INTERSPECIFIC HYBRIDIZATION IN LEDCAENA BENTHAN A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRONOMY AND SOIL SCIENCE DECEMBER 1987 By Charles T. Sorensson Thesis Committee Dr. James L. Brewbakerf Chairman Dr. Peter P. Rotar Dr. Richard M. Manshardt We certify that we have read this thesis and that in our opinion it is satisfactory in scope and quality for the degree of MASTER OF AGRONOMY in Agronomy and Soil Science. Thesis Committee V Z t t L C a I Dr. ,J. L. Brewbaker^ Chairman /i> Dr. p. p. Rotar h j Dr. R. M. Manshardt 11 ACKNOWLEDGEMENTS My undergraduate advisor at Colorado State University/ Dr. Wayne F. Keimr and Dr. Sheldon E. Ladd both encouraged me to stretch my focus to tropical agronomy. The members of my committee/ Dr. Peter P. Rotar/ Dr. Richard M. Manshardt/ and especially Dr. James L. Brewbaker/ encouraged me throughout the study and offered me excellent direction in the preparation of this thesis. Dr. Gerald D. Carr spent many hours patiently helping me in cytology and allowed me to use his equipment. Mr. George Linney helped with micropreparation and photo reproduction. Financial support for my graduate research assistantship came from the HITAHR Project 803 funded by Mclntyre/Stennis Forestry Research funds. International Breeding and Plant Germplasm Resources Center (IBPGR) funded a collecting expedition in 1985 to search for cold-tolerant leucaenas. My thesis is fondly dedicated to my mother and father/ Ann and Jan Sorensson. Many thanks also to my dear wife/ Melinda/ and to the many technicians and researchers who contributed to this work. I l l ABSTRACT Thirteen Leucaena species and subspecies were studied from 1982-1987. Their valid species status was previously determined from chromosome numbers/ geographical distributions/ ecology and morphology. Present studies include self-incompatibility/ interspecific hybridization/ and morphological analyses of FI interspecific hybrids. Self-incompatibility (SI) was tested by hand pollinating 184 flower heads (2/805 florets). Two tetraploid species (4x=104) were self-fertile of the three tetraploid and eight diploid species selfed. Selfed progenies of L. esculenta (2x=52) were discovered. L. retusa (2x=56) was weakly self- fertile. Possible selfed progenies of four other diploid species were grown. None of the species hybrids grown were self-compatible except those derived from mating self­ compatible species. A refined emasculation technique helped in hand pollinating 1420 inter- and sub-specific crosses (22/193 florets)/ thereby testing 135 of the 156 (86.5 %) species combinations in a 13 x 13 diallel. The genus was largely interfertile as 55 of the 64 species combinations (64/135 or 47.4 %) producing viable-appearing seed were grown and verified. In vitro techniques were used to grow two species hybrids from semi-abortive seeds. With reciprocal crosses combined/ 73 of the possible 78 (93.6 %) combinations in the diallel were tested/ and 47 (64.4 %) produced viable- iv appearing seed. Fourteen species combinations (14/135 or 10.4 %) produced abortive seeds. Two tetraploid hybrids resulted from the mating of a diploid (2x=56) x tetraploid (4x=104) mating. These tetraploid hybrids probably resulted from the union of a normal pollen of a tetraploid species (n=52) with an unreduced egg of a diploid species (2n=56)/ resulting in novel 4x=108-chromosome species hybrids. Growth rates rates of 50 species hybrids were determined. The fastest mean growth rate was 4.3 m/yr by L. divecigifolia X L. pallida (4x=104) . Seven hybrids had mean growth rates greater than 3 la/yr, and 23 hybrids had mean growth rates greater than 2 m/yr. Thirty hybrids had at least one tree which increased in height faster than 3 m/yr. The fastest growth rate of any tree was 6.2 m/yr by L. diversifolia x L. collinsii (2x=54). Psyllid resistance to Heteropsylla cubana Crawford in the interspecific FI hybrids was better than most L. leucocephala. Five hybrids had no observable damage from psyllids. One appeared to be heterotic for psyllid resistancef and one appeared to have poorer resistance than either parent. Glands were observed on pinnae rachises of L. esculenta leaves/ and appear to be producing mucilage which was implicated in psyllid resistance. Forty-one species hybrids reached sexual maturity/ but 14 (34.1 %) failed to produce any viable seeds from open- v pollination. Mean viable seed per pod production of the 27 hybrids setting pods was 37.4 %. Yellow and red floral color, gland shape and number per leaf, floral bract shape, and inflorescence diameter were useful markers for identifying species hybrids. All appeared to be inherited additively in the FI, and all appeared to exhibit dosage effects except for gland and