouted. AV watered seeds had sprouts of about 4 mm at 1
wk and 1 cm at 2 wks. After initial germination cotyledons
appeared and some leaves. 100% of the seeds germinated and
93% sprouted. Bottle water plants at 1 wk were 5 mm in root
length. They were about 1.2 cm at 2 wks. They grew root
hairs, cotyledons, and also leaves. All of the seeds had germinated and sprouted. At the beginning of the experiment, the
plants seemed to germinate and sprout at the same rate, but
by day 5 it was clear that the plants receiving bottled
water grew the best and the plants receiving the most
chromium positive water (that from Hinkley) were growing the slowest. Microscopically the root diameter was

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less & organelles large compared to AV and bottled water

roots. While germination was not stopped by the presence
of CrVI, there was no growth after the initial sprouting.
My results show that CrVI is harmful to plants, germination is slowed down, and overall growth stunted in plants
treated with CrVI rich water as opposed to chromium
negative water.
P-2034
Downregulation of MTHFR and COMT in Switchgrass
Decreases Lignin Content and Induces Lesion-mimic Cell
Death. SIJIA LIU1,2, Chunxiang Fu1,3, Jiqing Gou1, Yunwei
Zhang2, and Zeng-Yu Wang1. 1Forage Improvement Division,
The Samuel Roberts Noble Foundation, Ardmore, OK 73401;
2
Department of Grassland Science, College of Animal
Science and Technology, China Agricultural University,
National Energy R&D Center for Biomass (NECB), Beijing
100193, P. R. CHINA; and 3CAS Key Laboratory of Biofuels
and Shandong Provincial Key Laboratory of Energy Genetics,
Qingdao Institute of Bioenergy and Bioprocess Technology,
Chinese Academy of Sciences, Qingdao 266101, P. R.
CHINA. Email: sliu@noble.org
Methylenetetrahydrofolate reductase (MTHFR) catalyzes
the formation of S-adenosyl-L-methionine (SAM), which
is later consumed by lignin pathway enzymes. The maize
brown midrib 2 (bm2) phenotype is caused by MTHFR
mutation. Disturb the function of MTHFR in bm2 mutant
affected the expression of the genes involved in ethylene
and jasmonate pathways. Caffeic acid O-methyltransferase
(COMT) is one of the key enzymes involved in lignin
biosynthesis. It is also a defense-related protein associated
with resistance or super-susceptibility to pathogens infection. To date, there has been no report on the interaction of
MTHFR and COMT in plants. Here, we report the downregulation of MTHFR and COMT by RNAi to study the
function of these two genes and their relationships to lignin
biosynthesis and defense signaling in switchgrass, a potential bioenergy crop. Downregulation of MTHFR in COMTdeficient background decreased lignin content and altered
lignin composition. Interestingly, novel lesion mimic leaf
phenotypes were observed. Trypan blue staining showed
the leaf lesion cells were died. Lowest expression of
MTHFR (10-15%) in COMT-deficient lines showed high
density of lesions, moderate expression lines (20-60%)
had moderate density of lesions. Lesions were not found in
the lines with no change of MTHFR expression or single
MTHFR RNAi lines. The results indicate that the phenotype
was regulated by both MTHFR and COMT. Furthermore, the
density of the lesions is correlated with MTHFR expression.
DAB staining revealed the accumulation of hydrogen peroxide.
Microarray analysis demonstrated significant induction of genes

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involved in oxidative and defense-related processes. This study

provides a novel finding that MTHFR and COMT have interactive effects in hypersensitive response (HR)-mediated cell death.
P-2035
Preparing Future Brassica juncea Plant Towards Dry and
Saline Areas: Gene Pyramiding of Antioxidant Enzymes.
NEELAM PRABHA NEGI1, Vinay Sharma2, and Neera
Bhalla Sarin1. 1School of Life Sciences, Jawaharlal Nehru
University, New Delhi-110067, INDIA and 2Department of
Biotechnology, Banasthali University, Rajasthan, INDIA.
Email: sapphirenegi@yahoo.com
Reactive oxygen species (ROS) derived from molecular oxygen under biotic and abiotic stress such as salinity, drought,
extreme temperatures, heavy metals, UV radiation, atmospheric contamination, nutrient starvation and pathogen attacks, which have deleterious effects on cell metabolism.
The antioxidant metabolism protects cells from oxidative
damage caused by ROS, such as peroxidation of membrane
compounds, polysaccharide degradation, enzyme denaturation and DNA lesions Several enzymes act jointly to maintain redox status homeostasis. The antioxidative defense is
initated by SOD, which converts superoxide (O2-) radicals to
H2O2. The H2O2 that is also potentially harmful is converted
to non-toxic water and monodehydroascorbate by the APX
enzyme utilizing ascorbate as the electron donor. In the present study, genes for two different cytosolic antioxidant enzymes, superoxide dismutase (AhCuZnSOD) and ascorbate
peroxidase (AhcAPX) isolated from salt tolerant cell lines of
Arachis hypogaea were simultaneously over expressed in
highly important oilseed crop, Brassica juncea (Indian
Mustard) to check the effect on alleviation of abiotic stress.
Compared with untransformed control plants, transgenic
plants survived under longer period of water deficiency and
salinity stress and displayed improved recovery after rehydration. The enhanced levels of antioxidant enzymes in the
transgenic plants correlated with higher relative water content,
improved photosynthetic efficiency, less electrolyte damage,
elevated accumulation of compatible osmolytes, less
malondialdehyde as well as ROS accumulation and favorable
ionic balance under stress conditions compared to untransformed
controls. Our analysis showed that an increase in the activity of
antioxidant enzymes can significantly enhance salt and dought
tolerance in transgenic plants, suggesting an important role of
combined over expression of two antioxidant enzymes.
P-2036
Efficient Gene Delivery by Microinjection on Shoot Apical
Meristems of Watermelon. A. VINOTH1, R. Ravindhran1, G.
Ganesan2, M. Harikrishnan2, and Ajay Parida2. 1T. A. L. Samy

