WNHNWNUWUWWWWIWWW ;‘m" vanes 1.1313411! Michigan State University ., This is to certify that the thesis entitled Adventitious Shoot Induction Of Selected Explants 0f Kalanchoe Blossfeldiana Poellniz, Cultured _|_n_ Vitro presented by Robert Carl Karp has been accepted towards fulfillment of the requirements for M.S. degree in Horticulture [<. - // jam/2:5,! Major professor 9’, 7 _ r ' - "‘I ' :r‘ "I - Date { xqf/y ,/ > /f fi—f 0-7 639 ADVENTITIOUS SHOOT INDUCTION OF SELECTED EXPIANTS OF KALANCHOE BLOSSFELDIANA POELLNIZ. CULTURED IN_VITRO BY Robert Carl Karp A THESIS Submitted to MiChigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1977 ABSTRACT ADVENTITIOUS SHOOT INDUCTION OF SELECTED EXPIANTS OF KAIANCHOE BLOSSFELDIANA POELLNIZ. CULTURED IN_VITRO BY Robert Carl Karp The adventitious bud tedhnique when applied for mutation breeding 22:33232 may be a more efficient method than $2232X2 ones; thus, this study was undertaken to evaluate the potential of such systems for Kalanchoe blossfeldiana Poellniz. Six explants (leaf discs, stem segments, leaf petioles, flower buds, pedicels, and peduncle segments) from five cultivars of Kalanchoe blossfeldiana were tested on modified Linsmaier and Skoog media with the plant hormones 6-benzyl aminopurine (6-BAP) and naphthalene acetic acid (NAA) in all combinations at the following levels: 0.01, 0.1, 1.0, and 10.0 mg/l. After twelve weeks, each explant was evaluated for adven- titious shoot production followed by rooting of the plantlets which were subsequently grown to flowering for further evaluation. All explants from each cultivar produced adventitious shoots on at least one media combination of 6-BAP and NAA. There were extensive dif- erences in cultivar response relative to adventitious shoot production while in general, vegetative explants readily produced more shoots than reproductive ones. No single explant-media combination was found to be optimum for all cultivars but leaf discs at 1.0 and 10.0 mg/l 6-BAP in Robert Carl Karp in combination with 0.1 and 1.0 mg/l NAA was overall the best for the cul- tivars studied. ‘With 'Mace', leaf discs at 1.0 mg/l 6-BAP and 1.0 mg/l NAA was the best explant source while with 'Solferinopurpur', the least productive cultivar tested, leaf petioles at 10.0 mg/l 6-BAP and 1.0 mg/l NAA was the most efficient combination. Stem segments at 1.0 mg/l 6-BAP and 0.1 mg/1.NAA was the explant-media combination chosen for 'Sirius'. With 'Korall', stem segments at 10.0 mg/l 6-BAP and 1.0 mg/l NAA was the superior choice for adventitious plantlet production. Leaf discs of 'Granat' produced large numbers of shoots on many media combinations, the Optimum ones being 1.0 mg/l 6-BAP and 1.0 mg/l NAA, 10.0 mg/l 6-BAP and 0.1 mg/l NAA, and 10.0 mg/l 6-BAP and 10.0 mg/l NAA. Theoretically, the adventitious bud technique assumes that the plants originate from a single epidermal cell which assures that the re- generated plantlets are non-chimeric. In this study, no histology studies were undertaken to determine the origin of adventitious shoots but by observation of morphological plant characters, no visible chimeras were observed in the adventitious buds grown to flowering in the greenhouse. Thin-layer chromatography of flower pigment extracts and pollen viability tests revealed no significant differences between the parental plants and the regenerated ones thus indicating genotypic similarity which further supports the supposition that the adventitious plantlets in this study most likely were derived from a single cell or a few cells. An.i£ vitae system for mutation breeding with Kalanchoe blossfeldiana would appear to be a more efficient technique than in vivo systems in the production of new cultivars by requiring less time for shoot production, requiring considerably less Space, and producing higher numbers of shoots. To Kathy ii ACKNOWLEDGMENTS I would like to thank the members of my committee Dr. Robert Herner and Dr. P. S. Carlson for their assistance. Special thanks goes to Dr. K. C. Sink, Jr., my major professor, for his guidance and friendship. Special thanks to Mr. Jim Mikkelsen for the plant materials used in this study. My parents and especially my wife. iii List of Tables . Introduction . Literature Review Materials and Methods Results . . . Discussion . Appendix . . . Bibliography . TABLE OF iv CONTENTS Page 10 15 28 29 LIST OF TABLES Table Page 1. The effect of 6-BAP and.NAA combinations on shoot formation from each explant and cultivar . . . . . . . . . . ll 2. Optimal ig vitro plant organ-hormone combination of the cultivars tested of Kalanchoe blossfeldiana PoelJ—niz O O O O O O O O O O O O O O O O O O O O O O O O O 0 l6 3. The highest average and corresponding hormone combination of the cultivars tested grouped as plant parts . . . . . . . . . . . . . . . . . . . . . . . . 18 Al. Ig'vitro reaction, total number of shoots, and average of the plant organs of 'Mace' at all combinations of 6-BAP and.NAA tested . . . . . . . . . . . . 23 A2. IE vitro reaction, total number of shoots, and average of the plant organs of 'Sirius' at all combinations of 6-BAP and.NAA tested . . . . . . . . . . . . 2h A3. In vitro reaction, total number of shoots, and —average of the plant organs of 'Granat' at all combinations of 6-BAP and.NAA tested . . . . . . . . . . . . 25 Ah. In'vitro reaction, total number of shoots, and average of the plant organs of 'Solferinopurpur' at all combinations of 6—BAP and NAA tested . . . . . . . . 26 A5. In vitro reaction, total number of shoots, and —average of the plant organs of 'Korall' at all combinations of 6-BAP and.NAA tested . . . . . . . . . . . . 