Flower metabolic engineering projects

image1I am looking for a thesis student (spring 2019 for 6 months) that is interested in metabolic engineering of secondary metabolites by CRISPR, RNAi, and/or overexpression of target genes. Background with biotechnology, and/or molecular biology and cloning is essential. You will clone, transform plants, analyse transgene expression and measure metabolites.

If you are interested, please send a short CV with motivation to julian.verdonk@wur.nl

 

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Postharvest Technology Course 2018

The 5th edition of the Postharvest Technology course was held in Wageningen last week. Course leaders Ernst Woltering and Julian Verdonk, lecturers, Rob Schouten, Eelke Westra, Leo Lukasse, Jan van Kan, Marcel Wenneker, Jan Verschoor, and guest course leader Mary Lu Arpaia, were put together in four days of lectures, demo’s and pitches, and and excursion. We hosted participants from industry and education from all over the world, to teach them about product physiology and the cold chain of a large variety of fruits, vegetables and ornamental plant products.  Continue reading

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plasmid isolation, alkaline lysis (minprep)

introduction

The isolation of plasmids from bacteria, is one of  the simplest biochemical excersize in a molecular biological lab. It is a beautiful illustartion where the pH and differences in solubility of molecules are used to separate them from each other. The wole procedure can be done in about 30 min, quick and clean. The method is desribed in Birnboim and Doly, (1979) and also on wikipedia. Continue reading

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Oranje Boven (Orange on top) Is there mutual exclusion between anthocyanins and carotenoids in petunia flowers?

IMG_6724

Orange GMO Petunia. Photo credit Teemu Teeri

Nov 2017: In the 5th round of the Graduate School Tuinbouw & Uitgangsmaterialen, the proposal of Sara Abdou and Julian Verdonk was granted.

It is a collaboration with Francesca Quattrocchio of the University of Amsterdam and Teemu Teeri from the university of Helsinki, and industry partners from Deliflor, Dümmen Orange, Florensis, Gene Twister and Hudson River Biotechnology. Continue reading

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DNAse-free RNA isolation (for qRT), the Citrate-Citric Acid method

introduction

plant RNA isolations often requires the of use unhealthy organic solutions like phenol or beta-mercaptoethanol, and although I still prefer the TriZOL method or the SDS-TriZOL combo for when I need large quantities of great RNA, you have to wonder if it will ever be needed for me to do a northern blot. This method described here is for when you dont need that much RNA, for example because you’re going to do qRT-PCR. Here is a method to quickly and more importantly cheaply isolate good RNA from relatively small amounts of tissue (30-50 mg/a few 0.5 cm leaf discs) using only a low pH from citric acid  to inactivate the RNAses. The method is described by Oñate-Sánchez and Vicente-Carbajosa (2008). Continue reading

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SDS-TRIzol Combo RNA isolation from high carbohydrate containing tissues

Introduction

For simple tissues, I prefer to isolate RNA with the Citrate-Citric Acid method, but the isolation of good quality RNA of fruits and other plant tissue with a lot of sugars and/or other inhibiting crap in it can be quite a challenge. The TRIzol method is not always the best option because these compounds sometimes have similar biochemical characteristics as the nucleic acids we try to isolate. This method was used on maize aleurones (Reyes et al., 2011) based on Holding et al., (2007). You can do it all in eppendorf tubes, use about 0.2 g/sample, which is enough for a high enough yield and quality for RNA seq. Continue reading

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Julian C. Verdonk

Academic PositionPasphoto may 2014

  • Assistant Professor (1.0 FTE, tenure track) of Horticulture and Product Physiology, Plant Science Group, Wageningen University

 Research experience

  • 2013. Research Associate: Universidad de Andrés Bello, Centro de Biotecnología Vegetal, at Santiago, Chile. Principal Investigator: Ariel Orellana
  • 2012. Research Scientist: University of Wisconsin, Department of Botany, at Madison, WI, USA. PI: Hiroshi Maeda
  • 2010. Plant Molecular Research Geneticist: Cell Wall Biology & Utilization Research Unit, US Dairy Forage Research Center, ARS-USDA at Madison, WI, USA. PI: Mike L. Sullivan
  • 2008. Post-doctoral fellow: Great Lakes Bioenergy Research Center, Department of Genetics University of Wisconsin at Madison, WI, USA. PI: Patrick Masson
  • 2006. Post-doctoral fellow: Department of Environmental Horticulture, Institute of Agricultural and Food Science at the University of Florida. PI: David Clark
  • 2001. PhD: Universiteit van Amsterdam, Dept. of Plant Physiology. PI: Robert Schuurink

 

Education

  • 2006. Universiteit van Amsterdam, PhD. Biological Sciences. Promotors: Michel Haring & Arjen van Tunen; Co-promotor: Robert Schuurink. Dissertation: Floral Scent Production by Petunia hybrida
  • 2001. Universiteit van Amsterdam, MSc. Biotechnology

 

