in»... . . (f 1 ‘ 4 .r . , , . . .3“ fawwnxyfin . , ,fiwwmwfiw by? Jaw, .. 32.4.3. I.) ’1'}... I x: 3... to. 3. 8! 7 “Hr: c 4 A (inrm Wm J . fimmm :finfi. -Z. .l fit‘la 1 V ‘ 35.11093. 1.! 3. a“ :1. a)»: .32. (3v .1 )5» I I. i “at“ .. r... t‘mmuwu .. ‘ ‘ .w . , hart}... . : agaflfi gufigfiw; J1 . u... 22;? .‘Iny~ Linda, ‘ N r H wrunfl.‘ .wquHSawfi. MICHIGAN STA ”1 II II III IIIIII IIIIII’III II IIII'I‘IIII LIBRARY Michigan State University This is to certify that the thesis entitled CytogenefREvaluation and Foliage Color Inheritance Within Interspecific Begonia Inbreds presented by Yue Sun has been accepted towards fulfillment of the requirements for Master ' 3 degree in Horticulture Mfigmf Major professor 8/ 8/75 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE DI RETURN BOX to remove We checkout from your record. TO AVOID FINES return on or before dete due. DATE DUE DATE DUE DATE DUE MSU le An Affinnetive Action/Bond Opportunity lnetituion W pea-on CYTOGENETIC EVALUATION AND FOLIAGE COLOR INHERIT AN CE WITHIN INTERSPECIFIC BE GONIA INBREDS BY YueSun A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1996 ABSTRACT CYTOGENETIC EVALUATION AND FOLIAGE COLOR INHERITANCE WITHIN INTERSPECIFIC BEGONIA INBREDS By Yue Sun The cytogenetic investigation of the germplasm used in this research showed the plant material to be tenaploid with about 64 somatic chromosomes. A dominant gene, Ru (R), is hypothesized to control the red underfoliage color inheritance in the tetraploid fibrous-rooted Begonia x semperflorens—cultorum Hort. germplasm of this study. This dominam gene also affects the intensity of the foliage color with RRRR and RRRr giving dark red color on the underside of the leaves. The combination of RRrr and Rrrr gives intermediate red coloration, and homozygous recessive rrrr gives all-green foliage. Age of plants, daylength, and temperature also affect underfoliage color. Test crosses and analyses of F2 generations were used to determine inheritance information, and hybrids also were evaluated for potential commercial value. After nine generations, three triplex plants were selected as a base to develop a dominant homozygous inbred line through selfing and sib mating. ACKNOWLEDGMENTS I would like to express my sincerest appreciation to my major professor, Dr. Lowell C. Ewart, for his guidance, encouragement, and constant friendship throughout this study, and for his invaluable time and assistance in the preparation of this manuscript. Your friendship, professional and personal, will remain with me for a lifetime. I would also like to thank the other members of my committee, Dr. Joanne Whallon, who gave me tremendous help with cytogenetic research, and Dr. Jack Kelly, whose counsel and suggestions were invaluable during this research. I also thank the many other people who helped me in many ways with this study. Finally I sincerely thank my wife Jing Gao for her understanding and love. iii TABLE OF CONTENTS PAGE List of Tables .................................................................................................................... vi List of Figures ................................................................................................................ .viii Introduction ....................................................................................................................... 1 Materials and Methods ...................................................................................................... 14 Plant Materials .................................................................................................................. 14 Seed Handling .................................................................................................................. 14 Hybridization Procedure ................................................................................................... 15 Color Classification .......................................................................................................... l5 Statistical Analysis ............................................................................................................ 15 Pollinations Performed ...................................................................................................... 16 Seed Germination Test and Seedling Vigor Rates ............................................................ 16 Pollen Fertility Test ................. 18 Cytological Techniques .................................................................................................... 19 Results ............................................................................................................................... 22 Cytogenetic Evaluation ..................................................................................................... 22 Red Underfoliage Color Inheritance Patterns and Selection of Homozygous Red-underfoliaged Plants ................................................ 29 The Possible Effect of Age of Plants, Daylength, and Temperature on Red Underfoliage Coloration ............................................................................................ 48 iv Germination Percentage and Vigor Ratings ...................................................................... 57 Discussion ......................................................................................................................... 59 Summary ........................................................................................................................... 66 List of References ............................................................................................................. 67 LIST OF TABLES TABLE PAGE 1. Chromosomal numbers in Begonia fiom previous investigations ................................ 6 2. Expected frequency of gametic types from chromosomal and maximum equational segregation in tetrasomics or tetraploids heterozygous at a single locus ........................................................................................ .8 3. Expected ratios of different genotypes for selfed plants for the number of individuals required to possibly find one homozygous dominant individual (red-underfoliaged plant) according to Muller's formula (Muller, 1923) and from the different type of segregation by Bumham (1962) for underfoliage color inheritance within interspecific Begonia inbreds .......... 10 4. Pollinations performed for the underfoliage color inheritance study within interspecific Begonia inbreds ......................................................................................... 17 5. The overall raw data from Begonia root tip chromosomal counts of the germplasm examined in the underfoliage color inheritance study ................................ 27 6. Somatic chromosomal numbers of the germplasm examined for underfoliage color inheritance within interspecific Begonia inbreds .................................................. 28 7. Segregation ratios and statistical analyses of F2 populations from SCV x CV for underfoliage color inheritance within interspecific Begonia inbreds ....................... 30 8. Segregation ratios and statistical analyses of selfed inbred populations of SCV obtained by selecting plants with the darkest red underfoliage for the underfoliage color inheritance study within interspecific Begonia germplasm ............. 32 9. Segregation ratios and statistical analyses of test-cross populations obtained by crossing selections with the darkest red underfoliage from SCV inbred 93BB-5 with the homozygous green-foliaged CV inbred for underfoliage color inheritance within interspecific Begonia inbreds .................................................. 34 vi 10. Segregation ratios and statistical analyses of test-cross populations obtained by crossing selections with the darkest red underfoliage from SCV inbred 93BB-9 with the homozygous green-foliaged CV inbred for underfoliage color inheritance within interspecific Begonia inbreds ............................ 35 11. Segregation ratios and statistical analyses of test-cross populations obtained by crossing selections with the darkest red underfoliage from SCV inbred 93BB-10 with the homozygous green-foliaged CV inbred for underfoliage color inheritance within interspecific Begonia inbreds ............................ 