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Unit for Plant Tissue Culture and Molecular Biology,

Department of Plant Biology and Biotechnology, Loyola
College (Autonomous), Nungambakkam, Chennai 600 034,
INDIA and 2M. S. Swaminathan Research Foundation,
Biotechnology, Taramani, Chennai 600113, INDIA. Email:
vinoth.loyolite07@gmail.com, raviloyola1998@gmail.com
Direct gene transfer by microinjection of shoot apical meristems (SAM) was established for the production of transgenic
watermelon (Citrullus lanatus cv. Arka Manik). Successful
transformation was achieved by the use of different
Agrobacterium strains, pre-culture period and acetosyringone
(AS). SAM explants from 6-d-old in vitro germinated seedlings
were transformed with Agrobacterium strains EHA 105 and
LBA 4404 harboring the binary vector pCAMBIA 1301 containing hygromycin phosphotransferase gene (hpt) as the selectable marker and -D-glucuronidase (GUS) as the reporter gene.
The highest frequency of transformants (90.5%) was obtained
by pre-culturing the explants for 1 day in shoot regeneration
medium (SRM) containing MS salts and vitamins supplemented with 8.88 M BAP+2.85 M IAA+9.0 M AgNO3 and cocultivating with EHA 105 strain for 2 days in SRM containing
100 M AS. Thirty eight putative transgenic shoots developed
within 6 weeks of culture. Stable integration of the transgene
was confirmed by PCR and histochemical GUS staining assay.
GUS expression frequency of 79.2% was recorded from the
leaves of transformed shoots. Developed protocol in the present
investigation is simple and efficient for rapid, high-frequency
production of transgenic watermelon plants. This improvement
in the transformation frequency accelerates the incorporation of
novel traits into commercially important watermelon cultivars.
P-2037
Using the CHAID Data Mining Algorithm for Tissue Culture
Medium Optimization. MELEKSEN AKN1, Ecevit Eyduran2,
and Barbara M. Reed3. 1Oregon State University, Department
of Horticulture, ALS 4017, Corvallis, OR 97331; 2 Igdir
University, Agricultural Faculty, Department of Animal
Science, Biometry Genetics Unit, Igdir-TURKEY; and
3
USDA-ARS, National Clonal Germplasm Repository, 33447
Peoria Rd, Corvallis, OR 97333. Email: akinm
@oregonstate.edu
Tissue culture medium optimization is challenging, due to the
many chemical interactions and also from genotype variability. To make optimization processes practical, statistical approaches are needed that are more accurate and easier to interpret. The Chi-Squared Automatic Interaction Detection
(CHAID) data mining algorithm constructs simple visual decision tree diagrams through multi-directional node splitting
of heterogeneous data in homogeneous subgroups. The trees
show the significant salt cut-off concentrations and how these

values affect the importance of the other independent variables on the plant response. CHAID also allows analysis of
responses of several genotypes simultaneously, which could
contribute to developing a common tissue culture medium
rather than several genotype-specific media. The CHAID algorithm was used to analyze growth of shoots in a hazelnut
(Corylus avellana L.) tissue-culture media experiment. Driver
and Kuniyuki Walnut medium (DKW) salts (NH 4NO 3,
Ca(NO3)2.4H2O, CaCl2.2H2O, MgSO4.7H2O, KH2PO4 and
K2SO4) were varied from 0.5 to 3 DKW concentrations
with 42 combinations in a IV-optimal design. CHAID analysis
using a tree approach indicated that NH4NO3 at >2.75
DKW were required for good shoot quality and length.
NH4NO3 at <0.5 DKW and Ca(NO3)2.4H2O at <1.725
DKW were essential for higher multiplication and less callus.
The optimal growth medium for C. avellana was projected to
be: 0.5 NH4NO3, 2.8 KH2PO4, with the other basal salt
factors set at 1.0 DKW.
P-2038
Improving In Vitro Growth of the Endangered Dryland
Species, Arctomecon humilis. VALERIE C. PENCE 1 ,
Randall P. Niedz2, Linda Finke1, and Radhika Bhargava1.
1
Center for Conservation and Research of Endangered
Wildlife (CREW), Cincinnati Zoo & Botanical Garden,
3400 Vine Street, Cincinnati, OH 45220 and 2USDA-ARSU. S. Horticultural Research Laboratory, 2001 South Rock
Road, Ft. Pierce, FL 34945-3030. Email: valerie.pence
@cincinnatizoo.org
Arctomecon humilis (Apocynaceae, Dwarf bear-claw poppy)
is a federally endangered species found at only seven sites in
southwestern Utah. In vitro methods have been applied to this
species to increase numbers, but culture of A. humilis seedling
tissues initially produced only a bud-forming callus. A Design
of Experiments (DOE) approach was used in six experiments
to test the effects of nitrogen and other macronutrients (as
defined by MS medium), BAP, gelling agent, venting, and
light on callus growth, color, and bud production, and, as
shoots were produced from buds, on shoot growth of
A. humilis in vitro. Factors that had the greatest effects on
these responses were as follows: callus growth, bud color,
bud productionNO3-, and PO43+ and an NH4+ x NO3- interaction effect; bud and shoot hyperhydricity NO3- and
venting. Based on these results, and an additional comparison
of two bud-forming media, a shoot production protocol was
formulated. The bud-forming callus is grown on a modified
MS medium with no NH4+, 10 mM NO3-, 0.1 mg/L BAP,
Gelrite gel, and vented lids to produce buds, which are then
moved to a WP medium with 0.2 mg/L BAP, agar, and vented
lids to obtain growth of the buds into shoots. Rooting has been
attempted, but when these shoots were cultured in the