27 A6. Highest average of each plant part of each cultivar and the corresponding hormone combination . . . . . . . . . . . . . . . . . . . . . . . . 28 INTRODUCTION Kalanchoe blossfeldiana Poellniz. is currently increasing as a commercial flower crop in the United States. It is asexually propagated by stem.tip cuttings and new cultivars are produced by conventional hybridization or mutation breeding. For mutation breeding, leaf petiole cuttings appear to be the apprOpriate plant organs to utilize. This is due to the fact that shoots originating from the petiole base are believed to have their origin from a single epidermal cell which leads to non-chimeric mutants. The concept of adventitious shoots arising from a single cell has been reported for over 350 species, including both monocots and dicots. And, when implemented as the basis for mutation breeding has successfully resulted in new cultivars of a wide range or ornamentals including Begoniaceae, Compositae, Crassulaceae, and Gesneriaceae. For example, Broertjes (1969) showed with Streptocarpus that the adventitious technique was possible with leaf cuttings. Mutated and nonemutated shoots arose from single, epidermal cells but it was difficult to determine by histology precisely how many cells were involved. Of the mutants produced, 9h.8 percent were solid or non-chimeral. Broertjes interpreted the re- sulting high mutation frequency and wide spectrum obtained to indicate reduced diplontic selection; thus inferring that one or a few initial cells gave rise to the adventitious buds. The chimeras probably resulted from the instability of the genome induced by irradiation. 2 Recently various explants of plant species have been placed in vitro for mutation breeding to give more efficient results in terms of shoot yields. Broertjes et. a1. (1976) compared.ip_vivo and in vitro muta- tion breeding systems in Chrysanthemum morifolium. On detached leaves, shoots originated from callus at the petiole base or occasionally on callus formed on the upper part of the roots. Following irradiation with X-rays, the few shoots obtained from the main adventive shoots on laterals were chimeric indicating that they originated from more than one callus cell. Pedicels were chosen as the explant for iplvitrg_irradiation after screening various explants since their adventitious shoots came directly from the epidermis and 100 percent of the explants irradiated or not, produced high numbers of shoots. Of all plants produced, only one was chimeric. In_vivo mutation breeding has been accomplished with Kalanchoe blossfeldiana utilizing the adventitious bud technique with leaf petiole cuttings (Broertjes and Leffring, 1972). Young leaves produced the highest number of adventitious shoots but shoot production was low and even lower (below 1) following irradiation. No chimeras were observed after consider- able backpruning and histological observations providing further evidence that adventitious shoots were formed from single epidermal cells. The present study was undertaken to determine for ip:vit£g.muta- tion breeding, which explant and.ip;vitrg_media combination would.promote Optimum adventitious shoot production for Kalanchoe blossfeldiana. As well, to determine if desirable genetic variability possibly present in chimeric form, exists in intact plants that could be released through in'vitro cul- ture of various plant organs and their respective histogenic layers. LITERATURE REVIEW Kalanchoe blossfeldiana Poellniz. is a pot plant crop com- mercially propagated asexually by stem tip cuttings and leaf petiole cuttings for mutation breeding. The number of shoots from leaf petiole cuttings varies between cultivars, is generally'low, and a $1 w propaga- tion method but more importantly, for a mutation breeding project the adventitious plantlets appear to originate from a single, epidermal cell (Broertjes and Leffring, 1972). There are over 350 species, both monocots and dicots, capable of forming adventitious buds from leaves with the list being incomplete since with many species the technique has not been applied (Broertjes et. al., 1968). There is great potential for mutation breeding systems and already within the following families of ornamentals the adventitious bud technique has been used for producing new cultivars: Begoniaceae,- Compositae, Crassulaceae, and Gesneriaceae. Broertjes (1969) with Streptocarpus and by using the adventi- tious bud technique with leaf cuttings was able to induce mutations and lend evidence of single cell and epidermal origin of the shoots. It was difficult to determine histologically the number of cells involved in the formation of adventitious buds but through mutation induction and a high number of solid mutants, 9h.8 percent, it was felt that the shoots were single cell and epidermal in origin. Further proof of the solid.mutants resulted.when the mutants reproduced by leaf cuttings did so true to type. The high mutation frequency and wide mutation spectrum indicated reduced h diplontic selection which points to one or a few cells involved in adven- titious bud formation. The instability of the genome was the explana- tion for the chimera formation. Broertjes (1972a) working with Achimenes, a member of the Gesneriaceae, used the adventitious bud technique to produce mutants. With Achimenes, depending on the time of year and the dose of irradiation, detached leaves will form adventitious shoots, shoots and rhizomes, or rhizomes only and regardless of whether shoots or