Publications

H-index: 10. Google Scholar profile

  • Rob E. Schouten, Julian C. Verdonk, and Uulke van Meeteren. (2018). Re-evaluating the role of bacteria in gerbera vase life. Postharvest Biology and Technology 143, 1–12.
  • Rob E. Schouten, Luka van Dien, Arwa Shahin, Sjoukje Heimovaara, Uulke van Meeteren, and Julian C. Verdonk. (2018). Combined preharvest and postharvest treatments affect rapid leaf wilting in Bouvardia cut flowers. Scientia Horticulturae 227, 75–78.
  • Patricio Olmedo, Adrián A. Moreno, Dayan Sanhueza, Iván Balic, Christian Silva-Sanzana, Baltasar Zepeda, Julian C. Verdonk, César Arriagada, Claudio Meneses,
    Reinaldo Campos-Vargas. (2018). A catechol oxidase AcPPO from cherimoya (Annona cherimola Mill.) is localized to the Golgi apparatus. Plant Science 266, 46–54.
  • Nur Fariza M Shaipulah, Joelle K Muhlemann, Benjamin D Woodworth, Alex Van Moerkercke, Julian C Verdonk, Aldana M Ramirez, Michel A Haring, Natalia Dudareva, Robert Schuurink (2015). CCoAOMT downregulation activates anthocyanin biosynthesis in petunia. Plant Physiology 2015 Nov 30. pii: pp.01646.2015
  • Iván Balic, Troy Ejsmentewicz, Dayan Sanhueza, Christian Silva, Tamara Peredo, Miriam Barros, Julian C. Verdonk, Claudio Meneses, Ariel Orellana, Bruno G. Defilippi, Reinaldo Campos-Vargas (2014). Molecular and Physiological Study of Firmness of Table Grape Berry. Postharvest Biology and Technology 93, 15-23
  • Julian C. Verdonk, Ronald D. Hatfield, and Michael L Sullivan (2012). Proteomic analysis of cell walls of two developmental stages of alfalfa stems. Front Plant Sci. 3: 279
  • Julian C. Verdonk and Michael L Sullivan (2012). Micro RNA induced gene silencing in alfalfa (Medicago sativa). Botany 91 (2), 117-122
  • Alex Van Moerkercke, Carlos S. Galván-Ampudia, Julian C. Verdonk, Michel A. Haring and Robert C. Schuurink (2012). Regulators of floral 1 fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals. J Exp Bot. 2012 Feb 15
  • Thomas A. Colquhoun, Michael L. Schwieterman, Ashlyn E. Wedde, Bernardus C.J. Schimmel, Danielle M. Marciniak, Julian C. Verdonk, Joo Young Kim, Youngjoo Oh, Ivan Gális, Ian T. Baldwin, David G. Clark (2011). EOBII Controls Flower Opening by Functioning as a General Transcriptomic Switch. Plant Physiol. 2011 Jun; 156 (2) :974-84
  • Thomas A. Colquhoun, Julian C. Verdonk, Bernardus C.J. Schimmel, Denise M. Tieman, Beverly A. Underwood and David G. Clark (2010). Petunia floral volatile benzenoid/phenylpropanoid genes are regulated in a similar manner. Phytochemistry 2010 Feb; 71 (2-3) : 158-67
  • Julian C. Verdonk*, Kenichi Shibuya*, Holly M. Loucas, Thomas A. Colquhoun, Beverly A. Underwood, and David G. Clark (2008). Flower specific expression of the Agrobacterium tumefaciens isopentenyltransferase gene results in radial expansion of floral organs in Petunia hybrida. Plant Biotechnol J. 2008 Sep; 6 (7) : 694-701
  • Rickard J. Dexter*, Julian C. Verdonk*, Beverly A. Underwood, Eric A. Schmelz and David G. Clark (2008). Tissue-specific PhBPBT expression is differentially regulated in response to endogenous ethylene signals. J Exp Bot. 2008 ; 59 (3) : 609-18.
  • Julian C. Verdonk, Michel A. Haring, Arjen J. van Tunen and Robert C. Schuurink (2005). ODORANT1 Regulates Fragrance Biosynthesis in Petunia Flowers. The Plant Cell 17 (5): 1612
  • Julian C. Verdonk, C.H. Ric de Vos, Harrie A. Verhoeven, Arjen J. van Tunen, Michel A. Haring and Robert C. Schuurink (2003). Regulation of floral scent production in Petunia revealed by targeted metabolomics. Phytochemistry 62, 997-1008.
  • Ana M. Laxalt, Bas ter Riet, Julian C. Verdonk, Lisa Parigi, Wladimir I. L. Tameling, Jack Vossen, Michel Haring, Alan Musgrave and Teun Munnik (2001). Characterisation of five phospholipase D cDNAs: rapid and specific expression of LePLDβ1 on elicitation with xylanase. The Plant Journal 26 (3), 237-247.