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presence of IBA, only a few thin, aerial roots were produced,

suggesting that additional modifications are needed to normalize the shoots further and to induce rooting. These studies
demonstrate the value of the systematic approach to media
analysis provided by DOE, and provide improved methods
for studies directed at providing plants for restoration of this
species.
P-2039
Light Intensity Stress as the Limiting Factor in
Micropropagation of Sugar Maple (Acer saccharum Marsh.).
AMRITPAL S. SINGH, A. Maxwell P. Jones, and Praveen K.
Saxena. Department of Plant Agriculture, University of
Guelph, Guelph, Ontario, N1G 2W1, CANADA. Email:
amritpal@uoguelph.ca
Sugar maple (Acer saccharum Marsh.) is an important hardwood of North America and is the basis for the $350 million
maple syrup industry in Canada. The energy to concentrate
sap accounts for about 40% of syrup production costs and has
a significant carbon footprint (Huyler & Garret, 1979).
Developing elite germplasm with higher sap sugar content
(SSC) to reduce this cost/impact has been the focus of much
research, and while SSC is under genetic control, selection
and propagation of sweet trees has not been feasible due
to difficulties in vegetative propagation. Attempts of clonal
propagation through tissue culture have been largely unsuccessful and the basis for this longstanding problem has
remained unknown (Hanus and Rohr, 1984, Connolly et al.,
1998, Brassard et al., 2003 and Sutano and Stasolla, 2010). In
the present study, we report that the critical factor limiting
sugar maples response to in vitro propagation is light intensity. We used custom made growth chambers equipped with
adjustable intensity LED lights of two spectra to demonstrate
low light is required for in vitro multiplication. The effect of
light quality on the electron transport chain in the in vitro
leaves and various other stress parameters based on chlorophyll fluorescence imaging are discussed. Development of an
efficient propagation system will help improve yields and reduce the carbon footprint of maple syrup production. Further,
while this study is limited to maples, requirement of specific
light intensity is likely the cause of recalcitrance in other
woody species with similar ecological roles.
P-2040
Meta-topolin Combined with Liquid Culture Improves
Micropropagation of the Recalcitrant Species Black Walnut
(Juglans nigra). MICAH E. STEVENS 1 and Paula M.
Pijut2. 1Purdue University, Dept. of Forestry and Natural
Resources, Hardwood Tree Improvement and Regeneration
Center (HTIRC), 715 West State St., West Lafayette, IN

S67

47907 and 2 USDA Forest Service, Northern Research

Station, HTIRC, 715 West State St., West Lafayette, IN
47907. Email: steven31@purdue.edu, ppijut@purdue.edu
The addition of meta-topolin (MT) to established black
walnut (BW; Juglans nigra) tissue culture protocols combined with the novel use of a liquid medium has led to the
development of a rapid shoot multiplication system. BW
has long been prized for its timber, leading to significant
breeding efforts for improving marketable traits. Tissue
culture propagation is an efficient and complementary
method to traditional techniques such as rooted cuttings,
or grafting for the mass production of clonal stock. BW
however, remains recalcitrant to both ex vitro and in vitro
techniques. The goal of this research was to develop a
reproducible micropropagation protocol for elite BW genotypes. In vitro shoot cultures were established from nodal segments cultured on semi-solid Driver and Kuniyuki
walnut (DKW) medium supplemented with 8.9 M
benzyladenine (BA), 0.005 M indole-3-butryic acid
(IBA), 200 mg L-1 casein hydrolysate (CH), 50 mg L-1
adenine hemisulfate (AS), 2 ml L -1 Plant Preservative
Mixture (PPM), and 4.1 M MT. Rapid proliferation
of microshoots was achieved when nodal segments of
in vitro grown shoots were cultured in liquid DKW medium containing 8.9 M BA, 0.005 M IBA, 200 mg L-1CH,
50 mg L-1 AS, 2 ml L-1 PPM, and 4.1 M MT in 3 L
polycarbonate Fernbach-style flasks on a rotary shaker
(100 rpm) under a 16 h photoperiod at 25C. Lateral buds
of individual nodes quickly proliferated and elongated
forming a large mass of microshoots. Elongated
microshoots (5-7 cm in length) were rooted in a slurrytype medium composed of half-strength DKW medium
with 0.11% (w/v) Phytagel and coarse vermiculite (2:1,
v/v) supplemented with 30 M IBA for 5 wk. The combination of MT with liquid culture significantly improved the
reliability of BW micropropagation, but rooted shoots did
not survive acclimatization to the greenhouse.
P-2041
Effect of Plant Growth Regulators on Micropropagation and
In Vitro Conservation of Black Pepper. VIRENDRA M.
VERMA. Micronesia Plant Propagation Research Center,
Kosrae Agricultural Experiment Station, Cooperative
Research and Extension, College of Micronesia-FSM,
Kosrae, MICRONESIA. Email: vmv_vmv@hotmail.com
Black pepper (Piper nigrum L.) is a flowering vine of
Piperaceae family, cultivated for its berry. Berries are usually
dried and used as a spice and seasoning. Piper nigrum is
native to the humid jungles of the Malabar Coast of
Southwestern India. Pepper is currently cultivated in the

