MSU LIBRARIES .-_. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES wil] be charged if book is returned after the date stamped be10w. mm FIIIUTY 0F BETA WIS. L- RNETS IRIVED FROMMADVENTITIOUS BUDS 0N SHOOT CULTURE PETIOLES By Taha Hassan Abdel-Latif A THESIS Submitted to . Michigan State University in partia] fulfiliment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1985 ABSTRACT GENETIC FIDELITY OF BETA VULGARIS L. RAMETS DERIVED FROM ADVENTITIOUS BUDS ON SHOOT CULTURE PETIOLES By Taha Hassan Abdel-Latif Genetic fidelity of adventitious buds rising during ig_yjtgg_ shoot culture of Beta vulgaris L. was studied, based on the comparison between 417 axillary bud derived ramets and 1108 adventitious bud derived ramets. The characters of pollen shedding, seed set, germness, leaf shape, chromosome number, amd guard cell length were compared. The segregation of the characters; hypocotyl color, annualism, and germness was studied in the S1 progeny of some adventitious bud derived ramets and their heterozygous axillary bud derived counterparts. No evidence for genetic infidelity was seen in most of thousand ramets derived from 141 adventitious buds from 24 original clones, or in S1 progeny from 50 of those adventitious buds, although two aspects were observed. Tetraploid root tips were growing with diploid ones on the same ramets found to be diploid from guard cell Ineasurements. Different concentrations ofS-benzyladenine on this aspect did not result in any corresponding change of tetraploid frequency. Also, three conspicuously narrow leaf ramets from table beet were observed, but in no case were all ramets from an adventitious bud narrow leafed. This narrow leaf aspect did not persist through the second cycle of shoot culture propagation. TAB LE OF CONTENTS LIST OF TABLES ......................... LIST OF FIGURES...... . . . . . . . . . . . .. INTRODUCTION. . ... . . .. ..... ... ............. MATERIALS AND METHODS ...... ... ............... Genetic materials .. .................... Some working definitions used in this study ........ Culture methods ......... . ............. Establishment of shoot culture ............... Experiment 1 ....................... Experiment 2 ....................... Experiment 3 ....................... Experimnt 4 ....................... Experiment 5 ....................... Experimnt 6 ....................... RESULTS ............................ Experimnt 1 ....................... Experimnt 2, the field study ............... Experimnt 3 ....................... Experiment 4 ....................... Experiment 5, the genetic study of S progeny ....... Experimnt 6, the effect of benzylad nine on root tip chromosome number ............. ....... DISCUSSION .................. ' . . g ...... BIBLIOGRAPHY ...... ’ .................... ii Page iii 67 69 74 TABLE 10 11 LIST OF TABLES Page Hurashige-Skoog medium (MS). Inorganic Salts ...... 8 The number of ramets obtained from each genotype from both axillary and adventitious bud derived shoot culture ....... . . . . . ............. 15 Number Of ramets fOr the characters: flowering,pollen shedding, and seed setting and occurrence of mono- or multigerm in axillary and adventitious ramets ...... 30 Determination of leaf shape through width/length ratio of three leaves per ramet fOr both axillary and adventitious bud derived ramets in the field. . . ... . 34 Test of Significance for the deviation of the mean for the comparison of each axillary and its adventitious derived ramets as compared by their grand means for leaf shape character .................. 38 “t' test for the comparison between each adventitious isolate with its axillary counterpart for some table beet and sugarbeet genotypes .............. 43 Determination of the significance of the narrow leaf ramets as compared with their normal leaf ones ..... 45 Number of diploid and tetraploid cells and the percentage of tetraploid-containing root tips of axillary and adventitious bud derived ramets ...... 48 The average of the guard cell lengths measured by calibrating lens in light microscope of five randomly chosen fully expanded leaves of axillary and adventitious bud derived ramets ............. 51 The standard deviation and the deviation from the mean for the leaf shape character of the second cycle ramets and their counterparts normal leaf ramets ........ 56 The deviation Of segregation ratios of hypocotyl color TABLE 12 13 14 Page as tested by x2 for the S progeny fOr 50 5 seeds of two sugarbeet genotypes add their adventitidus bud derived .ramets ..................... 60 The segregation of germness and the deviation 0 segregation ratios of annualness as tested by x for the S progeny of two sugarbeet genotypes and their adventitious bud derived ramets ..... z ....... 63 Proportion of tetraploid root tips of axillary derived ramets produced from media containing différent benzyladenine concentrations. ............. 68 The analysis of variance of regression between benzyladenine concentrations and frequency of tetraploid cells. ................... 69 iv LIST OF FIGURES FIGURE ' . Page 1 Adventitious buds grown on a table beet leaf of the genotype EH-53 on the shoot culture medium (M20) ..... ll 2 Potted table beet ramets propagated by shoot culture. . .12 3 Seed stalks of some sugarbeet ranets propagated through shoot culture ...................... 14 4 . A narrow leaf ramet of the genotype EH-l Ax as compared with its neighbor normal leaf of the same genotype. . . .21 5 (a) A nonmal diploid cell of a sugarbeet genotype (FC701/5-116) propagated by shoot culture.(b) tetraploid cell of the same genotype ........... 22 INTRODUCTION Sugarbeet (Beta vulgaris L. ssp saccharifera) is an Outstanding example of a product of plant breeding. It was developed in Europe less than two hundred years ago from fodder beets by mass selection (Palmer, 1918). Since that time, sugarbeet has been bred to increase sugar percentage (from 7-10 to 13-20%). As a wind facilitated, cross pollinated, largely self- incompatible plant, beets are highly heterozygous. Both Mendelian and cytoplasmic male sterility are available (Smith, 1980). Mendelian male sterility is commonly used in improving populations of self fertile germplasm, while cytoplasmic male sterility is the method of choice for hybrid development and commercial seed production. Sugarbeet behaves as a biennial crop: the first year in the field it grows vegetatively as a rosette producing a large root with a high percentage of sucrose. If carried over to a second year after undergoing overwintering, it onld develOp a floral stalk. a process also known as bolting. Cuttings of the floral stalk of individual beets can be used for vegetative propagation fOr breeding purposes, as can Split roots, but the detrimental effect of this on seed quantities‘ produced must be considered. Survival of beet plants in the field following seed bearing is an uncommon occurrence. Sugarbeet breeding programs primarily utilize mass selection, recurrent selection, and family line breeding(Poehlman, 1979). All these different systems are geared to producing hybrid cultivars, either diploid or triploid. Hass selection is used primarily to increase disease resistance, root size, root shape. crown height. and. in sone programs. sucrose percentage. Recurrent selection is used to concentrate genes for particular quantitative characters, and as it involves progeny performance, favors more readily transmissible traits. Although this system involves some visual selection it differs from mass selection in that the visually selected roots are individually test crossed or intercrossed, then their ramets or selfed seed are used to intercross the best combiners from that test. In family line breeding, the remnant half sib seed of open pollinated mother beets with superior progeny performance such as resistance to disease and high sugar content. is