Books and Book Chapters

  • Laura M. Vaughn, Katherine L. Baldwin, Gengxiang Jia, Julian C. Verdonk, Allison K. Strohm, and Patrick H. Masson (2011). The Cytoskeleton and Root Growth Behavior. The Plant Cytoskeleton, Advances in Plant Biology, 2011, Volume 2, Part 3, 307-326
  • David G. Clark, Eran Pichersky, Julian C. Verdonk, Natalia Dudareva, Michel A. Haring, Ulrich Klahre, and Robert C. Schuurink (2009). Benzenoids dominate the fragrance of Petunia flowers. Petunia Evolutionary, Developmental and Physiological Genetics (2nd Ed). Tom Gerats and Judy Strommer (Eds.). Springer Life Sciences
  • Julian C. Verdonk. Floral scent production in Petunia hybrida (2006). PhD thesis, Universiteit van Amsterdam, Amsterdam, The Netherlands
  • Julian C. Verdonk, Michel A. Haring, Arjen J. van Tunen and Robert C. Schuurink (2006). Targeted Transcriptomics to Elucidate the Regulation of Benzenoid Synthesis in Petunia hybrida. Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues (1st Edition). Jaime A Teixeira da Silva (Ed). Global Science Books, Ltd., London, UK

Patents

  • Julian C. Verdonk, Michel A. Haring, Arjen J. van Tunen and Robert C. Schuurink (2004). Novel regulatory Protein. ODORANT1 Regulates Fragrance Biosynthesis in Petunia Flowers. US Patent App. 11/661,758. WO Patent 2,006,041,280. EP Patent 1,789,555

 

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RNA isolation with homemade TRIzol reagent

Introduction

My all time favorite RNA isolation, and most used protocol is the TRIzol method. Here I give you the basic protocol, including the recipe to make it yourself, which will save you a lot of money.

Method

  • Grind ~100 mg tissue in LN2 in a 10 ml polypropylene tube and a glass rod
  • Transfer powder to 1.5 mL tube (approx 1/4th of the tube filled with powder)
  • Add 1.2 ml TRIzol (TRIzol is best 1:10 (1 g 10 ml), but 1:5 and 1:2 works also fine)
  • Mix well by inverting, and make sure the mixture is completely molten
  • Shake vigorously 15” Don’t vortex! (shears gDNA, and can cause higher gDNA contamination)
  • Incubate 5’ @ RT
  • Add 0.3 ml CHCl3
  • Shake vigorously 15’’
  • Spin 5’ @ max speed
  • Optional: Perform a Phenol: CHCl3 (1:1) treatment to the (upper) aqueous phase followed by a CHCl3 treatment.
  • Transfer aqueous phase (approx 0.5 ml) to new tube
  • Add 1 volume (0.5 ml) isopropanol to precipitate
  • Mix well by inverting
  • Precipitate 10’ @ RT
  • Spin 5’@ max speed
  • Wash RNA pellet with 70% EtOH
  • Dry pellet
  • Dissolve in appropriate vol. of RNAse free MQ
  • Quantify on gel (1µl) and spectrophotometrically

Preparing TRIzol:

composition:
38%     Phenol (pure phenol from crystals, i.e. SIGMA P1037-500G)
0.8 M  Guanidine Thiocyanate (118.16 g/mol)
0.4 M  Ammonium Thiocyanate (79.12 g/mol)
0.1 M  Sodium Acetate (82.03 g/mol)
5 %      Glycerol

  • It is easiest to buy 500 g Phenol crystals, melt it in a water bath @ ~50 °C and then calculate the rest accordingly with 500 g phenol as 38%.
  • The density of phenol is 1.07 g/cm³, so if 500 g is 38%, this will be 500/1.07 = 467.3 ml, and therefore the total volume will be 467.3 *(100/38)= 1.23 liter.
  • Now you can calculate the rest:
    • 1.23 liter 0.8 M Guanide Thiocyanate: 0.8 M x 118.16 g/mol x 1.23 l = 116.3 g
    • 1.23 liter 0.4 M Ammonium Thiocyanate: 0.4 M x 79.12 g/mol x 1.23 = 38.9 g
    • 1.23 liter 0.1 M Sodium Acetate:1.23 x 0.1)/3 = 41 ml 3 M NaAc pH 5
    • 1.23 liter 5% Glycerol: 1.23/20 = 61.5 ml 100% glycerol
  • Make the aqueous solution (the salts and the glycerol) first, and then when the phenol is molten, add the phenol to it, mix well, aliquote (unless you have 1.23 L bottle), and store it in a cool dark place. Just make sure you dont shoot over te endvolume of 1.23 liter
  • The preparation of 1.23 liter TRIzol will cost you about 10 times less than buying the reagent
  • This TRIzol is not pink, but that is not important, just remember that Chloroform is heavier than water

Remarks:

  • If you expect a lot of contamination of sugars (for example if you are working with fruit) a less-stringent precipitation can help although the yield will also decrease
  • Instead of 1 volume isopropanol precipitate using 0.5 volume isopropanol and 0.5 volume of 0.8 M sodium citrate : 1.2 M NaCl (1:1)
  • There is a better method to get rid of the sugars: the SDS-TRIzol combo method
  • This protocol was previously published on my blog

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