Internaonal Conference

Plant Cells In Vitro: Fundamentals &Applications II

Programme and Abstracts

Vienna, Austria June 26-27, 2017

International Conference

Plant Cells In Vitro:

Fundamentals &

Applications II

Programme and Abstracts

Vienna, Austria

June 26-27, 2017

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Organizing Committee

Local Organizing Committee International Organizing Committee

Alisher Touraev (Austria, VISCEA) Anne B. Britt (USA)

Klaus Palme (Germany) Lin Xu (China)

Matthew S. Grasso (USA)

Momoko Ikeuchi (Japan) Pedro P. Gallego (Spain) Sacco De Vries (The Netherlands) Thomas Widiez (France) Traud Winkelmann (Germany) Yuling Jiao (China) Yuxin Hu (China)

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Dear Friends! Dear Colleagues!

Welcome to the 2nd International Conference on “Plant Cells In Vitro: Fundamentals &

Applications”!

Welcome to Vienna!

Plant cell, cultured in vitro, are able to regenerate into the complete fertile plants under the appropriate

culture and environment conditions, e.g. plant cells are totipotent. Currently, the technology is not only

the great tool in basic research, such as cell biology, genetics, biochemistry and biotechnology, but has

also the direct commercial importance in the mass propagation, production of doubled haploids,

secondary metabolites, genetic transformation, etc.

The 2nd International Conference “Plant Cells In Vitro: Fundamentals & Applications“ will discuss wide

range of modern in vitro plant cell and organ culture technologies, fundamental aspects of plant cell

totipotency, differentiation, regeneration, embryogeneisis, and practical applications of in vitro

technologies for crop improvement.

The main aim of the 2nd International Conference “Plant Cells In Vitro: Fundamentals & Applications” is

to provide leading academy and industry scientists a platform to communicate recent advances in

“Plants Cells In Vitro”, and an opportunity to establish multilateral collaboration.

The program of the event combines plenary lectures, poster sessions, a unique Conference Dinner Party

and sightseeing tours of Vienna.

Vienna is located in the heart of Europe on the banks of the Danube River, and considered as one of the

most important economic, cultural and touristic large cities of central Europe. Apart from providing top

science, the Conference will capture the spirit of the city thanks to the central location of the venue

offering a multitude of cultural events.

Prof. Klaus Palme (University of Freiburg, Chair, Scientific Programme)

Prof. Alisher Touraev (VISCEA, Local Organizer)

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Table of Contents

Scientific Programme …………………………………………………………… 7

Abstracts of Oral Presentations ……………………………………………. 10

Abstracts of Posters Presentations ………………………………………. 27

List of Poster Presentations …………………………………………………. 41

List of Participants ……………………………………………………………….. 42

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SCIENTIFIC PROGRAMME

June 26 (Monday)

08.00 - 17.00 Registration and Poster Mounting

09.00 - 09.20 Opening

Welcome address by Klaus Palme (Conference Chair, Germany)

Welcome address by Alisher Touraev (Local Chair, Austria)

09.20 - 10.20 (+10) Keynote Lecture:

Sacco De Vries (The Netherlands): The Fountain of Youth in Plants

10.30 - 11.00 Coffee Break + Poster Mounting

11.00 - 12.30 Session I: Plant Cell Totipotency & Differentiation

Chairs Lin Hu (China) & Momoko Ikeuchi (Japan)

11.00 - 11.20 (+5) Lin Xu (China): WOX11-Mediated De Novo Organogenesis in Plants

11.25 - 11.45 (+5) Momoko Ikeuchi (Japan): Emergency Response and Safety Locking System of

Plant Cell Differentiation

11.50 - 12.05 (+5) Vaclav Motyka (Czech Republic): Endogenous Phytohormone Profiling during

Norway Spruce Somatic Embryogenesis

12.10 - 12.25 (+5) Kin-Ying To (Taiwan): In Vitro Regeneration, Genetic Assay of Chalcone Synthase,

and Metabolic Engineering of Flavonoid Biosynthesis in the Economically

Important Medicinal Plant Echinacea Pallida

12.30 - 14.00 Lunch + Poster Session (all numbers)

14.00 - 15.30 Session II: Plant Cell Organogenesis & Regeneration

Chairs Yuxin Hu (China) & Kin-Ying To (Taiwan)

14.00 - 14.20 (+5) Yuxin Hu (China): Restricting the Arabidopsis Regeneration Capacity by VLCFAs

14.25 - 14.45 (+5) Klaus Palme (Germany): BioSystems Analysis of Plant Single Cells

14.50 - 15.05 (+5) Marcela Morato Notini (Brazil): The Acquisition of Competence for Shoot

Induction in Tomato is Driven by a Fine-Tuned Endogenous Auxin Status

15.10 - 15.25 (+5) Marek Kucera (Czech Republic): Dynamics of Small Noncoding RNAs Pools During

Dedifferentiation of Tobacco Cells and Plant Regeneration

15.30 - 16.00 Coffee Break

16.00 - 17.30 Session III: Somatic Embryogenesis

Chairs Traud Winkelmann (Germany) & Sacco De Vries (The Netherlands)

16.00 - 16.20 (+5) Traud Winkelmann (Germany): Somatic Versus Zygotic Embryogenesis: Learning

from Seeds

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16.25 - 16.45 (+5) Kim Boutilier (The Netherlands): A Chemical Enhancer (C1) of Arabidopsis

Somatic Embryogenesis

16.50 - 17.00 (+5) Sandra Correia (Portugal): Totipotency Acquisition in Plant Somatic

Embryogenesis – Solanum Betaceum, a Case Study

17.05 - 17.15 (+5) Gnanaraj Muniraj (India): Polyethylene Glycol Promoted In Vitro Somatic

Embryogenesis in Green Gram [Vigna Radiata (L.) Wilczek]

17.20 - 17.35 (+5) Anneke Horstman (The Netherlands): Get in Shape! Histone Deacetylation and

Auxin Biosynthesis Control Microspore Embryo Patterning

17.40 - 19.00 Welcome Reception + Poster Session (all numbers)

June 27 (Tuesday)

08.00 - 17.00 Registration

09.00 - 10.30 Session IV: Haploid & Doubled Haploid Production Technologies

Chairs Anne B. Britt (USA) & Thomas Widiez (France)

09.00 - 09.20 (+5) Anne Britt (USA): EMS-Inducible Mutations in Cenh3 Create Haploid Inducers

09.25 - 09.45 (+5) Thomas Widiez (France): How to Make Maize Seeds that Look “Not Like Dad”:

New Insights in Double Fertilization and Prospects for Novel Breeding Tools

09.50 - 10.05 (+5) Patricia Corral-Martinez (The Netherlands): Too Much Stress is Never Good!

10.10 - 10.25 (+5) Mehran E. Shariatpanahi (Iran): Assessment of Different Androgenesis Systems

for Production of Doubled Haploids in Sweet Pepper (Capsicum Annuum L.)

10.30 - 11.00 Coffee Break

11.00 - 12.30 Session V: Plant Protoplasts: Modern Application

Chairs Matthew S. Grasso (USA) & Tom Broeckx (Belgium)

11.00 - 11.25 (+5) Matthew S. Grasso (USA): Engineering Mechanically Tunable Microenvironments

for Individual Plant Cells

11.30 - 11.45 (+5) Tom Broeckx (Belgium): Analysis of SnRK1 Regulation in Leaf Mesophyll

Protoplasts

11.50 - 12.05 (+5) Henrik Svennerstam (Sweden): Organic Nitrogen in Plant In Vitro Culture

12.10 - 12.25 (+5) Fatima Aissa Abdi (France): A Defect in Primary Metabolism Promotes

Morphogenesis

12.30 - 14.00 Lunch + Poster Session (all numbers)

14.00 - 15.30 Session VI: Plant Micropropagation Technologies

Chairs Pedro P. Gallego (Spain) & Yuling Jiao (China)

14.00 - 14.20 (+5) Pedro P. Gallego (Spain): Artificial Intelligence Tools to Better Understand and

Model Plant Processes

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14.25 - 14.45 (+5) Yuling Jiao (China): Regulation of a Meristematic Cell Population Acting in Shoot

Branching

14.50 - 15.05 (+5) Vladan Ondrej (Czech Republic): In Vitro Micropropagation and Polyploidization

Using Oryzalin of Thymus Vulgaris

15.10 - 15.25 (+5) Natalya V. Romadanova (Kazakhstan): Optimization of Barberry

Micropropagation and Cryopreservation

15.30 - 16.00 Coffee Break

16.00 - 17.30 Session VII: Miscellaneous

Chairs Kim Boutilier (The Netherlands) & Takashi Okamoto (Japan)

16.00 - 16.15 (+5) Takashi Okamoto (Japan): In Vitro Fertilization System with Isolated Gametes: For

Basic and Applied Plant Sciences

16.20 - 16.35 (+5) Yuliya Fedorova (Russian Federation): Medicago Truncatula WOX Genes in the

Regulation of Somatic Embryogenesis

16.40 - 16.55 (+5) Barbara Wójcikowska (Poland): Trichostatin A (TSA), an Inhibitor of Histone

Deacetylases, Induces Somatic Embryogenesis in Arabidopsis Explants via Auxin-

Related Pathway

17.00 - 17.15 (+5) Robert Tripepi (USA): Optimization of Shoot Organogenesis from Litchi Tomato

Leaves

17.20 - 17.45 Closing Ceremony & Conference Photo

19.00 - 21.30 Conference Dinner Party

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The Fountain of Youth in Plants Radoeva, T., Albrecht, C., Weijers, D., Sacco de Vries Laboratory of Biochemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen, the Netherlands. Correspondence to: sacco.devries@wur.nl

The potential to initiate embryogenesis in plants is not restricted to the fertilized egg cell and has been reported to occur from many somatic and reproductive cell types, even in plant cell cultures; clearly the mythical fountain of youth providing eternal life for plants is already amongst us!1 A fundamental, yet unresolved question is whether the path to embryogenesis is genetically constrained by a dedicated gene regulatory network. If so, all genes that promote the initiation of embryogenesis should belong to the same network and converge on a common set of embryo genes. Alternatively, the embryogenic state may represent a cellular ‘default’ state to which cells revert to when under particular stresses3. Local inhibition of the response to the plant signaling molecule auxin by the expression of a mutant BODENLOS/Aux/IAA12 (bdl) transcriptional inhibitor protein, causes suspensor cells to lose their identity, convert to embryogenic cell fate and produce twin seedlings at low frequency2. This inducible switch between suspensor and embryo cell fate provides an experimental system to dissect the mechanisms that underlie the initial fate change towards embryogenesis. A major advantage is that the response is highly predictable and occurs only in a few cells. 1. De Vries, S.C., et al. (1988) GenDev 2, 462 2. Rademacher, E.H., et al. (2012) DevCell 22, 211 3. Radoeva, T. and Weijers, D. (2014) TIPS 19, 709

WOX11-Mediated De Novo Organogenesis in Plants Lin Xu Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences. Correspondence to: xulin01@sibs.ac.cn

De novo organogenesis, which gives rise to adventitious roots and shoots, is a type of plant regeneration for survival after wounding. In Arabidopsis thaliana, two main cell fate transition steps are required to establish the root primordium during de novo root organogenesis from leaf explants. The first step from regeneration-competent cells, i.e. procambium and vascular parenchyma cells, to root founder cells involves activation of WUSCHEL-RELATED HOMEOBOX 11 (WOX11) and WOX12 expression by auxin. In the second step, WOX11 and 12 directly activate WOX5, WOX7 and LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16) for initiation of root primordium. During tissue culture, WOX11 and WOX12 are also involved in establishment of founder cells for callus initiation. During initiation, WOX5 and LBD16 are highly expressed in callus cells, indicating that callus could be a group of root primordium-like cells. WOX11 and WOX12 are intermediate-clade WOX (IC-WOX) genes. We hypothesize that the IC-WOX-mediated root initiation mechanism was borrowed and developed for callus initiation in seed plants.