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tropics. In Micronesia, it is gaining commercial importance as

an important cash crop. A study was undertaken to develop an
economically feasible, efficient, rapid and reproducible
micropropagation and in vitro conservation protocol for black
pepper (Piper nigrum). Pepper plantlets were
micropropagated from shoot meristem. Murashige and
Skoog, 1962 medium (MS) was used throughout the study.
The cultures were initiated on MS medium augmented with
various concentrations and combinations of cytokinins and
auxins. Best results were observed on MS medium augmented
with 5M 6-benzylaminopurine (BAP) and 100mgL-1 ascorbic acid. Ascorbic acid was added to the medium to prevent
browning of explants. For further growth and subsequent multiplication, the cultures were first transferred on MS medium
augmented with 1M 6-benzylaminopurine (BAP) and then
on MS medium without any growth regulators. MS medium
without any growth regulators proved best for in vitro conservation. The number of multiple shoots produced from each
explant after two subcultures varied from 8 to 20. A 16-h
photoperiod with a temperature of 24C day and night, light
intensity of 40mol m-2 s-1, and 60% relative humidity were
maintained for multiplication. Multiple shoots were transferred on MS medium augmented with 2M indole-3-acetic
acid for rooting. Rooted plants were transferred to soil: vermiculate (1:1, v/v) mixture in pots and acclimatized with 40%
survival rate. Fully acclimatized plants were planted in the
field.
P-2042
In Vitro Multiplication of Commercially Important Pineapple
Cultivars. VIRENDRA M. VERMA. Micronesia Plant
Propagation Research Center, Kosrae Agricultural
Experiment Station, Cooperative Research and Extension,
College of Micronesia-FSM, Kosrae, MICRONESIA.
Email: vmv_vmv@hotmail.com
The pineapple (Ananas comosus L. Merr) is a tropical plant
with edible multiple fruit consisting of coalesced berries. It is
the most economically significant plant in the Bromeliaceae
family. A study was undertaken to develop an economically
feasible, efficient, rapid and reproducible multiplication protocol for two local commercially important pineapple cultivars
(Ananas comosus cv. Kosraean and Ananas comosus cv.
Hawaiian). An in vitro multiplication method for Ananas
comosus cv. Kosraean and Hawaiian was developed by using
apical and lateral meristems. Murashige and Skoog, 1962 medium (MS) was used throughout the study. The cultures were
initiated on MS medium augmented with various combinations and concentrations of 6-benzylaminopurine (BAP) and
1-naphthaleneacetic acid (NAA). The medium augmented
with 4.5M BAP and 2M NAA proved best for culture
establishment. For further growth and subsequent

multiplication, established cultures were first transferred on

MS medium augmented with 9M BAP and 3.5M NAA
and later on MS medium without any growth regulators. The
number of multiple shoots produced from each explant after
two subcultures varied from 18 to 35. A 16-h photoperiod
with a temperature of 25C day and night, light intensity of
80mol m-2 s-1, and 60% relative humidity were maintained
for multiplication. Many shoots formed roots in the same medium in which they were multiplied. However, maximum
rooting was observed on MS medium augmented with
0.5M NAA and 0.5M BAP. Rooted plants were transferred
to soil: compost (10:1, v/v) mixture in pots and acclimatized
with 93% survival rate. Fully acclimatized plants were planted
in the field.
P-2043
Somatic Embryogenesis in Ackee (Blighia sapida): A
Preferred Regeneration Pathway for Improving Traits of
Agronomic and Nutritional Importance. SEYMOUR A.
WEBSTER 1,2 , Marcia Roye 2 , and Sylvia Mitchell 2 .
1
College of Agriculture, Science and Education, Faculty of
Agriculture; Passley Gardens P. O. Box 170, Port Antonio,
Portland, JAMAICA and 2 The University of the West
Indies, The Biotechnology Centre, St Andrew, Mona
Campus, JAMAICA. Email: sawebster68@hotmail.com
Ackee (Blighia sapida) is considered to be the cornerstone of
the agro-processing industry in Jamaica; and in 2010
accounted for more than J$1Billion in export earnings
(STATIN, 2014). Despite its potential as a stellar foreign exchange-earner, there are lingering doubts surrounding its
GRAS-compliant status (Webster, 2012; Bowen-Forbes
et al., 2011; Webster et al., 2006). The presence of
Hypoglycin-A (HG-A)a non-proteinogenic amino acid
is the single most important threat to its compliance with
GRAS standards. The biochemistry of HG-A and its analogues has been the subject of extensive research (Blake
et al., 2006; Meda et al., 1999; Golden et al., 1984; Hassall
& Reyle, 1955; Hassall et al., 1954). However, the physiological functions of HG-A in the plant and the mechanisms,
which regulate its biosynthesis in the arillus are large unknown. We have developed a repetitive somatic embryogenesis system for ackee, as a first step, towards addressing these
issues. Repetitive somatic embryogenesis was established
from primary somatic embryos generated from nodular callus
(Webster, et al., 2006). Explants cultured on BM supplemented with TDZ at 0.4M, produced most nodular callus (80
5.0%; p=0.34) at an average of 8.01.90. Proliferation and
maturation of ackee somatic embryos were achieved on BM
supplemented with 2.40 M TDZ and 1.8M ABA. Primary
somatic embryos were used to establish two cycles of repetitive embryogenesis, which resulted in a 76.5 % (26.54.5; p <