36 12. Segregation ratios and statistical analyses of test-cross populations obtained by crossing selections with the darkest red underfoliage from SCV inbred 9333-9 and 9383-10 with the homozygous green-foliaged CV inbred for underfoliage color inheritance within interspecific Begonia inbreds ..... 38 13. Segregation ratios and statistical analyses of F, SCV inbred populations obtained by selfing red-foliaged inbreds the darkest red underfoliage from SCV F7 inbreds for foliage color inheritance within interspecific Begonia inbreds ............................................................................................................. 4O 14. Segregation ratios and statistical analyses of test-cross populations obtained by crossing six selections with the darkest red underfoliage from SCV F, inbreds 94BB-13, 94BB-14, 94BB-15, and 9433-16 with the homozygous green-foliaged CV inbred for underfoliage color inheritance within interspecific Begonia inbreds .................................................. 42 15. Segregation ratios and statistical analyses of F9 SCV inbred populations obtained by selfing inbreds with the darkest red underfoliage from SCV F3 inbreds for foliage color inheritance within interspecific Begonia inbreds ................... 45 16. Segregation ratios and statistical analyses of sib-cross populations obtained by crossing the two selections with the darkest red underfoliage from SCV F9 inbreds for underfoliage color inheritance within interspecific Begonia inbreds ......................................................................................... 47 17. Seed germination tests giving percentage of germination and vigor from various types of pollinations for underfoliage color inheritance within interspecific Begonia inbreds ......................................................................................... 58 vii LIST OF FIGURES FIGURE PAGE 1. Scheme for determining the theoretical maximum equational segregation for a triplex. Two alleles at a single locus, designated A, a are shown with crossovers between the locus and the centromeres, numbered 1 to 4. By assuming equal frequencies of alternate and two adjacent segregations of the centromeres, l + 4/2 + 3, 1 + 3/2 + 4, and 1 + 2/3 + 4, respectively, the gametic ratio may be determined as 13AA: lOAa : aa ................................................ 9 2. Germplasm development and relationship of inbred individuals with the darkest red underfoliage in the foliage color inheritance study from the interspecific Begonia cross B. Schmidtiana x (B. x semperflorens—cultorum cvs. Charm x Vodka) SCV ......... 13 3. Begonia root tip cell from inbred SCV (B. Schmidtiana x CV) (2n = 4x = 62-64; x3,128). .......................................................................................... 23 4. Root tip cell from SCV x B. x semperflorens-cultorum inbred ‘Pink Avalanche’ (2n = 2x = 32; x1,100) ................................................................... 23 5. Begonia root tip cell from SCV B. x semperflorens—cultorum inbred ‘Pink Avalanche’ (2n = 3x = 48; x1,lOO) ................................................................... 24 6. Root tip cell from Begonia x semperflorens-cultorum cv. Charm (2n = 2x = 32-34; x5,244) ........................................................................................... 24 7. Root tip cell from Begonia x semperflorens—cultorwn cv. Vodka (2n = 2x = 40-42; x3,600) ........................................................................................... 25 8. Root tip cell from Begonia x semperflorens-cultorum inbred developed fi'om ‘Charm’ x ‘Vodka’ (CV) (2n = 4x = 68-72; x3,848) ........................................ 25 9. Root tip cell from Begonia Schmidtiana (2n = 4x = 62-64; x1,100) .......................... 26 viii 10. Begonia pollen mother cell from inbred SCV (Begonia Schmidtiana x CV) (2n = 4x = 62-64; x5,167; indicates univalent) ...................................................... 24 11. Variation in Begonia underfoliage color determination over time for the test-cross population 9438-42 (a possible triplex SCV plant x CV) ......................... 50 12. Variation in Begonia underfoliage color determination over time for the test-cross population 9433-45 (a possible triplex SCV plant x CV) ......................... 50 !3. Variation in Begonia underfoliage color determination over time for the test-cross population 9433-50 (a possible triplex SCV plant x CV) ......................... 51 14. Variation in Begonia underfoliage color determination over time for the test-cross population 95BB-61 (a possible triplex SCV plant x CV) ......................... 51 15. Variation in Begonia underfoliage color determination over time for the test-cross population 95BB-63 (a possible triplex SCV plant x CV) ......................... 52 16. Variation in Begonia underfoliage color determination over time for the test-cross population 95BB-69 (a possible triplex SCV plant x CV) ......................... 52 17. Variation in Begonia underfoliage color determination over time for the F9 SCV inbred population 9SBB-40 (F9 from a possible triplex plant) ...................... 54 18. Variation in Begonia underfoliage color determination over time for the F9 SCV inbred population 958348 (F 9 from a possible triplex plant) ....................... 54 19. Variation in Begonia underfoliage color determination over time for the F 9 SCV inbred population 9SBB-49 (F 9 from a possible triplex plant) ...................... 55 20. Variation in Begonia underfoliage color determination over time for the F8 SCV inbred population 94BB-l3 (F 8 from a possible duplex plant) ..................... 55 21. Variation in Begonia underfoliage color determination over time for the F, SCV inbred population 94BB-l4 (F 8 from a possible duplex plant) ...................... S6 22. Variation in Begonia underfoliage color determination over time for the F, SCV inbred population 94BB-15 (F 8 from a possible duplex plant) ..................... 56 ix INTRODUCTION An interspecifrc Begonia cross between B. Schmidtiana and B. x semperflorens— cultorurn produced a population that segregated several plants having deep green color in the top tissue of the leaves and red color in the underside tissue of the foliage, making an attractive color contrast. The plants also had large, drooping stems ideal for hanging basket use. However, red underfoliage does not breed true by seed. Inheritance of this type of begonia foliage coloration has never been reported. The primary objective of this research was to investigate red underfoliage color inheritance patterns, and introduce a true inbred line that could be used to produce a cultivar with new leaf color and possible value for hanging basket production. Preliminary test crosses and selfs of red-underfoliaged plants indicated a dominant inheritance pattern and possibly a tetraploid chromosome number. To analyze the problem in more detail, increased numbers of selfs, test crosses, and sib matings were performed along with cytological analysis and seed germination studies. The genus Begonia is a large group with perhaps 2,500 species (Thompson, 1981). Before the genus name was established, species of Begonia were discovered under other names.