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Emergency Response and Safety Locking System of Plant Cell Differentiation Momoko Ikeuchi RIKEN CSRS Cell Function Research Team 1-7-22 Suehiro-cho, Tsurumi-ku Yokohama-shi, Kanagawa 230-0045 Japan. Correspondence to: momoko.ikeuchi@riken.jp

Plant cells have remarkable plasticity and regenerative potentials. Wounding serves as a primary trigger of callus formation and organ regeneration but how wounding stimuli activate cellular proliferation and reprogramming remain elusive. To address these questions, we study wound-induced callus formation in Arabidopsis thaliana. Our high-resolution time-course transcriptome analyses revealed that cytokinin synthesis genes are rapidly induced by wounding. Hormone measurement further showed that cytokinins massively accumulate before the onset of cell proliferation. Cytokinin synthesis or signaling mutants are significantly impaired in callus formation, together underscoring the importance of wound-induced activation of cytokinin synthesis and signaling in callus formation. Furthermore, we identified novel roles of AP2/ERF transcription factors in callus formation, which are already described to control meristem development. Although the ability of somatic cells to undergo cellular reprogramming and start cell proliferation is important to exert high regenerative capacity, the cellular plasticity needs to be tightly controlled in the context of multicellular development. We have shown that a histone modifying enzyme, POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), serves as a safety locking system to prevent unwanted cellular de-differentiation of mature root cells. Altogether, I will present our latest schemes of how plant cell reprogramming is controlled.

Endogenous Phytohormone Profiling During Norway Spruce Somatic Embryogenesis Zuzana Vondrakova, Petre I. Dobrev, Bedrich Pesek, Lucie Fischerova, Vaclav Motyka Institute of Experimental Botany CAS, Rozvojova 263, CZ-165 02 Prague 6, Czech Republic. Correspondence to: vmotyka@ueb.cas.cz Somatic embryogenesis (SE) in conifers is governed by a complex network of hormonal metabolic and signaling pathways. Taking advantage of advanced HPLC-ESI-MS/MS, we analyzed changes in the patterns and levels of endogenous phytohormones including auxins, cytokinins (CKs), abscisic acid (ABA), jasmonates and salicylic acid during SE in Norway spruce (Picea abies). The peak in concentration of endogenous ABA and its inactive catabolite, dihydrophaseic acid, at the start of maturation reflected the presence of exogenous ABA in the medium and showed its efficient uptake and deactivation by embryos as a prerequisite for their further development. The concentration maxima demonstrated for most of auxins, both indole and non-indole, in the third week of maturation indicated their role in embryo polarization. For the first time, endogenous jasmonates were reported in somatic embryos reaching their highest levels at germination. Aforementioned data together with quantitative and qualitative changes in concentrations of individual CK forms during Norway spruce SE suggested potential correlations between endogenous phytohormone profiles and particular embryo developmental stages. [Supported by Czech Science Foundation (16-14649S)].

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In Vitro Regeneration, Genetic Assay of Chalcone Synthase, and Metabolic Engineering of Flavonoid Biosynthesis in the Economically Important Medicinal Plant Echinacea Pallida Kin-Ying To Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan. Correspondence to: kyto@gate.sinica.edu.tw In vitro plant regeneration was established in Echinacea pallida, a plant that is commonly used as a folk medicine to treat the common cold, fevers, inflammation and so on. Conditions for callus induction, lateral root and shoot regeneration were determined. Subsequently, two vectors pCHS and pOSAG78, carrying different selection marker genes resistant to kanamycin and hygromycin, respectively, were independently used to transform leaf explants of E. pallida using an Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of the transgene and selection marker gene in obtained transgenic lines. Southern hybridization indicated that the T-DNA insertion in some transgenic E. pallida was single copy. Among them, transformants carrying Petunia chalcone synthase (CHS) were selected for further study. CHS is a key enzyme in the biosynthesis of diverse flavonoids including anthocyanin pigmentation. Here, we analyzed the roles and compared the gene expression of two clusters of CHSs, EpaCHS-A and EpaCHS-B (EpaCHS-B1 and EpaCHS-B2), isolated from E. pallida. Two of the genes, EpaCHS-A and EpaCHS-B1, were abundantly expressed in petals, whereas EpaCHS-B2 was expressed at high levels in leaves. The expression of EpaCHSs remained constant in leaves and roots of Petunia CHS transformants, while EpaCHS-B2 expression was changed in flowers of transgenic plants. The biosynthesis of caffeic acid derivatives, cichoric acid and caftaric acid, was increased in leaves and roots of CHS transformants, respectively, while the amount of echinacoside in roots of transgenic plants was decreased. This is the first report on genetic engineering of E. pallida. The information contained herein can be used as a tool for further study of the biological pathways and secondary metabolism of specific compounds from medicinal Echinacea species.

Restricting the Arabidopsis Regeneration Capacity by VLCFAs Yuxin Hu Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Correspondence to: huyuxin@ibcas.ac.cn The already differentiated organs in plants have a remarkable capacity to regenerate new individuals under culture conditions. The plant in vitro regeneration practically starts with the induction of a pluripotent cell mass named callus from detached organs on auxin-rich callus-inducing medium (CIM), which is generally required for subsequent regeneration of new bodies. Recent studies show that CIM-induced callus formation occurs from the pericycle or pericycle-like cells via a root developmental pathway, whereas the signals involved in governing callus-forming capacity of pericycle cells remain unknown. Recently, we showed that very-long-chain fatty acids (VLCFAs) play a critical role in confining the pericycle competence for callus formation and thus the regeneration capacity in Arabidopsis. By genetic screening, we identified the callus formation-related 1 (cfr1) mutant that could bypass the inhibition of callus-forming capacity in roots by the solitary-root (slr/iaa14). We demonstrated that CFR1 encodes the 3-ketoacyl-CoA synthase 1 (KCS1) that catalyzes a rate-limiting step of VLCFA biosynthesis. Our further biochemical and genetic analyses revealed that VLCFAs restricted the pericycle competence for callus formation, at least in part, by regulating the transcription of Aberrant Lateral Root Formation 4 (ALF4). Moreover, we also provided evidence that VLCFAs or their derivatives acted as cell-layer signals to mediate the pericycle competence for callus formation. Therefore, our findings define VLCFAs or their derivatives as the confining signals to mediate the pericycle competence for callus formation and thus the regeneration capacity of plant organs, which might also have a potential application to manipulate plant regeneration capability.

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BioSystems Analysis of Plant Single Cells

Klaus Palme

Institute of Biology II/Molecular Plant Physiology, Faculty of Biology, Centre for Biological Signalling Studies,

Freiburg Institute for Advanced Studies, Center of BioSystems Analysis, Albert‐Ludwigs‐University of Freiburg,

79104 Freiburg. Correspondence to: klaus.palme@biologie.uni-freiburg.de

Production of doubled haploid (DH) plants is an important method to speed up breeding by generating

homozygous plant varieties. Although this approach significantly decreases time and resource

investments in generation of new varieties, its rational use is limited due to poorly understood

molecular basis of the phenomenon. We pursue a comprehensive systems analysis of tobacco and

rapeseed single cell models to identify key molecular mechanisms and players relevant for DH

production. A multidisciplinary strategy has been adopted by successfully integrating recent advances in

engineering, laboratory automation and microscopy with time resolved pharmacogenetic studies of

developing microspore cultures. An automated screening platform enables large‐scale data capture for

predictive modeling and iterative coupling experiments on reprogramming cells with state‐of‐the art

bioinformatics and computational approaches. This image analysis pipeline forms the basis for

phenotypic studies interconnected with transcriptome profiling and pharmacogenetic analysis of the

reprogramming events.

The Acquisition of Competence for Shoot Induction in Tomato is Driven by a Fine-Tuned Endogenous Auxin Status Marcela Morato Notini1; Vicente MH1, Meyerowitz EM2, Peres, LEP1 1University of Sao Paulo ESALQ/USP, Brazil; 2California Institute of Technology, USA. Correspondence to: marcelanotini@gmail.com; marcelanotini@usp.br Tomato (Solanum lycopersicum L.) cv. Micro-Tom is a suitable model plant that shows in vitro organogenic pathway different from Arabidopsis. Although cytokinin supplementation is sufficient for tomato shoot induction the regeneration efficiency is improved by RIM but not by CIM pretreatments during the acquisition of competence phase. Here we analyzed the spatiotemporal expression of cytokinin (pARR5), auxin (pDR5) response promoters and auxin efflux carrier (pPIN1-PIN1) fused to reporter genes during the acquisition of competence for shoot organogenesis in different media incubation. The cytokinin response was similar among explant incubated on auxin- or cytokinin-rich media during the acquisition of competence phase, inducing pARR5::GUS expression all over the explant. RIM preculture induced an intense and wide expression of auxin response in the parenchyma and vascular tissues of proximal and distal cut ends of the explant. Conversely, cytokinin supplementation led to less intense pDR5::GUS staining restricted to the proximal pole. Additionally, PIN1 was not induced during the first stage of shoot organogenesis regardless the medium composition. Our results indicate that RIM preculture in tomato has similar properties of CIM preincubation in Arabidopsis, although bypassing the negative effect of callus formation. The extensive and intense auxin response induced by RIM improves the explant competence to produce shoots under posterior cytokinin induction.

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Dynamics of Small Noncoding RNAs Pools During Dedifferentiation of Tobacco Cells and Plant

Regeneration

Marek Kucera, Jana Lunerova, Ales Kovarik

Institute of Biophysics of the CAS, Brno, Kralovopolska 135, Czech Republic. Correspondence to: kucera@ibp.cz

Small noncoding RNAs (ncRNAs) are important regulators of gene expression in plants. Despite this fact, their role in formation of callus culture is still poorly understood. Here we analyzed the expression of ncRNAs in tobacco (Nicotiana tabacum) during plant dedifferentiation, callus culture cultivation, and plant regeneration. We used Illumina sequencing to obtain expression profile of ncRNAs. We identified several microRNAs (miRNAs) downregulated and upregulated during dedifferentiation. We hypothesize that miRNAs play a role in callus formation by regulating expression of various transcription factors and enzymes. However, their specific mRNA targets need to be identified. Most of the miRNAs reverted to the levels observed in parental plants during regeneration. We also observed 3’->5’ spreading of small interfering RNAs (siRNAs) in transgene locus carrying a neomycin phosphotransferase II gene linked to the 35S cauliflower virus promoter (P35S) in callus culture. A small fraction of the 21nt siRNAs appeared de novo in the P35S that became hypermethylated and eventually inactivated during callus growth and in regenerated plants. We propose that callus-specific ncRNAs may leave a heritable epigenetic imprint in the form of altered DNA methylation patterns. Somatic Versus Zygotic Embryogenesis: Learning from Seeds Traud Winkelmann Leibniz Universität Hannover, Institute of Horticultural Production Systems, Herrenhaeuser Str. 2, D-30419 Hannover, Germany. Correspondence to: traud.winkelmann@zier.uni-hannover.de

The term somatic embryo refers to the striking similarity to zygotic embryos regarding morphology and development. However, whereas zygotic embryogenesis and seed germination are highly organized and controlled processes, for somatic embryogenesis several limitations and anomalies, such as asynchronous development, premature germination, malformations, low germination rates, or strong genotypic differences in efficiency, have been observed in several species impeding its use for commercial mass propagation and as a tool for breeding. From comparisons between both types of embryos on the morphological/histological, transcriptomic, proteomic and metabolomics level, a better understanding of regeneration via somatic embryogenesis can be deduced. Surveying literature, besides the similarities important differences of between both types of embryos become obvious: For several species, a higher stress level in somatic embryos has been described compared to their zygotic counterparts. Moreover, somatic embryos lack a proper maturation phase that on the one hand results in accumulation of storage reserves, but on other hand is important for a clear separation of the developmental programmes of embryogenesis and germination. Based on these findings, in vitro culture conditions for somatic embryogenesis should be adjusted to mimic the conditions zygotic embryos are exposed to within the seed.