rhizomes were formed almost all were non-chimeric. Ninety-six percent of the mutants were solid adding support to the assumption that the plantlets were produced from single cells. The in 2232 adventitious bud technique has been utilized by Broertjes (1972b) with Saintpaulia leaves in an extensive mutation breeding project. Using two types of irradiation, few chimeras were pro- duced and histological work suggested that the adventitious plantlets were single cell in origin. Broertjes felt that diplontic selection was restricted in the adventitious bud technique since even slow growing cells were able to express themselves as dwarf mutants which represented a large percentage of the mutated.population. Another mutation breeding project utilizing the adventitious bud technique with Saintpaulia was undertaken by Sparrow et. al. (1960). The results obtained were similar to Broertjes (1972b) in that after irradiating with X-rays, 15H mutants were obtained from leaf petiole cuttings and all but one being non-chimeric. The authors felt that from these results one cell forms a meristem which forms a whole plant. No histological study or propagation of mutants was undertaken to completely identify the nature of the mutants. "I ”)I - ",( . '.i' ‘) II' . ' l' I I' j ' ' ‘ ‘ '""‘ ' ' J! 3 IT] - ' I" "rll I l‘ \) WI“ 'il|:'l(““l-"'_i 7 “ «I ' ',~r ~ 3 . .A .3 ’l. l 'I P ‘> ‘ )_| ' 7 r‘ 3 I ‘r‘: I" \ ‘ .fl.\ :. _-. rr‘ ‘1 II.' ' r‘ ° “I! ‘ C" I"? ‘rh r' I ‘- '> W 'o I" - v " ' “ ‘ ' =' ‘ ‘- “7 " - II-. "I ‘ , ‘l' ",I I" “‘l" “' "r ‘ “ ‘ 1" ‘ ’I :1: .- ‘ ."2 I" 3’): -I“- .. ~' “ " “- P ‘ f! F‘ ‘ 'J." >I-‘,l . 1. " r l _r;“ 7.. ‘ - - l ‘ I” '> IrI _r. 1) . \ |_ \IIIV‘I r x - r -I,. l 1 ) ‘. ._ .. ~ l‘l r1 rim 5 ‘. I I *4) ‘.I [III I I' ‘ ~ ~ " --r‘ “a e. 1'", II', ': I‘ll-I :.'I «H. ' .-- .. w -' “ I I ' "H I r. I '1 I , I"; 1 . s | , , '. - . l I)| ’ II '(: l: ‘ l I ‘ ' I I ‘ “I ‘ "l|) Ir’~ l1: \’) )‘ll 'YII ~' I. 'va ‘ ' m. 1'”): .. tl ,rI ‘ ' s. i ~ . " -‘ III I -| ' II I l I ‘ ' I I .. I, ' . I ' W‘. .I ‘ l I \ I' ll .I.’ . I | . ' ' I ' I ' ~ . r I \ . . x |-' Lil ‘;l - 1 ) I .. II " I - I (' l- l - I. II , -I I II ._ -| u ‘ I II I. ' "‘ 'l -| l- ‘ \II I).";' .) " ~'.‘. 1"“ ‘ " ‘ "" I (.i I ) ' ' .i" I‘ I . 'I "I. " l - IF "I'I' l .' I ."'-I 5 Warfield (1973), using EMS, a chemical mutagen, induced muta- tions using the adventitious bud technique and Saintpaulia leaves. His results agreed with Broertjes (1972b) and Sparrow et. al.'s )1960) work though no discussion of chimeras was included. Begonia hiemalis Fotsch. is pr0pagated mainly by leaf petiole cuttings utilizing the adventitious bud technique which Doorenbos and Karper (1975) have used in mutation breeding experiments by exposing intact leaf petiole cuttings to X—rays. Doorenbos and Karper (1975), at the levels used, found only 0.0——0.h percent chimeras varying with cultivars which led them to assume that both mutated and non-mutated plants originated from single cells. Spontaneous mutation in a later stage of development was the explanation for chimera formation. A comparison of the adventitious bud technique both.i2 vivo and in vitro in a mutation breeding system of Chrysanthemum morifolium Ramat., 'Bravo' was done by Broertjes et. a1. (1976) showing that with detached leaves (in vivo), the shoots originated from callus at the base of the petiole or occasionally on callus formed on the upper part of the roots. After irradiation with X-rays, it was found that the few shoots obtained, either the main adventive shoot or its laterals, were chimeric in nature which led the authors to believe that more than one callus cell was involved in the formation of a shoot. After pre- liminary screening, pedicels were chosen as the explant source in_vitro because they formed adventitious shoots directly from the epidermis, 100 percent of the explants, irradiated or not, produced shoots, and they were produced in relatively high numbers. The control produced a high 6 average of adventitious shoots per explant. Only one plant was chimeric and along with mutants being propagated by shoot cuttings ( .12 vivo) or by i_n vitro using pedicel explants resulting in true to type progeny which lends support to the single cell to whole plant theory. IO .5: f") .CGL adcvrfe "-111ij 10' .mfp If ”‘35 ' 3 (‘1. y _rr-x’ '3':: '.'_n Ii :rr'. .rfJ-‘SI n" _r '3 I'oni-'-o "Il'r‘I"-'-' «(i _|‘l"'):) of 1: r~ off; A ' ': 1]- MATERIALS AND METHODS Five cultivars of Kalanchoe blossfeldiana were used in this study. 'Mace' which has very large obvate leaves with slight serration and small reddish-orange flowers; 'Korall' and 'Granat', hybrids that have serrated leaves with red flowers and less serrated leaves and red flowers, respectively. 'Sirius' is a hybrid that has leaves similar to 'Granat' but with orange flower color and 'Solferinopurpur' has small, roundish leaves, is self branching and has magenta colored flowers. Vegetative and reproductive explants were used as the explants in this study. Vegetative explants were obtained from plants main- tained under artificial long days by providing supplemental incadescent light 200 lux from 10 P.M. to 2 A.M. nightly from September 15 to April 15. The vegetative plant tissues were 12 mm leaf discs punched with a cork borer from the second to third leaf subtending the shoot apex; using two leaf discs from each leaf from five plants to give ten discs per test medium. Leaf petioles were obtained from one to four leaves sub- tending the shoot apex of the plant. Five petioles were tested.per media combination; each originating from a different leaf and the slices were approximately 10 mm.long and 5-10 mm in diameter. They were placed basal end down on the culture media. Stem segments, from 8-12 mm in diameter, were also used as an explant by placing nine internode segments on each test mediumt Young, healthy green tissue was chosen and the position of stem.segments on the intact plant was noted for experimental purposes. 