subjected to further selection for subsequent cycles or is increased for testing as a parental line. Family line breeding. like recurrent selection, has the continual problem of producing enough seed on the mother beet to adequately test. In Europe, triploid or anisoploid sugarbeet cultivars have been successfully used for many years (Hornsey, 1975). when commercial hybrid sugarbeet seed is the goal, either diploid or triploid is the main breeding objective. In these programs either single cross, three way cross, or double cross can be used. The three way cross is mainly used in producing commercial seed in the United States. Male sterility. particularly cytoplasmic male sterility, is utilized in the seed parent since seed production is not necessary for sugar production in the farmers' fields. The male sterile line can be maintained by crossing with its equivalent male fertile line, from which it was derived by backcrossing. During a breeding program, it may be quite inoortant to maintain unique individual genotype over years, as in the case of cytoplasmic male sterility maintainers (O-types) and elite general combiners. Because beets rarely survive until even preliminary evaluation of their_progeny is complete,.and becauSe;self sterility precludes maintenance of a heterozygous superior transmitter's gene combination intact through its seeds, there has been a need for an efficient means of vegetative propagation in beets to preserve intact gene combinations until evaluation is complete. Recently. 19.1319. propagation of crop plants through tissue culture has become widely used. Shoot culture is successfully used in sugarbeet. A variety of'jgyyitgg methods for multiplication has appeared for beets (Hussey and Hepher, 1978; Margara, 1977; Coumans- Gilles et al, T981; Saunders, 1981; and Harms et al, 1983). Seedlings, axillary buds, and the terminal parts of seed stalks are Used to initiate the shoot cultures. Some procedures will be explained in detail in the materials and methods section. By definition, most shoots arising in shoot culture propagation are derived from axillary buds; but in some species such as Bgtg_ vulgaris L., Exbury azaleas (Fordham et al, 1982), and Helianthus saggy; (Paterson, 1984) adventitious buds also arise, and can Often be indistinguishable from axillary buds. At one time, it was presumed that the ranets Of both axillary and adventitious Shoot origin were genetically identical to the source genotype. However, reports of genetic variation arising from single cell regenerated plants (Cassells et al, 1983; Larkin and Scowcroft, 1981), protoplast regenerated plants (Karp et al. 1982), callus and embryo explants (Patel and Berlyn, 1981). and , moreover, adventitious bud derived plants (Evans and Sharp, 1983; Norris et al, 1983; FOrdham et al, 1982; Hermsen et al, 1981; Patel and Berlyn. 1981; and Van Harten et al, 1980) suggest that complete genetic fidelity through shoot culture adventitius buds should not be presumed. The origin of the adventitious buds in beets has been studied by Harms et al (1983), who suggested that in table beet adventitious buds arise from single cells of the upper epidermis of petioles. 0n the other hand Norris et al (1983) emphasized the multicellular origin of the adventitious buds from their work on plant chimeras in African violet. The main purpose Of this study is to test the genetic fidelity of the ramets of in vitrg adventitioUs shoot Origin. It is extremeiy important to determine the reliability of using shoot culture derived ramets in plant propagation during the breeding program. This is particularly so in the context of the somaclonal variation derived from jg yitgg culture (Larkin and Scowcroft, 1981). The fOllowing main objectives have been established for this study: T 1. To examine the stability of chromosome number by screening fOr polyploidy or aneuploidy through root tip chromosome counts, guard cell length measurement, plant morphology, pollen production, and seed set. 2. To study the genetic fidelity with regard to the Mendelian characters hypocotyl color, germness, annualness, and any other traits that might arise in the original clones and/or their 51 progenies. . To study the effect of the 6-benzyladenine in the shoot media on the frequency of any chromosome instability. MATERIALS AND METHODS Genetic materials: Both sugarbeet and table beet clones (Beta vulgaris L.).were used in this study. - The table beet seeds were from a single cultivar, Early Honder, which. phenotypically, has heavily pigmented hypocotyl and roots (RR YY), and is biennial (bb) and multigerm (NM). The histological development Of prolific adventitious bud fonpation has been studied in another table beet cultivar (Harms et al, 1983). - The biennial sugarbeet clones were obtained in the form of shoot culture. They represent both adapted as well as exotic germplasm. The following are the established shoot culture clones which were used. .‘ EL 36-18: A randomly sanpled individual clone from EL 36, a monogerm (nun), type 0 (ie, cytoplasmic male sterile maintainer) cultivar bred at East Lansing. This particular clone has been in a continuous shoot culture for five years from seedling origin. The clone has red pigmentation of some plant parts (R-. yy). . G335-18E: A randomly sampled monogernl(nn0 individual with some red pigmentation (Rr, yy) from an East Lansing breeding population. It has also been in continuous shoot culture for five years from enbryo origin. . 80-66: Elite monogerm (mm) individual from East LanSing breeding population. Lacks red pigmentation (rr, yy). . P-13: Nultigerm (NM) from Poland seed lot segregating fOr female sterility. Probably has bolting resistant background. It has also red pigmentation (R9, yy). . 2-294: Monogerm (mm) from East Lansing breeding population. Lacks red pigmentation (rr). . 6925—0-3: Monogerm (mm), 0-type from parental line SP6926-0. Has red pigmentation (R-). . J-4: Nultigerm (M), unstable stigmoid characteristic from Japan. Has red pigment (RR). . FC701/5-116: Random multigerm (114) sample from Rhizoctonia solanum tolerant line FC701/5. Lacks red pigmentation (rr). . 82 J1-13 and 82 J2-11: Two self-fertile clones from crosses of biennial R- partial stigmoid mutants from Japan with an annual rr genetic stock from East Lansing. Both are Rr 8b. Some working definitions used in this study: Ramet: Each plant derived by asexual propagation from the same original seedling or adventitious bud and propagated by shoot culture; asexual propagule. Clone: A collective term for shoots and ramets derived asexually from the same original plant. - Isolate: A collective term fOr adventitious bud and all Shoots or ramets derived from it by axillary shoot propagation. Each may be identical to the original source genotype. Germness: Nultigerm or monogerm. Culture methods: Murashige-Skoog (1962) basal medium (Table 1) was used with 3% sucrose, 0.9% DIFCO Bacto agar, 0.1 mg/l thiamine.HCl, 0.5 mg/l pyridoxine.HCl, 0.5 mg/l nicotinic acid, and 100 mg/l myo-inositol. This MS + 0.25 6-benzyladenine (BA) was used to multiply the shoot culture and to induce and maintain the adventitious buds. This medium has the lab code M20 and was used in 20 x 100 mm Falcon Optilux disposable Petri dishes sealed with parafin film strips. Plates were kept in 2sec-1) fluorescent light at continuous low intensity (20 to 50 Em. 24 t 2°C in growth chambers. Table 1.--Murashige-Skoog medium (MS). Inorganic Salts. Composition Amount mg/l " RN03”. 