‘ Earlier history records the names Totoncaxoxo coyollin (Mexican) and Qiu-hai- tang (Chinese). The name Begonia was founded by Charles Plumier, a Franciscan Monk 2 and botanist, in 1690. Plumier discovered six species on the Antilles Islands and named the genus in honor of Michael Begon, Governor of Santo Domingo (Thompson, 1981). Begonia is native to tropical and subtropical areas, with the greatest mrmber having been discovered in the Americas. The plant stems are succulent or woody. Some stems grow in an erect or semi-erect fashion, while others creep or climb. Leaves are alternate and asymmetrical with many shapes. Leaf sizes range from less than 2 to more than 45 centimeters in diameter. The leaf surfaces vary from glabrous to densely hairy and felted. Flowers are characteristically monoecious. Most often the staminate flowers have either two or four petals and the pistillate flowers have two to five petals. Usually the ovaries have two or three locules with axil placentae. The flower color ranges from white to red. Purple and blue are the only two colors not found. Blooming times vary greatly, most being seasonal (Thompson, 1981). Until 1800, only about five species were cultivated, but after that time the number increased rapidly. After 1850, there were four major developments in Begonia cultivation. First, in 1856, B. rex was introduced into England and evolved into the Rex Cultorum Group. Second, in the 1860s, six tuberous species that led to the development of Tuberhybrida group were discovered. Sometime between 1814 and 1821 , B. cucullata var. Hookeri (BCH), formerly called B. semperflorens (Bailey, 1978), accidentally was introduced into the Berlin Botanical Gardens by Ferkinand Sello. It was found growing in soil brought in from Brazil with other collected plants. This germplasm was not recognized until it was crossed with a newly discovered species, B. Schmidtiana, which was introduced by Haage and Schmidt in 1878. This cross was the third major 3 development in Begonia cultivation and was the beginning of the intensive hybridization that resulted in today's very important Begonia group, Begonia x sempedlorens-cultorum Hort (BSC). The fourth major development occurred in 1880, when the winter-flowering bulbous B. socotrana was found, from which the Hiemalis and Cheimantha groups originated (Thompson, 1981). Nearly all BSC come from South America, and all the plants are bushy with glossy or hairy leaves. The BSC group grown commercially today as fibrous-rooted begonias has similar characteristics. The plants have glossy, smooth, and sometimes hairy foliage. Leaf colors are green, bronzy-red, dark mahogany, and variegated. Begonia x sengoerflorens-cultorum Hort usually blooms throughout the year. Flowers are single, semidouble, or double. The colors of the flowers range from white to deep wine red, and some varieties are bi—colored. Fl hybrids of BSC were introduced commercially early in the twentieth century. The cultivar Primadonna, introduced in 1909, is listed for being the first F, hybrid ever introduced commercially (Ewart, 1995). Today in the United States, the named cultivars number more than 200 (Thompson, 1981). Fibrous-rooted begonias have been among the top five crops in the bedding plant mix since 1984. Their popularity is due to their versatility and beauty in beds, hanging baskets, window boxes or pots (Ewart, 1995). In 1994 begonias were third in the bedding plant production crop mix, accounting for an average 7.3 % of the total bedding plants produced (Bebe and Walker, 1994). The bedding plant industry significantly increased sales by 9% from 1993 to 1994, reaching $1.28 billion (Agricultural Statistical Board, 1995). 4 Zeilinga (1962) studied the inheritance of dwarfness in BSC and found it to be recessive. Holley (1945) studied several different characters in BSC, including doubleness, flower color, plant habit, and foliage color. He suggested that a dominant gene S controlled flower singleness but he could not explain adequately the inheritance of double and semidouble flowers. He also could not explain fully the inheritance of flower and foliage color, and he felt that two or three factors determine flower color, white being double recessive and red being homozygous dominant. In studying foliage color, he found that dark foliage (bronze leaf) was dominant to green foliage. A dark red-leaved cultivar, Carmen, was crossed with several green-leaved cultivars, and all the hybrids had dark foliage. The F2 progenies, however, segregated into approximately 50% dark foliage, 25 % intermediate-red foliage, and 25 % green foliage. He gave no further explanation as to the possible reason for this segregation. With regard to plant habit, Holley thought that a spreading, branched habit was recessive to upright growth, {the relationship being monogenic. Matsuura and Okuno (1943) described several advantages for Begonia as a subject for cytogenetic studies: existence of a number of species and cultivars, abundance of seeds per capsule and rather rapid growth from seed to flower, monoecious condition that facilitates making crosses, and striking diversity in nuclear organization, even within a single species. In spite of these advantages, only a few cytological studies lave been conducted on Begonia. Pastrana (1932) described a sex chromosome in B. Schmidtiana and observed 13 chromosomes in the sporophytic tissue. Matsuura and Okuno (1943) determined the chromosome numbers for 20 species and stated that chromosomal numbers 5 in Begonia showed wide variation. They also suggested 6, 7, or 13 as the possible basic number, 13 being of secondary origin. They noted two plants morphologically different from B. Schmidtiana, and both plants had a somatic chromosome number of 32. In pollen mother cells (PMCs) the chromosomal configuration was 15,, + 2,. The researchers studied chromosomal numbers of seven different BSC plant groups, but did not mention any cultivar names. Finally, they counted 66 chromosomes in a BSC cultivar that had normal meiotic configuration (Table 1). Zeilinga (1962) described the development of another group from BSC called B. x semperflorens-cultorum var. gracilis. It was the result of a hybrid between lBSC x B. Schmidtiana back-crossed with BSC. He studied the historical aspect of cultivar development of BSC, as well as the chromosomal numbers of the different cultivars. He then made crosses between different ploidy levels and reported the cultivars of the BSC group to be diploid. On the other hand, the BSC var. gracilis group was triploid or tetraploid as a result from back-crossing with diploid BSC. Zeilinga (l962)found that chromosomal numbers for BSC var. gracilis were 2n = 4x = 68; for Lwninosas compacta cv. Scarlet, 2n = 4x = 66. He also found that the dense cytoplasm and the very small chromosomes made counting chromosomes in Begonia very difficult. He observed that cell divisions were scarce in the small root tips, with extremely low numbers of metaphase plates available for study. Doorenbos and Legro (1968) conducted a cytological analysis of a winter-flowering Begonia called ‘Konkurrent’ type. They felt that this type was derived somehow from an interspecific cross between B. socotrana (2n = 28) and B. dregei (2n = 26). They made 6 Table 1. Chromosomal numbers in Begonia from previous investigations. Species Chromosomal numbers reported by Cultivar names (Group) Matsuura Zeilinga Doorenbos dregei 13(n) 26 socrotrana 28 28 Schmidtiana’ 32 32 No. I’ semperflorens‘ 60 No. my semperflorens‘ 60 No. Vy senyoerflorens" 33 No. VI’ semperfloremil 36 No. VIP semperflorens‘ 33 HORT rose versaliensis 33 Ball's Red sempery‘lorens’l 34 Christmas Cheer semperflorens" 34 Rosea sempery‘lorens’l 32 Vernon sempedlorens‘ 34 Luminosa gracilis 68 Luminosa compacta gracilis 66 Indian Maid gracilis 66 Organdy gracilis 66 Rosa Wunder Jracilis 66 ’Pastrana (1932) gave 2n = 13 for B.Schmidtiana. yMatsuura and Okuno (1943) studied chromosomal numbers of seven different B. x semperflorens-cultorum plant groups, plants No. II and N 0. IV were lost during research. "B. x semperflorens—cultorum Hort. 7 a cross between B. socotrana and B. dregei and used colchicine to obtain tetraploid . seedlings. The tetraploid plants (2n = 4x = 54) were fertile and produced triploid plants . when pollinated by B. socotrana. The triploid plants had the same appearance as the ‘Konkurrent’ type. They also found that counting Begonia chromosomes was very difficult, and that intraplant chromosomal numbers varied frequently in this interspecific hybrid group. Some of the chromosomal determinations from these studies are shown in Table l. The previous cytogenetic studies from Zeilinga (1962) and Doorenbos (1968) suggest that many tetraploid cultivars have been derived from diploid hybridization involving similar genomes with high fertility. In most cases, diploids were used in back— crosses with tetraploids to produce new triploid cultivars. Such a developmental strategy suggests the possibility of autotetraploid development, because the genomes of these