8 The floral organs were initiated on greenhouse grown plants by providing a short day treatment with black cloth from 5 P;M. to 8 A.M. each day for a five week period. Kalanchoe blossfeldiana has a dichasial floral structure and the influorescence is supported by the peduncle. The peduncle explants, 5-10 mm.thick, were placed basal end down, nine per test culture medium. Pedicel sections were used as nine explants per test medium each.placed on its epidermis. Nine immature un0pened flower buds were also cultured.per test medium. All plant tissues or organs were surface disinfected in 20% commercial sodium.hypochlorite solution for fifteen.minutes and rinsed twice in sterile distilled.water. Prepared explants were placed on Linsmaier and Skoog (1965) medium with 2% sucrose, 0.8% agar, adjusted to pH 5.5-—5.6 prior to autoclaving. The growth regulators tested included 6-benzyl aminopurine (6-BAP) and naphthalene acetic acid.(NAA) at all com- binations of 0.01, 0.1, 1.0, and 10.0 mg/l. Penicillin-G (5 mg/l), gentamicin sulfate (10.0 mg/l), and tetracycline (h mg/l) were filtered, sterilized and added to the cooled.medium.(hOOC) after autoclaving to con— trol bacteria (Power et. al., 1976). The autoclaved.medium was added, 25 mls, to sterile plastic petri dishes, 100 x 15 mm, and the explants were subcultured every four to five weeks. The dishes were placed under 1000 lux continuous cool white fluorescent illumination at room temperature (260 0:2) for twelve weeks. The adventitious shoots were directly rooted in Jiffy Mix in covered glass containers in about three weeks under 10,000 lux of cool white fluorescent light, sixteen hours per day. Rooted plantlets were placed in cell packs filled with Jiffy Mix for two weeks under the same environmental conditions \~—\ -\-"II J _- ‘1 1.. \ .. . ‘-‘ ,. _‘_|l 9 used for rooting. The plantlets were subsequently placed on the mist bench for one week and to greenhouse conditions for further growth. To induce flowering of the plantlets, six weeks of nine-hour daily photo- period was provided by blackcloth treatment. Evaluation of regenerated plantlets was by visual inspection of morphological characters and thin-layer chromatography of flower pigment extracts. The pigment extract was made by grinding 0.5 g of freshly opened corolla tissue with a mortar and pestle in 5 mls of acidi- fied methanol. The supernatant was collected and the remaining tissue was resuspended and pulverized in 5 mls of acidified methanol. The super- natant was again collected and both portions passed through a glass filter by vacuum. The extract was centrifuged 2000 x g, reduced in volume and stored at 100C. Twenty'rds of the extract were spotted, dryed, and run on cellulose thin-layer plates in butanolzformic acidzwater (h0:10:50) solution using the upper phase. Pollen viability was determined by placing dehisced anthers on a microscope slide and adding 1 percent aceto-carmine stain. Only round, dark stained grains were scored as viable. at an”: .- "awr -f'ra.ero-"r_rI* on- “of . _IEJ . :. '..I-3_rr'. .‘r’ _ "C": D" (\j‘ ifi- l . 99!]: fl ‘7 ' --r--‘I.:' rim" -.-_n nus _r 7"9'1 --.~ ~-:sI.-.. ..5"‘-').'. r'.-l1‘ Iv-j:_2-"‘- = J l' I-ni (' “r- "ford I ' ‘ ."7 ‘ "1 I ’ '1 Ir .- r‘ f!" 1:." I... r: J " I7 ‘I ur'II‘ ( 7.“. n I": 1 Il') E‘I.‘_ri:' as .r. -. I-JJ' "II-5 '9:— r arr‘j'rn‘rr‘ ['IPC' "Ir ' '3 .rerr .l hoorI'I-r): _ - 'H')? '-I'_I'I _r .. ow -o' r ' 319' " J") -r.':r~"_'-['r‘u '_m .I' n- {r. V) _‘r .2: -~,'.- 'ler f !‘- I "I 3""?9 I I [”31 'H " Wr-lil ‘ I r"- 't .' I RESULTS Shoots were produced from all plant tissues and organs obtained from the five cultivars studied. With 'Mace', leaf discs pro- duced the highest average number of shoots, 5.6, at 1.0 mg/l 6-BAP and 1.0 mg/l NAA followed by stem segments with an average of 3.5 on 0.01 mg/l 6-BAP and 0.1 mg/l NAA medium (Table 1, see Appendix Table 1). Leaf petioles produced the next highest number of shoots and averaged 2.7 on 0.1 mg/l 6-BAP and 0.01 mg/1.NAA medium, 'Mace' peduncle sections averaged 1.5 plants per section on 1.0 mg/l 6-BAP and 1.0 mg/l NAA levels. Flower buds produced an average of 1.5 at 10.0 mg/l 6-BAP and 0.01 mg/l NAA with pedicel sections the highest average of 0.h was pro- duced on the medium 0.1 mg/l 6—BAP and 1.0 mg/l NAA. Stem segments explants of 'Sirius' yielded shoots on seven different media combinations of 6-BAP and.NAA; the highest average was 16.2 from 1.0 mg/l 6-BAP and 0.01 mg/l NAA (Table 1, see Appendix Table 2). Leaf discs produced shoots on six different media combinations with an average of 7.6 per leaf on 10.0 mg/l 6-BAP and 0.1 mg/l NAA. The other vegetative explant, leaf petiole discs, produced shoots on only one combination, 1.0 mg/l 6—BAP and 10.0 mg/l NAA with an average of 2.1+. The floral explants also produced shoots with pedicels producing shoots on six different media combinations and the highest average 2.6, on 1.0 mg/l 6-BAP and 1.0 mg/l NAA. Peduncle tissue produced shoots at one 10 l]. meowpoom oHOQSbom N am .ucsoo 0 on: woodwoom Hecaoma H mm a pmasa 00» p59 omahoy who: muoonm **** moan Hosoak H mm . n I noaoapoa mesa u an noesaaxo oobeoasseeooes co senses fiasco . mucosa Hence I omeno>< ass assesses amps u mm coapoahom poonw oz ** nomad Mama u 94* s.o o.a H.o em a.m o.oa m.o o.m m.o m.o m.a mm w.m m.H o.a m.m s.o mm m.a s.o o.sH o.m m.m :.H mm :.ma m.s s.m m.H H.m H.o H.H o.a m.m m.o mm w.o m.