1900.000 NH4N03 1650.000 CaClz°2H20 440.000 MgSO4'7H20 370.000 KH2P04 170.000 Na'EDTA-ZHZO 37.300 FeSO4-7H20 27.800 MnSO4'4H20 22.300 ZnSO4-7H20 8.600 ~ H3803 6.200 KI 0.830 Na2M004'2H20 0.250 CoClz-GHZO 0.025 CuSO4'5H20 0.025 . All salts were reagent grade unless otherwise noted. In these experiments the medium was adjusted to the pH 5.95 9 with a few drops of 1N KOH or 1N HCl before adding the agar. Three to five shoots were placed into each plate containing 35-40 m] of nedium. TO induce roots on the shoots, MN-19 medium (MS + 3.0 mg/l naphthaleneacetic acid) was used in 125 ml Erlenmeyer flasks capped by foam stoppers and aluminum foil. Rooting flasks were kept in 80-100 HEM-25-1 fluorescent light at 24 t 2°C in the growth chamber. The same MN-19 medium without the agar was used for culture of isolated roots in flasks shaken mechanically in the dark at room temperature (about 22°C). Establishment of Shoot culture: Shoot cultures can be established from seedlings, embryos, axillary or adventitious bads, or the terminal parts of floral stalks. Sugarbeet clones were already established mostly from lateral buds of floral stalks. Shoot cultures of table beet were established in the fOllowing way: In August 1982, seeds of Early Wonder were germinated in peat soil in the greenhouse. After ten days most of each cotyledon was trimmed off the seedling and each hypocotyl was cut to give a piece up to two cm long containing the cotyledonary node, small true leaves, and the primary growing point. These pieces were Surface sterilized using 15% chlorox and 0.01% sodium laurylsulfate (as a wetting agent). Surface sterilization was made twice each for twenty minutes, then rinsed 5-6 times with sterile distilled water. These shoots were then placed on M20 shoot culture medium. All procedures were performed in a laminar flow hood to give aseptic conditions. The experiments 10 were begun with 70 different Early Wonder seedlings (seventy individual table beet genotypes) in addition to the previously established sugarbeet shoot cultures. Every individual genotype with its subsequent ramets were designated saparately, EH1 Ax, EH2 Ax, ... etc. Only twenty four individual table beet genotypes from the original seventy seedlings were both uncontaminated and growing as jg_yitgg, shoots after fOur weeks. Shoots were then sudivided if axillary buds had grown out and subcultured on M20 for further multiplication with axillary shoots. The adventitious buds that gave rise to the ramets in this study were fOund on leaf petioles in the shoot cultures (Figure IL Frequently several adventitious buds were on a single leaf. Adventitious shoots were isolated from the petioles and transferred onto M20 fOr multiplication by axillary outgrowth. Each adventitious bud and subsequent shoot cultures were given a unique isolate number in addition to its axillary number (for example, EH1 PAl. EH1 PAZ; PA= petiole adventitious). In several cases adventitious buds found in shoot cultures derived from previous adventitious buds were isolated to constitute secOnd cycle, and potentially différent, genotypes. These second cycle isolates were designated with PZA‘ Throughout the study the axillary (Ax) derived ramets were used_as control plants. Both axillary and adventitious shoots that multiplied sufficiently were taken out and separated into smaller shoots of 3-5 leaflets and put onto rooting medium (MN-l9). After the development of the roots (3-6 weeks), ramets were transferred to potting mix in Jiffy peat pots and left in the greenhouse for in vivo growth (Figure 2). Some of Early wonder .axillary bud derived ramets were utilized 11 Figure 1.--Adventitious buds grown on a table beet leaf of the genotype Eli-53 on the shoot culture medium (MZO). - u Figure 2.--Potted table beet ramets propagated by shoot culture. 13 without any comparable adventitious bud derived ramets in order to better sample preexisting variation within this cultivar. Table 2 shows the number of ramets of different starting clones for both sugarbeet and table beet and their adventitious shoots for experiments 1 and 2. Uneven rooting and plant establishment in pots as well as some contamination problems account fOr the uneven number Of ramets among different isolates. Experiment 1: Potted ramets were vernalized in a cold room (4°C) for three months after reaching a height of 10-15 cms. These ramets were placed on a greenhouse room under several incandescent lights for two months to obtain flower stalks (Figure 3). The reproductive characters such as pollen shedding, seed set, and germness were recorded. After this, seed stalks of about 5 ramets of each axillary and adventitius isolate were cut Off and reversion to vegetative state was achieved by removal of the incandescent lights during the winter months. Root tip sampling procedures such as described for experinent 3 were then performed. Experiment 2: (Potted ramets were transplanted in the field for vegetative growth in June 1983 (Table 2)., The purpose of this experiment was to study some morphological characters and to Observe whether there was any easily detectable mutation or unusual growth. Leaf length and leaf width of three fully expanded leaves fOr each ramet were measured. Leaf shape, the examined character in this experiment, was determined as the ratio between leaf width to leaf length. Figure 3.--$eed stalks of some sugarbeet ramets propagated through shoot culture. 15 Table 2.--The number Of ramets obtained from each genotype from both axillary and adventitious bud derived shoot culture. Starting clone Number of ramets Number Of ramets Axillary Isolate in experiment 1 in experiment 2 Eli-1 Ax 20 8 EH-l PA 3 1 EH-3 Ax ‘EH-3 PA 1 1 Eli-9 Ax ' e EH-9 PA 2 1 EH-9 PA 3 2 EH-9 PA 5 EH-10 Ax 8 6 EH-10 PA 3 2 2 EH—10 PA 6 2 6 EH-10 PA 7 2 EH—10 PA 8 1 2 EH-10 PA 9 2 3 EH-10 PA 10 2 EH-10 PA 12 3 3 EH-10 PA 13 3 3 EH-10 PA 14 4 EH-10 PA 15 2 EH-10 PA 17 8 EH-11 Ax 14 EH-ll PA 2 2 EH-ll PA 3 3 16 Table 2.--Continued. Starting cflone Number of ramets Number of ramets Axillary Isolate in experiment 1 in experiment 2 EH-20 Ax 25 7 EH-20 PA 5 4 EH-20 PA 6 2 EH-20 PA 7 3 1 EH-20 PA 9 2 EH-ZO PA 10 EH-20 PA 11 EH-20 PA 17 EH-20 PZA 2-6 1 EH-ZO PZA 7-1 10 EH-20 PZA 7-2 3 EH-20 PZA 7-4 4 EH-21 EH-25 3 EH-ZS PA 1 3 EH-25 PA 4 1 EH-ZS PA 12 1 EH-ZS PA 13 1 EH-37 EH-39 2 EH-39 PA 2 1 EH-39 PA 3 1 EH-39 PA 22 4 17 Table 2.--Conti-ued. Starting clone Number Of ramets Number Of ramets Axillary Isolate in experiment 1 in experiment 2 Ew-4O Ax 2 Eli-40 PA 4 4 Ev-4O PA 5 3 EH-40 PA 11 2 EH-40 ‘ PA 12 2 EH-44 Ax 15 EH-44 PA 1 1 Ew-44 PA 2 1 EH-44 PA 3 1 EH-44 PA 4 1 EH-53 Ax 8 EH-55 Ax 6 1 EH-55 PA 7 2 Ew-ss PA 8 63 EH-55 PA 9 1 Ew-59 Ax 16 3 Eli-68 Ax 2 EH-68 PA 2 Eli-70 Ax s 1 Ew-TO PA 3 EH-70 PA 7 Table 2.--Cont inued. Starting clone Number of ramets Number of ramets Axillary Isolate in experiment 1 in experiment 2 EL 36-18 Ax 13 30 EL 36-18 PA 2 2 6 EL 36-18 PA- 3 19 13 EL 36-18 PA 4 1 6 EL 36-18 PA 5 9 15 EL 36-18 PA 6 6 EL 36-18 PA 18 5 16 EL 36-18 PA 23 6 P-13 PA 2 12 11 P-13 PA 3 5 P-13 PA 4 22 6 P-13 PA 5 25 4 80-66 Ax 4 80-66 Ax PA 3 3 3 80-66 PA 4 3 8 80-66 PA 5 2 5 80-66 PA 7 2 80-66 PA 8 3 2-294 Ax 12 9 2-294 PA 1 17 18 FC701/5-116Axr 5 11 FC701/5-116 PA 1 9 6 FC701/5-116 PA 2 3 11 FC701/5-116 PZA 1-1 5 19 Table 2.--Continued. Starting clone Number of ramets Number of ramets Axillary Isolate in experiment 1 in experiment 2 6926-0-3 Ax 6926-0-3 PA 1 3 6926-0-3 PA 5 2 6335-18E Ax ' 21 G335-18E PA 1 2 G335-18E PA 2 3 G335-18E PA 3 1 G335-18E PA 4 3 Total number of ramets 356 370 20 Three rants with conspicously narrow leaf shape (Figure 4) were transferred along with their normal counterparts to the greenhouse to be examined further. Exarignt 3: The examination of root tip chromosoee mater and guard cell length of a samling of the ramets from both axillary and adventitious shoots.was the object of this experiIent. Root tip sales were collected in eid-mrning (between 8:00 and 9:00 AM during saver and between 9:00 and 10:00 an during winter). About 10-15 fresh. robust root tips from each ranet were placed in vials containing 0.01% cyclohexieide for 1.5 hours in the refrigerator in order to contract the chronosomes. Following this, the solution in each vial was replaced with 3:1 absolute alcohol to glacial acetic acid and the vials left for24 hours in the refrigerator to fix the chromsones in their stages. Both 1N and 3N HCl were used for ' hydrolysing the roots. The solution was used for 10 Innate: at 60°C in a water bath. 3N HCl gave better results. Following hydrolysis, roots were kept in Feulgen stain at least two hours. The phase contrast microscope with oil lens was used'for chronosome counting, which was achieved at the pro-netaphase stage ( Figure 5 aw). . These procedures for chromosone counting were also used for the five ramets of each isolate noted in experiment 1. Data for all chromosome counting were pooled. Because of the appearence of some tetraploid cells in root tips, microscopic examination of guard cell length was conducted to indicate ploidy of the leaves. A simple ligit microscope with 40x lens was used 21 '1’,". " .