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Carbohydrate Status of in Vitro Grown Trillium Rhizomes

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Carbohydrate Status of in Vitro Grown Trillium Rhizomes CARBOHYDRATE STATUS OF IN VITRO GROWN TRILLIUM RHIZOMES by David W. Opalka A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Plant and Soil Science Winter 2006 Copyright 2006 David W. Opalka All Rights Reserved UMI Number: 1432293 UMI Microform 1432293 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 CARBOHYDRATE STATUS OF IN VITRO GROWN TRILLIUM RHIZOMES by David W. Opalka Approved: __________________________________________________________ S.L. Kitto, Ph.D. Advisory Professor and Professor of Horticulture in the Plant and Soil Sciences Department Approved: __________________________________________________________ Donald L. Sparks, Ph.D. Departmental Chair of the Plant and Soil Sciences Department Approved: __________________________________________________________ Robin Morgan, Ph.D. Dean of the College of Agriculture and Natural Resources Approved: __________________________________________________________ Conrado M. Gempesaw II, Ph.D. Vice Provost for Academic and International Programs ACKNOWLEDGMENTS Thanks: Sherry L. Kitto, Ph.D. For your guidance over the past two years. I’ve appreciated your ability to talk about so many different subjects and your relentless skill for generating ideas. John Boyer, Ph.D, Jeanne Frett and Robert Griesbach, Ph.D. For helpful ideas and availability throughout the course of this research. Hugh Frick, Ph.D. and Thompson Pizzolato, Ph.D. For discussions and guidance on the finer points of anatomy and physiology, and for help interpreting observations and developing assays. Mark Roh, Ph.D and Deric Picton For your generosity in not only allowing me to analyze samples on your HPLC, but by making it as easy as possible for me to do so. Jesse Sinanan and Christen Pollinger For all of your help with general lab duties and setting up experiments. I was so spoiled by not having to do any of my own dishes. This manuscript is dedicated: In memory of Matthew Swanson. A true encourager, intellectual and friend who was not afraid to ask the hard questions that lead to the shaping of character. To Tammy L. Opalka for her amazing support and patience. Your encouragement has given me the attitude for endurance that I so often needed over the past two years. iii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….…viii ABSTRACT ..................................................................................................................ix Chapter 1 – Introduction & Literature Review………………………………………...1 1.1 Trillium………………………………………………………………………1 1.1.1 Botany……….…………………………………………………………1 1.1.2 Micropropagation for conservation and maintenance……..…………4 1.1.2.1 Proliferation of plant material…………………………………6 1.1.2.1.1 Growth regulators…………………………………..6 1.1.2.1.1.1 Auxin……………..…..………………….6 1.1.2.1.1.2 Cytokinin………………………………...6 1.1.2.1.1.3 Gibberellic Acid……………..…………..7 1.1.2.2 Re-establishment………………..……………………………..7 1.1.2.3 Acclimation and stratification……………...………………….8 1.1.2.3.1 Field growth……….………………………………..8 1.1.3 Analysis of rhizome carbohydrate status………………….….……….9 1.1.3.1 Starch stain method……………...…………………………….10 1.1.3.2 Starch quantitation method…………...…..……………………11 1.1.3.3 HPLC method………………………...…..……………………12 Chapter 2 - In vitro culture…………………………………………………………..13 2.1 Introduction……………………...…………………………………………..13 2.2 Materials and Methods…………………………...………………………….14 2.2.1 Plant Material………………..………………………………………..14 2.2.2 Tissue culture media……………....…………………………………..14 2.2.3 Re-establishment protocol………...…………………………………..15 2.2.4 Rhizome harvest protocol………………..……………………………15 2.2.5 Processing rhizomes for quantitative carbohydrate assays……………16 2.2.5.1 Colorimetric assay for starch………...…...…………………….16 2.2.6 Methods Development…………………..……………………………18 2.2.6.1 Starch stain……………………………………………………..18 2.2.6.1.1 Titration of iodine stain to determine affects of starch density……………………………………….18 2.2.6.1.2 Qualitative starch determination for rhizomes iv under varied conditions…………………19 2.2.6.2 Quantitative colorimetric assay for starch ……..…………….19 2.2.6.2.1 Comparison of starch content in rhizomes…………19 2.2.6.3 HPLC assay for sugars……………………………………….20 2.2.6.3.1 In vitro assessment of carbohydrate at 4-day intervals for 1 month……………………………….20 2.2.7 Proliferation…………………………………………………………..21 2.2.7.1 Bead sterilizer vs. flame sterilization ………..………………21 2.2.7.2 Rhizome shoot excision experiment……………..…………..21 2.2.8 Influence of growth regulators on rhizome carbohydrates……..……..22 2.2.8.1 In vitro experiment with auxin (IBA & NAA) ..