bract shape. Red flower color was recessive to yellow in matings of yellow x red flowers. Numbers of leaflets per pinna and pinnae per leaf and leaflet lengths and widths in 50 interspecific hybrids and their parents were counted or measured. Parental and FI hybrid data for leaf characteristics were linearized when plotted as the natural log of the data. Dosage effects occurred in triploid hybrids. Most (148/200 or 74 %) predicted hybrid leaf traits were predicted within 20 % of actual hybrid measurements; predictability would have been higher if data were used only from healthy mature trees. Only L. retusa x L. collinsii had leaflets which were not intermediate in size between than of the parental species. Leaf trait analysis was helpful in determination of parents of an open-pollinated species hybrid. Meiotic chromosomes were studied in two species hybrids and one species. L. collinsii K450 had 28 II, L. retusa K280 X L. collinsii K450 had 4 II + 48 I and L. diversifolia ssp. trichandra K399 x L. collinsii had 26 II + 2 I. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ..................................... iii ABSTRACT ............................................. iv LIST OF TABLES ....................................... xi LIST OF FIGURES ...................................... xvi LIST OF PLATES ....................................... xviii CHAPTER 1. INTRODUCTION .............................. 1 CHAPTER 2. LITERATUREREVIEW .......................... 6 2.1. Taxonomy of the Genus Leucaena Bentham ........... 6 2.2. Chromosome Counts of Leucaena Species ............ 8 2.3. Self-Incompatibility Status of the Leucaena Species 9 2.3.1. Leucaena Species Tested for Self-Incompatibility 9 2.3.2. Genetics of Self-Incompatibility in Leucaena .. 10 2.4. Interspecific Hybridization ...................... 12 2.4.1. Emasculation Techniques ...................... 12 2.4.2. Open-Pollinated Natural Interspecific Leucaena Hybrids ...................................... 12 2.4.3. Species Compatibility in vitro ............... 14 2.4.4. Inheritance of Morphological Traits .......... 15 CHAPTER 3. MATERIALS AND METHODS ..................... 16 3.1. Maintenance of Leucaena Accessions at Hawaii ..... 16 3.2. Emasculation Technique ........................... 16 3.3. Pollination and Harvest Technique ................ 17 3.4. Planting Procedures .............................. 19 3.5. In vitro Seed Rescue ............................. 21 3.6. Microsection and Staining Technique .............. 21 3.7. Pollen Analyses .................................. 22 3.8. Meiotic Analyses ................................. 22 3.9. Analyses of Leaflet Morphology ................... 23 vi1 CHAPTER 4. SELF-INCOMPATIBILITY ........................ 25 4.1. Results of Hand Self-Pollinations ................ 25 4.2. Possible Selfs Grown From Species Hybrid Seed .... 26 4.2.1. L. retusa Selfs ............................... 26 4.2.2. L. shannon! Selfs ............................. 27 4.2.3. L. lanceolata ssp. lanceolata Selfs ............ 29 4.2.4. L. pulverulent? Selfs ......................... 30 4.3. Possible Selfs of Self-Incompatible Species Found Among Open-Pollinated Progeny ..................... 30 4.3.1. L. esculenta Selfs ........................... 30 4.3.2. L. QOlliusii Selfs............................ 33 4.4. Discussion of Competition Interaction and Self- Compatibility ..................................... 33 4.5. Mechanisms Accounting for Occasional Selfing in Self-Incompatible Leucaena Species ................ 35 4.6. Summary .......................................... 38 4.7. Research Needs ................................... 39 CHAPTER 5. INTERSPECIFIC HYBRIDIZATION ................. 40 5.1. Introduction to Interspecific Hybridization ...... 40 5.2. Compatibility of Interspecific Cross-Pollinations .. 41 5.3. Expected Production of Viable Interspecific Hybrid Seed .............................................. 45 5.4. Verifiable Interspecific Hybrids Grown at Waimanalo, Hawaii ............................................ 47 5.5. Insect Pests Which Decreased Hybridization Success . 47 5.6. Floral Abnormalities Which Decreased Hybridization Success ........................................... 50 5.7. Verification of Species Hybrids. 5.7.1. Overview ...................................... 50 5.7.3. Verification of L. retusa K280 x L. collinsii K450 .......................................... 53 5.7.3. Verification of the Ploidy Level of Tetraploid Species Hybrids Produced from Matings of Diploid X Tetraploid Species .......................... 59 5.8. Possible Maximum Interspecific Compatibility Among Leucaena Species ................................. 60 5.9. Possible Mechanisms to Account for a High