Creating a somatic embryogenic system to study resistance traits to the white pine weevil (Pissodes strobi Peck.) in Sitka spruce (Picea sitchensis (Bong.) Carr). by Natalie Annastasia Prior B.Sc., University of Victoria, 2008 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in the Department of Biology Natalie Annastasia Prior, 2010 University of Victoria All rights reserved. This thesis may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author. ii Supervisory Committee Creating a somatic embryogenic system to study resistance traits to the white pine weevil (Pissodes strobi Peck.) in Sitka spruce (Picea sitchensis (Bong.) Carr). by Natalie Annastasia Prior B.Sc., University of Victoria, 2008 Supervisory Committee Dr. Patrick von Aderkas, (Department of Biology) Supervisor Dr. Barbara Hawkins, (Department of Biology) Departmental Member Dr. Jürgen Ehlting, (Department of Biology) Departmental Member iii Abstract Supervisory Committee Dr. Patrick von Aderkas, (Department of Biology) Supervisor Dr. Barbara Hawkins, (Department of Biology) Departmental Member Dr. Jürgen Ehlting, (Department of Biology) Departmental Member A somatic embryogenic system was created using material from the British Columbia Ministry of Forests and Range’s Sitka Spruce (Picea sitchensis (Bong.) Carr) breeding program for resistance to the white pine weevil (Pissodes strobi Peck.). The goal was to provide a system that could aid in understanding the phenotypic and genotypic variation that exists in these traits. Embryogenic lines were derived from controlled crosses of parental genotypes previously ranked for the abundance of three physical bark traits: sclereid cells, constitutive resin canals and traumatic resin canals. The number of filled seeds per cone from controlled pollinations was low, with a mean of 9.4 ± 6.8 (mean ± SD), compared to open-pollinated material, which had greater than 40 seeds per cone. The mean induction rate (to embryogenic cultures) was 7 %, ranging from 0 % to 56 % by cross. Of 135 genotypes, 88.1 % produced mature embryos. The number of embryos produced varied by culture. Nearly all (44 of 45) genotypes germinated, with a mean germination rate of 80 %. The overall conversion rate of somatic embryos to plants was 5.5 %. A novel method of cryopreservation that used a temperature pretreatment but did not require dimethyl sulfoxide was tested. Embryogenic cultures were recovered from 31 % of genotypes (n = 112). Genotypic and phenotypic variation were observed during each stage of the somatic embryogenic process. This project demonstrated that somatic embryogenesis and cryopreservation can be used to create a system to study phenotypic and genotypic variation in Sitka spruce. iv Table of Contents Supervisory Committee.......................................................................................................ii Abstract ............................................................................................................................. iii Table of Contents ...............................................................................................................iv List of Tables......................................................................................................................vi List of Figures ...................................................................................................................vii List of Abbreviations..........................................................................................................ix Acknowledgments ...............................................................................................................x Chapter 1: Introduction ......................................................................................................1 Weevils and Resistance ...............................................................................................2 Tree Breeding for Weevil-Resistance .........................................................................5 Somatic Embryogenesis and Tree Breeding ...............................................................8 Introduction to Phenotypic Variation ........................................................................10 Causes of Phenotypic Variation ................................................................................12 Phenotypic Variation in Conifers ..............................................................................16 Project Aim................................................................................................................22 Chapter 2: Creating a Somatic Embryogenic System ......................................................23 Introduction ...................................................................................................................23 Methods and Materials ..................................................................................................26 Breeding ....................................................................................................................26 Induction....................................................................................................................27 Maturation .................................................................................................................28 Germination...............................................................................................................29 Acclimatization .........................................................................................................30 Growth.......................................................................................................................31 Cryopreservation .......................................................................................................32 Histology ...................................................................................................................33 Statistical Methods ....................................................................................................35 Results ...........................................................................................................................36 Breeding and Induction .............................................................................................36 Maturation .................................................................................................................52 Germination...............................................................................................................58 Acclimatization and Growth .....................................................................................63 Cryopreservation .......................................................................................................68 Developmental Series................................................................................................76 Discussion .....................................................................................................................86 Phenotypic and Genotypic Variation ........................................................................86 Breeding ....................................................................................................................89 Induction....................................................................................................................92 Maturation .................................................................................................................96 Germination.............................................................................................................100 Growth.....................................................................................................................102 Cryopreservation .....................................................................................................104 v Development ...........................................................................................................106 Conclusions .................................................................................................................108 Chapter 3: Perspectives ..................................................................................................110 Literature Cited................................................................................................................113 Appendix .........................................................................................................................126 vi List of Tables Table 1. Number of filled seeds per cone for controlled-pollinations of Sitka spruce.....37 Table 2. Number of seeds collected per cone for open-pollinated Sitka spruce...............40 Table 3. Induction rates for somatic embryogenic cultures from Sitka spruce................42 Table 4. Responses of individual genotypes to maturation treatment..............................53 Table 5. Observed responses of genotypes (n=112) to 5°C pretreatment and their survival responses to cryopreservation...............................................................74 vii List of Figures Figures 1 - 4. Responses of Sitka spruce genotypes to induction treatment............. 48 Figure 1. Successfully induced zygotic embryo explant with translucent aggregations of immature somatic embryos............................................ 48 Figure 2. Non-embryogenic green callus...............................................................