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0.0001) increase in the number of somatic embryos produced

in the third generation (3G) compared with that of the first
generation (1G). RAPD or ISSR analyses of
cotyledonary-stage somatic embryos of ackee, suggest
that this regeneration system might be genetically stable.
P-2044
Plant-derived t-PA and DSPAa1 Dissolve Fibrin and Blood
Clots. J. PETERS, Y. D. Hall, L. Tull, T. Skorupski, and K.
Wang. Northeastern State University 3100 E. New Orleans
St., Broken Arrow, OK 74012. Email: peters32@nsuok.edu
Currently, human tissue plasminogen activator (t-PA) is the
only drug approved by FDA to treat stroke patient. Though
effective, it has some side effects, such as hemorrhage or neuron damage. Desmodus salivary plasminogen activator
(DSPAa1) is an enzyme found in vampire bat saliva is more
specific to break down fibrin. . In this study, both t-PA and
DSPAa1 were codon-optimized for tobacco preference. A
germinivirus-based DNA replicon system was used transiently express t-PA and DSPAa1 in tobacco leaves via agro-infiltration. The proteins were also targeted to seeds under seedspecific Phaseolin (Phas) promoter. Purified proteins from
either infiltrated leaf or seed can dissolve fibrin and break
down blood clots. The study demonstrated that plant-derived
t-PA or DSPAa1 has potential to treat ischemic stroke disease.
The project was supported by the National Institutes of Health
(8P20GM103447 and R03NS095246).
P-2045
Novel Designer Glycopeptides as a Molecular Carrier Boosts
Secreted Protein Yields in Plant Cell Culture. NINGNING
ZHANG, Maria Gonzalez, Brett Savary, and Jianfeng Xu.
Arkansas Bioscience Institute, Arkansas State University,
Jonesboro, AR Email: Ningning.zhang@smail.astate.edu
Plant cell culture in controlled environment has been demonstrated to be a safe and cost-effective bioproduction platform
for therapeutic proteins. However, Low-yield protein production remains the most significant economic hurdle with plant
cell culture technology. Expression of recombinant proteins as
fusion with a designer hydroxyproline-O-glycosylated glycopeptide (HypGP) tag have consistently boosted secreted protein yields as high as 1500-fold, due to the function of the
HypGP tag as a molecular carrier to promote efficient transport of tagged-proteins into culture media. This prompted us
to study the process development of this technology.
Transgenic tobacco BY-2 cells expressing EGFP as fusion
with a glycopeptide tag comprised of 32 repeat of Ser-Pro
dipeptide, or (SP)32, was used as a model system to study cell
growth and protein secretion, manipulation of cell culture

S69

media and establishment of perfusion cultures for continuous

production. The BY-2 cells accumulated low levels of cell
biomass (~7.5 g DW/L) in SH medium but secreted high yields
of (SP)32-tagged EGFP (125 mg/L). The basal salts of culture
media, specifically, the nitrogen supply greatly impacted the
glycosylation of the (SP)32 tag and subsequent protein secretion. Modified MS medium with reduced nitrogen supply increased the secreted EGFP yields to 170 mg/L. Successful
operation of a cell perfusion culture for 30 days was achieved
under the perfusion rate of 0.25 and 0.5 day-1, generating a
protein volumetric productivity of 17.6 and 28.9 mg/day/L,
respectively. This research demonstrates the great potential of
the designer HypGP engineering technology for use in commercial production of valuable proteins with plant cell cultures.
P-2046
The Effect of Absolutely Constant Energy Source on Bitter
Melon. LIZA CHEN, Y. T. Lin, and Y. Lin. Chinese
Association for the Human Evolution, Shuilin Township,
Yunlin County, TAIWAN. Email: lhchen2001@hotmail.com
Momordica charantia, commonly known as bitter gourd is
widely grown in Taiwan. The bitter melon produces an abundance of male flowers each day but only a limited number of
female flowers can be found on the same plant. Apparently,
the gourd yield is determined by the number of successfully
pollinated female flowers. Since the seedlings of the bitter
melon are flood-intolerant, it will be better for them to be
grafted onto the flood-tolerant luffa root stock. The objective
of this study is to investigate if the Absolutely Constant
Energy Source (ACES) can affect the flowering behavior of
the bitter melon and what advantage ACES can associate in
marketable yield. Eight bitter gourd seedlings were grafted on
luffa root-stock, planted in the greenhouse and evenly grouped
into two lots: one for the control group (without treatment) and
the other for experimental group to be treated by ACES. When
the first female flower bud was appeared on April 14 (4/14)
but not yet blossomed, the lot with this first female flower bud
was assigned as the treatment group and the other lot without
female flower bud was assigned as the control group. The four
plants in the treatment group were then activated by the
ACES.Female flowers were pollinated early in the morning.
Seven days after pollination, the gourd was manually weighed
on a hand-held balance, until the gourd reach maturity. The
first female flower appeared on 4/18 in the treatment group
while the first appearance of female flower appeared on 4/30
in the control group, by which time there were already 7 female flowers born in the treatment group. For the treatment
group, a maximum of 18 female flowers were found on 5/24
and, in the meanwhile, there were also 5 matured gourds harvested and their total weight was 1860 gram. As the plants in
the control group had 10 more days of growth than the