diploids are very close. Bumham (1962) demonstrated two types of segregation in autopolyploids: chromoSomal segregation and maximum equational segregation. With chromosomal segregation, genes closely linked with the centromeres. An autotetraploid with a triplex AAAa genotype form only AA and Aa gametes, which occur in a ratio of 1:1. When crossovers occur between the gene locus and the centromere, the two pairs of resulting sister chromatids pass to the same pole in anaphase I, and aa gametes can be expected. This behavior has been termed maximum equational segregation (Figure 1). This segregation type requires quadrivalent formation and random separation of the chromatids at anaphase II. Table 2 shows the different segregation ratios between these two segregation types. Table 2. Expected frequency of gametic types from chromosomal and maximum equational segregation in tetrasomics or tetraploids heterozygous at a single locus. Chromosomal Maximum equational Genotype segregation aa (%) segregation aa ( %) AAAa AA+Aa 0 13AA+10Aa+aa 4.2 AAaa AA+4Aa+aa 16.7 2AA+5Aa+2aa 22.2 Aaaa Aa+aa 50.0 AA+10Aa-i-l3aa 54.2 Muller (1923) developed a formula for determining the number of individuals required at various probabilities to avoid missing a particular genotype: = -loge F (P-l/2) n = number of offspring to be raised F = a chance of failure P = reciprocal of the chance that any one individual will be of the desired type According to this formula, with a 99% chance of success and 1% chance of failure, F = 0.01. If an autotetraploid plant with a triplex (RRRr) genotype is selfed in an attempt to find one possible homozygous-dominant individual, 16 plants would need to be raised for chromosomal segregation: P = 4, F = 0.01, n = 16 (Table 3). Based on maximum equational segregation, thirteen plants would be needed. If, however, a duplex (Rrrr) A82 .Easfism :m unawE Eot 3:625 an n «<2 ” < K O < EVA H a. N It. < in < < X 7% I+|_ t o _ q 10 823388 .8883 88882. .eouawonwom .88o8o80: 69.88 on 8 actuate Co 3:85? 6.88». .«o 3826 <.n .25 :03 Co on :9: 1823—8 28 8a 38 35:0 08 .8 308883 on: ad 84 8: SN e2 8 . a .8 .52 _ a _ .oaosaoao Ex 8 8.: 8.2 4 cm mm cm 4 .8 .82 4: 23. mm _ w a m _ .uaaoeoao :8. a :3 an _ om we :8 as .8 .52 2 ad 4 _ N _ .uaaoaoao 82 .a a a :8 Ex 58 .88 gm Banana 2888 he 25. .855 5.888 058235 as? 8:825 .28 882.58: 8 a8: 8.88 .3 833338 mo 25 888.5. 08 89c 83 Gna— .._o==<6 «88.8 93:32 9 $8888 can—n coma—83.88.35 3:238: 888cc -meowmno8on one one bemmom 8 38:02 £32368 .8 .5388 05 88 38:9 eoaom 88.. £80m 8288 Co 888 Boomxm .m oEaH 11 individual is selfed, 144 plants would be needed at F = 0.015 to find one homozygous dominant plant for chromosome segregation , but only 86 plants would be needed at F=0.01 for maximum equational segregation. Thus, if a triplex dominant plant could be identified, theoretically the possibility of finding a homozygous-dominant individual would be greater. In this study, the desired mrmber of plants for both selfs and test crosses was 144, which used three flats of 48 cells. This number for each population was workable for the space and growing containers available and the number of populations to be analyzed. This number also would yield a good possibility of finding a homozygous-dominant plant from a self of any triplex plant. The number of plants did vary because of seed availability and plant mortality. The germplasm for red underfoliage color used in this research was developed by the ornamental breeding research program at Michigan State University. The germplasm, designated SCV, was generated from the interspecific cross between B. Schmidtiana and a BSC plant selected from a cross of the BSC cvs.Charm and Vodka. B. Schmidtiana has pink flowers and dark green leaves with red underfoliage, and the plants are upright in habit with rather hairy stems. The BSC cv. Charm has variegated leaves, long stems, light red flowers, and is vegetatively propagated. The hybrid BSC cv. Vodka has dark bronze- colored leaves with bright red flowers, a compact upright habit, and is seed-propagated. A green-foliaged inbred plant, now designated as CV, selected from a cross of ‘Charm’ and ‘Vodka’ in the F2 generation, was used to make the cross with B. Schmidtiana. The CV inbred germplasm selected for this foliage inheritance study has bright red flowers, an 12 upright habit, and it breeds true for all-green foliage. It was used in test crosses with SCV inbred selections to get underfoliage color segregation information. Such crosses also could be considered Fl hybrid crosses, because both parents are inbreds. Several such populations were planted in the summer of 1995 for potential commercial application. The SCV red-underfoliaged inbred plants used in this study have red flowers, long drooping stems, and are seed-propagated. After several generations of selfing (F6 to F9), three inbred individuals that did not segregate all-green—foliaged plants after selfmg were found. An all-green-foliaged inbred that was developed from the hybrid cultivar Pink Avalanche was used in a special crossing sequence to help determine polyploidy of the germplasm for red underfoliage. Germplasm development and relationship of the darkest red-underfoliaged inbred individuals in this study are shown in Figure 2. 13 .>Um¢c_vo> x EEO .26 EESEG-EEoEMiEa x .3 x 028.3850- .m 320 8.63% 05009808. 05 Eoc 88m 00582.8 8—8 0825 05 8 $38.0???» «000:8 05 .23 382205 005.: Co 85:33? 8:. 8083.050 83.9800 .N 0.85 :18? 8.83 gamma II. 8-83 II 2.83 8.88 II 8.88 8-83 enema ”7mm? 318.3 a o o o e ”5.83 "—3-88 5-8.8 III Mac-meg II. 8.708.... 8788 II 8:88 8:88 ,rll. 8488 ll <:._mm8 m2._mm8 IIL 5:33.88 Czsc-mmg 3:52-88 c...Um A§3> x 885v SSEo-EESbuQEBx .m x 388.85% .m an .m a...— on .m MATERIALS AND METHODS Plant Materials The development of the germplasm involved in this research is described in the introduction. Seeds from two F6 inbred plants provided by Dr. Ewart were used to start this research. Seed Handling All seeds were sown in 20-row, plastic germination trays using a peat-lite planting mix. The seeds were covered with a very light dusting of fine vermiculite. After sowing and watering, clean clear plastic covers were placed on top of the seed trays for moisture control, and the trays then were placed in a germination room at 80'F. Radicle emergence took place five to seven days after sowing. After 10 days, the plastic covers were removed. After three weeks, seed flats were moved to a cooler greenhouse with 65'F and 55'F day/night (DIN) temperatures respectively. Cool-white fluorescent or high-pressure sodium (I-IPS) lamps were provided to encourage rapid growth. The seedlings were fertilized with 0.13% KNo3 every 10 days. Approximately seven weeks after sowing, the seedlings were transplanted to 48- cell flats and 0.2% KNO3 was applied every two weeks. Standard greenhouse practices were followed for disease and insect control. 14 15 Hybridization Procedure Selected plants from the transplant flats were planted directly into five—inch standard plastic pots. As plants came into flower, the required pollinations were made. To avoid unwanted fertilization, female flowers just at the verge of opening were used. Tweezers were used to handle the male flowers, using direct flower-to-flower pollen transfer. All greenhouse crosses and selfs were performed in a screened house. Four to six weeks. were required for the seeds to mature. After cleaning, the seeds were placed in a 5 ' C, 35 % relative humidity (RH) seed storage room for at least two weeks before sowing. Color Classification Underfoliage coloration was categorized as dark red, intermediate red, and green for inbred and test-cross populations. Foliage color segregation data were collected about 25 days after transplanting and every seven to nine days for another two months to note any changes over time. At each generation of red-tmderfoliaged inbreds, five to 10 of the darkest red-underfoliaged plants were selected and transplanted into five-inch standard plastic pots. Statistical Analysis Chi-square and goodness-of-fit analyses were used to test the segregating generations to Mendelian ratios. The non significant null hypothesis was accepted when 16 the probability was equal to or greater than 