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A Chemical Enhancer (C1) of Arabidopsis Somatic Embryogenesis Chen Baojian1, Fiers M1, Zhao Y2, Ligterink W1, Joosen R1, Zhu J-K2, Stekelenburg T1, Angenent G.C.1 and Kim Boutilier1 1Wageningen University and Research Centre, Wageningen, the Netherlands; 2Purdue University, West Lafayette, USA. Correspondence to: baojian.chen@wur.nl; kim.boutilier@wur.nl

Somatic embryogenesis (SE), in which embryos are derived from vegetative cells of the plant, is generally induced by exposing explants to exogenous growth regulators. SE can be induced by 2,4-D in arabidopsis from different explants with varying efficiency. We used a chemical genomics approach to identify genetic factors that enhance 2,4-D mediated SE from germinating seeds in arabidopsis. Screening of the LATCA library identified several enhancers, of which 4-chloro-N-methyl-N-(2-methylphenyl) benzenesulfonamide, named C1, was the most powerful, increasing the number of responding explants from 20% in the control to 70% after C1-treatment. The timing and location of somatic embryo formation did not appear to be different between C1 treatment and control. Moreover, microarray analysis showed that a short C1 treatment induced changes in gene expression that were also observed after 2,4-D treatment, including the expression of many genes involved in seed specific ABA pathways. Together this data suggests that C1 and 2,4-D impinge on the same developmental pathway. Here we report on the role of the ABA receptor PYL10 in 2,4-D/C1-mediated somatic embryogenesis.

Totipotency Acquisition in Plant Somatic Embryogenesis – Solanum Betaceum, a Case Study Sandra Correia Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calcada Martim de Freitas, 3000-456 Coimbra, Portugal. Correspondence to: sandraimc@ci.uc.pt; sandra.correia@uc.pt

Plant somatic embryogenesis (SE) is a developmental pathway in which a somatic cell acquires

totipotency and evolves into an embryo. During the last years our group has developed effective

protocols for the establishment and cloning of the solanaceous tree tamarillo (Solanum betaceum)

through different in vitro culture techniques. Tamarillo’s SE is particularly relevant since it has allowed

successful developments in cryopreservation and genetic transformation protocols. Also, it has several

advantages for experimental embryology approaches. SE induction in this species is achieved by

exposing leaf segments or mature zygotic embryos to an auxin and high sucrose concentration and then

transferring the induced embryogenic masses (EM) to auxin-free medium to allow somatic embryos

development. The EM formed can be isolated from surrounding non-embryogenic calli (NEC) and

subcultured, and protocols for the proliferation of tamarillo EM cell suspension cultures were achieved.

Based on this system a comparative proteomic profile of tamarillo’s EM and NEC was obtained, and a

protein with a putative inhibitory role in the acquisition of embryogenic competence was isolated and

characterized. Besides the easy in vitro manipulation of this woody plant, the establishment of a

protoplast isolation protocol is also an important tool for functional genomics studies in tamarillo,

particularly for cell-type-specific transcript profiling. The results obtained so far have given good

indications about tamarillo’s SE use to understand the mechanisms of plant totipotency.

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Polyethylene Glycol Promoted In Vitro Somatic Embryogenesis in Green Gram [Vigna Radiata (L.) Wilczek] Gnanaraj Muniraj, Udhayakumar Nagan & Manoharan Kumariah Department of Plant Morphology and Algology, School of Biological Sciences, Madurai Kamaraj University, Tamil Nadu, India. Correspondence to: mgnanam1987@gmail.com Green gram [Vigna radiata (L.) Wilczek] is a protein rich (24% protein) and nutritionally important pulse crop cultivated in India and other Asian countries. Usually legumes are recalcitrant in culture and it is very difficult to re enerate plants in vitro. Here, We developed a high frequency somatic embryogenesis system of green gram. The age of the source seedlings for explants and type of explants were found to influence the callusing response. Explants were cultured in a semi solid MS medium supplemented with various concentrations and combinations of NAA, IBA, 2,4-D, picloram and BAP. Embrogenic callus was developed from 3 day old cotyledons in MS media with 4µM picloram. 3 day old cotyledons or callus derived from such explants were exposed to dehydration stress imposed by polyethylene glycol (2-8%). Subsequent to stress incubation, the cultures (PEG 4%) formed only early stage somatic embryos. Subsequently, the early stage somatic embryos developed into torpedo and cotyledonary somatic embryos in MS liquid medium supplemented with picloram (2 μM) + BAP (0.2 μM). The matured somatic embryos were converted into plantlets in hormone-free half-strength MS semisolid medium and successively regenerated into plants. Our protocol assures a high frequency somatic embryogenesis, maturation and plantlet convertion.

Get in Shape! Histone Deacetylation and Auxin Biosynthesis Control Microspore Embryo Patterning Anneke Horstman, Hui Li, Gerco Angenent, Kim Boutilier Wageningen University and Research, Bioscience; Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. Correspondence to: anneke.horstman@wur.nl Brassica napus microspores (immature pollen) can be induced to form haploid embryos in culture after a heat stress treatment. Previously we showed that histone deacetylases (HDACs) repress totipotency in microspore cultures. Here we show that HDACs also control embryo morphology in microspore culture. Apical-basal embryo patterning becomes progressively more compromised when older stages of pollen are used for microspore culture. This effect can be partially complemented by the HDAC inhibitor trichostatin A (TSA), which is accompanied by increased expression of the DR5 auxin response marker. Older pollen have lower expression of auxin biosynthesis genes and blocking auxin biosynthesis in microspore cultures causes similar defects as using older pollen stages. TSA treatment increases the expression of auxin biosynthesis genes in microspore culture. Therefore, we hypothesize that TSA rescues the morphology phenotype by enhancing auxin biosynthesis.

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EMS-Inducible Mutations in Cenh3 Create Haploid Inducers Sundaram Kuppu, Han Tan, Luca Comai, Simon Chan, and Anne Britt 1 Shields Ave, Dept. Plant Biology, C Davis, Davis, CA USA 95694. Correspondence to: abbritt@ucdavis.edu

CENH3 is a histone H3 variant. The position of CENH3-containing nucleosomes determines the position of the centromere. In 2010 Chan and Maruthachalam reported that Arabidopsis plants expressing a transgenic, highly modified CENH3 protein, termed “GFP-tailswap”, produced haploid progeny when crossed by wild-type plants. The resulting haploids, produced at approximately 50% of progeny, carry chromosomes derived only from the wild-type parent, and are produced as mature seeds. Here we report on our investigations into the effects of single amino acid substitutions in CENH3. We selected candidate sites that are A) conserved in both monocot and dicot crops and B) potentially capable of induction via EMS mutagenesis. We tested these mutations for haploid-inducing effects (when crossed by wild type pollen), introducing them as transgenes in a cenh3 defective background. We found that the majority of the mutations tested result in healthy, fertile plants that produce haploids progeny when crossed by plants carrying the wild-type CENH3 locus (at between 1 to 12%, depending on the amino acid change. This suggests that extant EMS-mutagenized populations of crop species might already include plants that carry mutations that may result in a haploid-induction when crossed by wild-type.

How to Make Maize Seeds that Look “NOT LIKE DAD”: New Insights in Double Fertilization and Prospects for Novel Breeding Tools L. Gilles, A. Khaled, J.B. Laffaire, S. Chaignon, G. Gendrot, J. Laplaige, H. Bergès, G. Beydon, V. Bayle, P. Barret, J. Comadran, J.P. Martinant, P.M. Rogowsky, Thomas Widiez Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, Lyon, France. Correspondence to: thomas.widiez@ens-lyon.fr

Mixing male and female genetic information during sexual reproduction is considered as the basis of plant breeding. Sexual reproduction in flowering plants involves double fertilization, characterized by two separate fusion events between the male and female gametes. A maize line first reported in 1959 deviates from this classic pattern. Crosses using pollen from this so-called haploid inducer line, trigger the development of the egg cell into a haploid embryo with only the maternal genome, a process known as in vivo gynogenesis. Map based cloning restricted a major QTL responsible for gynogenesis to a zone containing a single gene coding for a patatin-like phospholipase A, which was named NOT LIKE DAD (NLD) because haploid embryos do not have paternal contribution. In all surveyed haploid inducer lines NLD carries a 4 pb insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction as complementation with wildtype NLD abolishes the haploid induction capacity. Translational NLD::CITRINE fusion protein localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wildtype NLD protein. Thus, an intact sperm-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing haploid breeding tools in numerous crops.

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Too Much Stress is Never Good! Patricia Corral-Martinez and Kim Boutilier Wageningen University and Research, Wageningen, Netherlands. Correspondence to: patriciacorralmartinez@gmail.com; patricia.corralmartinez@wur.nl In plants, the male gametophyte or pollen can be induced to develop directly into haploid embryos when exposed to stress treatments in vitro. This process is called microspore embryogenesis (ME). Heat stress is used to induce ME in Brassica napus. After the heat stress, cells either arrest, continue pollen development, or form histodifferentiated haploid embryos or embryogenic callus. Cytoplasmic cleaning mediated by autophagy and excretion to the apoplast is specifically associated with the haploid embryo pathway, and is thought to be necessary for the progression to haploid embryogenesis to remove proteins and organelles damaged by heat stress. We are examining the role of the autophagy pathway and the related ER-stress and cell death pathways in haploid embryo initiation using B. napus genotypes that differ in their ability to form haploid embryos. We show that the heat stress used to induce ME induces endoplasmic reticulum stress, followed by autophagy or cell death. Embryogenic structures that successfully clean the cytoplasm survive to become histodifferentiated embryos, while those that fail to induce sufficient cytoplasmic cleaning or undergo massive autophagy form callus-like structures and eventually die. Our data suggest that the ability of dividing cells to modulate cell cleaning and autophagy responses underlies the competence of different genotypes for haploid embryo induction.

Assessment of Different Androgenesis Systems for Production of Doubled Haploids in Sweet Pepper (Capsicum Annuum L.) Mehran E. Shariatpanahi, Ali Akbar Heidari Zefreh Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. Correspondence to: mehran.shariatpanahi@abrii.ac.ir; m_shariatpanahi2002@yahoo.com

Three culture systems including anther culture, isolated microspore culture and shed-microspore culture were compared in sweet pepper. 21 hybrid pepper cultivars were used as donor plants. The ploidy level of regenerants were analyzed by both cytometric and cytogenetic tests. The efficiency of anther culture was not satisfactory and in the best conditions, the average of regenerants obtained from a flower bud was 1.2. In isolated microspore culture, the best results obtained when isolated late uni-cellular microspores were cultured in B5 medium supplemented by 3% sucrose and stressed at 32ᵒC for 4 days. In this conditions, the most responsive varieties showed 3.6 regenerants per flower bud. The most efficient androgenesis system was shed-microspore culture. Maximum embryos derived from shed microspores were produced when flower buds were pretreated with cold shock (4ᵒC) and then excised anthers cultured on the double-layer medium and stressed by heat shock(32ᵒC). In addition to embryos, more regeneration (11.03 regenerated plants per flower bud) was also observed. Ploidy level analysis of 100 regenerated plants showed that 54% of regenerants were spontaneous doubled haploids. Finally, 50 DH pepper lines were produced.

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Engineering Mechanically Tunable Microenvironments for Individual Plant Cells Matthew S. Grasso & Lintilhac P.M. University of Vermont, Dept. Plant Biology, 63 Carrigan Drive, Burlington, 05819 Vermont, USA. Correspondence to: msgrasso@uvm.edu

Protoplast technologies and in vitro culturing techniques have facilitated a great deal of progress in studies of plant development. However, these techniques still do not offer researchers the ability to control the physical environment of individual plant cells. The development of hydrogel microbeads and microcapsules is of significant interest with applications in many fields including bioengineering and biomedicine. This technology has not found significant application in the field of plant biology however. In this study hydrogel microcapsules containing living plant protoplasts were produced. Individual cells were isolated by cell wall digestion from a suspension culture of BY-2 tobacco cells. Microdroplets around 70 micrometers in diameter were generated from a stream of liquid agarose containing plant protoplasts using a commercial microdroplet chip. These droplets were then solidified into microbeads and coated with a shell of poly(sodium 4-styrenesulfonate) (PSS) and poly(diallyldimethylammonium chloride) (PDADMA). Producing multiple layers of these shells on microbeads makes the shell thickness tunable. This method has the potential to conduct novel studies in plant cell biomechanics and may continue to benefit from technical advances in the field of microsphere fabrication.

