Gladiolus breeding for rapid generation cycling for potted plant production and the discovery of gladiolus genes, UPSTREAM OF FLOWERING LOCUS C (UFC) and FLOWERING LOCUS C EXPRESSOR (FLX) A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Jaser A S A Aljaser IN PARTIAL FULFILMENT OF THE REQUIRMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY Neil O. Anderson, Advisor July 2020 © Jaser A S A Aljaser 2020 Acknowledgments I would like to give many thanks and acknowledgements for the following people who helped me in the research project and guided me through experiments: • Dr. Neil Anderson (Primary Investigator) for allowing me to work in this project, for his overall guidance during the whole research, providing materials, equipment, comments on the dissertation and encouragement in graduate school. • Dr. Alan Smith, Dr. Neil Olszewski and Dr. Changbin Chen (Committee) for their guidance in experiments, advise and commenting on the dissertation. • Dr. Andrzej Noyszewski for his help in the lab experiments procedures and guidance in the lab analyzing the DNA results. • Dr. Neil Anderson’s lab members for their help in providing greenhouse suitable environment for gladiolus cultivation. i Dedication • Kuwait University for offering me the scholarship and taking care of the financial support for my graduate studies in horticultural at the University of Minnesota. • My parents and siblings for their love, encouragement and support to help me overcome the challenges. ii Jaser Aljaser Abstract Gladiolus (Gladiolus ×hybridus Rodigas) is a geophytic floriculture crop, cultivated for cut flower and garden ornamental uses. It is a perennial crop and is characterized to have a long juvenile period of up to 5 years, with tall plant height which is undesirable for potted production and poorly understood in flowering pathway. In this research, the constant selection in gladiolus breeding at the University of Minnesota for rapid generation cycling (RGC) resulted in reducing the juvenility time from 3 – 5 years into a range of 3 – 11 months from germinating seedlings, recurrent selection breeding method was significantly early in flowering time (21.63 weeks). Also, our gladiolus genotypes have reduced dormancy, are able to re-sprout after senescence, which is a new trait in gladiolus. These traits in RGC genotypes were tested further with the application of ancymidol, a gibberellin inhibitor to control height in potted plants. The high concentrations of ancymidol can inhibit flowering for commercial cultivars. However, the RGC lines had significant reductions in plant height to a more acceptable plant size within the aesthetic ratio and the ability to flower while commercial cultivar comparisons failed to flower at higher concentrations of ancymidol. All this knowledge drove us to test for the existence of flowering genes in gladiolus. Herein we were able to discover the existence of UPSTREAM OF FLOWERING LOCUS C (UFC) and FLOWERING LOCUS C EXPRESSOR (FLX) in gladiolus. UFC is an adjacent gene to FLOWERING LOCUS C (FLC), the floral repressor in Arabidopsis thaliana, while FLX gene upregulates FRIGIDA (FRI) which upregulates FLC expression. The amino acid sequence of UFC has up to 57% identity to Musa acuminata. The discovered FLX gene in gladiolus is partially completed which is missing 2 exons and shows up ~65% of identity of FLX to Ananas comosus. These two, newly discovered genes in gladiolus, can provide better iii understanding of the flowering and vernalization response in ornamental geophytes and begin clarification of the effects of breeding for RGC on flowering genes / alleles as well as the application of plant growth retardants for production of dwarf gladiolus for potted plant production iv Table of contents Acknowledgements ………………………………………………………………………………. i Dedication ………………………………………………………………………………………... ii Abstract …………………………………………………………………………………………. iii Table of contents …………………………………………………………………………………. v List of tables ……………………………………………………………………………………. vii List of figures ……………………………………………………………………………………. ix List of Appendices …………………………………………………………………………….... xii 1.0 Literature review ………………………………………………………………………… 1 1.1 Gladiolus, Taxonomy and distribution ……………………………………………… 1 1.2 Botanical description ………………………………….…………….……….……… 2 1.3 Crop Production and sales …………………………………………………….….…. 3 1.4 Pests and diseases …………………………………………………………………… 4 1.5 Gladiolus breeding ………………………………………………………………….. 5 1.6 Gladiolus genetics …………………………………………………………………... 7 1.7 Gladiolus and the use of plant growth regulators …………………………………… 8 1.8 Flowering models and genes ………………………………………………………. 10 1.9 Literature cited ……………………………………………………………….….… 14 2.0 Gladiolus breeding for rapid generation cycling and reduced dormancy ………….…... 