> m.: m.o m.H mu _Haohox. m.o m.o pm **so.o m.o m.m m.a s.o o.H :.o w.m m.o mm m.o H.o m.o m.o H.H ma m.oa m.o m.m m.a sssso.o ****o.o o.m mg :.m m.a :.m m.o o.H :.H m.o m.o mm o.m o.o o.H m.o m.o on .ssassaoeasomaom. m.m m.m m.o m.o m.o 2m o.ma m.o 0.0 m.: :.o m.H w.o mm m.ma m.o s.a m.o H.m m.o mm s.m sssso.o m.m 0.3m o.H s.H H.o m.a m.o an m.m m.o H.o s.a mm H.o :.mH s.m o.m m.ma m.w m.s m.m s.: :.H as .psesao. 5.0 m.o m.H m.o o.H H.o on m.o s.o o.m m.m :.o mm m.H m.a :.H m.o H.o mg :.m mg s.a m.a m.oa H.m w.m s.m mm 0.0 0.5 m.H w.m m.H mg .msfiuflm. m.H H.H om m.o H.o 1.0 mm m.H H.o H.o ma m.o s.m mg H.o m.o m.m m.o m.a asam.m mm H.m m.o m.m o.m 1.: H.o s.H m.H m.o ** mg .oosz. o.H H.o Ho.o 0.0H o.H wa Ho.o 0.0a mhw www Ho.o o.oa o.H H.o Ho.o seemso sasapaso 0.0H o.oa o.oa o.a o.a o.a o.H a.o H.o H.o H.o Ho.o Ho.o Ho.o Ho.o sedan A<< .Hw>apddo new pcmamxm Sumo Bong coprEMOQ poonm co meowprHQEOQ «<2 cow m3 " 3‘“! . I IV .‘ .. I\ I I I -‘ ' I-' I I" 4 . "I 1 -','~ "I I "I: 3 \ - . I ". I I" ‘J ‘I n ”H Ir' 1'2 ll " I I ‘1 II FI‘ ‘I - ' I 4' '1 I a II." n If "i I zI ‘. 'II‘ "f"‘ r [I . ‘ '1'r.r' . I "- .. I ' I ’)[' -.I_ .\nn~f 'f‘i' 13 giving shoots at only two levels with 1.0 mg/l 6-BAP and 0.01 mg/l NAA being optimum with an average of 0.8. Four of the six optimal concen- trations of the 'Solferinopurpur' plant organs had 10.0 mg/l 6-BAP with various NAA levels; the other two levels were at 0.01 mg/l and 1.0 mg/l 6-BAP. 'Korall', similarly, produced shoots from both vegetative and floral explants (Table 1, see Appendix Table 5). 0f the vegetative ex- plants, the stems produced shoots on the most combinations 10, and was the most prolific at 10.0 mg/l 6-BAP and 1.0 mg/l NAA averaging l9.h. Leaf petioles averaged 17.0 shoots per explant at 1.0 mg/l 6-BAP and 10.0 mg/l NAA. Shoots were also produced on six other combinations with leaf petiole discs. The levels 1.0 mg/l 6-BAP and 1.0 mg/l NAA averaged 7.8 from leaf discs while shoots were formed on five other combinations. 'Korall' pedicels produced shoots on eight media combinations with 10.0 mg/l 6-BAP and 0.01 mg/l NAA averaging 10.0 per explant. Adventi- tious buds occurred on six different media combinations when flower buds were used; the combination 10.0 mg/l 6-BAP and 1.0 mg/l NAA pro- ducing an average of 5.8. Of all the explants, peduncle tissues produced shoots on the fewest combinations, three, and the lowest average, 1.0, on 1.0 mg/l 6-BAP and 0.1 mg/l NAA. In four of those six combinations the NAA level was either 1.0 or 10.0 mg/l. Visual evaluation of the flowering plantlets revealed no visible modification in plant morphology when compared to the parents. Leaf shape, amount of leaf serration, plant habit, flower shape, and flower color were considered during evaluation and no differences were found. EI _l'\\_ JV.) I'm". " F—D I'\'_n II ("'I No.9] r~ — Du-nn I tJ n 'I‘ 1H5 To incl . 1 ‘r ‘- :JM HJI- ' {wa |\' l.‘I “.H ‘I':Ee fir I 'Tfl TU nIf- |\' ‘Ll “: ' I\- r3.) :3' of: mister r-'1 Jorfc .' ‘ "x r. 1’910' [”01‘ I- r”' a. - m" "axy'm'; - l' ‘ I" --\ r~| I’)[' -- «i' I'( ' 'I" “ ' .....j I\- II ‘II .. '\. )_'II I-I huuuWru “on""jfi TIIIW‘J 13: l I IN” II I I I, . » I I"-’7 '1 '.-")I.I I I I ‘ I"' ‘I - I .h' I 'I "1' I ' ‘ .. [.11' Ipl' I I we, .- I 'I I “ ", . I . If I ' l ' ' 1h By using a completely randomized design, choosing three adventitious plants per organ per three different hormone combinations, it was found with 'Granat' that there were no significant differences in pollen viability. Likewise with the thin-layer chromatography (TLC), all pig- ment extracts were freshly prepared for each replicate and three replicates were made of all the plant organs of 'Granat' and no qualita- tive differences in the resolved TLC pattern of the pigments was observed. I-AII DISCUSSION The primary objective of the study was to determine which ex- plant of Kalanchoe was most suited for in vitro mutation breeding. There are several criteria which need to be considered as guidelines in establishing the appropriate explant. Firstly, to identify the explant that would yield the highest number of adventitious shoots on a given media combination. The average shoot production in this study was the total number of shoots arising divided by the number of uncontaminated explants that did or did not form shoots. Secondly, consistency of shoot production, as indicated by a low standard deviation which is cal- culated from the shoot forming explants, is as important as the first criterion. Preference is given to the plant tissue or organ that pro- duces shoots on a high percentage of the explants and with even distribution. Thirdly, the growth stage of the shoots after a period of time, as reflected in the percentage rooted, is important since those plantlets which are relatively more developed will root readily thus ensuring a high survival rate. The five Kalanchoe cultivars studied herein varied considerably with respect to these criteria. Using the above criteria, each cultivar warrants individual discussion since they did not have identical optimum explant-media com- binations. As seen in Table 2, with all the cultivars the choice of the explant is evident, one being leaf discs of 'Mace’ at 1.0 mg/l 6-BAP 15 ”h l6 mpoeamxo dopeowaepnoons Hepop m mpoonm Hdep u owmho>< * case see sooa n.m H. o.mH one o.oa one o.oH neon oeneso .I omen awn eon w.ma+ n.ma nae H.o one o.oa neon reneno .I omen ewe sooa m.m + m.mH baa o.H one o.H neon reneno I. pooawom emm ens m.ma+ n.ma oma o.a one o.oH norm onenom I. somewow ems soon H.HH+ m.oH Hm no.0 one o.H norm ennnnm I. oHowpom new so: n.e + m.oa Hm o.H one 0.0H neon annnnnonanonaom .I omflm new sooa m.m + o.m me o.H one o.H neon ooez epoonm nuns anoonm «an dnmIe oouoom mpoeamxm coopeo>om mo amoeba AH\wav pnem ubmoammh PG mo hmm 68.35de *mwdhm>< HmpOB COHPGQHPSOO pdwflm Hw>flpgo ocoenom ooflocoaem mo oopmop mHo>HpHdo map 90 ooapeoflbeoo oc08hofluoemno penam ospflb so Hoaflpmonn.m canoe .Nflcoaaom womMWHoMmmoap -) . .