Analysis of SnRK1 Regulation in Leaf Mesophyll Protoplasts Tom Broeckx, S. Hulsmans, and Filip Rolland* Laboratory of Molecular Plant Biology, Biology Department, KU Leuven, Kasteelpark Arenberg 31, 3001 Heverlee, Belgium. Correspondence to: tom.broeckx@kuleuven.be; filip.rolland@kuleuven.be

As sessile and autotrophic organisms, plants are challenged by a continuously changing environment and constantly need to adjust their metabolism and growth to the available resources. Cellular carbon and energy status is an important point of integration of both developmental and environmental signals and the SnRK1 kinases (orthologs of yeast SNF1 and animal AMPK) act as key cellular fuel gauges. These heterotrimeric complexes (with catalytic alfa and regulatory beta and beta gamma subunits) are activated in response to energy-depleting stress conditions, maintaining homeostasis for optimal growth and survival. How exactly the plant kinases are regulated by metabolic status is still elusive. Transient expression in leaf mesophyll protoplasts in combination with target gene promoter-luciferase and direct protein phosphorylation reporters is an excellent tool to investigate both the conserved and plant-specific upstream regulatory mechanisms and cell-autonomous downstream functions. In addition to complex formation and localization, different types of post-translational modifications contribute to activity regulation. SnRK1 activity also appears to be redox-regulated. Reactive oxygen species are byproducts of various types of stress and metabolic dysfunction. Consistently, expression of antioxidant enzymes in leaf cells significantly affects metabolic stress and SnRK1 signaling. Present research focuses on the exact mechanisms involved and their genetic confirmation in plants.

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Organic Nitrogen in Plant In Vitro Culture

Henrik Svennerstam and Johanna Carlsson

Dept. of Forest Gen. and Plant Phys. Swedish Uni. of Ag. Sci., Umea, Sweden. Correspondence to:

henriksvennerstam@hotmail.com; henrik.svennerstam@slu.se

Organic nitrogen (N) is common in plant in vitro culture, often supplied as amino acids (AAs) or AA

mixtures such as casein. The question is why organic N improves in vitro performance and through

which mechanisms? We have studied the growth and N metabolism of Norway spruce cell culture, with

the intention of specifically addressing the importance of glutamine (Gln) by: (I) Measuring the uptake,

assimilation and impact on growth, of ammonium, nitrate and Gln. (II) Assessed if Gln can be a source of

energy (directly and through assimilation cost savings). (III) Investigated the possibility that Gln is

alleviating metabolic constraint(s). We found that nitrate uptake was comparably small and assimilated

at even lower amounts (10 % of AA N) despite constituting 57 % of media N. For Gln the opposite was

observed, 67 % of AA N vs. 27 % of media N. Although Gln carbon (C) was respired and found in cell

biomass, our results do not suggest Gln C to be important for C-metabolism. Metabolic profiling

revealed high levels of alanine and trace amounts of ethanol, two indicators of low oxygen stress. Our

results suggest that, under current lab conditions, Norway spruce cell culture seem to be under stress,

making Gln indispensable for functional metabolism, possibly acting as a N donor in order to avoid

fermentation.

A Defect in Primary Metabolism Promotes Morphogenesis Fatima Aissa Abdi1, E. Fernandez1, M. Quadrado1, O. Bouchabke-Coussa1, M. Hernould2, F. Delmas2, H. Mireau1, P. Hilson1 1Institut Jean-Pierre Bourgin, INRA, CNRS, Universite Paris-Saclay, Versailles; 2UMR 1332 Biologie du Fruit et Pathologie, INRA, Universite de Bordeaux, France. Correspondence to: aissa.abdi.fatima@gmail.com; fatima.aissa-abdi@inra.fr

Shoot regeneration is a complex process involving poorly understood regulatory mechanisms. We

identified an Arabidopsis thaliana mutant in which a defect in the complex I (cI) of the mitochondrial

electron transport chain (METC) promotes in vitro shoot regeneration on protoplast-derived calli.

Correlation between respiratory defect and caulogenesis was confirmed with a cI-specific inhibitor,

rotenone. On tomato, a major crop plant, rotenone doubles the number of buds on cotyledon-explants.

Furthermore, the analysis of different Arabidopsis mutants affected in cI activity revealed that growth

retardation is positively associated with shoot regeneration. To understand how METC perturbations

promote organogenesis, we compared Arabidopsis gene expression profiles in cI mutants and in calli

treated with rotenone and showed that it induces an oxidative stress and inhibits cell proliferation. We

are currently tracking changes occurring in the regeneration process with a redox-sensitive biosensor.

Altogether, our results will help explain how a respiratory defect promotes shoot regeneration and may

lead to alternative ways to promote in vitro organogenesis.

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Artificial Intelligence Tools to Better Understand and Model Plant Processes Pedro Pablo Gallego Applied Plant & Soil Biology, Faculty Biology, University of Vigo, Vigo, Spain. Correspondence to: ppgallegoveigas@gmail.com; pgallego@uvigo.es

Artificial intelligence has been used to analyze and interpret information and to make decisions from complex process in engineering, medical or ecological fields. Plant micropropagation also is complex in their nature to be elucidated by a particular formula or a simple algorithm. In vitro propagation can be only understood as the interaction of multiple elements governed by non-deterministic rules and influenced by internal (genotype, culture media…) and external (culture conditions) factors. Thereby, several variables (inputs and outputs) should be simultaneously evaluated for understanding the obtained responses. Our research group has been pioneer in using of artificial neural networks and fuzzy logic within the field of plant tissue culture. In this talk we will review how those tools can be applied into plant cell culture where multidimensional datasets, frequently composed by different type of data (nominal, discrete, continuous) are employed, and how we were able of building models for properly describing and understanding those complex biological interactions. Particularly, we will talk about the application of neural networks in woody plants micropropagation; the approaches to understand the effect of light and sucrose on kiwifruit micropropagation, to optimize the rhizogenesis or acclimatization of grapewine, to design new media composition for efficient apricot in vitro multiplication or to predict optimal culture media for pistachio.

Regulation of a Meristematic Cell Population Acting in Shoot Branching Yuling Jiao Institute of Genetics and Developmental Biology, 1 West Beichen Road, Beijing, China. Correspondence to: yljiao@genetics.ac.cn

Shoot branching requires the establishment of new meristems harboring stem cells; this phenomenon raises questions about the precise regulation of meristematic fate. In seed plants, these new meristems initiate in leaf axils to enable lateral shoot branching. Using live imaging of leaf axil cells, we show that the initiation of axillary meristems requires a meristematic cell population continuously expressing the meristem marker SHOOT MERISTEMLESS (STM). The maintenance of STM expression depends on the leaf axil auxin minimum. Ectopic expression of STM is insufficient to activate axillary buds formation from cells that have lost STM expression, suggesting an irreversible cell fate commitment. In more mature leaves, REVOLUTA (REV) directly up-regulates STM expression in leaf axil meristematic cells, but not differentiated cells, to establish axillary meristems. Finally, leaf axil cytokinin signaling pulse, likely resulting from the enhanced STM levels, de novo activates local WUSCHEL (WUS) expression promote axillary meristem formation. In particular, type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), transcriptional activators in the cytokinin signaling pathway, directly bind to the WUS promoter to activate its expression. Cell type-specific binding of REV to the STM region, and ARRs to the WUS region, correlate with epigenetic modifications. Our data favor a threshold model for axillary meristem initiation, in which low levels of STM maintain meristematic competence and high levles of STM lead to cytokinin signalling pulse, WUS expression, and axillary meristem formation.

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In Vitro Micropropagation and Polyploidization Using Oryzalin of Thymus Vulgaris Michaela Svecarova, Vladan Ondrej, Bozena Navratilova Department of Botany, Faculty of Science, Palacky University in Olomouc, Slechtitelu 27, Olomouc-Holice, 783 71. Correspondence to: vladan.ondrej@upol.cz; michaela.svecarova@upol.cz

Thyme (Thymus vulgaris L.) is a subshrub and medicinal perennial herb from the Lamiaceae family with plants that are rich in essential oils and antioxidative phenolic substances. T. vulgaris is a well-known herbal medicine that has been used for thousands of years to treat alopecia, dental plaque, bronchitis, inflammatory skin disorders and gastrointestinal distress. Several chemotypes of thyme contain essential oil including thymol, linalool, borneol, geraniol, carvacrol etc. The major constituents of T. vulgaris oil is the thymol, which has shown antibacterial, antifungal and anti-inflammatory effects, accounting for the medicinal uses of T. vulgaris. The micropropagation in vitro, that is a good method for the multiplication of selected genotypes and chemotypes medicinal and aromatic plants. The aim of this work was to develop an in vitro micropropagation protocol for the Thymus vulgaris cultivar Varico to obtain sufficient identical plant material for in vitro polyploidization using oryzalin. The analysis of DNA ploidy level was evaluated by flow cytometry using the method of internal standardization. In conclusion, after in vitro polyploidization using different concentrations of oryzalin some plants became tetraploid. Acknowledgement: This work was supported by the grant QJ1510160 (NAZV, Ministry of Agriculture, Czech Republic).

Optimization of Barberry Micropropagation and Cryopreservation Natalya V. Romadanova, S. A. Mishustina, L. N. Karasholakova, S. V. Kushnarenko Institute of Plant Biology and Biotechnology, Timiryazev str. 45, 050040, Almaty, Republic of Kazakhstan. Correspondence to: nata_romadanova@mail.ru

Eighty two barberry accessions were collected: Berberis amurensis, Berberis iliensis, Berberis integerrima, Berberis koreana, Berberis nummularia, Berberis oblonga, Berberis sibirica, Berberis sphaerocarpa, Berberis thunbergii, Berberis vulgaris, 2 hybrids Berberis x ottawiensis Schneid, 2 cultivars Berberis x ottawensis 'Superba' and Berberis thunbergii 'Kelleriis'. The influence of 26 combinations of the nutrient medium composition was investigated in order to create an in vitro collection of 51 barberry accessions at a temperature of 24°C and 4°C. The best nutrients to obtain sufficient quality of plants was: Murashige and Skoog (MS) medium + 166 mg/l CaCl2, 30 g/l sucrose, 0,8 mg/l 6-benzylaminopurine, 0,1 mg/l gibberellic acid, 0,02 mg/l indole-3-butyric acid, 1 mg/l ascorbic acid, 2 mg/l calcium pantothenate, 1,75 g/l gelrite, 4 g/l agar, pH 5.7. Cryopreservation requires a hardening of donor plants at alternative temperatures (-1°C 16 hours/+22°C

8 hours) for 4 weeks and cultivation of shoot tips: 1) on MS medium with 0,3 M sucrose for 2 days; 2) in

2 M glycerol in 0,4 M sucrose for 20 min at 0°C; 3) in PVS2 cryoprotectant for 80 min at 0°C. Shoot

regrowth for this vitrification protocol composes from 50 to 82.3% for barberry species studied. A

cryobank of barberry seeds was established at 4°C, -20°C and -196°C. A cryobank of shoot tips is in

progress.

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In Vitro Fertilization System with Isolated Gametes: For Basic and Applied Plant Sciences Takashi Okamoto, Erika Toda, Katsuyuki Kakeda, Yukinosuke Ohnishi Department of Biological Sciences, Tokyo Metropolitan University, Tokyo, Japan; PBIL, RIKEN, Yokohama, Japan; Grad. Sch. of Biores., Mie Univ., Mie, Japan. Correspondence to: okamoto-takashi@tmu.ac.jp; erika.toda@riken.jp

Fertilization and subsequent events in angiosperms, such as embryogenesis and endosperm development, occur in the embryo sac deeply embedded in ovular tissue. Because male and female gametes are specialized for cell/gamete fusion, cell walls are poorly developed and the gametes exist as protoplast-like cells in embryo sac like. Therefore, isolated gametes have been used for electro-fusion to produce zygotes for addressing post-fertilization processes, such as karyogamy, egg activation, paternal chromatin decondensation and zygotic genome activation. In addition to investigations for the fertilization-induced events at basic plant scientific level, the IVF system has been applied to establish new breeding system. Procedures for isolating viable gametes have been established in a wide range of plant species. Since IVF system can bypass barriers in sexual reproduction, interspecific zygotes can be prepared from gametes of desired plant species. In addition, polyploidy plants can also produced by repeated gamete fusions. The IVF system described here might become an important technique for generating new cultivars with desirable characters as well as for investigating post fertilization events.