0.05. Analysis of variance (AN OVA) was used to analyze md germination and vigor rates. If F tests showed a significant difference, the least significant difference (LSD) was used for comparing the results in the seed germination and vigor experiment. Pollinations Performed The types of pollinations performed and the reason for each self, sib, and test cross are given in Table 4. Each new selfed population was obtained by selfmg the darkest red-underfoliaged individuals selected from the previous inbred populations. Each test-cross population was obtained by crossing the darkest red-underfoliaged selections from inbred populations with the homozygous all-green-foliaged CV inbred. To increase inbred vigor and possibly help hedge against missing any homozygous red-underfoliaged plants caused by inbreeding depression resulting from repeated selfmg, sib-mating was performed among some of the dark red-underfoliaged individuals. Seed Germination Test and Seedling Vigor Rates Seed germination tests were performed on various populations. The purpose was to check germination in the various populations representing different genotypes on the assumption that any unusual results might help explain unexpected ratios, should they occur in the material. These tests were handled by counting out 50 seeds of each line to be tested and spreading the seeds on filter paper in lOO-x-15 mm glass petri dishes in 17 Table 4. Pollinations performed for the underfoliage color inheritance study within interspecific Begonia inbreds. Parental Types Objective Red-underfoliaged irTbreds selfed: To continue the Tmes, to get segregation 91BB-llB, 91BB-15A counts, to obtain a homozygous red- 93BB-9-D,E,G,J underfoliaged individual. 93BB-10-B,E 94BB-l3-A,B,E,I 94BB-l4—A,B,F 9433-15-B,E,F 94BB—16-B,C Test-cross pollination of individuals To obtain segregation ratio counts, to selected for red-underfoliaged plants determine if any inbred plant homozygous with homozygous all-green-foliaged for red underfoliage was possible to obtain. inbred. Sib pollination within individual lines To increase vigor, to check segregation and between sister lines of red- ratios, to make further selections for underfoliaged inbreds with the possible homozygous red-underfoliaged darkest red color. individuals. 18 which enough distilled water had been added to moisten the paper thoroughly. The petri dish covers were sealed with Parafilm’, and the dishes were placed in a tissue-culture room where the temperature averaged 25'C with a light intensity of 12 to 14 uE.M'2.S". Germination counts were made one week from the time of sowing. The counts are shown in Table 17. Seedling vigor rating was on a scale of 1 to 5 (poor to excellent), which was determined on seedling vigor qualities, qualities of seedling tissue, and development of seedling structure. Pollen-Fertility Test Empty or poorly stained pollen grains are an indication of poor fertility. A pollen-fertility test was conducted on a plant selected from an apparently sterile hybrid population. The stain used was cotton blue (Darlington and LaCour, 1976). Prepared by adding equal parts of water, 85% liquified lactic acid, 88% liquified phenol (liquified carbolic acid), and U. S. P. glycerine, in that order, and mixing well before each addition (Ewart, 1963). The anthers were removed from flower buds, placed in a drop of cotton blue on a slide, cut in half, and mixed with the stain, and the excess debris was removed. A cover slip then was placed over the drop of stain containing the pollen. The slide then was observed under an Olympus binocular microscope at x200 magnification. 19 Cytological Techniques In order to get chromosome number information, root tips and pollen mother cells were used. The stain used was aceto-carmine (Smith, 1947). It was prepared by boiling an excess of carmine in 45% acetic acid for five minutes, which then was cooled and filtered. Three pretreatment methods were attempted on root tips: 1) cold treatment, keeping root tips at 0°C overnight (Singh, 1993; Whallon, 1993 a), 2) placing the root tips in paradichlorobenzene (Meyer, 1945; Palmer and Heer, 1973) for four hours at room temperature, 3) placing the root tips in 0.1% colchicine for three to four hours at room temperature (Singh, 1993; Whallon, 1993 a). These pretreatments were tried to prevent spindle fiber formation and keep the chromosomes from clumping. Methods 2 and 3 gave the best results, but none of them were outstanding. The root tips usually were harvested between 8:00 and 10:00 am. during the summer and a little later during the fall and winter. The root tips were fixed for 24 hours in Farmer’s Solution (Carnoy’s Solution 1), which consisted of one part glacial acetic acid and three parts absolute or 95% ethanol (Singh, 1993; Whallon, 1993 a). The root tips were maintained in 70% ethanol until they could be analyzed. The root tips were placed in a drop of aceto-carmine on a slide, which then was placed on a dissecting microscope at x10 to remove the root caps. Next the tip portion wasmaeeratedandsmearedusingadissectingneedleandaspear-point needle. After the cover slip was in place, further smearing was obtained by pressure on the cover slip applied with the slide between folds of a paper towel. At this point two small drops of 20 stain were added to the edge of the cover slip, and the whole preparation was heated gently. Besides being used to determine chromosomal number, pollen mother cells were examined to try to determine chromosomal meiotic configurations. Aceto-carmine again was used. Small male flower buds approximately 5 to 7 mm long usually were picked between 8:00 and 10:00 am. The flower buds were fixed 18 to 24 hours in a propionic acid-alcohol solution that consisted of one part propionic acid, three parts absolute ethanol and one gram ferric chloride (FeCl3) per 100 ml of fixative (Swaminathan et al. , 1954) without any pretreatment. The male flower buds then were washed with two changes of 70% ethanol and stored in 70% ethanol until used. The anthers were removed from flower buds and placed in a drop of aceto-carmine on a slide and cut in half using a dissecting needle and spear-point needle. The macerated anthers were pmsed to force the pollen mother cells out into the stain. The excess debris then was removed and the material covered with a cover slip. The procedures above were performed under a dissecting microscope. Further spreading of the cells was obtained by applying pressure on the cover slip with the slide between folds of a paper towel. Two small drops of stain then were added to the edge of the cover slip and whole preparation was heated gently. Observations for root tips and pollen mother cells were made using an Olympus binocular microscope at x200 to 1,000. Photomicrographs for root tips were made at x1,000. Because the chromosomes were extremely small, a Zeiss Laser Scanning Confocal 21 Microscope (LSM) was used to take photomicrographs from x4,000 to x10,000. The LSM improves contrast by removing of out-of-focus light in the final image, resulting in increased resolution. With LSM, the magnification can vary from x100 to a high of x16,000 with combinations of objectives and zoom values, which is more than ten times the magnification of a conventional light microscope (Whallon, 1993 b). RESULTS Cytogenetic Evaluation The overall raw data from root-tip chromosomal counts of the germplasm examined in this study ranged from 32 to 72 (Figures 3-9, Table 5), which indicated that ploidy levels ranged from diploid to tetraploid. The clearest observed somatic chromosomal numbers and the suggested ploidy level are shown in Table 6. The interspecific inbred SCV is polyploid (tetraploid) with 60 to 64 chromosomes (Figure 3). A BSC inbred derived from the cultivar Pink Avalanche was diploid with 32 chromosomes (Figure 4). When this inbred was crossed with the interspecific inbred SCV , the resulting hybrid had 48 chromosomes in somatic cells (Figure 5), and the plants were sterile. The cotton blue test revealed empty pollen grains. Self and test-cross pollinations were unsuccessful. These results indicate that this hybrid was a triploid, reinforcing previous indications that SCV is tetraploid. Begonia x sentoetflorens-cultonan cv. Charm had 32 to 34 chromosomes (Figure 6) and was considered diploid, whereas BSC cv. Vodka had 40 to 42 chromosomes (Figure 7). The hybrid of these two cultivars (CV) was polyploid with 68 to 72 chromosomes and self-fertile (Figure 8). Begonia Schmidtiana had 62 to 64 chromosomes (Figure 9). Pollen mother cells also were used to determine chromosomal mimbers and meiotic configurations, but proved to be even more unsatisfactory than root tips. It was 22 23 Figure 3. Begonia root tip cell from inbred SCV (B. Schmidtiana x CV) (2n = 4x = 62—64; x3,128). Figure 4. Root tip cell from B. x semperflorens—cultorum inbred ‘Pink Avalanche’ (2n = 2x = 32; x1,100). 