— . u . .I I I .. . . I. . I I v. _ I. II,.. I .I ..I .. I. I .. In I .II. . I C .I 1...; ..I ... . . .. . .. I m u . ..I .._ . I 1. ill. l7 and 1.0 mg/l NAA since the average was the highest for the explants tested with a low standard deviation 3.2. All the explants produced shoots and a substantial number of the adventitious plantlets rooted (81%). With 'Solferinopurpur' (Table 2), though shoot production was low, leaf petioles at 10.0 mg/l 6-BAP and 1.0 mg/l NAA was the best combination with an average of 10.2, standard deviation 6.h, but a low percentage of explants with shoots (MO%) and the percent rooted was high, 8h%. With 'Sirius' (Table 2), stem segments at 1.0 mg/l 6-BAP and 0.01 mg/1.NAA was the choice since there was a high average of 16.2 but also a high standard deviation of 11.1. All the explants produced shoots and 78% of the plantlets rooted. 'Korall' presents a clear choice (Table 2) since no other plant organ approaches the high average, l9.h, though the standard deviation is high but a high percentage of the explants produced shoots and the rooting problem should be corrected by minor modifications in the rooting procedure. Leaf discs of 'Granat' are the explant choice and the media combination is not critical. The three most productive are represented showing that the averages are high (Table 2), the standard deviation fairly low with a high percent of explants with shoots. By comparing the same explants of the different cultivars, trends in response to the media formulation is evident, the most obvious being that explant variation occurred. This was not unexpected since there were differences in shoot production and their response to the various media combinations. Leaf discs had averages from 3.6 to 19.h (Table 3) and.most of the shoot production occurred on the 1.0 and 10.0 mg/l 6-BAP combination with either 0.1 or 1.0 mg/l NAA. Stem segments, 1" ”I " ' fl - \ “9" ! g . ‘3' . ‘) re'n ‘Lrizu - '“J'j I ‘r' . - (f.- . 2.1T i‘lr ,_‘ . l I I‘ i.' In . ‘f ’ ... , . .‘i. . .. I II | l ' v. ! ‘ .1 ‘. ,n .) .5f' ’3 I ' .11 "I' . . :I, - -' l'\1. \j' .‘u'ufl' | I 1' ' 'i-5 ".f "I". 1 .. q H ‘ [\ . 18 muawamwo vopdadadpcoocfi mo Hones: Hdpop m mpoonm prop u mmduoh<* b o a a o \o.H .Haanom. m.o Ho.o \o.a .Hsnnsmoaanunaom. m.m o.H \o.H .vdawnc. m.H H.o \o.H .msanam. m.H o.H \o.H .man. mucoawom aflocsbmm o.oa Ho.o \o.oH .Haaaom. m.m o.oa\ao.o .nsnnsnocanmmaom. o.ma o.H \o.oa .pacano. o.m o.H \o.H .msfiaam. we 1 on 3.2 .082. lawman f m.m o.a \o.oa .Haaaom. :.N o.oa\o.oa .HfimhnmocwHOMHom. m.ma o.H \o.oa .pucanu. :.H 0.0H\0.H .mswnwm. m.H Ho.o \o.oa .moaz. mesm amzoam o.sa o.oa\o.a .Hawgom. m.oa o.H \0.0H .Hsmhsmocano%aom. 0.:m o.H \o.a .pacauw. :.m o.oa\OeH .msaaam. m.m o.oa\o.oa .oowz. moHoapmm Mama :.ma o.H \o.oa .Hamaom. :.m H.o \0.0H .Hamhnmocahmmaom. m.m o.oa\o.H .pdcwhw. m.ma Ho.o \o.a .msanam. m.m H.o \Ho.o .oomz. mucoawmm scum m.> o.H \m.a .Haanom. o.m o.oa\o.oa .nsmnsmocaumwaom. :.ma H.o \o.0H .paaanw. m.s H.o \o.oa .msfinam. (m.m o.H \o.H .moaz. woman mama «az mam-m Ad>wpdao scam AH\wav mo *omwnm>< pmmnmam soapdcfipsoo ocoauom Hw>HpH50 sumac pawam h "I" mhwbwpddo on» 90 cowpwcfinaoo ocoahon h wdficcommmhhoo vow mmdho>w pmmnwan mnann.m magma 19 in general, were not as productive as leaf discs and there is no signi- ficant trend in media response where all appear to produce plantlets. Except for 'Granat', there was less production at 10.0 mg/l 6-BAP and all combinations of NAA. Petioles, the explant used by Broertjes and Leffring (1972) for Kalanchoe were the Optimal explant to use in vivo but produced few shoots as was the case with the in vitro work where they “were the least productive of the vegetative explants. In this work, low levels of BAP and low levels of NAA at the high 6-BAP levels inhibited shoot production. 6-BAP at 1.0 and 10.0 mg/l in combination with 0.1 and 1.0 mg/l NAA were the combinations which produced shoots. On all but one cultivar 'Granat', there was low shoot production with flower buds and the hormone levels which produced adventitious shoots was from the 1.0 mg/l 6-BAP and 0.1 mg/l NAA through the 10.0 mg/l 6-BAP and 10.0 mg/l NAA levels. The highest average of each cultivar utilizing pedicel ex- plants ranged from 0.6 to 12.0 which occurred at the 1.0 and 10.0 mg/l 6-BAP levels in combination with 0.1 and 1.0 mg/l NAA. Peduncle explants generally produced few shoots with the high of each cultivar ranging from O.7-5.5, mostly below 1.0 with the best response occurring at 1.0 mg/l 6-BAP in combination with 0.1 and 1.0 mg/l NAA. Of the floral organs, pedicels were the best explant source but when compared to the optimal vegetative plant organ, leaf discs, the vegetative one is the Choice over- all. For the cultivars tested, the highest average was with leaf discs at 1.0 and 10.0 mg/l 6—BAP in combination with 0.1 and 1.0 mg/l NAA. Differences in shoot production between the cultivars was ap- parent when all the plant organs of the cultivars are compared (Appendix Table 6). With 'Mace', leaf discs were the only productive explant source, [A " | -\ I \ I .. n. v .9“ 3"|""" 20 the other plant parts being similar in average to one another. The highest average of 'Sirius' was 16.2 with stem.segments while leaf discs produced an average of 7.6 with the four remaining plant organs averaging between 1.0 and 2.0. 