Medicago Truncatula WOX Genes in the Regulation of Somatic Embryogenesis Yuliya Fedorova, Varvara Tvorogova, Ludmila Lutova Department of Genetics and Biotechnology, Saint Petersburg State University, 7/9 Universitetskaya emb, Saint Petersburg, Russia. Correspondence to: julchen.fedorowa@gmail.com

Somatic embryogenesis (SE) is a process in which somatic cells change their developmental program and become embryogenic. Plant hormones and transcription factors (TF) are believed to play a central role in this process. We demonstrated that expression of Medicago truncatula STENOFOLIA gene, belonging to the WOX genes family, is associated with SE. Overexpression of STF gene can increase SE intencity and leads to decrease in expression levels of MtGH3.6 and MtHB1 genes in embryogenic calli. These genes are involved in auxin metabolism and abscisic acid response, respectively. STF loss-of-function mutants show increased level of MtHB1 and also increased level of another SE-associated WOX gene, MtWOX9-1. Nethertheless STF loss-of function mutantse do not demonstrate reduction of embryogenic capacity. We suppose that this TF is not strictly necessary for inducing SE and may influence SE in different pathways. To unravel the mechanism of these pathways, we are going to identify STF targets by transcriptome analysis. This work was supported by RSCF grant 16-16-10011 and RFBR grant 17-04-01708 A.

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Trichostatin A (TSA), an Inhibitor of Histone Deacetylases, Induces Somatic Embryogenesis in Arabidopsis Explants via Auxin-Related Pathway Wójcikowska Barbara, Morończyk J, Wójcik A, Gaj MD Department of Genetics, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland. Correspondence to: barbara.wojcikowska@us.edu.pl; barbaragzyl@gazeta.pl

Somatic embryogenesis (SE) induced in in vitro cultured explants exemplifies developmental plasticity of already differentiated somatic cells of plants. In most of plants species, including Arabidopsis, the embryogenic programme is induced in response to auxin, mostly 2,4-D, treatment of the cultured tissue. We showed, that the use of a deacetylase inhibitor, trichostatin A (TSA), instead of 2,4-D, also results in the SE induction in Arabidopsis explants. The aim of the study was to get insights into the molecular mechanism involved in the TSA-induced SE. The results provided evidence on the SE-promoting effect of TSA that include: (1) the significantly stimulated expression of the genes encoding transcription factors of essential role in SE (LEAFY COTYLEDON1,2 FUSCA3, BABY BOOM, AGAMOUS LIKE 15, PHABULOSA, PHAVOLUTA and MYB118); (ii) the high level of the reactive oxygen species (ROS) and lipids found in the TSA-induced explants. In addition, the YUCCA genes involved in auxin biosynthesis pathway were activated and in accordance to this observation, a level of endogenous auxin was increased in TSA-induced culture. These results imply that the TSA- and 2,4-D-induced genetic mechanisms that governs SE induction share some similarities. Given that TSA-treatment is expected to impact gene expression via changes in histone acetylation level it is tempting to speculate that 2,4-D might control SE induction through control of the epigenetic processes, including histone acetylation. Further analysis are required to verity this hypothesis.

Optimization of Shoot Organogenesis from Litchi Tomato Leaves

A. Knerr, Robert Tripepi, S. Royals, and J. Kuhl

Dept. of Plant Sciences, University of Idaho, Moscow, ID 83844-2339, USA. Correspondence to:

btripepi@uidaho.edu

Rapid multiplication via shoot organogenesis has been used for some solanaceous species. The objective

of this study was to develop a shoot organogenesis protocol, which consistently generated high

numbers of quality shoots from Solanum sisymbrifolium (litchi tomato) leaves. Leaf explants (~1 cm2)

from established shoots were placed on an MS-based callus induction medium with BA and NAA for 6

days and then moved to an MS-based shoot induction (SI) medium with BA and GA3 for 6 weeks

(control) or moved to fresh SI medium after 1, 2 or 3 weeks or moved after 1 week and again 3 weeks

later. After 6 weeks, shoots were excised then maintained 4 additional months. Numbers of shoots and

the percentage of healthy, abnormal or dead shoots were determined for each treatment. All explants

formed shoots. The fewest shoots formed (18.5 shoots per jar) if explants remained on the original SI

medium for 6 weeks, whereas moving leaf explants after 2 or 3 weeks increased shoot regeneration by

3.3 fold and doubled the percentage of healthy shoots compared to the control treatment. Additional

genotypes were tested and responded well to the new protocol. This study demonstrated that moving

litchi tomato leaf explants to fresh SI medium after 3 weeks significantly improved shoot organogenesis.

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New England Biolabs GmbH New England Biolabs GmbH Brüningstrasse 50 Geb.B852 65926 Frankfurt am Main Tel: +49/ (0)69/305-23140 Fax: +49/ (0)69/305-23149 Email: info.de@neb.com Web: www.neb.com Founded in the mid-1970s as a collective of scientists committed to developing innovative products for the life sciences industry, New England Biolabs (NEB) is now a recognized world leader in the discovery, development and commercialization of recombinant and native enzymes for genomic research. NEB is renowned for consistently providing exceptional product quality and unsurpassed technical support. For over four decades, NEB has been shaping the landscape of bioscience research by discovering, developing and supporting innovative and superior research reagents and optimized enzymatic workflow solutions. NEB is continuously expanding its product offerings into new areas including Next-Generation-Sequencing, Epigenetics and Cellular Imaging.

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Poster № 1: Dose-Dependent BABY BOOM Function Mengfan Li, Anneke Horstman, Gerco Angenent, Kim Boutilier* Wageningen University and Research, Netherlands. Correspondence to: kim.boutilier@wur.nl; mengfan.li@wur.nl

AINTEGUMENTA-LIKE (AIL) transcription factors are key regulators of cell proliferation and meristem identity that can induce pluripotency (organogenesis) and totipotency (embryogenesis) when overexpressed. It was previously suggested that the PLT2 protein regulates root meristem size and maintenance through a protein concentration gradient, with high, intermediate and low AIL concentrations instructing stem cell fate, cell division and differentiation, respectively. Here we show that the AIL protein BABY BOOM (BBM) promotes cell proliferation in a dose-dependent manner, with a high dose inducing somatic embryogenesis, a medium dose inducing shoot organogenesis, and a low dose inducing cell dedifferentiation. PLT2 also directs the same dose-dependent overexpression phenotypes as BBM. These data suggest that AIL protein dose drives the developmental fate of regenerating tissues. We are determining the molecular basis for this dose-dependent regeneration by identifying and characterizing the genes that are directly regulated at different BBM doses. The results of these experiments will be presented.

Poster № 2: Optimization of Shoot Organogenesis from Litchi Tomato Leaves

A. Knerr, Robert Tripepi, S. Royals, and J. Kuhl

Dept. of Plant Sciences, University of Idaho, Moscow, ID 83844-2339, USA. Correspondence to:

btripepi@uidaho.edu

Rapid multiplication via shoot organogenesis has been used for some solanaceous species. The objective

of this study was to develop a shoot organogenesis protocol, which consistently generated high

numbers of quality shoots from Solanum sisymbrifolium (litchi tomato) leaves. Leaf explants (~1 cm2)

from established shoots were placed on an MS-based callus induction medium with BA and NAA for 6

days and then moved to an MS-based shoot induction (SI) medium with BA and GA3 for 6 weeks

(control) or moved to fresh SI medium after 1, 2 or 3 weeks or moved after 1 week and again 3 weeks

later. After 6 weeks, shoots were excised then maintained 4 additional months. Numbers of shoots and

the percentage of healthy, abnormal or dead shoots were determined for each treatment. All explants

formed shoots. The fewest shoots formed (18.5 shoots per jar) if explants remained on the original SI

medium for 6 weeks, whereas moving leaf explants after 2 or 3 weeks increased shoot regeneration by

3.3 fold and doubled the percentage of healthy shoots compared to the control treatment. Additional

genotypes were tested and responded well to the new protocol. This study demonstrated that moving

litchi tomato leaf explants to fresh SI medium after 3 weeks significantly improved shoot organogenesis.

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Poster № 3: Regulation of Somatic Embryogenesis in Medicago Truncatula Varvara Tvorogova, Yulia Fedorova, Tatiana Vashkevich, Ludmila Lutova Department of Genetics and Biotechnology, Saint Petersburg State University, 7/9 Universitetskaya emb, Saint Petersburg, Russia. Correspondence to: krubaza@mail.ru; varvaratvor@gmail.com Somatic embryogenesis is widely used in plant biotechnology, but there are a lot of undiscovered molecular mechanisms underlying this process. WUSCHEL-related homeobox (WOX) family transcription factors and PIN auxin transporters are shown to play important roles in plant development. The aim of our research is to find new somatic embryogenesis regulators among the members of these gene families in Medicago truncatula. We found three M. truncatula WOX family genes (STENOFOLIA (STF), MtWOX9-1 and MtWOX11-like) and one PIN family gene, SMOOTH LEAF MARGIN 1 (SLM1), whose expression is associated with somatic embryogenesis. In embryogenic calli, promoters of STF, MtWOX9-1 and SLM1 genes are active in somatic embryos and also in adjacent zones of calli. Futhermore, overexpression of MtWOX9-1 gene leads to increased embryogenic capacity of calli and correlates with changes in expression levels of several embryogenesis-associated genes, including MtWOX11-like and SLM1. Further uncovering of mechanisms of WOX and PIN genes functioning in somatic embryogenesis should help to improve the protocols for plant regeneration in vitro. This work was supported by RSCF grant 16-16-10011 and RFBR grant 17-04-01708 A.

Poster № 4: A Chemical Enhancer (C1) of Arabidopsis Somatic Embryogenesis Chen Baojian1, Fiers M1, Zhao Y2, Ligterink W1, Joosen R1, Zhu J-K2, Stekelenburg T1, Angenent G.C.1 and Kim Boutilier1 1Wageningen University and Research Centre, Wageningen, the Netherlands. 2Purdue University, West Lafayette, USA. Correspondence to: baojian.chen@wur.nl; kim.boutilier@wur.nl

Somatic embryogenesis (SE), in which embryos are derived from vegetative cells of the plant, is generally induced by exposing explants to exogenous growth regulators. SE can be induced by 2,4-D in arabidopsis from different explants with varying efficiency. We used a chemical genomics approach to identify genetic factors that enhance 2,4-D mediated SE from germinating seeds in arabidopsis. Screening of the LATCA library identified several enhancers, of which 4-chloro-N-methyl-N-(2-methylphenyl) benzenesulfonamide, named C1, was the most powerful, increasing the number of responding explants from 20% in the control to 70% after C1-treatment. The timing and location of somatic embryo formation did not appear to be different between C1 treatment and control. Moreover, microarray analysis showed that a short C1 treatment induced changes in gene expression that were also observed after 2,4-D treatment, including the expression of many genes involved in seed specific ABA pathways. Together this data suggests that C1 and 2,4-D impinge on the same developmental pathway. Here we report on the role of the ABA receptor PYL10 in 2,4-D/C1-mediated somatic embryogenesis.

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Poster № 5: The Influence of Fluorescent Light and LED Light on the Shoot Multiplication in Amelanchier Alnifolia var. Cusickii Júlia Hunková1,2, Gabriela Libiaková2, Alena Gajdošová2

1Comenius University, Faculty of Natural Sciences, Department of Genetics, Ilkovičova 6, 84215 Bratislava, Slovakia; 2Plant Science and Biodiversity Center SAS, Institute of Plant Genetics and Biotechnology, Akademická 2, 95007 Nitra, Slovakia. Correspondence to: julia.hunkova@savba.sk

The aim of our work was to analyse the influence of two light sources on the shoot multiplication in vitro in Amelanchier alnifolia var. cusickii. White fluorescent light (FL, Tracon Electric) and red-blue LED light (80% red, 20% blue, Phillips) represented the light sources. 30 explants were cultivated 3 months in growth chamber under each light source at 22°C day/night temperature, 16h photoperiod with light intensity 50 μmol.m-2.s-1. Shoot proliferation ability for both variants was evaluated by number of shoots/explant (multiplication coefficient Q). The results have shown that higher multiplication rate was achieved after cultivation under FL (Q = 3.21). The shoot growth under LED light was less effective (Q = 2.08). Overall appearance of plants was healthy and green, but they differed morphologically. Plant cultivated under FL had tall, thin stems with smaller leaves. However, plants cultivated under LED light developed short thick stems with bigger leaves. Thus, white fluorescent light can be recommended for A. alnifolia micropropagation.

