This is to certify that the thesis entitled VARIATION AMONG TOMATO SEEDLINGS AND SOMACLONES IN RESPONSE TO INOCULATION WITH TOMATO MOSAIC VIRUS presented by KRISTIN AMY BARDEN has been accepted towards fulfillment of the requirements for _I‘L_S_._degree in W PLANT PATHOLOGY 24%, 2/ 2WM%‘ Major professor Date W 0-7639 MS U i: an Afiman’ve Action/Equal Opportunity Institution IVISSI_I RETURNING MATERIALS: Place in book drop to LJBRAfiJES remove this checkout from .“ your record. FINES will be charged if book is returned after the date stamped below. VARIATION AMONG TOMATO SEEDLINGS AND SOMACLONES IN RESPONSE TO INOCULATION WITH TOMATO MOSAIC VIRUS By Kristin Amy Barden A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1985 VARIATION AMONG TOMATO SEEDLINGS AND SOMACLONES IN RESPONSE TO INOCULATION WITH TOMATO MOSAIC VIRUS By Kristin Amy Barden Somaclones regenerated from tomato stem or leaf sections were inoculated with tomato mosaic virus (ToMV) to determine whether variation in response to the virus occurred. Seedlings and somaclones of five commercial tomato lines presumed to be susceptible to the virus were screened with ToMV-ZA. and somaclones of a known-susceptible genotype from the Glasshouse Crops Research Institute (GCRI. Littlehampton, UJQ) were screened with TMV-Flavum or TMV—YA, a yellow aucuba strain of ToMV. GCRI near-isogenic tomato lines homozygous for various ToMV resistance genes were inoculated*with'TMV—2A.and‘TMv— Flavum, but the host resistance genes were not overcome by these virus strains. All progeny tested from a selfed somaclone of a commercial line and from one selfed GCRI-26 somaclone gave negative or low readings in the enzyme-linked immunosorbent assay following inoculation with the virus strains ToMV-ZA.and TMV-Flavum. respectivelyu Further testing is necessary to determine the stability of this putative resistance. This thesis is dedicated to my loving parents. 11 ACKNOWLEDGEMENTS I would like to thank Dr. Harry Murakishi for his guidance and the kindness which he extended to me. and Rita Harris. Cathy Brandi and Sandy Smith for their help and encouragement. Also. Dr. Norman Good and Dr. Joseph Saunders deserve thanks for their counsel and the time they devoted to being on my graduate committee. iii TABLE OF CONTENTS Page LIST OF TABLES .............................................. vi LIST OF FIGURES ............................................ vii LISTOF ABBREVIATIONS ..................................... viii INTRODUCTION AND LITERATURE REVIEW ........................... 1 MATERIALS AND METHODS ........................................ 7 Sources and purification of viruses ...................... 7 Tomato lines and seed irradiation ........................ 7 Somaclones ............................................... 8 Virus characterization by genotype interaction .......... 11 Visualization of ToMV coat protein by gel electrophoresis ....................................... 11 Enzyme-linked immunosorbent assay (ELISA) ............. 12 Inoculation of seedlings or regenerated plants .......... 14 in vitro screening ...................................... 17 Protoplast isolation and culture: GCRI—26 ............... 17 RESULTS ..................................................... 20 Optimization of hormone concentration in shoot— regeneration medium ..................................... 20 ELISA detection of viruses .............................. 20 Virus characterization by genotype interaction and determination of ELISA threshold for resistance screening ............................................... 20 Commercial lines: disease reactions to inoculation with ToMV—ZA ................................................. 27 Selection of putatively resistant plants .............. 27 Virus inoculation of detached leaves. cuttings and progeny of selected plants ............................ 32 Somaclones of GCRI—26: disease reactions to inoculation with ToMV ............................................... 41 Method of screening ................................... 41 Selection of somaclones ............................... 47 Virus inoculation of detached leaves. cuttings and progeny of selected somaclones ........................ 