‘ .j v fin-“I, .F'": 'h, I. ",: ._'.,: ' , 1 . a}... I “ . 3‘;'1‘.-:.,;'(.§€‘§’%‘\5 ' : . H r‘ 'i' _’:~_.5'i’IA’:e‘3. ’93.. ‘:x_*«m...‘.-5' . :9 Figure 4.--A narrow leaf ranet of the genotype Ell-1 Ax as compared with its neighbor normal leaf of the same genotype. 22 (b) Figure 5.--(a) A normal diploid cell of a sugarbeet genotype (FC701/5-116 Ax) propagated by _ shoot culture. (b) A tetraploid cell of the same genotype (some chromosomes are not clear because of the focusing. 23 and its scale units were calibrated. The procedure was executed by peeling the lower epidermis, spreading it on a slide with a drop of water, covering with a glass cover slip, and measuring the longtudinal diameter of the guard cell. The test was always conducted in the afternoon. Thirty readings of five fully expanded leaves for each ramet so tested were taken.. For comparison with guard cell lengths of known diploid. triploid, and tetraploid sugarbeet cultivars. the range and the average of a total 100 guard cells of 10 plants of each ploidy were calculated. It was found that: for Zn: guard cell length ranged from 7-10 units, for 3n: ' " " A " . " 10-12 units, and for 4n: guard cell length ranged from 12-15 units. The same 2n, 3n, and 4n sugarbeet plants were used also in the cytological studies as mentioned above to determine any mixture of ploidy levels in root tips, as was noticed fOr certain ramets derived from axillary and adventitious buds. Experiment 4: In this experiment, investigation was concentrated on the narrow leaf aspect observed in three ramets mentioned in experiment 2. The three ramets were taken from the field together with their normal counterparts which did not have narrow leaves. Three kinds of analysis were done: a. Cytological examination of root tips. b. Flowering and fertility studies. c. Reestablishing shoot cultures from these ramets to test the persistence of this narrow leaf character after a propagation cycle. _ 24 a. The cytological studies: The three abnormal ramets were from the following clones; Eu-l Ax, Eli-10 PA 17, and Eli-20 92A 7-4. The hunter 1 was added to the designation of each normal leaf ramet and the number 2 to the designation of each narrow leaf ramet. The six ramets were potted in fresh soil to induce new roots, which were available in ten days. Samples of about 10 root tips were taken and prepared as before. b. The flowering and fertility studies: The six ramets were put in a cold room (4°C) for three months for vernalization. The plants were then moved into the greenhouse to complete flower induction. Plants grew and flowered normally. All of them shed polled. Because of the great infection of aphids no seed set could be obtained from the six plants. Lack of seed set even from the three normal leaf plants eliminated the possibility of detecting female infertility. c. Reestablishing shoot cultures: The last step which was done to these plants was the test of the narrow leaf character persistence for the same clones throughout the other propagation cycle. This was achieved by reestablishing shoot cultures once again, this time from the terminal parts of flower stalks. These parts were surface sterilized as usual and put first on M20 medium either in petri dishes, or in vials for several days prior to transfer to petri dishes. Because of a contamfination problem with these parts, they were subcultured several times fOllowing repeated disinfection attempts before being rooted and potted. Ramets which grew well in the M20 were cut and transferred to MN19 fOr rooting. 25 After that, rooted plants were transferred to the pots containing fresh' soil mix to obtain intact plants. Leaf length and leaf width measurements were recorded. I Experiment 5: The purpose of this experiment was to compare the segregation behavior of the axillary and adventitious derived ramets in their 51 generation. and to detect any recessive-like mutations in the S1 - progeny. Two different sugarbeet genotypes that gave considerable numbers of adventitious buds were used; 82 02-11 and 82 J1-13. initially available as axillary shoot cultures. 82 J2 11 is self-fertile, annual, red hypocotyl and monogerm. It is known to be heterozygous at both 8 locus (fer annualness) and R locus (for red hypocotyl and partially red petioles and stems). 82 J1-13 is self-fertile. annual. red hypocotyl and multigerm. It is also known to be heterozygous in both 8 and R loci. From existing shoot cultures, the maximum nunb‘er of adventitious buds was isolated and transferred to shoot culture medium (M20). A total of 50 adventitious buds was obtained from the two genotypes. After both axillary and adventitious buds had multiplied in shoot culture, at least nine shoots of each adventitious or axillary clone were transferred to MN19 medium for rooting. The plantlets of each clone then were transferred to 3” peat pots after developing roots. Ramets were later transferred to 6" plastic pots and kept in the greenhouse for flowering. Being heterozygous fer the dominant annualism allele. these ramets flowered after exposure to only incandescent bulb light. Just prior to first flower opening, bags 26 were placed over the inflorescences to insure self pollination. Selfing was achieved by the fbllowing steps: 1. Just before the first flower Opened the plant was sprayed by strong jets of water to clean the surface from foreign pollen and any aphids. 2. The plants were bagged for 3 weeks. Every bag was shaken every 2 days to distribute the pollen inside. 3. After three weeks,the bag was taken off and the plant was left. after cutting and eliminating the floral stalk tips with unfertilized flowers. to mature the seeds. 4. The seeds were harvested after they ripened (i.e. dried to a brown color) and the seeds of different ramets of the same isolate were bulked together. Seeds were then sown in a greenhouse room and transplanted into beds. After this, the plants were grown in the sumner greenhouse under incandescent lighting. Segregation data were recorded for the following characters : 1. The hypocotyl color (red or green). 2. Annualism or biennialism. 3. Germness (multigerm or monogerm). This experiment had two different objecttVes: First, to detect any change in segregation pattern which might have resulted from a mutation- like event at the B, M, or R loci. This would be eSpecially true if the mutation arose as a part of chimera and was not expressed in the ramets of the adventitious clones. Second, to detect by segregation any recessive mutations which would not be expected to be seen in the /. ramets themselves of the adventitious isolates. 27 Experiment 6: when it was found from comparison of guard cell lengths of Zn, 3n, and 4n plants of seedling origin that ramets of axillary and adventitious bud origin did not differ in ploidy level, the question was raised whether there is any effect of the medium components on producing tetraploid root cells. Therefore, an experiment was conducted to test the effect of various concentrations of benzyladenine (BA) in the shoot culture medium on the presence of tetraploid cells in the roots of subsequently rooted and potted diploid plants. Two different genotypes were used: 1. FC701/5-116 which showed some completely tetraploid root tips from both axillary and adventitious ramets; 2. 