Poster № 6: Heterologous Protein Expression in Carnivorous Plants: a Potential New Drug-Delivering System Raphael Kolano1, Paula Lätsch1, Karin Fester1, Tobias Bonitz1

1Department of Pharmaceutical Biology, Institute of Pharmacy, University of Leipzig, 04103 Leipzig, Germany. Correspondence to: raphael.kolano@outlook.de

Carnivorous plants bear the special ability to produce and secrete well-defined digestive fluids consisting of pharmacological active secondary metabolites and digestive enzymes. Our aim is to produce pharmaceutical relevant substances in carnivorous plants by modifying plant-derived secondary metabolites or the expression of heterologous proteins. Furthermore, by interfering in plant secondary metabolite or signal transduction pathways we can gather information about biosynthetic and regulatory cascades of the plant. Here we present the transformation of several genera of carnivorous plants with the marker protein GFP (green fluorescent protein) using the method of Hirsikorpi et al. (2002). [1]Merja Hirsikorpi, Terttu Kämäräinen, Teemu Teeri, Anja Hohtola Agrobacterium-mediated transformation of round leaved sundew (Drosera rotundifolia L.) Plant Science 162 (2002) 537-542

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Poster № 7: Development of an Improved In Vitro Ovary Culture Protocol for Doubled Haploid Production in Cucumber (Cucumis Sativus L.) David Downey, Tatjana Sretenovic Rajicic, Joe Palys Monsanto, Paraje Romeral y Atochares s/n, 04720 La Mojonera Almeria, Spain. Correspondence to: David.dg.Downey@monsanto.com

Provision of low cost inbred lines to breeding programs in a reliable and routine manner is of fundamental importance in a modern breeding program. In an industrial environment one always looks to optimize methods to maximize output and reduce cost. Here we present improvements to known cucumber DH protocol that have led to a two-fold increase in DH production. The addition of the polyamine spermidine to the culture medium of immature unfertilized cucumber fruits improved gynogenic response for the genotypes tested. Induction and regeneration media supplemented with spermidine produced significantly more embryos compared to media lacking this component. Additionally, the expansion of the fruit picking range substantially increased the amount of available embryogenic material. A new protocol was implemented with spermidine supplemented medium and expanded fruit range, as a consequence of which, DH cucumber production was increased two-fold.

Poster № 8: Germination of Oat (Avena Sativa L.) Haploid Embryos Depending on the Growth Regulators Ilona Czyczyło-Mysza, Edyta Skrzypek, Marzena Warchoł, Angelika Noga, Kinga Dziurka, Kamila Kapłoniak, Izabela Marcińska The Franciszek Górski Institute of Plant Physiology Polish Academy of Science, Niezapominajek 21, 30-239 Krakow, Poland. Correspondence to: i.czyczylo@ifr-pan.edu.pl

The purpose of the study was to analyze the germination ability of oat haploid embryos depending on the growth regulators added to the regeneration medium. Twenty one oat genotypes (17,904 florets) derived from Strzelce Plant Breeding Ltd. were used for the wide crossing with maize (Zea mays L.). Oat and maize plants were grown in a greenhouse (16 h photoperiod, 21/17°C day/night). Haploid embryos were transferred to 190-2 regeneration medium with combination of growth regulators: (A) - 0.5 mg/dm3 kinetin (KIN) and 0,5 mg/dm3 naphthaleneacetic acid (NAA); (B) - 1 mg/dm3 zeatin (ZEA), 0.5 mg/dm3 NAA or (C) - 1 mg/dm3 dicamba (DIC), 1 mg/dm3 picloram (PIC) and 0.5 mg/dm3 kinetin (KIN). Germinated embryos were transferred on ½ MS medium. Seven hundred haploid embryos were obtained from all genotypes and 133 germinated. The most of haploid embryos (19%) germinated on medium (A) and the least (11%) on medium (C). The haploid plants were acclimatized to the natural conditions. After chromosomes doubling with colchicine, 50 DH lines were obtained. The research was funded by the National Centre for Research and Development, PBS3/B8/17/2015

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Poster № 9: Recovery of Oat (Avena Sativa L.) Doubled Haploid Plants Marzena Warchoł, Edyta Skrzypek, Angelika Noga, Kinga Dziurka, Ilona Czyczyło-Mysza, Kamila Kapłoniak, Izabela Marcińska The Franciszek Górski Institute of Plant Physiology Polish Academy of Science Niezapominajek 21, 30-239 Krakow, Poland. Correspondence to: m.warchol@ifr-pan.edu.pl

Biotechnological methods such as generating in vitro doubled haploid (DH) lines reduce significantly time of new cultivars production. The purpose of the study was to analyze the germination ability of oat haploid embryos depending on their size. The experiment was carried out on about 700 haploid embryos obtained from wide crosses with maize. The isolated haploid embryos were divided into four groups according to their size and transferred into 190–2 regeneration medium. Fifty of 700 embryos (0.7%) were smaller than 0.5 mm. There were 158 haploid embryos in the 0.5–0.9 mm group (22.6%). The largest group (323 embryos; 46.1%), was 1.0–1.4 mm. One hundred and sixty-nine haploid embryos (24.1% of all) were bigger than 1.5 mm. The germination of embryos was depended on their size. The embryos bigger than 1.5 mm shown the highest germination ability, while smaller than 0.5 mm did not germinate. Almost all germinated embryos developed into haploid plants. The percentage of germinated embryos and haploid plants per emasculated floret was similar (ca. 0.67%). The research was funded by the National Centre for Research and Development, PBS3/B8/17/2015

Poster № 10: Development of Culture Media for Pollen Germination and Tube Growth of Sunflower Thitiporn Machikowa and Sarinporn Sukmee Institution of Agricultural Technology, Suranaree University of Technology, 111 University Ave., Suranaree district, Muang, Nakhon Ratchasima, Thailand, 30000. Correspondence to: machiko@sut.ac.th

In developing a diploid plant from pollen tissue, pollen germination and pollen tube growth are very important. The pollen germination and tube growth are usually evaluated on artificial media. However, there are limited reports of suitable media for pollen germination and pollen tube growth. The aim of this study was to develop suitable media with the combination of B, sucrose and plant growth regulator for sunflower pollen culture. The combination of two concentrations of H3BO3 (100 and 200 ppm), two concentrations of sucrose (10 and 15%) and plant growth regulator (NAA and GA) were tested. Sunflower pollen grains were cultured on the different media and incubated at 25°C. After 6 hours, pollen germination and pollen tube growth were evaluated. The results revealed that, both sucrose and B affected the pollen germination and tube growth. The highest percentage of pollen germination (95%) was recorded in the media containing 10% sucrose, 200 ppm H3BO3 and NAA, while the maximum pollen tube length (8 mm) was observed in 15% sucrose media supplemented with 100 ppm H3BO3 and NAA. Key words: Pollen germination, pollen tube growth, boron, Helianthus annuus L.

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Poster № 11: Diploid Protoplasts of White Cabbage – Effectiveness of Electrofusion Agnieszka Mrzygłód, Agnieszka Kiełkowska, Adela Adamus Institute of Plant Biology and Biotechnology, University of Agriculture in Krakow, Al. 29 Listopada 54, 31-425 Kraków, Poland. Correspondence to: a.mrzyglod@ogr.ur.krakow.pl

Protoplasts electrofusion is one of the methods allowing to generate somatic hybrids combining useful traits. To use this method in practice an important factors are both proper methodology (electrical parameters of fusion, culture and regeneration) and the genetic properties of fusion donors. The aim of this study was to test the effectiveness of fusion of diploid protoplasts isolated from accessions possessing agronomically useful traits such as cms, resistance or self-compatibility. We used five diploid accessions of Brassica oleracea var. capitata: commercial cultivar Kilaton F1 and four breeding lines: MS, IF1, 2023, ZKI13. Protoplasts were enzymatically isolated from leaves of in vitro-derived plants, suspended in a fusion medium (0.4 M mannitol) and stained with fluorescent dyes (FDA and rhodamine B) in order to identify electrofusion products. Fused protoplasts were immobilized in calcium alginate layers and cultured in liquid medium. In this study the overall frequency of fusion and frequency of heterokaryons exibiting double (red-green) fluorescence was scored. The viability and mitotic activity of electrofusion products was investigated. Callus formation was observed in all fusion combinations. The research project was financed by the Polish Ministry of Agriculture and Rural Development

Poster № 12: Harnessing FACS for the Evaluation of Pollen Viability and Flavonoid Content Luria Gilad, Lazar Eitay, Miller Gad The Mina and Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan, Israel. Correspondence to: giladellu@gmail.com

The majority of food supply originates from sexual reproduction in flowering plants. Reproductive development, especially the involvement of the male gametophyte (pollen grains) is highly sensitive to stress, particularly temperature stress. Therefore, it is of great importance to be able to accurately assess pollen quality for crop improvement. Several techniques and protocols are available for the determination of pollen viability, but most of them are inaccurate, laborious and species-dependent. We present a flow-cytometry based method for fast and reliable pollen viability assays using the reactive-oxygen-species responsive fluorescent dye, H2DCFDA.The stain reliably distinguishes between live and dead pollen grains and between pollen of different ecotypes. By pre-treating the pollen grains with caffeic acid (antioxidant) and paraquat (oxidative stress inducer) we prove that the fluorescence emitted from the dye directly corresponds to the redox state of the cells. Moreover, we present an elegant way of assessing flavonoid content in pollen using FACS and a fluorescent probe. In summary, this novel approach provides an easy, rapid, robust and reliable way for detecting viable pollen grains and for testing their redox state and flavonoid content.

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Poster № 13: In Vitro Cultures and Polyploidization of Blue Bugle (Ajuga Reptans L.) Bozena Navratilova, Michaela Svecarova, Vladan Ondrej Department of Botany, Faculty of Science, Palacký University in Olomouc Šlechtitelů 27, Olomouc-Holice, 783 71. Correspondence to: vladan.ondrej@upol.cz; michaela.svecarova@upol.cz; bozena.navratilova@upol.cz

Ajuga reptans (L.), commonly known as blue bugle is a perennial flowering herb native to Europe. It is also cultivated in several cultivars as an ornamental plant. Ajuga herbs contain tannins, organic acids and etheric oils. Thus, it has been used in traditional medicine, mostly, in the treatment of respiratory diseases. Recently, Ajuga, in relation to its medical potential, became more attractive for breeding and improvement to increase amount of biological active compounds. Here, we reported derivation of in vitro cultures of the bugle (donor plants from collection of Crop Research Institute, Prague) including culture medium and conditions. We also derived protocol of the polyploidization based on oryzalin. Treatment of the plantlets cultivated in vitro by oryzalin procedure leds to obtaining of bugle tetraploids. The analysis of ploidy level of these plants was checked out by the flow cytometric analysis using the method of internal standardization. After in vitro polyploidization using different concentration of oryzalin, we obtained some tetraploid plants. Acknowledgement: This work was supported by the grant QJ1510160 (NAZV, Ministry of Agriculture, Czech Republic).

Poster № 14: Auxin Dependent ARF/bHLH Module Regulates Extra-Embryonic Identity During Arabidopsis Embryogenesis Tatyana Radoeva, Cristina Llavata-Peris, Annemarie Lokerse, Jos Wendrich & Dolf Weijers Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands. Correspondence to: tatyana.radoeva@wur.nl The plant hormone auxin is required for many aspects of plant growth and development. Recently, it was found that inhibition of auxin response in suspensor cells induces transformation of suspensor cells to embryonic cells (Rademacher, E. et al., 2012). Using a transcriptomics approach, a small set of bHLH genes was identified that are: a) upregulated upon auxin response inhibition and normally expressed in the proembryo or b) downregulated upon auxin inhibition and normally expressed in the suspensor. Detailed investigation of gain-of-function and loss-of-function mutants of these genes strongly suggests that their function is required for controlling normal suspensor and hypophysis development. Strikingly, ectopic expression of one of these is by itself sufficient for inducing embryo-like structures in suspensor cells. Moreover, we show that these genes act downstream of auxin signaling, and proper auxin signaling is indispensable for the regulation of these genes. Hence, this work identifies a novel ARF-bHLH module that operates in parallel to the previously identified ARF5/MP-TMO5/TMO7 module that operates in patterning the embryo (Schlereth, A., et al., 2010; De Rybel, B., et al., 2013). We conclude that auxin-ARF-bHLH modules are conserved elements that mediate auxin-dependent plant development.