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treatment group, they carried a larger leaf area to support a

maximum number of 21 gourds between 5/22 and 5/25, whereas in the treatment group a smaller leaf area was developed and
only a maximum number of 18 gourds were found on 5/24
since the plants were forced to initiate flowering at an earlier
time. In order to support the existing fruit on the vine, there was
no occurrence of new female flower between 5/26 and 6/17 in
the control group, but this happened in the treatment group only
between 5/24 and 6/4. The observation of new female flower
would be terminated for both the control group and the treatment group when the total female flowers reached 45 on 7/1. It
is interesting to observe that bitter gourd has their internal
mechanism to regulate the occurrence of female flowers since
the photosynthetic reserves can sufficiently support the existing
gourd. The Absolutely Constant Energy Source can induce the
early flowering, increasing mature gourd yield, but the total
number of female flowers may not be affected. The accumulated weight of gourd in treatment group was found always surpass that in the control group during the experiment. From the
experimental data, we can observe that the number and the
appearance of female flowers seem to be determined by the
size of leaf area or the photosynthetic reserve and that early
flowering results in higher marketable yield.
P-2047
Mechanical Integrity of Gelling Agents. ANDREW J.
DILLON, David S. Hart, Gary R. Seckinger, and
Kenneth C. Torres. PhytoTechnology Laboratories, 9245
Flint St., Overland Park, KS 66214-1739. Email:
tech@phytotechlab.com
Gelling agents, such as agars and gellan gums, are widely used
in plant, animal cell, and microbial growth media. These products are typically thought to have long shelf-lives. However,
recent evidence indicates these agents, when stored dry, may
experience a reduction in gelling strength with time. This work
examines the mechanical integrity of agars from Gracilaria
and Gelidium species. as well as gellan gums from bacterial
fermentation. The peak compression force necessary to break
the surface of agars and gellan gums prepared with Murashige
and Skoog (MS) media was measured using a digital force
gauge. The results indicate that the newer lots of agars required
significantly greater force to break the gel surface relative to
the older lots. Older lots of gellan gums also show a similar
trend, however, not as substantial as was seen with agar. This
evidence implies that effectiveness of gelling agents break
down with time, and may necessitate shorter shelf-lives.
P-2048
Generation of Human-Mouse Hybridomas Secreting
Antibodies to Tomato and Japanese Cedar Pollen Allergens.

SEIYA CHIBA, Haruka Yokoyama, Yasunori Naganawa, and

Hiroshi Shinmoto. Graduate School of Agricultural Science,
Tamagawa University, Machida, Tokyo, 194-8610 JAPAN.
Email: chbse1sa@agrs.tamagawa.ac.jp
Patients allergic to Japanese cedar pollen allergens sometimes
have oral allergy syndrome (OAS) to tomato fruit allergens. In
this study, we generated human-mouse hybridomas secreting
antibodies reactive to both Japanese cedar pollens and tomato
fruits by transformation of B-lymphocytes from allergic patients with Epstein-Barr virus followed by cell fusion with
mouse myeloma cells. Several human-mouse hybridoma
clones secreting IgM class human monoclonal antibodies to
both Japanese cedar pollen and tomato extracts were
established. Three clones were found to secrete antibodies to
Japanese cedar pollen allergen Cry j2. Pin-Peptide ELISA
revealed that those human monoclonal antibodies reacted to
Cry j2 overlapping icosa-peptides synthesized on the multipin apparatus. One epitope seemed to be identical to an IgE
epitope reported by Tamura et al. Epitope analysis with decaand octa-peptides showed detailed B-cell epitope sequence of
Cry j2. Further analysis of tomato fruit allergens with those
human monoclonal antibodies will be needed. Y. Tamura,
et al. Hum. Antibodies, 22(3):73-76 (2013)
P-2049
Montana State University Seed Potato Certification Program.
WHITNEY HARCHENKO1, Nina Zidack2, and Jessica
Rupp1. 1Department of Plant Sciences and Plant Pathology,
Montana State University, Bozeman, MT and 2Montana State
University Seed Potato Certification Program, Bozeman, MT.
Email: whitney.harchenko@msu.montana.edu
In Montana, efforts to provide the North American commercial potato industry with genetically pure, disease-free certified seed begins at Montana State University (MSU). Since
1921, the MSU Seed Potato Certification Program has been
known for its rigorous certified seed program. The MSU Seed
Potato Certification Programs goal is to work with growers to
produce disease free, high quality seed for re-certification in
Montana and other states, and to produce quality certified seed
for commercial potato production throughout the country. The
program is responsible for the production of all pre-nuclear
tissue culture plantlets and microtubers. Any new potato lines
entering the program are tested for an extensive array of viral
and bacterial pathogens before entering the motherstock collection and propagated to produce disease-free cuttings. As
many as 120 unique lines are grown in Montana, including
grower selections that are maintained in tissue culture. MSU
certified seed potatoes are grown and handled in accordance
with strict disease tolerances and production standards to ensure only the highest quality of seed is produced. MSU seed

PLANT POSTERS

potato certification is 100% grower supported. Montana is a

closed system, with no seed stock originating from outside the
state, and has a unique advantage due to geographic isolation
and lack of significant commercial potato production. The
seed potato certification program is responsible for visual field
inspections, summer leaf testing on all early generation material, and winter post-harvest testing, which is conducted in
Hawaii. The MSU Seed Potato Certification Program collaborates closely with the Montana Potato Improvement
Association, extension/outreach personnel at MSU, and other
universities.