……………22 2.2.8.2 In vitro experiment with cytokinin(BA)…………………….22 2.2.8.3 Gibberellic acid………………………………………………24 2.2.8.3.1 Preliminary GA experiment…………….…………..24 2.2.8.3.2 In vitro experiment with GA………....…………….24 2.3 Results………………………...……………………………………………..25 2.3.1 Methods development…………………………...……………………25 2.3.1.1 Starch stain……………………...……………………………25 2.3.1.1.1 Titration of iodine stain to determine affects of starch density……………………………………...25 2.3.1.1.2 Qualitative starch determination for rhizomes under varied conditions……...……………………….28 2.3.1.2 Quantitative colorimetric assay for starch..…….……………..31 2.3.1.2.1 Comparison of starch content in rhizomes…..………31 2.3.1.3 HPLC assay for sugars………………………..………………31 2.3.1.3.1 In vitro assessment of carbohydrate at 4-day intervals for 1 month………………………………..31 2.3.2 Proliferation…………………………………………………………..33 2.3.2.1 Bead sterilizer vs. flame sterilization………...……………….33 2.3.2.2 Rhizome shoot excision experiment……………...…………..33 2.3.3 Influence of growth regulators on rhizome carbohydrates……..……..34 2.3.3.1 Auxin…………...…………………………………………….34 2.3.3.2 Cytokinin ………………….…………………………………39 2.3.3.3 Gibberellic acid………………………………………………44 2.3.3.3.1 Preliminary GA experiment……………...………….44 2.3.3.3.2 In vitro experiment with GA…………..…………….44 2.4 Discussion………………………..………………………………………….49 Chapter 3 - Ex vitro re-establishment………………….……………………………..53 3.1 Introduction…………………….……………………………………………53 3.2 Materials and Methods…………..…………………………………………..54 v 3.2.1 Plant Material…………………..……………………………………..54 3.2.2 Tissue culture medium……………………………..……………………54 3.2.3 Re-establishment protocol…………………….………………………54 3.2.4 Processing rhizomes for analysis………………..…………………….55 3.2.5 Starch stain………………………….…………………………………55 3.2.6 Starch colorimetric assay……………….……………………………..55 3.2.7 HPLC assay for sugars…………………..…………………………….55 3.2.8 Re-establishment………………………………………………………55 3.2.8.1 Acclimation…………………..………………………………..55 3.2.8.1.1 Rhizomes at 4°C for 12 wks/ greenhouse for 6wks ...55 3.2.8.1.2 Comparison of different greenhouse temperatures ....55 3.2.8.1.3 Comparison of 4°C coldrooms ………….....……….56 3.2.8.1.4 Rhizomes at 4°C for 9wks……………..……………56 3.2.8.2 Temperature stratification………………..…...………………57 3.2.8.2.1 Rhizomes in preliminary experiment to examine an offset stratification scheme ……………………57 3.2.8.2.2 Rhizomes put through two offset stratification regimens for varied time intervals ...…..57 3.3 Results……………………………………………………………………….58 3.3.1 Re-establishment………………….…………………………………..58 3.3.1.1 Acclimation………………...………………………………….58 3.3.1.1.1 Rhizomes at 4°C for 12 wks/ greenhouse for 6wks..…58 3.3.1.1.2 Comparison of different greenhouse temperatures ..…58 3.3.1.1.3 Comparison of 4°C coldrooms………………....…….59 3.3.1.1.4 Rhizomes at 4°C for 9wks……………………………59 3.3.1.2 Temperature stratification………………….…………………64 3.3.1.2.1 Rhizomes in preliminary experiment to examine an offset stratification scheme ……………………...64 3.3.1.2.2 Rhizomes put through two offset stratification regimens for varied time intervals..…….68 3.4 Discussion…….……………………………………………………………..72 Chapter 4 – Field Growth………...……..…………………………………………….75 4.1 Introduction………………………………………………………………… 75 4.2 Materials and Methods………………………………………………………75 4.2.1 Rhizome harvest…………..…………………………………………..75 4.2.2 Starch stain……………………...…………………………………….76 4.2.3 Starch colormetric assay………...…………………………………….76 4.2.4 HPLC assay for sugars…..………….…………………………………76 4.2.5 Field growth…………….…………………………………………….76 4.2.5.1 Series of field grown rhizomes (Trillium discolor)……..…….76 4.3 Results………………………………………………………………………76 vi 4.3.1 Field growth ……………………..…………………………………...76 4.4 Discussion………………………..…………………………………………80 Conclusion……………………………………………………………………………83 References……………………………...……………………………………………..85 vii LIST OF FIGURES Figure 1: The worldwide distribution of Trillium ………………………...…………...2 Figure 2: Indication of excision area for removal of apical shoot……….…………...23 Figure 3: Lugol’s iodine stain titration images…………………………..……....26&27 Figure 4: Lugol’s iodine stain of rhizomes from varied treatments…….….…….29&30 Figure 5: Comparison of starch concentrations of rhizomes direct from tissue culture and those stored at 4°C for 1 year………………….………..32 Figures 6A to D: The effect of auxin (IBA and NAA) on carbohydrate profile....35-38 Figures 7A to D: The affect of varied levels of cytokinin (BA) on carbohydrate profile……………..…………………..……………………..40-43 Figures 8A to D: The effect of gibberellic acid (GA) on carbohydrate profile......45-48 Figures 9A to D: Changes in carbohydrate over 9 weeks………….…..…………60-63 Figure 10A: Lugol’s iodine stain of rhizomes harvested from Mt. Cuba Inc. at 24 weeks after planting……………………………….…………………65 Figure 10B: Lugol’s iodine stain of rhizomes harvested
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