'Granat' plant organs, except for stem segments, had relatively high averages, above 6.0. Leaf discs pro- duced large numbers of adventitious shoots at many combinations but all the plant organs of the cultivar were productive. With 'Solferinopurpur', all plant organs were consistent regarding shoot production, between 2.0 and 3.0, except for peduncle tissue. AS‘With 'Solferinopurpur', all plant organs of 'Korall', except peduncle tissue, adventitious shoot production was between 5.5 and 19.4 consistent within the cultivar. Adventitious shoot production between cultivars varied which was not un- expected. Within each cultivar there was consistency of shoot production even between vegetative and floral plant tissues except for outstanding examples. With Kalanchoe, the response of a particular plant organ to a specific combination of growth regulators in the media was not always con- sistent. Parts of this study were repeated by utilizing explants taken from parent plants grown at different seasons in the greenhouse that plant tissues respond somewhat differently'EELZEEEQ. The results indicated where temperature and light intensity affected the physiological condition of the parent plants that this could account for the dif- ferences in EE.XEE£2.ShOOt production. This parallels Mikkelsen's work (1976), where differences in shoot production of Begonia hiemalis Fotsch. both 'Peach' and 'Rose' was apparent between the winter and summer months. Physiological age of the parent plants is also a prime concern since older uf 'm n e'Ir "Ir . 7 ' ' I H I I I I n" n-'—\ J -' . l I n' w‘ I' .: -' j" ._.r '5‘. r -I 1 “mr M- ‘E- |.l .I I} . ~_ .- r 7"I‘f‘r ‘ I ',-...' _ _ _ [I' ‘I I n HM, _) "D -'~,r:“"l 5' I "In-I T ' I I 9 M‘ I E :II'1 ‘ .- $1. ' ' ' .I.I' rI 5: 3" ‘ ‘M .I H~n\ Hln.= ’ I '1! ‘II . fl on: __‘I_ _-__1_' "" 'I 7‘1" r 'l " h." -'.'I- I I‘--- -\ i‘ I ’r' nnhllf 7‘ 'P" 5" l \‘ ‘ h ‘ \- ' .1. . '.~, .. , II I' ' r 1‘." .' f‘ r"- - “..rf ' a ‘--r .-n " -r~ -. ---\ - ~ : .._ a. '7‘ I I n w'l". ‘ ~§ . . _ .. . \ I I I. I I l I m- I _.._.II I‘I'sflfl": was “_A ~ II'_"' f ' *- I f _ .-. '7‘ I‘ll." ' ._ 'I'fl" ‘ 1 ..I --."‘ 4 21 tissues tend to produce fewer adventitious shoots. As Kalanchoes age, the plants tend to become day neutral (Schwabe, 1969) causing flowering which could alter the endogenous hormone levels resulting in a varied response. There is a need to find the Optimal environmental conditions and age of parent plants to secure a consistent media combination response. New Kalanchoe cultivars are predominantly produced through hybridization followed by asexual propagation when a desirable seedling is found. This shows that most Kalanchoe cultivars are probably very heterozygous genetically Which is advantageous for a mutation breeding program for new potential combinations of germplasmw In Broertjes and Leffring's in 1319 work (1972) with leaf petioles of Kalanchoe no chimeras were found and in this in vitro work no variability was re- leased regardless of explant origin (epidermis, vascular, or cortex). With the observation of morphological Characters, thin-layer chromato- graphy of flower pigment extracts, and pollen viability, all the plants appeared genetically similar to their respective parents. As reported by Skirvin and Janick (1976) with scented Pelargonium spp., an asexually propagated crop, variation can be released dependent on the ex- plant being derived from histogenic layers. Thus, leading one to believe that after years of asexual propagation, complex chimeras have formed along with inherent cell variation. With Kalanchoe, though not the primary aim of this study, the explants utilized represented two histogenic layers in origin and they released no variability through in vitro culture. Although an asexually propagated crop, the original hybrid plants are of seedling origin from plant breeding programs. Thus, ' fl‘ 22 because they are of recent origin, the histogenic layers are most likely identical and complex chimeras have not arisen either by point mutation or by ploidy changes. The overall results indicate that an in vitae mutation breeding system may be feasible with Kalanchoe. Even cultivar dif- ferences in terms of shoot response and.variation with respect to Optimum media required, the number of shoots was generally greater than in 3222 systems. Along with less space needed for propagation of adventitious shoots and less time to produce a plantlet to flower, preliminary screening of cultivars, in addition to those tested, indicated that an ‘in vitro mutation breeding system for Kalanchoe blossfeldiana should be a practical system for producing new cultivars. 