80-66 which did not show any tetraploid cells. The axillary shoots of these two genotypes were cultured on the Murashige-Skoog shoot culture medium with different concentrations of benzyladenine as follows: 0.10 mg/l, 0.25 mg/l, 0.50 mg/l, 0.75‘mg/l, and 1.0 mg/l. After the two genotypes were cultured on the prepared media long enough to have some shoots that can be separated, shoots were transferred to rooting medium (MN19). At the time shoots had roots, they were potted in soil in peat pots. After about three to four weeks, root tip samples were taken from 5 ramets for oath BA level 28 for microsc0pic examination and previously mentioned fixing and staining procedures were followed. Five plants from each treatment for each genotype were used. RESULTS Table beet and sugarbeet materials were treated under the same conditions. It was found in general that the table beet, at least the cultivar Early wonder, is less adapted for shoot culture than sugarbeet: shoots are slower in growth rate, proportion of survival shoots from all cultured buds or shoots is less in table beet than in sugarbeet, and they seem to be less vigorous. However, in general it produces more adventitious buds which are the main object in this research. Although no data was collected on survival after transplanting, rooted table beet shoots were notably weaker for this. Experiment 1: ~Table 3 shows no difference has been detected between axillary derived ranets and their adventitious derived counterparts for pollen shed, seed set, or germness. Some of the sugarbeet ramets of the starting clones; P-13 PA 4, P-13 PA 5, EL 36-18 Ax, EL 36-18 PA 3, and EL 36-18 PA 5 did not flower with the same proportion as the other clones or as table beet clones, although all ramets underwent vernalization at the same time. P-13 is thought to have a bolting resistance selection background from Poland. Pollen shedding and seed setting would eliminate the possibility of male or female sterility. The germness readings indicate that there has been no change between the axillary shoots and their adventitious ones. 29 3O Iable 3.--Number of ramets fer the characters: flowering, pollen shedding, and seed setting and occurrence of mono- or multigerm in axillary and adventitious ramets. Starting clone Total Nunber of ramets nunber . - Germness Axillary Isolate of Flowering Pollen seed set * ranets shedding EH-l Ax 20 15 15 15 multi EH-3 Ax 2 2 2 2 " EH-3 PA 1 1 1 1 1 “ EH-9 Ax 6 5 5 5 " EH-9 PA 3 2 2 2 2 " EH-9 PA 5 1 1 " EN-lo Ax 8 8 8 8 " EH-10 PA 3 2 2 2 2 " Eli-10 PA 6 2 2 2 2 " EH-10 PA 8 1 1 1 1 " Eli-10 PA 9 2 2 2 2 " EH-IO PA 12 3 3 3 3 " EH-10 PA 13 3 3 3 3 " EH-ll Ax 14 14 14 14 " EH-ll PA 2 2 2 2 2 " EH-ZO Ax 25 21 21 21 " EH-ZO PA 5 4 4 4 4 " EH-ZO PA 6 2 1 1 1 " EH-ZO PA 7 3 3 3 3 " EW-ZO PA 10 5 5 5 5 " 3.1 Table 3.--Continued. Starting clone Total Number of ramets number Germness Axillary Isolate of Flowering Pollen seed set‘* ramets shedding EH-ZO PA 11 2 2 2 2 multi EH-ZI Ax 5 5 S 5 “ “‘25 AX 4 4 4 4 u EH—37 Ax 3 1 1 1 " Eli- 39 Ax 1 1 1 1 " EH-40 Ax 2 - - - - EN-44 Ax 15 9 9 9 " EH-44 PA 1 1 1 1 1 " EH-44 PA 2 1 1 1 1 ' EH-44 PA 3 1 1 1 1 “ EH-44 PA 4 I 1 I 1 " EN-53 Ax 8 . 8 8 8 " EH-55 Ax 6 5 5 5 " EH-59 Ax 16 13 13 13 " EH-70 Ax 6 6 6 6 " EL 36-18 Ax 13 4 4 4 " Table 3.--Gontinued. Starting clone Total Number of ramets nunber Germness Axillary Isolate of Flowering Pollen Seed set * ramets shedding EL 36-18 PA 2 2 1 1 1 mono EL 36-18 PA 3 19 4 4 4 " EL 36-18 PA 4 1 - - - - EL 36-18 PA 5 9 1 1 1 " EL 36-18 PA 18 5 5 5 " P-13 PA 2 12 11 11 11 multi P-13 PA 3 5 4 4 4 “ P-13 PA 4 22 13 13 13 " P-13 PA 5 25 5 5 5 “ 80-66 Ax 4 4 4 4 mono 80-66 PA 3 3 3 3 3 “ 80-66 PA 4 3 3 3 3 " 80-66 PA 5 2 2 2 2 " 2-294 Ax 12 12 12 12 " 2-294 PA 1 17 17 17 17 " FC701/5-116Ak 5 5 5 ‘ 5 multi FC701/5-116 PA 1 9 9 9 9 " FC701/5-116 PA 2 3 3 3 3 " FC701/5-116 PZAI-I 5 5 5 5 " * In all cases seed set was more than 50%. 33 Experiment 2,;the field study: Table 4 summarizes the leaf shape as indicated by the width/ length ratio. It appears that leaf shape in table beets is narrower than in sugarbeets. However, within the counterpart axillary and adventitious pairs, there are no significant differences (Table 5). Data of EH-l Ax, EN—IO PA 17, and EH-ZO PZA 7-4 in table 4 did not include the narrow leaf ramets of 0.225, 0.232, and 0.241 means, respectively. When the mean and the standard deviation of these three starting clones were calculated considering the narrow leaf ramets, the values were; i 8 0.355 and 6 = 0.183, 32 8 0.417 and 6 = 0.83, and SE = 0.533 and a =-0.195 for these three clones, respectively. Testing the - comparison between the adventitious bud derived ramets and their counterparts axillary derived ramets for this character has been demonstrated by "t" test for each isolate. Table 6 shows that there were few differences between the adventitious derived ramets and axillary derived ramets. Only two values which represent less than 0.06 of all values exhibited significance. These two values can be' statistically significant but biologically insignificant. In Table 7 the significance of the three narrow leaf ramets is shown as compared with their normal leaf sisters. -Significance was demonstrated at 95% confidence. The study of this character completes the greenhouse study and indicates no difference between the axillary derived ramets and their adventitious derived ramets. The three abnormal ramets were investigated further to detect whether they have any genetic difference. They were all diploid and produced normal quantities of pollen. Insect infestation prevented seed set. 34 Table 4.--0etermination of leaf shape through width/length ratio of three leaves per ramet for both axillary and adventitious bud derived ramets in the field. Values are the mean of all ramets of each clone*. NL means narrow leaf ramet. Starting clone TOtal number Mean ratio of ramets of clone 6 Axillary Isolate EH-I Ax 7 0.373 0.056 EH-I Ax ML 1 0.225 EN-l PA 3 0.461 EN—9 PA 2 1 0.458 EH-lo Ax 6 0.467 0.065 EN-IO PA 3 2 0.501 0.067 EN-IO PA 6 6 0.409 0.100 EH-IO PA 7 2 0.433 0.049 EH—IO PA 8 2 0.462 0.001 EH-IO PA 9 3 0.398 0.035 EH—IO PA 10 2 0.433 0.019 EN-IO PA 12 3 0.326 0.079 EN-IO PA 13 3 0.484 0.036 EH-IO PA 14 4 0.395 0.093 EH-lO PA 15 2 0.396 0.020 EH-IO PA 17 7 0.442 0.039 EN-IO PA 17NL 1 0.232 EH-II 1 0.538 EH-Il PA 2 3 0.586 0.035 Ew-II PA 3 3 0.569 0.109 EH-ZO Ax 7 0.618 0.057 35 Table 4.--Continued. Starting clone Total number Mean ratio of ramets of clone 6 Axillary Isolate EH-ZO PA 7 1 0.647 EH-20 PA 9 2 0.629 0.053 EH-20 PA 17 3 0.531 0.051 EH—20 P2A2-6 1 0.616 EN-20 P2A7-1 10 0.533 0.075 EH-20 P2A7-2 3 0.507 0.061 Ew-20 P2A7-4 3 0.631 0.019 EH-20 P2A7-4NL 1 0.241 EH-ZS 3 0.590 0.067 EW-ZS PA I 3 0.699 0.121 EH-ZS PA 4 1 0.441 EN-25 PA 12 1 0.717 EH-ZS PA 13 1 0.564 EN-39 2 0.704 0.202 EN-39 PA 2 1 0.609 EH-39 PA 3 1 0.695 EH-39 PA 22 4 0.639 0.081 Ew-40 PA 4. 4 0.508 0.121 EH-40 PA 5 3 0.756 0.178 EN-40 PA 11 2 0.826 0.188 EH-40 PA 12 2 0.461 0.055 EH-SS 2 0.458 0.052 'EW-SS PA 7 2 0.469 0.033 EN-55 PA 8 6 0.511 0.106 Table 4.--Continued. Starting clone Total number Mean ratio of ramets of clone 6 Axillary Isolate EH-55 PA 9 1 0.444 EH-59 Ax 3 0.492 0.033 EH-68 Ax 2 0.656 0.044 EH-68 PA 2 1 0.494 Eli-70 Ax 0.574 EN-70 PA 3 0.710 EN-70 PA 7 0.506 EL 36-18 Ax 30 0.845 0.073 EL 36-18 PA 2 6 0.902 0.034 EL 36-18 PA 3 13 0.904 0.059 EL 36-18 PA 4 6 0.898 0.037 EL 36-18 PA 5 15 0.888 0.053 EL 36-18 PA 6 6 0.805 0.051 EL 36-18 PA 18 16 0.837 0.066 EL 36-18 PA 23 6 0.864 0.029 9-13 PA 2 11 0.640'” 0.046 P-l3 PA 6 0.704 0.058 P-13 PA 5 4 0.631 0.091 80-66 PA 3 3 0.678 0.031 80-66 PA 4 8 0.685 0.052 80-66 PA 5 5 0.659 0.043 80-66 PA 7 2 0.693 0.079 37 Table 4.