24 Figure 5. Begonia root tip cell from SCV x B. x semperflorens—cultorum inbred ‘Pink Avalanche’ (2n = 3x = 48; x1,100). Figure 6. Root tip cell from Begonia x semperflorens—cultorum cv. Charm (2n = 2x = 32-34; x5,244). 25 Figure 7. Root tip cell from Begonia x semperflorens-cultorum cv. Vodka (2n = 2x = 40-42; x3,600). Figure 8. Root tip cell from Begonia x semperflorens-cultorum inbred developed from ‘Charm’ x ‘Vodka’ (CV) (2n = 4x = 68-72; x3,848). 26 Figure 10. Begonia pollen mother cell from inbred SCV (B. Schmidtiana x CV) (2n = 4x = 62-64; x5,167; 1‘ indicates univalent). 27 Table 5. The overall raw data from Begonia root tip chromosomal counts of the germplasm examined in the underfoliage color inheritance study. I8''p7c'i'i8sflI-_'——_73_ountable cells ’1 i if i 939m fl‘ mm fig; 8 p91. 1 || (cultivar) observed Maximum Minimum Mean B. Schmidtiana 3 68 57 64.3 Charm2 4 34 32 32 Vodkaz 6 50 40 44.4 CVy ll 72 58 64.7 SCVy 17 68 57 65.2 Avalanchew 14 34 26 g 31.4 SCV x Avalanche 5 49 42 46.8 ‘Begonia x semperflorens-cultorum cvs. yThe inbred developed from B. x semperflorens-cultorum cvs. Charm x Vodka. "The inbred developed from B. Schmidtiana x CV. "The inbred developed from B. x semperflorens-cultorum cv. Pink Avalanche. 28 Table 6. Somatic chromosomal numbers of the germplasm examined for underfoliage color inheritance within interspecific Begonia inbreds. Species (cultivar) Chromosomal Numbers Ploidy level Begonia Schmidtiana 62-64 4x Begonia cultivarz Charm 32-34 2x Begonia cultivar” Vodka 40-42 3x CVy 68-72 4x SCVx 64 4x Begonia cultivarz Pink Avalanche 32 2x SCV x B. cultivar‘ Pink Avalanche 48 3x zBegonia x semperflorens-cultorum. ’The inbred developed from Begonia x sempelflorens-cultorum cv. Charm crossed with Begonia x semperflorens-cultorum cv. Vodka. "lnbred developed from B. Schmidtiana crossed with an F2 plant from a B. x sempetflorens-cultorum cross between cultivar Charm and Vodka. 29 extremely difficult to spread chromosomes. Figure 10 shows meiotic chromosomes of SCV. About 30 bivalent and four to six univalent chromosomes were found at diakinesis. Red Underfoliage Color Inheritance Patterns and Selection of Homozygous Red-Underfoliaged Plants Crossing B. Schmidtiana (red underfoliage color) with the all-green-foliaged inbred CV (B. x semperflorens—cultomm cv. Charm x B. x semperflorens-cultorum cv. Vodka) produced 46 plants, all of which had red under-foliage. A dominant gene, now designated (Sun and Ewart) ‘Ru’ (R) with its recessive allele ‘ru’ (r), is hypothesized to control the red underfoliage color inheritance in the germplasm used in this research. Table 7 shows the segregation ratios and statistical results from the F2 population of three red-underfoliaged plants and one all-green-foliaged plant. These three red- underfoliaged plant selections segregated out red-, intermediate red-, and all-green-foliaged plants. The all-green-foliaged plant, when selfed, produced all-green-foliaged plants. These results showed that the all-green foliage color is recessive to the red underfoliage character. Tables 8 through 16 present the Chi-square determinations of the various population categories studied. We hoped that some homozygous dominant red-underfoliaged plants would be found during this research. Based on chromosomal segregation, any selfed triplex, duplex, and simplex plants should segregate 0%, 2.8% and 25% recessive genotype individuals, respectively, and 30 09080» 028008 mo 08800.89. .383 B8:e.82»-=<. .858 woma__ot0862 08608085: .3582?» 808.50 8832. as... 888025-83. dons—88 03 do... 53 8380808 .805 50.500808 .56 8 mod n m .a 80288 .8 80058832 II o I. ”2 23.8— e .2 23.8— e 8 .F: :88 82-9. :38 3 Ex :84. :8 :3 :8 8 :83 :8 :3 a .2 8 2: w... o 3 3-88 .88 8:8. :38 :m 8 :83 :8 :3 :8 8 :83 :8 :3 m .2 8 «.8 on w... 8 8888 28.8 8:5. :38 :m E: :84. :8 :8 :8 8 :2; 3 :8 :3 a .7. 8 m8 8 8 8 8.88 28.8 8:5. 23.8 :n 8 :88 :8 :8 :8 8 :83 :8 :3 m .2 8 N8 8 3. 8 8.88 .33 2:88 5. e8 .33 2838 3. 28 8888 $3 806 .820 .88 .5 .3. 888... 808.8 8888 0388 8800a .828 3:88 0500.835 £55 0088an .830 08:88.08 08 >U x >Um Eat «coca—ace an .«o mom-m8 302888 .08 828 850888 8 05$. 31 0.174%, 4.94%, and 29.34% if based on the maximum equational segregation system. Therefore, in this research the statistical analysis was conducted by combining the dark and intermediate red- underfoliaged plants to represent the dominant genotype. The percentage of all-green-foliaged plants whose genotype was recessive in various pollination _ populations was calculated and analyzed. Table 8 shows observed segregation ratios obtained from three F7 populations of dark red underfoliage of SCV germplasm. The percentage of all-green-foliaged plants of 9338-9 is 10.5, not significantly different from the expected duplex, based on maximum equational segregation, but significantly different based on chromosomal segregation. In 93BB-10, the percentage of all-green-foliaged plants is 7.7, which was not significantly different from the expected level for a duplex segregation ratio, either for the chromosomal or maximum equational segregation systems. The results of the statistical analysis suggest that these two populations were derived from a duplex plant with RRrr genotype. The all-green-foliage segregation percentage is 18.1 for the population of 93313-5, a result that does not fit any expected genotype. However, the closest fit was for Rrrr (simplex) based on chromosomal segregation. This was significant at 0.05. Possibly some of the all-green-foliaged plants were classified incorrectly, since coloration changed over time. Ten of the darkest red-underfoliaged plants from the three populations (93BB-5, -9 and -10) were selected and selfed to produce seed for the next generation. These plants also were test-crossed with a homozygous all-green-foliaged inbred to check for selections homozygous for the red underfoliage character. The test crosses also can be considered .onboaow 36802 no ems—.889 .353 Bws_o§o.w-__<_. .afia $3853.88 «58:58, .883 coma—pro???“ x89. . «60595600.. .839? scammouwom 385950 825882» .832:ch 28 5 832 825988 238$ $8380qu .86 8 mod n m 8 885:me 8 .fiocfimfiaoz: .. .mz :vmdn mofihov baa fim PE :36 YR :wd finm Em xvi .o finbm zed m =< DEM fiE F. >2 mm 5m Wanna :deN $3.9. :93 fin EM 236. 32. En." fimn Eb— :vt .o fimnm :od M =< g 56 2 we 3 fl Edmma :vmdm aofibov :odu fim E 23.9 v": 3”." fimn Em :vS .o u “mum ..o.c a =< g m .2 S E. en fin— mémmm .33 03833— .3. ovum .Afim ozmmooom hm ovum 09085? 33 520 .3020 ,3.— .5 atom .50 cv.—wagon a; gag 0368“— g h 839:8» 5.83% 0989325 £65 :83 85:85: .880 owfiotovs. 05 8a nag—crown: 82 38.58 05 .23 853 958—8 an .8598 >Um .8 823.88 385 3:8 .8 «8358 .8393 98 8:8 cocamoumom .w 038% 33 hybrids because the two parents were inbreds and were, therefore, selfed for F2 segregation ratio data (Table 7). Seed was obtained successfully from six individuals of 9388-5: 93BB-5A, 5B, 5C, SD, SE, and 5H, which were test-crossed with the all—green—foliaged inbred and yielded six populations (Table 9). The results of these six populations were combined according to underfoliage color classification to represent the segregation ratio of 93BB-5. The all- green-foliaged segregation percentage was 49.4, not significantly different from the expected simplex segregation ratio, based on chromosomal segregation. The results indicate that 9SBB-5 was simplex with a genotype of Rrrr, which correlates with the information for 93BB-5 in Table 8. Since the data indicated all the selections from 93BB-5 were simplex, and it was not possible to select any homozygous-dominant individuals, the inbred line was abandoned. Tables 10 and 11 show the results from the eight successful test-cross populations from 9BBB-9 and nine test-cross populations from 93BB-10, respectively. Both results show that the all-green-foliaged segregation percentage is higher than expected (16.7 to 22.2% for a duplex genotype), probably because some simplex plants were selected from 9388-9 and 9383-10. When these possible simplex selections were test-crossed with an all-green-foliaged inbred, they produced more all-green-foliaged plants than would be produced from duplex or triplex plants. Since the segregation numbers of these populations were combimd for analysis, the all-green-foliaged percentage would be higher than expected. These results also show the difficulty in selecting for the various dominant genotypes based on the red color intensity. 