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2 0 0 2 0 0 2 0 0 2 0.0 2 2.0 0 0 2 H.0 000 H0.0 0 0 2 0 0 2 0 0 2 0 0 2 0.0 2 2.0 0 0 2 20.0 000 H0.0 .0>< 090090 2029000m .0>< 000090 0029000m .0>< 090090 0029000m .0>< 090090 2029000m .0>< 090090 0020000m **.0>< 09009m *0022000m <0 000 .000090 20 209850 20000 .00220002 0202> mMru.2< 02902 27 002200 n 0 20220002 02 n x 02002 n 2 .02202220 00202228200025 20 202050 m 020020 20202 n 0m020>< ** 0025002 2002:029 020 20020u02m n 2 020020 u 0 * o 0 2.0 0.0 00 2.0.0 0.0 2H 2.0.0 N.H 0 2.0 o 0 0 2.0 0 2.0.0 0.0H 000 0.0H o 0 2 H.0 mm 2.0 0.0 mm 2.0 0.H 0 2.0.0 2.0H 00H 2.0 0.0 0 2.0 o.H 000 0.0H 0 0 2 0 0 2 0 0 2 2.0 m 2.0 0 0 2 o 0 2 H.0 000 0.0H 0 0 2 0.0H 00 2.0 m.H 2H 2.0 0 0 2 0 0 2 0 0 2 H0.0 000 0.0H 2.0 0 2.0 0.0 0 2.0 o.H m 2.0 0.2H H0 2.0.0 0.2 0H 2.0.0 0 0 2.0 0.0H 000 o.H o o 2 0.0 20 2.0 0.0 mm 2.0 0.0 mm 2.0.0 2.0 20 2.0 0.2 22 2.0 o.H 000 o.H o.H m 2.0 0.0 2 2.0 0 0 2 0.0 H0 2.0 0.H 0H 2.0 0.2 00 2.0 H.0 000 o.H 0 0 2.0 o 0 0 0 0 2 0 o 2 H.0 0H 2.0 o o 2 H0.0 000 o.H o 0 2.0.2 0.0 0 2.2.0 2.0 0 2.2.0 2.H 2 2.0 H.0 H 2.2.0 0.0 0 2.2.0 0.0H 000 H.0 .0 H 2.0 0 0 2 o 0 2 o 0 2 H.H 0 2.0 m.H 0 2.2.0 o.H 000 H.0 0 0 2 0 0 2 0 0 2 0 0 2 o.H 0 2.0 0 0 2 H.0 000 H.0 0 0 2 0 0 2 0 o 2 0 o 2 o 0 2.2 0 0 2.2 H0.0 000 H.0 0 0 2.2 o 0 2 0 0 2 0 o 2.2 0 0 2.2 o 0 2.2 0.0H 000 H0.0 0 0 2 m.H m 2.0 0 0 2.2 0 0 2.2 0 0 2 0 0 2.2 o.H 000 H0.0 o 0 2 0 0 0 o 0 0.2 0 0 2 0.0 mm 2.0 0 0 2 H.0 000 H0.0 o 0 2 0 0 2 0 0 2 0 0 2 0.0 H 0 o 0 2 H0.0 000 H0.0 .022 020020 20220002 .0>< 020020 20220002 .0>< 020020 2022000m .0>< 020020 2022000m .022 020020 2022000m **.0>< 020020 *20220002 <22 m0 000 0020020 20 209852 20202 .20220002 0222>_HMVI.0< 0290B 28 Table A6.--Highest average of each plant part of each cultivar and the corresponding hormone combination. Hormone Cultivar Plant Combination Average** Organ* (mg/l) 6-BAP NAA 'Mace' LD 1.0 and 1.0 5.6 SS 0.01 and 0.1 3.5 LP 0.1 and 0.01 2.7 PE 10.0 and 0.01 1.5 PE 10.0 and 1.0 0.6 PU 1.0 and 1.0 1.5 'Sirius' LD 10.0 and 0.1 7.6 ss 1.0 and 0.01 16.2 LP 1.0 and 10.0 2.h FB 1.0 and 10.0 l.h PE 1.0 and 1.0 2.6 PU 1.0 and 0.1 1.2 'Granat' LD 10.0 and 0.1 192 SS 1.0 and 10.0 2.8 LP 1.0 and 1.0 2h.0 PE 10.0 and 1.0 12.2 PE 10.0 and 1.0 12.0 PU 1.0 and 1.0 6.2 'Solferinopurpur' LD 10.0 and 10.0 3.6 SS 10.0 and 0.1 2.h LP 10.0 and 1.0 10.2 FB 10.0 and 10.0 2.h PE 0.01 and 10.0 5.8 PU 1.0 and 0.01 0.8 ’Korall' LD 1.0 and 1.0 7.8 ss 10.0 and 1.0 192 LP 1.0 and 10.0 17.0 FB 10.0 and 1.0 5.8 PE 10.0 and 0.01 10.0 PU 1.0 and 0.1 1.0 * LD = leaf disc SS = stem segment LP = leaf petiole FB = flower bud PE = pedicel PU = peduncle segment ** Average = total shoots ; total number of uncontaminated explants ' .I' '.f_rI~. I) .E -'l _I —--—. '5' .l ‘ I I” I I . I I ‘ . ' I ‘l! I. I I ‘l n ' I , . , _ BIBLIOGRAPHY BIBLIOGRAPHY Broertjes, C. 1969. Mutation breeding of Streptocarpus. Euphytica 18:333-339. Broertjes, C. 1972a. Mutation breeding of Achimenes. Euphytica leh8-63. Broertjes, C. 1972b. Use in plant breeding of acute, chronic, or fractionated doses of X-rays or fast neutrons as illustrated with leaves of Saintpaulia. Thesis Wagenigen: Agric. Res. Rep. 776, 7Hipp. Broertjes, C., B. Haccius, and S. Weidlich. 1968. Adventitious bud formation on isolated leaves and its significance for mutation breeding. Euphytica 17:321-3hh. Broertjes, C. and L. Leffring. 1972. Mutation breeding of Kalanchoe. EUphytica 2izui5-h23. Broertjes, C., S. Roest, and G. S. Bokelmann. 1976. Mutation breeding of Chrysanthemum.morifolium Ram. using in vivo and in vitro adventitious bud techniques. Euphytica 25:11-19. Doorenbos, J., and J. J. Karper. 1975. X-ray induced.mutations in Begonia hiemalis. EUphytica 2u;13-19. Linsmaier, E. M., and F. Skoog. 1965. Organic growth faCtor requirements of tobacco tissue cultures. Physiologica Plantarum 18:100-127. Mikkelsen, E. P. 1976. Histology of adventitious shoot and root formation on leaf-petiole cuttings of Begonia hiemalis Fotsch. cv. 'Aphrodite Peach'. M.S. thesis, Michigan State University. Power, J. B., E. M. Frearson, D. Gearge, P. K. Evans, S. F. Berry, C. Hayward, and E. C. Cocking. The isolation, culture, and regeneration of protOplasts in the genus Petunia. Plant Science Letters 7:51-56. 29 “m LancJ 3th ‘0 :rrboatu ICIF in .’>jI .93'jtor7' .DEE—EEF _F -I_I'-:i 'r' ,u ' 'Dffl”h U” 93r9.jfl3 'c If“39r6 ojfifidu .-23}I - an-J . = a :IsCfiE .8“- -‘ _r‘ )Iro'cI'q .9dno1 1o Irfiaavc dr'f If aaU €17 F “a 5 “h °-rtdy9r Jo'“ TC =~':- To “sac“ “9d”lCid>'TF “‘ 2Iwod' rIn” Erin ‘r ‘0 car f-t” W?“'ff>nflf‘ ”_ ‘I( l I\- 1‘: - ' ' -II 9' '3 oh: -- ~-.r->.—_ TS”T “unidij svh , ‘3j _dqfrmr) _ r 33: '0 fl?! LED. ecif "I- ' ~3u":)_r '_‘ ' .p HHEaITF.fF "Didrfi.nT "rfi355l -~ ' " I I -J I 3 II "L'r‘ 'r .fi'wosn' I ‘ rJ 3.5!} . ‘- “F ' ‘ '- '5‘.“ .Ei'gl- r3 \f'fitf [I ' -‘-(‘- ‘ r ICfri TLU ‘I'TF . (“Viv I _r_ _I l"_f_l n" '-_|'_|l h“ r":(“ r; N.H. -= 'I-' "Hi. '1 III“! II ' .eanr‘h r."3=).i " -.-Jo%. . ~n tavf 'Il-f" "rm I :3 fl'.' I 9‘ erofd in Wejub f '—' .33“! To :‘ ' I .noL-3"roT .‘l-Fr !(1 nwiI H !I ‘If .I II‘ 1‘ I Ich l flavor; II. Irfl P I '-).l .c:_l("_|'"-‘.\Ilrr"" _ ‘_)'.'n.i I'_II\ .‘II_I->"f ' (‘ \-'\ I‘I‘.I ' ".‘in I -I :IIIP-fi'.‘ l-f‘,[..\|| l rI'.'_ :i- [ 'ir‘CI "'I 'r-r‘rfp 9IJI~_I.Z I".:I \-' . r' 'll‘I-VI' ‘I' "I zilr-ejfl “IIPXW: -0 ‘ f'iuw olva‘ -‘- H r"'.'rrhr "I'T'Hl' 'lI ) I-iI'I‘TIF ‘ 9-" .I'I‘-.‘ 'ir . I . J . .--.-.-3.- ' ' ': --.. ‘. 931.3 IJJ '- IC‘ r.I Irv-‘r )[ 'IIIJi') III‘I; 'II" II "-"~ I 30 Schwabe, W. W. 1969. Kalanchoe blossfeldiana Poellniz. ‘lg L. T. Evans (ed.). The induction of flowering. MacMillan. Australia. pp. 227-2h7. Skirvin, R. M. and Jules Janick. 1976. Tissue culture-induced variation in scented Pelargonium spp. J. Amer. Soc. Hort. Science lOl(3):281-290. Sparrow, A. H., R. C. Sparrow, and L. A. Schairer. 1960. The use of X-rays to induce somatic mutations in Saintpaulia. African Violet Magazine 13:32-37. Warfield, D. 1973. Induction of mutations in african violet (Saintpaulia ionantha Wendl) by ethyl methanesulfonate. Hort. Science 8(1):29. ““'II‘IIIIIIIIIIIII‘III’IIIIII‘I‘IIIIIEs 1111111111111