--Continued. Starting clone Total number Mean ratio of ramets of clone a Axillary Isolate 80-66 PA 8 3 0.676 0.044 2-294 Ax 9 0.746 0.185 2-294 PA 1 18 0.852 0.081 FC701/5-116Ax 11 0.672 0.032 FC701/5-116 PA 6 0.698 0.040 FC701/5-116 PA 11 0.703 0.075 6926-0-3 Ax 2 0.970 0.035 6926-0-3 PA 1 3 0.979 0.019 6926-0-3 PA 5 2 1.061 0.033 6335-18E Ax 21 0.693 0.021 G335-18E PA 1 2 0.713 0.032 G335-18E PA 2 3 0.736 0.091 6335—18E PA 3 1 0.685 G335-18E PA 4 3 0.679 0.074 * The mean ratios of the clones marked above do not include the narrow leaf ramets . " - 33 Table 5.--Test of significance for the deviation of the mean for the comparison of each axillary and its adventitious derived ramets as compared by their grand means for leaf shape character. Starting clone Mean ratio Deviation from of clone a ** the mean Axillary Isolate EH-I Ax *** 0.355 0.075 0.053 EH-l PA 3 0.461 0.053 Grand mean 0.408 EN-IO Ax 0.467 0.048 0.040 EH-IO PA 3 0.501 0.074 EN-IO PA 6 0.409 0.018 EH-IO PA 7 0.433 0.006 EN—10 PA 8 0.462 0.035 EH-IO PA 9 0.398 0.029 EN-IO PA 10 0.433 0.006 EH-IO PA 12 0.326 0.101 EH-IO PA 13 0.484 0.057 EH-IO PA 14 0.395 0.032 EH-IO PA 15 0.396 0.031 EH-IO PA 17*** 0.417 0.010 Grand mean 0.427 EH-II Ax 0.538 0.024 0.026 EH-II PA 2 0.586 0.022 EH-II PA 3 0.569 0.005 Grand mean 0.564 EH-20 Ax 0.618 0.057 0.041 39 Table 5.--Continued. Starting clone Mean ratio Deviation from of clone 6 ** the mean Axillary Isolate' EN-20 PA 7 0.647 0.070 EN-ZD PA 9 0.629 0.052 EH-ZD PA 17 0.531 0.046 EH-ZD P2A2-6 0.616 0.039 EH-20 P2A7- 0.533 0.044 EH-ZD P2A7-2 0.507 0.070 EH-ZD P2A7-4*** 0.533 0.044 Grand mean 0.577 EH-25 Ax 0.590 0.112 0.012 EN-25 PA 1 0.699 0.097 EH-25 PA 4 0.441 0.161 EW-ZS PA 12 0.717 0.115 EN-25 PA 13 0.564 0.038 Grand mean 0.602 Eli-39 Ax 0.704 0.045 0.042 ‘ EH-39 PA 2 0.609 0.053 EH-39 PA 3 0.695 0.033 EH-39 PA 22 0.639 0.023 Grand mean 0.662 EN-40 PA 4 0.508 0.180 0.130 EH-40 PA 5 0.756 0.118 EH—40 PA 11 0.826 0.188 EH-40 PA 12 0.461 0.177 Grand mean 0.638 40 Table 5.--00ntinued. Starting clone Mean ratio Deviation from of clone 6 ** the mean Axillary Isolate EH-55 Ax 0.458 0.029 0.013 EH-SS PA 7 0.469 0.002 EH-SS PA 8 0.511 0.040 EH-55 PA 9 0.444 0.027 Grand mean 0.471 EH-68 Ax 0.656 0.115 0.081 EH-68 PA 2 0.494 0.081 Grand mean 0.575 EN-70 Ax 0.845 0.036 0.023 EH-70 PA 3 0.710 0.113 EH-70 PA 7 0.506 0.091 Grand mean 0.597 EL 36-18 Ax 0.574 0.104 0.023 EL 36-18 PA 2 0.902 0.034 EL 36—18 PA 3 0.904 0.036 EL 36-18 PA 4 0.898 0.030 EL 36-18 PA 5 0.888 0.020 EL 36-18 PA 6 0.805 0.063 EL 36-18 PA 18 0.837 0.031 EL 36-18 PA 23 0.864 0.004 Grand mean 0.868 P-13 PA 2 0.640 0.040 0.018 41 Table 5.--Continued. Starting clone Mean ratio Deviation from of clone 6 ** the mean Axillary Isolate P-13 PA 4 0. 704 0. 046 P-13 PA 5 0.631 0.027 Grand mean 0.658 80—66 PA 3 0.678 0.013 0.000 80-66 PA 4 0.685 0.007 80-66 PA 5 0.659 0.019 80-66 PA 7 0.693 0.015 80-66 PA 8 0.676 0.002 Grand mean 0.678 2-294 Ax 0.746 0.075 0.053 2-294 PA 1 0.852 0.053 Grand mean 0.799 .672 0.017 0.019 FC701/S-116Ax 0 FC701/5-116 PA 1 0.698 0.007 FC701/5-116 PA 0.703. 0.012 Grand mean 0.691 6926-0-3 Ax 0.970 0.050 0.030 6926-0-3 PA 1 0.979 0.021 6926-0-3 PA 0.061 0.061 Grand mean 1.003 G335-18E Ax 0.693 0.023 0.008 42 Table 5.--Continued. Starting clone Mean ratio Deviation from of clone 6 ** the mean Axillary Isolate G335-18E PA 1 0.713 0.012 G335-18E PA 2 0.736 0.035 G335-18E PA 3 0.685 0.016 G335-18E PA 4 0.679 0.022 Grand mean 0.701 * Significance was demonstrated as 99% confidence, i.e. deviation is compared with 36. ** The standard deviation is calculated for each group of axillary and adventitious derived ramets. ***The mean ratios of the clones marked above include the narrow leaf ramets. ' 43 Table 6.--”t” test for the comparison between each adventitious isolate with its axillary counterpart for some table beet and sugarbeet starting clones. Starting clone Total number Mean ratio "t" test of ramets of clone Axillary Isolate EH-IO Ax 6 0.467 EN-lO PA 3 2 0.501 0.654 EN-IO PA 6 6 0.409 1.115 EN-10 PA 7 2 0.433 0.654 EN-10 PA 8 2 0.462 0.357 EH-IO PA 9 3 0.398 0.566 EN-IO PA 10 2 0.433 2.429 EN-IO PA 12 3 0.326 2.820 EN-IO PA 13 3 0.484 0.463 EH-lO PA 14 4 0.395 1.330 EH-IO PA 15 2 0.396 5.071** EN-10 PA 17 7 0.442 0.806 EH-20 Ax 7 0.618 EN-ZO PA 9 2 0.629 0.216 EN-ZO PA 17 2 0.531 2.023 EW-ZO P2A7-1 10 0.533 2.429 EN-ZO PéA7-2 3' 0.507 2.643 EH-20 P2A7-4 3 0.631 0.342 EN-ZS Ax 3 0.590 EN-25 PA 1 3 0.699 1.124 EH-39 Ax 2 0.704 EH-39 PA 22 4 0.639 0.591 Table 6.--Continued. Starting clone Total number Mean ratio "t" test of ramets of clone Axillary Isolate EN-SS Ax 2 0.458 EN-SS PA 7 2 0.469 0.175 EH-SS PA 8 6 0.511 0.570 EL 36-18 Ax 30 0.846 EL 36-18 PA 2 6 0.902 1.750 EL 36-18 PA 3 13 0.904 2.522** EL 36-18 PA 4 6 0.898 1.625 EL 36-18 PA 5 15 0.888 1.909 EL 36-18 PA 6 6 0.805 1.281 EL 36-18 PA 18 16 0.837 0.409 EL 36-18 PA 23 6 0.864 0.563 2-294 Ax 9 0.746 2-294 PA 1 18 0.852 1.927 FC701/5-116Ax 11 0.672 FC701/5-116 PA 1 6 0.698 1.130 FC701/5-116 PA 2 11 0.703 1.148 6926-0-3 Ax 2 0.970 6926-0-3 PA 1 3 0.979 0.310 6926-0-3 PA 5 2 1.061 2.022 6335-18E Ax 21 0.693 G335-18E PA 1 0.713 1.176 6335-18E PA 2 0.736 2.263 G335-18E PA 0.679 0.737 ** Values are significantly different at 99% confidence. 45 Table 7.--Determinati0n of the significance of the narrow leaf ramets as compared with their normal leaf ones. Starting clone Mean ratio of Deviation from each ramet 6 the mean Axillary Isolate Eli-1 Ax 0.288 0.056 0.085 0.356 0.017 0.318 0.055 0.225 0.148 0.411 0.038 0.384 0.011 0.431 0.048 0.428 0.055 Grand mean 0.355 EH-ID PA 17 0.433 0.083 0.017 0.519 0.103 0.232 ” 0.184 0.437 0.021 0.430 0.014 0.457 0.041 0.427 0.011 0.395 0.021 Grand mean 0.416 Eli-20 P2A7-4 0.509 0.092 0.074 0.639 0.104 0.248 0.287 0.644 0.109 Grand mean 0.535 * Values are significantly different from the grand mean confidence,i.e. deviation is compared with 26 at 95% Experiment 3: Results (Table 8) for the microscopic examination have been recorded over the course of a year. They were pooled from three groups of ramets available at different times. All clones were expected to be diploid, based on the background from which they were chosen. Chromosome counting indicated that ramets derived from 24 of 35 clones were indeed diploid. In the eleven other clones, however, some of the root tips were entirely diploid and others were entirely tetraploid although both types of root tips were taken from the same ramet. Thus the same examined root tip showed either diploid or tetraploid cells. On the other hand, no root tip samples of the same ramet gave only tetraploid. That would suggest that the ploidy duplication might occur during the initiation of the secondary roots, possibly during root induction on the shoots on MN-19 medium. The frequency of tetraploid root tips was fairly high (between 12.82 and 42.00%). when a control group of 50 root tips from sugarbeet seed derived plants (5 plants of Zn, 4n, and 3h; parental lines and a hybrid respectively from Nickerson Seeds) were examined microscopically, no tetraploid cells were observed in 2n plants. This result may suggest the effect of the medium on inducing tetraploid cells. This possibility was studied by testing the effect of BA in the shoot culture media on the frequency of tetraploid root tips. The existence of tetraploid root tips raised the queStion whether the tap of the plant was chimeric or mixaploid. This question was approached by measuring the guard cell lengths.‘ Table 9 shows that the average of these lengths ranges from 7.75 to 9.47 units which is 47 within the diploid controls (from Nickerson Seeds) examined. These results did not indicate any occurrence of tetraploid cells in the shoot system. 48 me.- «ma hmvm owe wevmm me e m (a mmuxu m~.