4 3 .258 see 32:8 95:. 32a? 5.. 69.00:...» 03888 no amazoocoms .253 Bms_e.=83.=<. .353 Busstocfiéa 238.503.. .aouamoumom 3:33.50 Eng? .35; 833325-? “so. dean—:98 28 c8 33 :ouamocwom 380$ 50332—9: .86 8 mod n m 3 Enema—ma co Enema—$802.. . .. mz twin 2”: 29.3 :— E ..N.- NE :62 fin EM :~.v 38 :cd m =< H52 vdv 0mm Em 9. 38,—. 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The statistical results of the all-green-foliaged segregation percentage of these three test-cross populations are shown in Table 12. The 948846 and -48 populations were used for comparison. The results indicate that two of the three selections, 94BB-45 and 94BB- 50 (6.2% and 4.9% all-green-foliage, respectively) fit a triplex inheritance pattern based on the maximum equational segregation. The 9483-42 population had only one all-green- foliaged plant, and this segregation pattern does not fit any logical segregation system. It is possible that this all-green-foliaged plant is a mixed seedling. If it is, therefore, ignored, the segregation ratio of 9483-42 would fit the expected triplex inheritance pattern based on the chromosomal segregation. Consequently, the three individuals, 9333-96, 9388-91 , and 93BB-10E, that corresponded to the three test crosses with the lowest number of all- green-foliaged plants were selfed to produce seed for the next inbred generation, F8. Table 13 shows the segregation ratios for the inbred populations 94BB-13, -14, -15 and -l6', which were derived from the selfs of 93BB-9E, -9G, -9J, and 93BB-10E. The seeds from the test cross of 93BB-9E had poor germination, so no test-cross data were possible. However, 9BBB-9E was included here because of the intense red color of the underfoliage. The all-green-foliaged segregation percentages of 9488-14 and 94BB-15 were 6.25 and 8.3, respectively. The data show no significant differences from expectations for duplex segregation based on the maximum equational segregation. The statistical result indicates that the 94BB-14 and 94BB-15 populations were from duplex plants (93BB-9G, 9BBB-9J), which is contradictory to their test-cross segregation results 38 :mém 2”: :98 fig FE . «.3 NS Lue— fim .52 >0 x 25— ..~.v finu :od M =< and Van we 3 on $153 8&3 2“: :c.% _; Ex :Ndm Nun Lam: in Ema >0 x .9: 2N6 mama 39¢ a =< g «3. h E. on owing :ném m— H S 39.8 _n_ F2 :~.- Nun Lao— fim ham >0 and 5N6 mum" :9: a =< .55:— N6 a 2. mm mama :Ném m.— ” : :98 _n_ .EM :ndm Nun Lue— 1m hm”— >U x .3 :~.v fimm sod M =< g mod _ E. 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YE. :w.m fimm .52 .meémma :vS .c in? :96 a =< g _ . ~ _ 2 2. me am @333 LdeN 82 ”Se ..o.m~ _ ”m E 23.9 32. :wd finm E5— .2358 :v: .6 ~65 :od #— =< g m.w Q ow an an 2.9—3 :deN $3.9. :odm fin EM 223. 32.. .w.~ inn Emu— .Oaémmo :v: .o finbm rod a =< 552 must. a mm mm an— 3433 :den 33.9. :o.mN in F8 23.9 32. .w.m inn and“ .moémma :2.— .c _ ”mum .bd m =< g o. m w 2. no 6 mug—mg .33 02880“ hm 23¢ .33 0380qu .3— 85; 258% $3 520 9:020 .82 .5 :83— .50 0833; 333333 gang 033mg Magma .8055 383m 0588885 5.23 3.55:5 88° ems—8 .8.“ 8055 am >Um Sea “33:02: 8. 3.5 05 as, 82E 2%... B 3538 “832.8 855 >8 m. .6 Baas 38% Ba 8%: coummemom .2 2%... 41 (Table 12). The inbred population 94BB-16 produced an even higher percentage of all- green-foliaged plants than did 94BB-l4 and 94BB-15. Also the all-green-foliaged percentage shows that it does not fit any logical genotypic segregation. Some of the darkest red-underfoliaged individuals, however, were selected from this inbred population to be test-crossed and selfed to produce the next generation. The inbred population 94BB- 13 had the lowest percentage of all-green-foliaged plants (5.6%), but the statistical data suggest it may be a duplex. Either there were errors in identifying the red-underfoliaged plants, or other factors influence the intensity of this characteristic. Daylength, temperature, and age of plants influenced the intensity of the red under-foliage characteristic. These factors are discussed later. Six to ten of the darkest red-underfoliaged individuals were selected from 94BB-l3, 948B-14, 94BB-15 and 94BB-16 (F8 populations), and each selection was selfed and test- crossed with the homozygous all-green—foliaged inbreds. The test-cross results are shown in Table 14, and the selfed population results are shown in Table 15. Table 14 shows the segregation ratios of the six test-cross populations derived from 9438-13, 943B-14, 9433-15 and 94BB-l6. Two of them, 95BB-61 and 95BB-63, did not segregate all-green-foliaged plants and were not significantly different from the expected triplex segregation ratio. These two populations, however, were not large. Two other test-cross populations QSBB-60 and 9533-69 segregated only a few all-green-foliaged plants and were not significant for maximum equational triplex segregation. The statistical results indicate that these four selected plants, 94BB-13E, 9433-1416., 94BB-15F, and 94BB-16C, may be triplex. 42 393.53% I u I! 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Bu...o..o..§-..o. 228.53.... 62.03.30. 3.3.0.60 €5.82. .853 Ema—8.0.0560. 0:09 dons—.50.. 0:0 .8 80.. 5.33.3... .022... $3383.00. .56 .0 mod n m .0 .cuocfiww. .0 .aaocfiwmmaoz :- . 0. ml :30 m. u .. 2.8 E E... ..~.~N S .25.. .6 5... >0 x .00. an... :8 ac... .. ..< .5... 3.. n a. .m 03.08 .33 2.808.. 5. 2.3. .33 3.208... 5. 0...... 25250 A... 820 .520 .8. .5 .3. 8.0.8.. gaudy—Mug 03.08am Hag; 9.2.08 3 030,—. 44 In Table 15 the first three selfed lines correspond to the first three test-cross populations in Table 14. Two F8 inbred plants, 94BB-15F and 9433-1313, did not segregate any all-green-foliaged plants in their selfed populations, 9583-42 and 95BB-43. The all-green-foliaged segregating percentage fit the expected triplex segregation ratio, based on chromosomal segregation. The selfed 9SBB-4O population from 94BB-14A produced only one all-green-foliaged plant, and the segregation pattern does not fit any of the segregation systems. It is possible that this all-green-foliaged plant is a mixed seedling from other population. Therefore, without counting this all-green foliage, the segregation ratio of 95BB-40 was not significantly different from that expected for triplex segregation, based on chromosomal segregation. The statistical results indicate that these three F8 inbred plants, 94BB-15F, 94BB-13E, and possibly 94BB-14A, are triplex with RRRr genotype. The population from the self of 94BB-16C, because of poor seed germination, did not have enough plants to determine a segregation ratio properly. The two test-cross populations in Table 14, 9SBB—65 and 9588-68, which were derived from 94BB-13B and 9488-16B, showed a higher percentage of all-green foliage than the other four test-cross populations. The statistical results reveal no significant difference from the expected duplex segregation ratio, based on the chromosome segregation system. Table 15 also shows the selfed segregation ratios for the two F8 individuals, 94BB- 133 and 94BB-16B, which were involved in sib matings. 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Ba 3055 am >Um Soc nag—crown: R: 89.5. 05 55 8260—8 93 $883 .3 3538 3.333303 $80.98 Mo 83:38 .8233... 93 8:2 couawouwom .2 033. 