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Poster № 15: Influence of UV Illumination on Crithmum Maritimum In Vitro Cell Culture Julien Fouilland Biotechmarine, 22260 Pontrieux, France. Correspondence to: julien.fouilland@airliquide.com

Halophyte plants represent a small part of the biodiversity. These plants, growing on the seashore, are submitted to high stresses, such as oxidative stress from the UV and osmotic stress due to the high salinity of their environment. Therefore, these plants have developed a wide range of chemical responses to fight against these stresses, highly valuable because of their potential biological activities. On the other hand, plant cell culture is a good method for producing bioactive compounds. Furthermore, adjusting culture parameters sometimes permit to produce higher yield of bioactive compounds and even activate new metabolic pathways, ensuring to obtain a brand new phytochemical profile. Taking into account these two parameters, cell culture of halophyte plants can be very interesting for the cosmetic industry. Crithmum maritimum is a halophyte plant from Brittany seashore, which has been commonly used in cosmetic. Cell culture from Crithmum maritimum has been established by dedifferentiation of leaf explants. These cells have been cultivated in solid and liquid medium. This first experiment was conducted under white light with no photoperiod. A second experiment was conducted under UV illumination, with a replicate under white light. Again, no photoperiod was used. Growth curves, phytochemical fingerprint and phenolic profile were established for both culture conditions in order to see the UV influence. We found that the UV illumination had a negative effect on the growth of the cells, but also induced an over-production of the phenolic compounds. Half-dozen molecules have been detected and a flavonoid that was not produced in the cells under white light has also been detected in UV condition. This study proves the effectiveness of UV illumination in order to over-produce phenolic compounds in Crithmum maritimum dedifferentiated cells. Bibliography Antognoni F., Zheng S., Pagnucco C., Baraldi R., Poli F., Biondi S., « Induction of flavonoid production by UV-B radiation in Passiflora quadrangularis callus cultures », Fitoterapia, Vol.78, p. 345–352 (2007) Namdeo A. G., « Plant Cell Elicitation for Production of Secondary Metabolites: A Review », Pharmacognosy Reviews, Vol. 1, Issue 1 (2007) Parikrama R. and Esyanti R. R., « Effect of UV Elicitation on Callus Growth, Alkaloid and Terpenoid Contents in Eurycoma longifolia Jack », International Journal of Advances in Chemical Engineering & Biological Sciences (IJACEBS), Vol. 1(1) (2014) Ramakrishna A., Ravishankar G. A., « Influence of abiotic stress signals on secondary metabolites in plants », Plant Signaling & Behavior, Vol. 6(11), p. 1720-1731 (2011) Siracusa L., Kulisic-Bilusic T., Politeo O., Krause I., Dejanovic B., and Ruberto G., « Phenolic Composition and Antioxidant Activity of Aqueous Infusions from Capparis spinosa L. and Crithmum maritimum L. before and after Submission to a Two-Step in Vitro Digestion Model », J. Agric. Food Chem., 59 (23), p 12453–12459 (2011)

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Poster № 16: Auxin Control of De Novo Root Organogenesis in Arabidopsis Yachao Ge, Xiaomei Hu, Jie Yu, Lin Xu Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Correspondence to: geyachao@sibs.ac.cn

De novo organogenesis is a process through which wounded or detached plant tissues or organs regenerate adventitious roots and shoots. Auxin behaviors, including auxin biogenesis, auxin polar transport and auxin signaling had been proved to play key roles in de novo root organogenesis. Auxin is required for WUSCHEL RELATED HOMEOBOX11 (WOX11) and WOX12 activation that mediates the first-step cell fate transition from regeneration-competent cells to root founder cells. WOX11/12 directly activate WOX5/7 and LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) for the second-step cell fate transition from root founder cells to root primordium cells. Auxin acts upstream of WOX5/7, and auxin polar transport to the wounded region is required for activation of WOX5/7 by WOX11/12. We show that two related AUXIN RESPONSE FACTORs ARF6 and ARF8, are involved in de novo root organogenesis. Compared with those of the wild type, leaf explants from arf6 arf8 double mutant showed a slight delay in adventitious rooting. Expression levels of LBD16 and WOX5/7 were reduced in the arf6 arf8 double mutant background, resulting in rooting defect.

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Effect of Low Molecular Weigt Osmogenic Substanses And Solidifying Agent Of The Medium On Barley Haploid Production In Anther Culture In Vitro Bіlynska Olena V. Yuriev Plant Production Institute of National Academy of Agrarian Sciences of Ukraine, Moskovsky av. 142 Ukraine, Kharkiv, 61060. Correspondence to: bilinska@ukr.net

Osmotic pressure of nutrient medium, which depends on mineral salt composition, sugar concentration, type of gelling agent and, to a lesser extent, on other organic supplements, is known to be an important factor for morphogenesis in plant cell, tissue and organ culture in vitro. The effect of maltose concentration, osmotically active substance mannitol add and the impact of solidifying component of medium on embryo formation and plant regeneration in spring barley anther culture in vitro have been studied. DH00-126 line with a high androgenetic capacity was used as a model genotype. It has been shown that a decrease in maltose concentration from 9 to 6 % had a strong negative effect on these processes. At the same time, addition of 0.1 M mannitol to agar solidified medium containing 6 % maltose promoted sufficiently increase the efficiency of embryo formation and plant regeneration. The best result was obtained when agar was substituted for less costly chemically modified starch. Combination of starch preparation D-5aM with 9 % maltose and 0.1M mannitol resulted in increase green plant regeneration efficiency from 37 to 52 % per cultivated anthers.

Production of Doubled Haploid Lines in Durum Wheat : a Useful Tools of Selection Olfa Ayed Slama1, H. Slim Amara2

1Laboratory of Genetics and Cereal Breeding - Department of Agronomy and Plant Biotechnology - National Agronomic Institute of Tunisia, 43, Avenue Charles Nicolle 1082 -Tunis- Mahrajène, Tunis- Tunisie; 2Laboratory of Genetics and Cereal Breeding - Department of Agronomy and Plant Biotechnology - National Agronomic Institute of Tunisia, 43, Avenue Charles Nicolle 1082 -Tunis- Mahrajène, Tunis- Tunisie. Correspondence to: olfa.slama@planet.tn; olfayed@yahoo.fr

Doubled haploids production provides a strategy that offers complete homozygosity and phenotypic uniformity in one generation. The major problems of this approach for durum wheat are the low efficiency of regenerated plants and the albinism. In order to determine an efficient method in producing durum wheat green haploid plants, two haplomethods were assessed: isolated microspore and unpollinated ovaries cultures using two varieties (Khiar and Razzek). For the isolated microspore culture, Khiar regenerated 16% of haploid plants from repiquated embryos whose 10.38% are green while Razzek regenerated only 3% of plants all albinos. Using the gynogenesis technique, Khiar and Razzek produced from induced calli 28.38% and 6.66% of haploid plants respectively all of them green. In our conditions, Khiar variety showed the best performance using the two haplomethods. Compared to the microspore culture, gynogenesis has been shown to be the most efficient technique to produce green haploid plants and to ovoid albinism problem of durum wheat.

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Selection of Conditions for Germination of Astragalus Sieversianus Seeds in Laboratory Conditions Sevara Baboeva, Oksana Charishnikova, Yuliya Levitskaya Educational and Experimental Center of High Technologies, Talabalar shaharchasi 3a, Olmazor district, Tashkent 100174, Uzbekistan. Correspondence to: yv.levickaya@gmail.com

The genus Astragalus L. is one of the largest genera of vascular plants, numbering about 3,000 species, many of which are endemic plants. In the south of Uzbekistan this family has about 259 species, one of which is Astragalus sieversianus, widely used in ethnoscience. To preserve the natural range, an attempt was made to cultivate plants under laboratory conditions. Seeds of Astragalus sieversianus have very low germination due to the synthesis by sprouts of a large number of phenolic compounds. Seeds after sterilization were divided into 3 groups of 20 seeds each. In the first group, the seeds were cultivated in the Murishige Skooge medium ½ composition. Within a month, none of the seeds sprouted. In the second group, after soaking for 24 hours in the presence of a growth stimulant (plant hormone), 2 seeds swelled, but in the future a full-fledged sprout gave only one seed. In the third group, seeds after sterilization were subjected to mechanical action, and then 10 seeds were soaked in a solution of plant hormone and 10 in distilled water. Within a day, all the seeds swelled, but seeds soaked in solution with plant hormone sprouted on the third day, and seeds soaked in distilled water - only on the 14th day of the experiment. Thus, preliminary scarification and the presence of a plant hormone in the solution significantly increase seed germination and vitality of seedlings Astragalus sieversianus.

In Vitro Morphogenesis Anatomical Traits of Galanthus Nivalis L. and Galanthus Plicatus M. Bieb. Holubenko Andriy, Nuzhyna N., Golubenko A., Belava V., Svietlova N., Storozhenko V., Taran N. 60 Volodymyrska Street, 01033, Kyiv, Ukraine. Correspondence to: holubenko@yahoo.com; toddthewraithlord@gmail.com

The aims of our investigation was to study the morphogenesis and observe the main stages of microbulb regenerant formation of Galanthus nivalis L. and Galanthus plicatus M. Bieb. in vitro. Murashige and Skoog medium complemented with 1.25 mg/l BAP was the most effective for G. plicatus microbulbs development on which 6.75 regenerants per 1 chip were formed. For G. nivalis MS medium with 2 mg/l kinetin (3.67 bulbs/chip) was the most effective. For anatomical analysis there were taken morphogenic calli and microbulbs of G. plicatus and G. nivalis. Anatomical research demonstarate that the development of G. plicatus morphogenic callus is different then the one of G. nivalis. The growth speed of sheath leaves is a lot higher than the bud growth speed in comparison to G. nivalis. Visually it is observed as forming of a spherical bulb of G. plicatus from the center of which leafs appear only after 3 or 4 weeks and as almost simultaneous growth of leaves and the bulb of G. nivalis. In conclusion, the investigated species have substantial morphogenenic differences, that are manifested in individual reaction on cytokinin growth regulators composition in the medium, different development speed and order in which bulb parts form.

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Influence of Basic Salt Mixtures and Carbohydrate Source on Embryogenesis in Sweet Pepper Microspores In Vitro

Oksana Charishnikova1, Yuliya Levitskaya1, Alisher Touraev1,2

1Educational and Experimental Center of High Technologies, Talabalar shaharchasi 3a, Olmazor district, Tashkent 100174, Uzbekistan; 2Tashkent State Technical University, Department of Biotechnology, 100095, Tashkent, University str. 2. Correspondence to: charaoxa@gmail.com

Embryogenesis in vitro from isolated microspores is one of the most promising technologies for mass production of haploid and DH plants. We investigate whether the type of basal salt mixture used as a source of micro- and macroelements might affect the efficiency of microspore development. Microspores of sweet pepper pretreated in starvation media BS7 for 3 days at 33°C and then transferred to induction media supplemented with 0.25 molar maltose, 10 % PEG6000, 439 mg l-1 glutamine and 500 mg l-1, Ca(NO3)2.4H2O and different basal salt mixtures (NLN, NLN1/2, Nitsch&Nitsch, B5, MS) were tested. The development of microspores was evaluated after 2 weeks of culture. The best result is observed for half strength NLN1/2 and Nitsch&Nitsch , followed by full strength NLN basal salt mixture, then B5 and MS. Although the difference in the frequency of dividing microspores was not significant, half strength NLN1/2 basal salt mixture showed the best result with 13.4 % dividing microspores compared to Nitsch&Nitsch with 13.1 % and NLN full strength with 12.2 %. In addition microspores growing in NLN1/2, NLN and Nitsch&Nitsch salt mixtures gave rise to multiple calli structures. B5 and MS basal salt mixtures showed division frequency of 8.6 % and 8.3 %, but later on microspores failed to develop into callus or embryo structures. This showed that from all tested, NLN basal salt mixture is the most effective for microspore development and subsequent calli and embryo formation, with the most effective variation when half strength of NLN mixture is used.