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Ghazanfar Ali Khan3, and Satyajit D. Sarker4.

Department of Pharmacy, COMSATS Institute of
Information Technology, Abbottabad 22060, PAKISTAN;
2
Department of Chemistry, School of Natural Sciences,
National University of Science and Technology,
Islamabad 46000, PAKISTAN; 3 Drug Regulatory
A u t h o r i t y, I s l a m a b a d , 4 4 0 0 0 , PA K I S TA N ; a n d
4
Medicinal Chemistry and Natural Products Research
Group, School of Pharmacy and Biomolecular Sciences,
Faculty of Science, Liverpool John Moores University,
James Parsons Building, Byrom Street, Liverpool L3
3AF, ENGLAND. Email: abdulmannan_ka@yahoo.com
1

P-2050

The generation of large scale mutagenesis in Medicago

truncatula is very useful to study gene functions and to understand general legume biology. Tnt1 is an autonomous
5.3 kb long copia-like LTR element from tobacco that creates
a target site duplication of 5 bp upon insertion. Tnt1 is very
active and it randomly transposes into, on average 30-50 different locations during M. truncatula tissue culture.
Previously, we have successfully developed a Tnt1-based loss
of function mutant population in M. truncatula which is used
by researchers worldwide. Recently, we developed a novel
approach to exploit CRISPR-Cas9 based transcriptional activation in combination with Tnt1 mutagenesis. We developed
vector constructs containing catalytically inactive dCas9 protein fused with a transcriptional activation domain to target
LTR region of Tnt1 element using guide RNAs (gRNAs)
complementary to LTR sequence. We developed nine different combinations of gRNAs and dCas9 fused with activation
domains and transformed M. truncatula genotype R108 via
Agrobacterium-mediated transformation of leaf explants.
Through general tissue culture process, carried on a callusinducing phase followed by somatic embryogenesis and plant
regeneration, we were able to regenerate transgenic plants
expressing dCas9 and gRNA. Our recent findings in this project will be presented.

The aim of the current research work was to carry out the
fingerprint analysis of four fractions obtained from methanol
extract of roots of Trillium govanianum. These four fractions
(TGMF1, TGMF2, TGMF3, TGMF4) were prepared through
solid phase extraction (SPE) by using StrataTM C-18 cartridge
(20g). The obtained fractions were subjected to analytical
HPLC system consisted of an Alliance System 2695
(Waters). Reverse phase chromatography was performed on
a Phenomenex GeminiNX 5U C18 column (250 x 4.60 mm).
The column temperature was set at 25C including a variable
wavelength detector set at 220nm. An elution gradient was
used with solvent A (HPLC grade water with 1% formic acid)
and solvent B (methanol with 1% formic acid). The initial
mobile phase composition was 70% of A and 30% of B at 0
min, then linear gradient to 100% of B at time 0-30 min and
hold at that composition for 30-35min and linear gradient to
30% B for 35-40min at flow rate of 0.3 mL/min. The LC
system was connected to a quadrupole-time-of-flight mass
spectrometer (Waters Micromass LCT) equipped with an
electrospray ion source. Response was recorded in real time
by the mass spectrometer data system (Waters MassLynx).
Positive ion mode was operated with many cone voltage settings. About 29 characteristics peaks possessed identical molecular ion [M + H]+ and [M + Na]+ of different peaks at
similar or different retention times were primarily assigned
as flavonoids and steroidal saponins by comparing their MS
data in the literature. The current study provides the characteristic marker profile which facilitates the authentication of
the active moiety, which could be responsible for the biological outcomes for drug discovery. The LC-ESI/QTOF/MS
method has proven to be accurate and credible which could
be used for the quality control of roots of Trillium
govanianum.

P-2051

P-2052

Fingerprint Analysis of Methanol Extract of Trillium

govanianum by HPLC-Photodiode Array Coupled with ESIQuadrupole Time-of-Flight Mass Spectrometry. ABDUL
MANNAN1, Kashif Maqbool Khan1, Muhammad Arfan2,

Transcriptional Regulation of MicroRNA-targeting Genes in

Tomato Response to Drought Stress. B. CANDAR-CAKIR1,2,
O. Cakir2,3, E. Arican3, and B. Zhang2. 1Programme of
Molecular Biology and Genetics, Institute of Science,

Development of Tnt1 Based Activation Tagging Lines in the

Model Legume Medicago truncatula Using RNA-guided
Transcriptional Regulation Via CRISPR-dCas9 System. HEEKYUNG LEE, Upinder Gill, Babitha Chandrashekar, Janie
Gallaway, and Kirankumar S. Mysore. Plant Biology Division,
The Samuel Roberts Noble Foundation, 2510 Sam Noble
Parkway, Ardmore, OK 73401. Email: hklee@noble.org

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Istanbul University, Vezneciler, Istanbul, 34134, TURKEY;