48 either chromosomal and maximum equational segregation for 94BB-lGB. These results correlate with the results of test-cross segregations in Table 14, which indicates 94BB-13B and 948B-lGB are duplex. Table 16 shows the segregation ratios for two sib-mated populations. The 95BB- 84 was derived by sibbing 94BB-14F x 948B-IBB. The 95BB-85 population was derived from the sib cross of 94-16B and 94813-15E. No all-green-foliaged plants segregated out of these sib matings. The results also indicate that the two F8 inbred plants, 94BB-14F and 94BB-15E, are triplex. Otherwise, a few all-green-foliaged plants should have segregated out of the sib-mated populations. The population sizes, however, were rather small. Starting with F,5 germplasm, and after three additional generations of selection, five individuals, 948B-IBE, 948B-14A, 94BB14F, 94BB-15E, and MED-15F were selected and are possibly triplex. Two of them, 94BB-14F and 948B-15E, need further verification by test-crossing with an all-green-foliaged inbred. The Possible Effect of Age of Plants, Daylength, and Temperature on Red Underfoliage Coloration During this research, we found that the age of the plants, daylength, and temperature possibly affected under-foliage coloration. Figures 11 through 22 show the variation we encountered. Figures 11 through 13 show the underfoliage color changes over time along with age of plants for three test-cross populations, 94BB-42, 94BB-45, and 948B-50, from selected inbreds of F8 SCV in 1994. After 72 days from the time the seed was sown, the 49 plants were either all green or intermediate red. As the plants aged, the number with all-V green foliage decreased, and the dark red underfoliage started to appear. Figures 14 through 16 give the result of three test-cross populations, 9SBB-61, 95BB—63, and 9SBB—69, from selected inbreds of F9 SCV in 1995. In these populations the number of intermediate red- and all-green-foliaged plants also decreased, and the number of dark red-underfoliaged plants increased as the plants aged. The difference between the two years' results is that the dark red-underfoliaged plants in 1995 appeared earlier than in 1994. The difference may be due to daylength and temperature influences for the time of year. In 1995 the populations were grown from late June through late October. The first count was on September 6. Most of this experiment covered a considerable period with long days and warm temperatures. In 1994, the populations were grown from early August through early November, the first underfoliage color count occurring on October 21. During this period, the daylength was getting shorter and the greenhouse temperatures were lower. Longer daylength and warmer temperatures may, therefore, increase photosynthetic activity, which could influence the earlier pigment formation in the leaves. The same difference also was found in the selfed populations in 1994 and 1995. Figures 17 through 19 show the 1995 results over time for the F9 populations 95BB-40, 95BB-48, and 95BB-49 from the self-pollinations of three selected inbreds of F8 SCV for the underfoliage color changes. These populations were grown during the hot summer of 1995. At day 71 there were mostly dark red- and intermediate red- underfoliaged plants. Over time, the number of dark red-underfoliaged plants increased, 50 \\\\\\\\\\\\\\\\\\\\\\\\\\\\ as sowing (Aug 9. 1994) ................................... 79 seed il-m \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ 7 70* 80‘ 350‘ 10‘ 0 3.. -W ~640- -Inlermediabered -Green .lhrlrred Figure 11. Variation in Begonia underfoliage color determination over time for the test-cross population 94BB-42 (a possible triplex SCV plant x CV). ”0.. O C O a O m m m W c n O O o I O I O O o O M m m \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ \\\\\\\\\\ m . .... . ....a..\... . . ................................................................................................. iiiiiiigiu \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ \\\\\ _ ..\,.\.,_ ooooooooooooooooooooooooooooooooooooooooooooo nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 01 3 9 ‘4} 9 9 1 9 8 8 m m 7 ”Intermediateredtkeen -Ihrkred Figure 12. Variation in Begonia underfoliage color determination over time for the test-cross population 94BB-45 (a possible triplex SCV plant x CV). 51 72 79 88 my! trom need sowing (Aug 9. 1994) [-Dukmd .lnlzrmediabered -Green J Figure 13. Variation in Begonia underfoliage color determination over time for the test-cross population 94BB-50 (a possible triplex SCV plant x CV). 70 80 5 50 340 .330 71 78 81 86 91 98 [by- trom seed sowing (June 28. 1995) Elm-km qumm I Figure 14. Variation in Begonia underfoliage color determination over time for the test-cross population 95BB-61 (a possible triplex SCV plant x CV). 52 350 71 78 81 86 91 96 Days from seed sowing (June 26. 1995) librkl‘a‘l .[ntermediatered -Green I Figure 15. Variation in Begonia underfoliage color determination over time for the test-cross population 9SBB-63 (a possible triplex SCV plant x CV). 80 71 78 81 88 91 [km from seed lowing (June 28. 1995) [-Dukmd Figure 16. Variation in Begonia underfoliage color determination over time for the test-cross population 9588-69 (a possible triplex SCV plant x CV). 53 and the number of intermediate red- and all-green foliaged plants decreased. The 95138- 48 and 9SBB-49 populations at the end showed only intermediate red- and dark red- underfoliaged plants (Figures 18 and 19). These three populations are possibly from triplex plants. Figures 20 through 22 show three F3 SCV populations, 9438-13, 94BB-14, and 9433-15, in 1994 for underfoliage color change over time. The parent plants that produced these populations were possibly duplex. These populations were grown during the winter and did not show red underfoliage color until 98 days from sowing. At 114 days from sowing there were still some all-green-foliaged plants in the populations. The results obtained from these populations covered a period from November 15 to the middle of March under short days and cooler temperatures. Compared to the possible triplex 95BB-40, 95BB-48, and 9533-49 population results, the underfoliaged color changed much more slowly. Red-underfoliaged plants did not appear until 91 days from sowing, generally 20 days later than when grown at warm temperatures. Two all-green-foliaged plants from the F8 inbred population 94BB-13 were selected and potted. Within two weeks, both of these plants had a foliage change to intermediate red. This phenomenon might help to explain the contradictory results between Tables 12 and 13. It is possible that if the three populations represented in these tables had been saved longer, the number of all-green-foliaged plants would have decreased. These results strongly suggest that the determination for pigmentation of the leaves for the red-underfoliage character should be done during long and warm days in late spring and summer. 54 71 78 81 88 91 Days from seed sowing (June 28. 1995) {-narknd -lntermediatered -c.-een l Figure 17. Variation in Begonia underfoliage color determination over time for the F9 SCV inbred population 95BB-40 (F9 from a possible triplex plant). 71 78 81 88 91 my! from seed ”win; (July 8. 1995) r-hrkrd -lntermediatered -Green | Figure 18. Variation in Begonia underfoliage color determination over time for the F9 SCV inbred population 95BB-48 (F9 from a possible triplex plant). 55 71 78 81 8B 91 98 101 [bye from seed sowing (July 3. 1995) IIIIIDukIed isllnunnnmnnund llllkum I Figure 19. Variation in Begonia underfoliage color determination over time for the F9 SCV inbred population 9SBB-49 (F9 from a possible triplex plant). 100 so so a j 70 Ba. ‘6 g 50 20 10 o . . . 91 9B 105 114 Days from seed sowing (Nov 15. 1994) U!IIanIed fififllnhnnndhhsnd Illcnnn J Figure 20. Variation in Begonia underfoliage color determination over time for the F3 SCV inbred population 94BB-13 (F8 from a possible duplex plant). 100- 56 91 105 my: from seed sowing (Nov 15. 1994) ..... 0.00.0.0“.— 114 IIIthkred .Inlermedintered .(hen Figure 21. Variation in Begonia underfoliage color determination over time for the F8 SCV inbred population 94BB-14 (F,3 from a possible duplex plant). 100- owomouuuuu Inuuooomumnoo 80 ii 70' 80‘ .II 0 50' 3... i 80' 105 Day. from seed sowing (Nov 15. 1994) cocoon..." a... ....... tumoomoo ..... Woooooooo 114 ~‘