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List of Poster Presentations

Poster number. Author and title Page in abstract

book

Poster № 1. Mengfan Li. Dose-Dependent BABY BOOM Function 28

Poster № 2. Robert Tripepi. Optimization of Shoot Organogenesis from Litchi Tomato Leaves 28

Poster № 3. Varvara Tvorogova. Regulation of Somatic Embryogenesis in Medicago Truncatula 29

Poster № 4. Chen Baojian. A Chemical Enhancer (C1) of Arabidopsis Somatic Embryogenesis 29

Poster № 5. Júlia Hunková. The Influence of Fluorescent Light and LED Light on the Shoot Multiplication in Amelanchier Alnifolia var. Cusickii

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Poster № 6. Raphael Kolano. Heterologous Protein Expression in Carnivorous Plants: a Potential New Drug-Delivering System

30

Poster № 7. David Downey. Development of an Improved in vitro Ovary Culture Protocol for Doubled Haploid Production in Cucumber (Cucumis Sativus L.)

31

Poster № 8. Ilona Czyczyło-Mysza. Germination of Oat (Avena Sativa L.) Haploid Embryos Depending on the Growth Regulators

31

Poster № 9. Marzena Warchoł. Recovery of Oat (Avena Sativa L.) Doubled Haploid Plants 32

Poster № 10. Thitiporn Machikowa. Development of Culture Media for Pollen Germination and Tube Growth of Sunflower

32

Poster № 11. Agnieszka Mrzygłód. Diploid Protoplasts of White Cabbage – Effectiveness of Electrofusion

33

Poster № 12. Luria Gilad. Harnessing FACS for the Evaluation of Pollen Viability and Flavonoid Content 33

Poster № 13. Michaela Svecarova. In Vitro Cultures and Polyploidization of Blue Bugle (Ajuga Reptans L.)

34

Poster № 14. Tatyana Radoeva. Auxin Dependent ARF/bHLH Module Regulates Extra-Embryonic Identity During Arabidopsis Embryogenesis

34

Poster № 15. Julien Fouilland. Influence of UV Illumination on Crithmum Maritimum in vitro Cell Culture

35

Poster № 16. Yachao Ge. Auxin Control of De Novo Root Organogenesis in Arabidopsis 36

42

List of Participants

Agnieszka Mrzygłód University of Agriculture in Krakow, Kraków, Poland a.mrzyglod@ogr.ur.krakow.pl

Alicja Białachowska Kutno Sugar Beet Breeding, Kłodawa, Poland k.stankiewicz@khbc.pl

Anastasia Matrosova Swetree Technologies, Umeå, Sweden Anastasia.Matrosova@swetree.com

Andriy Holubenko Taras Shevchenko National University of Kyiv, Kiyv, Ukraine toddthewraithlord@gmail.com

Anne Britt University of California, Davis, Davis, United States abbritt@ucdavis.edu

Anneke Horstman Wageningen University and Research, Wageningen, Netherlands anneke.horstman@wur.nl

Baojian Chen Wageningen University and Research, Wageningen, Netherlands baojian.chen@wur.nl

Barbara Wójcikowska University of Silesia, Katowice, Poland barbara.wojcikowska@us.edu.pl

Beata Dedicova Syngenta Seeds AB, Landskrona, Sweden Beata.Dedicova@syngenta.se

Benoit Leroux Vilmorin Sa, La Ménitré, France benoit.leroux@vilmorin.com

Catherine Baillon Bayer Sas, Milly La Foret, France catherine.baillon@bayer.com

Coralie Hamon Biotechmarine, Pontrieux, France coralie.hamon@airliquide.com

Cosima Dufour-Schroif L'Oreal, Aulnay Sous Bois, France cdufour-schroif@rd.loreal.com

David Downey Monsanto, San Nicolas, La Mojonera, Spain David.dg.downey@Monsanto.com

Emilie Val SECOBRA Recherches, MAULE, France emilie.val@secobra.com

Erika Toda RIKEN, Yokohama, Kanagawa, Japan erika.toda@riken.jp

Fatima Aissa Abdi Institut National de la Recherche Agronomique (inra), Versailles, France Fatima.Aissa-Abdi@inra.fr

Gilad Luria Bar-ilan University, Ramat-gan, Israel giladellu@gmail.com

Gnanaraj Muniraj Madurai Kamaraj University, Madurai, India mgnanam1987@gmail.com

Gregory Barshtein The Medicine Faculty, Hebrew University of Jerusalem, Jerusalem, Israel gregoryba@ekmd.huji.ac.il

Hassan Hamasha Jerash University, Jerash, Jordan h.hamasha@jpu.edu.jo

Heike Gnad Saaten-Union Biotec GmbH, Gatersleben, Germany gnad@saaten-union-biotec.com

Henrik Svennerstam Swedish University of Agricultural Sciences, Umeå, Sweden Henriksvennerstam@hotmail.com

Hiroshi Asami Takii Seed Company, Konan-city, Japan hiroshi-asami@takii.co.jp

Ilona Czyczyło-Mysza The F. Górski Institute of Plant Physiology Polish Academy of Sciences, Kraków, Poland czyczylo-mysza@wp.pl; i.czyczylo@ifr-pan.edu.pl

Jie Yu Chinese Academy of Sciences, Shanghai, China yujie2016@sibs.ac.cn

Judith Leibiger Saaten-Union Biotec GmbH, Seeland OT Gatersleben, Germany leibiger@saaten-union-biotec.com

Júlia Hunková Plant Science and Biodiversity Center SAS, Nitra, Slovakia julia.hunkova@savba.sk

Julien Fouilland Biotechmarine, Pontrieux, France julien.fouilland@airliquide.com

Kamila Kozak-Stankiewicz Kutno Sugar Beet Breeding, Kłodawa, Polska k.stankiewicz@khbc.pl

Kim Boutilier Wageningen University and Research, Wageningen, Netherlands kim.boutilier@wur.nl

Kin-Ying To Academia Sinica, Taipei, Taiwan kyto@gate.sinica.edu.tw

Klaus Palme Albert-Ludwigs-Universität Freiburg, Freiburg, Germany klaus.palme@biologie.uni-freiburg.de

Kristina Kosmrlj Enza Zaden Research & Development Bv, Enkhuizen, Netherlands K.Kosmrlj@enzazaden.nl

Kucera Marek The Czech Academy of Sciences, Brno, Czech Republic marekkucerabio@gmail.com

Lin Xu Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China xulin01@sibs.ac.cn

43

Manuelle Bodin Vegenov, Saint Pol de Leon, France broquet@vegenov.com

Mara Dekel Evogene, Rehovot, Israel mara.dekel@evogene.com

Marcela Notini University of Sao Paulo, Piracicaba, Brazil marcelanotini@gmail.com

Marzena Warchoł The F. Górski Institute of Plant Physiology Polish Academy of Sciences, Kraków, Poland m. warchol@ifr-pan.edu.pl

Massimo Bosacchi KWS, St. Louis, United States massimo.bosacchi@kws.com

Matthew Grasso University of Vermont, Burlington, United States msgrasso@uvm.edu

Mehran E. Shariatpanahi Agricultural Biotechnology Research Institute Of Iran (ABRII)), Karaj, Iran mehran.shariatpanahi@abrii.ac.ir

Mengfan Li Wageningen University and Research, Wageningen, Netherlands mengfan.li@wur.nl

Michaela Švécarová Palacky University In Olomouc, Olomouc, Czech Republic michaela.svecarova@upol.cz

Momoko Ikeuchi Riken, Yokohama, Japan momoko.ikeuchi@riken.jp

Murielle Philippot Vegenov, Saint Pol De Leon, France broquet@vegenov.com

Nandkumar Kunchge Beejsheetal Research Pvt. Ltd., Jalna, India kunchge@yahoo.co.in

Naoki Takemori Japan Tobacco Inc., Shizuoka, Japan naoki.takemori@jt.com

Natalya Romadanova Institute of Plant Biology and Biotechnology, Almaty, Kazakhstan nata_romadanova@mail.ru

Norio Kato Japan Tobacco Inc., Iwata, Shizuoka, Japan norio.kato@jt.com

Okamoto Takashi Tokyo Metropolitan University, Hachioji, Japan okamoto-takashi@tmu.ac.jp

Oksana Charishnikova Educational and Experimental Center of High Technologies, Tashkent, Uzbekistan charaoxa@gmail.com

Oksana Varchenko ICBGE, Kiev, Ukraine oksanavarchenko@yandex.ua

Olena Volodymyrivna Bilynska Yuriev Plant Production Institute of National Academy of Agrarian Sciences of Ukraine, Kharkiv, Ukraine bilinska@ukr.net

Olfa Ayed Slama National Agronomic Institute Of Tunisia (INAT), Tunis, Tunisia olfayed@yahoo.fr

Patricia Corral Martinez Wageningen University and Research, Wageningen, Netherlands patricia.corralmartinez@wur.nl; patriciacorralmartinez@gmail.com

Pedro Pablo Gallego University of Vigo, Vigo, Spain pgallego@uvigo.es

Preeti Preeti Mahyco, Jalna, India prtojha61@gmail.com

Raphael Kolano University Leipzig, Leipzig, Germany raphael.kolano@outlook.de

Robert Tripepi University of Idaho, Moscow, United States btripepi@uidaho.edu

Sabine Vollenweider Givaudan, Duebendorf, Switzerland sabine.vollenweider@givaudan.com

Sacco De Vries Wageningen University, Wageningen, Netherlands sacco.devries@wur.nl

Sachin Bodkhe Beejsheetal Research Pvt Ltd., Jalna, India sachin.bodkhe@beejsheetal.in

Sandra Correia University of Coimbra, Coimbra, Portugal sandraimc@ci.uc.pt

Sunday Olumide Olupinla Hosanna Medical Hospital, Ikorodu Lagos, Nigeria olumiderule4ever@gmail.com

Tatyana Radoeva Wageningen University & Research, Wageningen, Netherlands tatyana.radoeva@wur.nl

Thitiporn Machikowa Suranaree University of Technology, Nakhon Ratchasima, Thailand machiko@sut.ac.th

Thomas Dobrenel Umeå Universitet, Umeå, Sweden thomas.dobrenel@umu.se

Thomas Widiez INRA at ENS de Lyon, Lyon, France thomas.widiez@ens-lyon.fr

Tom Broeckx Ku Leuven, Heverlee, Belgium tom.broeckx@bio.kuleuven.be

Traud Winkelmann Leibniz Universitaet Hannover, Hannover, Germany traud.winkelmann@zier.uni-hannover.de

44

Václav Motyka Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czech Republic vmotyka@ueb.cas.cz

Varvara Tvorogova Saint Petersburg State University, Saint Petersburg, Russian Federation krubaza@mail.ru

Vladan Ondrej Palacky University in Olomouc, Olomouc, Czech Republic vladan.ondrej@upol.cz

Wanchai De-Eknamkul Chulalongkorn University, Bangkok, Thailand dwanchai@chula.ac.th

Weiguo Liu Syngenta, Durham, United States weiguo.liu@syngenta.com

William Voermans Rijk Zwaan Breeding BV, Fijnaart, Netherlands d.van.noort@rijkzwaan.nl

Xianwen Ji HZPC Reserach B.V., Metslawier, Netherlands xianwen.ji@hzpc.com

Xiaomei Hu Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China xmhu@sibs.ac.cn

Yachao Ge Institute of Plant Physiology & Ecology, SIBS, CAS, Shanghai, China geyachao@sibs.ac.cn

Yue Sun East China Normal University, Shanghai, China ysun@bio.ecu.edu.cn

Yuling Jiao Institute of Genetics and Developmental Biology, Beijing, China yljiao@genetics.ac.cn

Yuliya Levitskaya Educational and Experimental Center of High Technologies, Tashkent, Uzbekistan. yv.levickaya@gmail.com

Yuliya Fedorova Saint Petersburg State University, Saint Petersburg, Russian Federation julchen.fedorowa@gmail.com

Yuriy Symonenko Icbge, Kiev, Ukraine yurisymonenko@hotmail.com

Yuxin Hu Institute of Botany, Chinese Academy of Sciences, Beijing, China huyuxin@ibcas.ac.cn

Zengyu Wang The Samuel Roberts Noble Foundation, Ardmore, United States zywang@noble.org