¢m H- “Nye" mew “mVNN “a m x<.mm.zu mam Hm Hm a x< mm-3m m~.mH mHN have mNe hmvmm 24 m AH wcmu use maowupucm>cm new agmppwxa we may» uooc m:_:vaacou-c_opaacuou mo omuucmucma any can mppmo cwopamcuou ucm evopavu we coneaz--.m m—amw 49 NmN om om m N (a a--m\NoNue oo.om mmN “meme nae “mean om m a «a aHH-m\~ONuu oo.Ne mNm hevgN mmm hmvaN om m x 33 11 1.090 82 02-11 PA 6 29 8 27.75 9. 25 0.224 82 02-11 PA 7 34 12 34.5 11.5 0.089 82 02-11 PA 8 30 12 31.5 10.5 0.280 82 02-11 PA 9 30 14 33 11 1.090 82 02-11 PA 10 33 12 33.75 11.25 0.066 82 02-11 PA 11 33 14 35.25 11.75 0.573 82 02-11 PA 12 34 9 32.25 10.75 0.378 82 02-11 PA 13 34 11 33.75 11.25 0.006 82 02-11 PA 14 30 12 31.5 10.5 0.285 82 02-11 PA 15 27 11 28.5 , 9.5 0.333 Total of ramets for 491 190 510.75 170.25 3.054 genotype 1 82 01-13 Ax 35 14 36.75 12.25 0.164 82 01-13 PA 1 33 15 36 12 1.000 82 01—13 PA 3 37 13 37.5 12.5 0.027 82 01-13 PA 4 37 10 35.25 11.75 0.348 82 01-13 PA 5 31 19 37.5 12.5 4.507 * Table 11.--Continued. 61 Starting clone Number of ramets x2 Axillary Isolate Observed Expected Red Green Red Green 82 01-13 PA 6 40 9 ' 36.75 12.25 1.149 82 01-13 PA 8 31 18 36.75 12.25 3.598 82 01-13 PA 9 36 12 36 12 0.000 82 01-13 PA 10 37 13 37.5 12.5 0.027 82 01-13 PA 11 38 11 36.75 12.25 0.171 82 01-13 PA 12 38 10 36 12 0.444 82 01-13 PA 13 34 16 37.5 12.5 1.307 82 01-13 PA 14 37 12 36.75 12.25 0.007 82 01-13 PA 15 32 15 35.25 11.75 1.198 82 01-13 PA 16 35 10 33.75 11.25 0.185 82 01-13 PA 17 39 9 36 12 1.000 82 01-13 PA 18 34 10 33 11 0.121 82 01-13 PA 19 31 15 34.5 11.5 0.420 82 01-13 PA 20 37 10 35.25 11.75 0.348 82 01-13 PA 21 38 9 35.25 11.75 0.869 82 01-13 PA 22 37 13 37.5 12.5 0.027 82 01-13 PA 23 38 16 40.5 13.5 0.617 82 01-13 PA 24 46 7 39.75 13.25 3.931 82 01-13 PA 25 35 ' 16 38.25 12.75 1.104 82 01-13 PA 26 37 15 39 13 0.411 82 01-13 PA 27 40 12 39 13 0.103 82 01-13 PA 28 38 12 37.5 12.5 0.027 82 01-13 PA 29 34 10 33 11 1.063 82 01-13 PA 30 36 14 37.5 12.5 0.240 82 01-13 PA 31 37 14 38.25 12.75 0.164 82 01-13 PA 32 37 13 37.5 12.5 0.027 82 01-13 PA 33 40 9 36.75 12.25 1.149 Table 11.--Continued. 62 Starting clone Nunber of ramets 2 Axillary Isolate Observed ' Expected Red Green Red Green 82 01-13 PA 34‘ 37 12 36.75 12.25 0.007 82 01-13 PA 35 43 11 40.5 13.5 0.617 82 01-13 PA 36 42 13 41.25 13.75 0.055 82 01-13 PA 37 41 12 39.75 13.25 0.157 Total of ramets of 1337 449 1339.5 446.5 0.019 genotype II Total number of ramets 1828 639 1850.25 616.75 1.071 * Ratio is significantly different at P= 0.05, where x" at df = 1 and P 2 0.05 is 3.84. 63 e oo<.m mN.HH mn.mm NH 2N mH <2 HH-Na Nm 2 mHm.o mm.oH mN.Nm NH 2N NH <2 HH-Na NN 2 22H.c mN.HH mN.mm mH em HH <2 HH-Na NN = oo<.HH m.oH m.Hm oN NN oH <2 HH-Na NN e 2omno muonomH xgmHHHx< 22255222 Nx 222522 mo 222522 mcoHu mcHu2mum .222522 um>22mu 2:2 msoHpHacm>uo 2.622 252 2mgauocmm 222222222 23» 22 2222222 H2 2:2 22» Nx an 222222 22 2222H22552 mo 22H222 :oHuaam2uom we coppav>mu 2:2 222 22225222 No 22.22a22mm2 2;»-.NH mHnep 64 2 2 222.2 NH 22 HN 2N NH <2 2H-H2 N2 2 .2 222.2 2.HH 2.22 2N 2N HH <2 2H-H2 N2 2 2 HHH.HH NH 22 NN 2N 2H <2 2H-H2 N2 2 2 2N2.2H 22.HH 2N.22 2N 2N 2 <2 2H-H2 N2 2 22H.2 2N.HH 22.22 2H 22 2 <2 2H-H2 N2 2 2 222.2 2N.NH 22.22 HN 2N 2 <2 2H-H2 N2 2 22222.HH 2.NH 2.22 2N 2N 2 <2 2H-H2 N2. 2 2NH.H 2.HH 2.22 2H H2 2 <2 2HuH2 N2 2 222.N NH 22 2H H2 2 <2 2H-H2 N2 2 2 2HH.2 NH 22 2N 2N H <2 2H-H2 N2 22.25 2 222.2 NH 22 2H 2N x< 2H-H2 N2 22222222 2 222.2N 22H 222 2NN 222 222 222522 22 H2222 2 2N2.N 2N.2 22.2N 2H 2N 2H <2 HH-N2 N2 2 2 222.2 2.2H 2.H2 2H 2N 2H <2 HH-N2 N2 H2222222 H2222< H2222222 H2222< 222222x2 22>22222 222222H 222HH2x< 22225222 Nx 222522 22 222522 22222 22222222 .222222222--.~H 22222 65 2 222.H NH 22 2H 22 2N <2 2H-H2 N2 2 H22.H 2H 22 2H 22 2N <2 2H-H2 N2 2 222.H 22.NH 2N.22 22 e2 2N <2 2H-H2 N2 2 222.2 2.NH 2.22 2H 22 2N <2 2H-H2 N2 2 2N2.2 2N.2H 22.22 2H 22 «N <2 2H2H2 N2 2 2N2.2 2H 22 2H 22 2N <2 2HuH2 N2 2 2N2.2 2N.NH 22.22 2H 22 NN <2 2H-H2 N2. 2 H2N.2 2.HH 2.22 2H 22 HN <2 2H-H2 N2 2 222.H 2N.HH 22.22 2H 22 2N <2 2H-H2 N2 2 2 222.2 22.2H 2N.N2 2H 2N 2H <2 2H-H2 N2 2 2 2H2.2 22.2H 2N.N2 2H 2N 2H <2 2H-H2 N2 2 2 222.2 22.HH 2N.22 2H 2N 2H <2 2HuH2 N2 2 22N.N 22.2H 2N.N2 2H 2N 2H <2 2H-H2 N2 2 222.2 22.HH 2N.22 2H 2N 2H <2 2H-H2 N2 2 2 2N2.2 22.HH 2N.22 2N 2N 2H <2 2H-H2 N2 22225 2 222.2 2N.NH 22.22 2H 22 2H <2 2H-H2 N2 H2222222 H2222< H2222222 H2222< 222222x2 22>22222 222—22H 222HH2x< 222252 22 Nx 222522 22 222522 222H2 22222222 .222222222--.~2 22222 22.2 22 22.2 n 2 222 2 u 22 22 x 22222 .22.2 a 2 22 222222222 2222222222222 22 22222 2 66 .w 2 22N.2HH 2.222 2.2H2H 222 N22H 222522 22 222522 H2222 HH 22222222 2 H2H.N2 2.222 2N.222H 222. 22HH 222 222522 22 22222 2 N2H.2 22.NH 2N.22 2H 22 22 <2 2H-H2 N2 2 222.2 2N.2H 22.22 2H 22 22 <2 2H-H2 N2 2 222.2 2N.2H 22.22 2H 22 22 <2 2H-H2 N2 2 222.2 2N.NH 22.22 2H 22 22 <2 2H-H2 N2 2 22H.2 2N.NH 22.22 HH 22 22 <2 2H-H2 N2 2 222.2 2N.NH 22.22 2H 22 N2 <2 2H-H2 N2 2 222.2 2.NH 2.22 2H 22 H2 <2 2H-H2 N2 2 HHH.2 NH 22 HH 22 22 <2 2H-H2 N2 22225 2 2N2.2 2H 22 2N N2 2N <2 2H-H2 N2 H2222222 H2222< H2222222 H2222< 222222x2 22>22222 222222H 222HH2x< 22225222 Nx 222522 22 222522 22222 22222222 .222222222--.~2 22222 67 ‘gxperiment 6. the effect of bengyladenine on root tip chromosome number: The frequency of tetraploid root tips and the absence of tetraploid cells in root tips from the sample of seed derived plants, prompted an experiment to test the effect of different levels of BA in the shoot culture medium. Two clones were used: 80-66, which had not shown any tetraploid roots before, and FC701/5-116, which.had produced some tetraploid roots in both axillary and adventitious clones(Table 8). Table 13 gives the results of this study. No tetraploid root tips were seen in clone 80-66, whereas FC701/5-116 ramets from three of the five treatments (0.25 mg/l, 0.75 mg/l, and 1.0 mg/l) had some tetraploid root tips. To study the effect of BA on tetraploid frequency. regression analysis was achieved. From the analysis of variance of regression (Table 14), we can run the F test for significance as follows: Calculated F = giggg- = 4.6, while the tabulated F(0.05) (1,4) = 7.71. Therefore, the calculated F is insignificant and we can conclude that data from this experiment suggests that there is no major effect of BA concentrations in the shoot culture medium on ploidy level of subsequent roots. I It is worthwhile to mention that during this investigation an attempt was made to culture roots jfl_xitgg in the liquid form of the root induction medium (MS plus 3.0 mg/l NAA). The purpose was to see if growth would be strong enough to provide material for chromosome counts. There was a 15-20 times increase in volume of roots, but vigorous potted plants were still thought to be better root tip sources. 68 ~fi~.o mu mum m~ - .,- . «mm omA F\me co.“ Hufi.o NR men mN - - okm om P\ms m~.o ooo.o - mmm om - - mfie om F\me om.o Nofi.o Ne mam NN - - own an P\ms mN.o ooo.o - o~¢ on - - mHN on P\ms ofi.o mppou mppmu m—pmu muoom mppmu muooa m__mu muooa mpm>wp n.opgmgumv u*o_nmxuuh u_o_a*o uPOPgmgpmp c_o.avo «cvcwcapz~=mm mo aucmaamgm oHH-m\~c~um mo-om .mcoFHngucoucou mcvcmuo—z~=on acmgmmmpu m:_=_uu:ou upcme sag» nouacosn mamas; um>Fgmc xgaF—Pxo mo nave poo; upopaogumu $o cowugoaogg--.mfi mpnm» 69 These cultured roots also were used in the microscopic studies and gave ceils compieteiy simiiar to their oriqina] ramets. Tab1e 14.--The anaiysis of variance of regression between benzyiadenine concentrations and frewuency of tetrapioid ceils. Source of df Sum of squares Mean of squares F variation Totai 4 0.038 Regression 1 0.023 0.023 4.6 Error 3 0.015 0.005 DISCUSSION 1311312 vegetative propagation through tissue culture has been increasingly used during the past ten years. One of the methods listed under tissue culture techniques is shoot culture, which involves artificial branching by axillary buds stimulated by cytokinin in the medium. There are references on shoot culture for various species of economic importance (Smith and Murashige, 1970; Murashige, 1974; Kartha, 1975; Pierik. 