31 2.1 Preface ………………………………………………………………………….… 32 2.2 Introduction ……………………………………………………………….….….… 33 2.3 Material and Method ………………………………………………………….…… 37 2.4 Results ………………………………………………………………….….….…… 39 v 2.5 Discussion ……………………………………………………………………….… 41 2.6 Literature cited ……………………………………………………………….….… 46 3.0 Effects of a gibberellin inhibitor on flowering, vegetative propagation and production of rapid generation cycling gladiolus for potted plant production ………………………... 69 3.1 Preface ……………………………………………………………………….….… 70 3.2 Introduction ……………………………………………………………….….….… 71 3.3 Material and Methods ……………………………………………………………… 74 3.4 Results …………………………………………………………………….….….… 77 3.5 Discussion ……………………………………………………………………….… 78 3.6 Literature cited ……………………………………………………………….….… 82 4.0 Discovery of UPSTREAM OF FLOWERING LOCUS C (UFC) and FLOWERING LOCUS C EXPRESSOR (FLX) Genes in Gladiolus ×hybridus and G. dalenii ………... 96 4.1 Preface ………………………………………………………………….………… 97 4.2 Introduction ……………………………………………………………….….….… 98 4.3 Material and Method ……………………………………………………………... 103 4.4 Results …………………………………………………………………….……… 107 4.5 Discussion ………………………………………………………………………... 109 4.6 Literature cited …………………………………………………………………… 114 5.0 General conclusion …………………………………………………………………… 138 5.1 Literature cited …………………………………………………………………… 141 Appendices …………………………………………………………………………….……… 143 vi List of tables Chapter 2.0 Gladiolus breeding for rapid generation cycling and reduced dormancy Table 1. Mean number of weeks of gladiolus seed germination, number of weeks of flowering from seed germination and percentage of flowering in every generation ………………………. 50 Table 2. Number of gladiolus seedling, number of re-sprouted seedling, dormant and percentage of re-sprouted gladiolus plants in every generation and 1:1 Chi-square (χ2) test ratios of the tested Gladiolus generations …………………………………………………………………………... 51 Chapter 3.0 Effects of a gibberellin inhibitor on flowering, vegetative propagation and production of rapid generation cycling gladiolus for potted plant production Table 1. Number of replicates / treatment (0, 100, 400 ppm A-Rest®) and commercial or breeding source of the tested Gladiolus genotypes. …………………………………………….. 87 Table 2. Influence of A-Rest® concentrations (0, 100, and 400 ppm) on the number of flowering plants (frequency of flowering) and 1:1:1 Chi-square (χ2) test ratios of the tested Gladiolus genotypes .………………………………………………………………………………………. 88 Table 3. Influence of A-Rest® concentrations (0, 100, and 400 ppm) on the number of flowering plants (frequency of flowering) and mean number of weeks reached to flowering in each genotypes of the tested Gladiolus genotypes …………………………………………………... 89 Table 4. Mean plant height (cm), flower stalk height (cm), number of stalks, leaf width (cm), no. of corms, fresh weight (FW) of corms (g), number of cormels, and fresh weight (FW) of cormels (g) for seven gladiolus genotypes corms treated with different concentrations of A-Rest® (0, 100, and 400 ppm) …………………………………………………………………………………… 91 vii Chapter 4.0 Discovery of UPSTREAM OF FLOWERING LOCUS C (UFC) and FLOWERING LOCUS C EXPRESSOR (FLX) Genes in Gladiolus ×hybridus and G. dalenii Table 1. The Gladiolus genotypes used for the study …………………………………………. 125 Table 2. The identity of amino acid sequences and number (%) of two UFC proteins (GhUFC-A, GhUFC-B) in two Gladiolus (genotypes 1 and 15) in relation to other species (Gene locus/ID) through pair alignment ………………………………………………………………………… 126 Table 3. Identity of amino acid sequences, number (%) of UFC proteins in the conserved domain DUF966 in Gladiolus genotypes in relation to other species through pair alignment ………… 127 Table 4. Number (%) of amino acid sequences of GhFLX protein in Gladiolus genotypes 3 and 6 (genotype 16 is identical to genotype 6) in relation to the other species through pair alignment ………………………………………………………………………………………………….. 128 viii List of figures Chapter 2.0 Gladiolus breeding for rapid generation cycling and reduced dormancy Figure. 1 A. University of Minnesota gladiolus breeding lines with unique flower colors and patterning ……………………………………………………………………………………….. 52 Figure 2. The breeding scheme for rapid generation cycling (RGC) gladiolus ………………… 53 Figure 3. The University of Minnesota gladiolus breeding lines have large capsule (fruit) as evidence of fertility ……………………………………………………………………………... 54 Figure 4. Representation of number of flowering plants in cycles (years) in respect of parent generation (Green line) and RGC-1 population (Blue line) …………………………………….. 55 Figure 5. Phenotypes of the rapid generation cycling (RGC-1) gladioli ……………………….. 56 Figure 6. Frequency of the number of flowering plants in the RGC-2 recurrent selection generation ……………………………………………………………………………………….