2
Department of Biology, East Carolina University, Greenville,
NC 27858; and 3Department of Molecular Biology and
Genetics, Faculty of Science, Istanbul University, Vezneciler,
Istanbul, 34134, TURKEY. Email: bilgincandar@gmail.com
Drought, as other environmental stresses, causes plant damages. Drought response is generally controlled by antioxidant
genes, transcriptional factors and signaling proteins. These
proteins are generally post-transcriptionally regulated by
miRNAs. For instance, auxin response factors (ARFs),
targeted by miR160 family, play a crucial role in auxin signaling and root development. NAC [NAM (no apical meristem)ATAF (Arabidopsis transcription activation factor)-CUC
(cup-shaped cotyledon)] and SPL (Squamosa-promoter binding protein-like) family transcription factors regulated by
miR164 and miR156/157, respectively, control plant development and abiotic stress response. Plantacyanins are the members of phytocyanin class blue-copper-binding proteins
(BCBs) and trancription of BCBs is regulated by miR408
enabling the copper homeostasis response to drought stress.
Another copper-related gene, copper/zinc superoxide dismutase (CSD) is targeted by miR398 and associated with oxidative stress tolerance. For this reason, we aimed to reveal
drought-responsive expressions of these genes in droughtsensitive (L. esculentum) and drought-tolerant
(L. esculentum var. cerasiforme) tomatoes. Firstly, we exposed 5% polyethylene glycol to tomato seedlings for 7 days
and collected root and upground tissues separately. Then, we
carried out qRT-PCR for ARF16, BCB, CSD, NAC and SPL
genes. The expressions of all genes were increased in root
tissues of two genotypes, but BCB was down-regulated in
tolerant one. For example, ARF16 and SPL were upregulated by 2.3- and 2.7-fold in root tissues of sensitive
genotype. In upground tissues, all genes were downregulated (ARF16, -2.5-fold; CSD, -2.6-fold; NAC,1.7fold and SPL, -2.8-fold) excluding BCB (up-regulated by
1.8-fold) in sensitive genotype, while the expression of
CSD did not change and the other genes were upregulated in tolerant one. The expression results indicated
that these genes have a function on drought tolerance of
tomato.
P-2053
Development of dandelion Weed as a Platform for
Triterpenes Production. M. MARTINEZ1,2, L. Jorquera1,2,
R. Chamy2,3, and P. Poirrier1,2. 1School of Biochemical
Engineering, Pontifical Catholic University of Valparaso,
Av. Brasil 2085, Valparaso, CHILE; 2Fraunhofer Chile
Research Foundation Centre for Systems Biotechnology
(FCR-CSB), Mariano Snchez Fontecilla 310, of. 1401, Las
Condes, Santiago, CHILE; and 3Ncleo de Biotecnologa de

Curauma, Av. Universidad 330, Curauma, Valparaso,

CHILE. Email: maria.martinez@fraunhofer.cl
Dandelion is a plant known as a common weed, but also
has an important background as a traditional medicine
related to its anti-inflammatory, antimicrobial and anticancer activity, among others (Martnez et al., 2015).
Some species of this genus has been used in the past as
a source of natural rubber, producing inulin as a
byproduct of this process (Fl et al., 2013); however,
several bioactives compounds has also been identify in
its tissue (Gonzlez-Castejn et al., 2012). Among these
compounds, triterpenes are one of the main responsible
for the medicinal properties of this plant and is our aim
to establish an in vitro culture for the selective production
of two triterpenes: amyrin and lupeol. A tissue culture
system for production of high quality planting material
was developed at our university in collaboration with the
Mnster University in Germany (WWU). A tissue culture
system was established for mass propagation of common
dandelion (T. officinale), in which different explants and
culture conditions were tested for callus induction.
Hypocotyls gave the best induction at every condition
tested (100%) using sucrose as carbon source in a suitable
combination of PGRs (NAA/BAP). Triterpenes were detected after 4-6 weeks of culture and directly related to the
maintenance conditions of callus.
P-2054
Innovative Breeding Technologies with Single Plant Cells
in Potato and Barley. J. WEYEN1, G. Jach1, N. Efremova1,
R. Lhrs2, J. Schondelmaier2, B. Schwier2, C. Einfeldt3,
and P. Welters1. 1Phytowelt GreenTechnologies GmbH,
Nettetal, GERMANY; 2 Saaten-Union Biotec GmbH,
Leopoldshoehe, GERMANY; and; 3Saatzucht Ackermann
G m b H & C o . K G , I r l b a c h , G E R M A N Y. E m a i l :
j.weyen@phytowelt.com
The induction of single plant cell growth and hole plant regeneration is known and under application in R&D and breeding since more than 100 years. Still many basics are not understood in plant cell totipotency and genomic research brings
more insights slowly. On the other side the possibility of new
breeding technologies demands efficient plant cell induction
and regeneration technologies. We used somatic hybridization
of protoplasts from winter barley to develop new genotypes to
make use of new combinations of CMS sources and restorer
genetics. Different tissues for the production of protoplasts
were used and successful regeneration of fusion products
was possible. Quality and quantity of fusions and regeneration
was different if protoplasts came from different sources.
Finally marker studies confirmed parental genotypes and

PLANT POSTERS

plantlets could be rooted and developed. Undesired properties

like for example allergy risk factors can be removed and desired qualities like the enrichment of certain ingredients can be
enhanced by TALEN. After the application of TALEN in isolated cells from Solanum tuberosum, a deletion of 16 base
pairs in the DNA sequence of the target gene (1 in 286 clones,
zeaxanthin epoxigenase gene, allele zep2) was detectable.
This leads to the loss of enzyme functionality and therefore

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to the accumulation of zeaxanthin. Unlike conventional

mutation-inducing methods via chemicals or radiation, this
state-of-the-art technology does not require the accumulation
of large populations for selecting desired mutants. Timeconsuming back crossings are not required either. Within in
6 months the mutation was induced and detected. We establish
new anther culture and isolated microspore culture protocols
in a set of spring and winter types of barley and wheat.