1975; Pieper and Zimmer, 1976; Sagawa, 1976; Arnold van and Eriksson, 1979; Jones et al, 1979; Skirvin and Chu; 1979; Konar and Singh, 1980; and Paterson. 1984). Various methods for cloningfbeets by shoot culture have been reported (Margara,1977; Hussey and Hepher, 1978; Coumans et al, 1981; Saunders, 1981; and Hanms et al, 1983). The adventitious buds that grow on the petioles of shoots during in yi§§g_pr0pagation would be a supplemental source for multiplication if they produce true copies of the original clone. Very few reports of clearly adventitious buds in shoot cultures exist (Fordham et al, 1982 in blue berry; Harms et al, 1983 in table beets; and Paterson; 1984 in sunflower). Adventitious buds were investigated in this research to determine if they present any danger of genetic change if they are used to increase the number of in 11339 propagated shoots. In general, adventitious buds can arise in five situations: on leaf parts isolated from intact plants, on leaf parts isolated from shoot culture, on intact plants, on callus, or on intact shoot cultures, which was the case of the material studied here. Larkin and Scowcroft (1981) have proposed the term "Somaclonal Variation” to describe the genetic infidelity.observed among plants 70 71 regenerated from adventitious shoots or somatic embryos. They reviewed the Species which have displayed this phenomenon: sugar cane. potato, tobacco, rice, oats, maize, barely, Brassica sp., pelargonium, carrots, Chrysanthemum, carnation, red clover, sorghum, pineapple, garlic, and lettuce. Genetic infidelity of the adventitious buds regenerated on isolated leaves has been reported. Hermsen et al (1981) found 84.7% of 425 plants of adventitious shoots grown on in 11352 cultivated rachis and petiole explants of the F1 hybrids of Solanum etubersum X S, pinnatisectum were scored as doubled or quadrupled chromosome number. Evans and Sharp (1983) regenerated plants from leaf explants of Lycopersicon esculentum. They obtained autotetraploid plants frequently. Moreover, they reported that there were several monogenic mutations segregating in the progeny of the regenerated plants. There have been no studies of the genetic fidelity of adventitious buds from shoot cultures in beets or any other species. This kind of study was undertaken in the investigation here. Reviewing the reports of somaclonal variation, three levels of sophistication can be noticed. The first level described types of morphological variation in the 1 plants regenerated from tissue culture. The second level involfied genetically transmissible variation and detection of recessive variation in self progeny (McCoy and Phillips, 1982). The third level has involved detection of linkage change in regenerated plants. The research described here on beets was done at the first and second levels. No persistent variation was fOund in plants derived from 141 individual adventitious buds from 24 original clones of 72 sources. However, this study is not meant to be a comperhensive answer to the question of somaclonal variation from adventitious buds in beet shoot cultures. Firstly, sample size of individual adventitious buds and wider germplasm sample should be larger, it was difficult to get almost equal or near numbers from different adventitious shoots at the . same time through tissue culture techiques because of the vast variation in growth on the media. Secondly, there could be further investigations to detect any possible change in the combining ability among the adventitious ramets or in the isozyme patterns which are useful as molecular markers. This study was executed by comparing the adventitious bud derived ramets with their counterpart ramets from the axillary buds through several approaches. Hhen morphological and fertility characters were monitored, no aneuploidy was detected, and only three variants in leaf morpholdgy were found. These narrow leaf types did not maintain that abnormal appearance through a subsequent cycle 6f shoot culture multiplication. In the field these narrow leaf individuals were noteworthy. One narrow leaf plant appeared unexpectedly among ramets of axillary bud origin. Additionally, other ramets derived from the same adventitious or axillary bud were normal in appearance. The narrow leaf character that does not persist might be explained as a carryover from some effect of the first shoot culture or as an epigenetic aspect. ' Hhen root tip chromosome number and guard cell length were examined, neither gave any polyploid or aneuploid plants. The fully tetraploid root tips which were found were accompanied by fully? 73 diploid root tips from the same ramet. This indicates spontaneous duplication in somatic cells (polysomaty), probably during the shoot culturing or the initiation of the secondary roots. Some important points must be mentioned. In spite of the occurrence of some tetraploid root tips in some otherwise diploid ramets, there was no indication of tetraploid guard cells in the shoot system. Both axillary and adventitious derived ramets behaved similarly, i.e. the tetraploid root tips were found in some adventitious derived ramets as well as in some . axillary derived ramets. One axillary clone displayed a mixture of diploid and tetraploid root tips in two different batches of axillary bud derived ramets. when S1 progeny of adventitious bud derived ramets were screened, no offtypes were found as segregates, as would occur if recessive mutants had arisen with the adventitious bud. Furthermore. no major change in segregation pattern of originally heterozygous characters was noticed. If this had happened, it would have indicated a mutation-like change at the heterozygous loci (B, b) and (R, r). No unusual segregation was noted in the families expected to breed true fer either monogermness (mm) or multigermmess (MM). Segregation for annualness which would be expected to be 3:1 annnual to biennial, did not give this ratio in most cases, with significant deviation (Table 12), even in the control as well as total ratio. The most likely- explanation of this deviation for annualness is because of the variability in the microenvironment as well as genetic background among the annual plants. They did not grow at the same rate and therefore, they had not started to initiate the flowering stalks at the same time. 74 Since the time of the experiment was limited, it is very likely some of the annual plants were classified as biennial ones. The hypocotyl color was closer to the expected ratio, however, some ratios deviated significantly. There was no promotive effect of higher benzyladenine concentrations used fbr shoot multiplication when frequency of tetraploid root tips was measured. The origin or maintaining of tetraploid cells in the rooting shoot is still unknown. However, Nitsch et al (1969) reported certain cytokinins such as the substituted phenylureas are effective in producing diploid shoots from haploid Nicotiana. In concolusion, there was no indication of any genetic infidelity in ramets or progenies derived from adventitious buds in most cases of characters studied in this research. Moreover, the study suggests that the adventitious derived ramets are probably identical to their counterpart axillary derived ramets and there is no reason to exclude these buds during the in vitro propagation of shoot culture. BIBLIOGRAPHY Arnold von, 5. and T. Eriksson. 1979. Bud induction on isolated needles of Norway Spruce ( Picea abies L. Karst) grown jg_ vitro. ~Plt. Sci. Letts. 15:363-372. Cassells, A. C., E. M. Goetz and S. Austin. 1983. Phenotypic variation in plants produced from lateral buds, stems, explants and single-cell—derived callus of potato. Potato Research 26:367-372. Coumans-Gilles, M. F., Cl. Kevers, M. Coumans, E. Ceulemans and Th. 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