47 iv In vitro screening ...................................... 54 Protoplast isolation and culture: GCRI—26 ............... 54 DISCUSSION .................................................. 56 Virus characterization by genotype interaction .......... 57 Variation in disease reactions among seedlings and somaclones of commercial lines .......................... 57 Variation in disease reactions among somaclones of GCRI—26 ................................................. 59 In Vitro screening ...................................... 60 Culture of GCRI-26 protoplasts .......................... 60 LITERATURE CITED ............................................ 63 Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table 16. Table 10. 11. 12. 13. 14. 15. 17. LIST OF TABLES Page Composition of EL rooting medium....................9 Composition of shoot-inducing medium....... ..... ...10 Composition of TM‘I rooting medium.................15 Composition of solutions for tomato protoplast iSOIationOOOOOOOIOOOOOOIOOOOOOOOOO0.0.0.... ...... .018 Composition of media for tomato protoplast cultureOOOOOOOO0.0.0.000...OOOOOOOOOOOOOOOO...0.0.019 Growth of leaf discs in media of varying BAzIAA concentrations......................... ..... 22 Comparison of host ranges..........................26 Number of seedlings and regenerated plants of five commercial lines screened for variation in disease reaction...................................34 Putatively resistant control (non-irradiated seedlings) plants of line 574......................36 ELISA readings of samples from selected commercial plants following initial virus screen..............38 Virus multiplication in detached leaves of selected commercial plants.................... ..... 40 Virus multiplication in cuttings of selected comerc1a1 plantBOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.43 Progeny tests of selected commercial plants........45 Percentages of plants from commercial lines in which virus was not detected upon completion of screeningOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.....46 ELISA readings of selected GCRI-26 somaclones......49 Virus multiplication in cuttings and detached leaves of selected GCRI somaclones.................51 Progeny tests of somaclones of GCRI-26.............53 vi LIST OF FIGURES Page Figure 1. Detection of virus strains TMV—Flavun and ToMV-2A by ELISA ................................ 24 Figure 2. SDS—polyacrylamide gel electrophoresis of GCRI near—isogenic lines inoculated with ToMV-ZA ..... 29 Figure 3. SDS-polyacrylamide gel electrophoresis of GCRI near—isogenic lines inoculated with TMV-Flavum..31 vii LIST OF ABBREVIATIONS BA ............. benzyladenine 2.4—D .......... (2',4'-dichlorophenoy)acetic acid EDTA ........... ethylenediamine tetracetic acid ELISA .......... enzyme—linked immunosorbent assay GA3 ............ gibberellic acid GCRI ........... Glasshouse Crops Research Institute IAA ............ indole acetic acid NAA ............ naphthalene acetic acid MES ............ 2-(N—morpholino) propanesulfonic acid MS salts ....... Murashige and Skoog salts PVP ............ polyvinylpyrrolidone TMV ............ tobacco mosaic virus ToMV ........... tomato mosaic virus viii INTRODUCTION AND LITERATURE REVIEW The yield of greenhouse-grown tomatoes has been reported to decrease following infection by strains of tobacco mosaic virus (TMV) (Broadbent. 1964). Disease symptoms include stunting, leaf distortion and mottling (Hall and Bowes. 1980), and internal browning of the fruits (Boyle and Wharton, 1957) TMV strains which infect tomatoes are designated ToMV, and are members of the tobamovirus group. Though typical strains are closely related to type strain TMV. ToMV’strains can be differentiated from all other tobamoviruses on the basis of host range. serological reactions. and amino acid composition of the coat protein (van Regenmortel. 1975. Thallings. 1976). Like other viruses in the tobamovirus group. ToMV particles are rod-shaped and about 300 x 18 nm in size (Hollings. 1976). Strains O. 1. 1.2. 2 and 22 of ToMV were numbered according to their ability to overcome known host resistance genes. These resistance genes have been backcrossed into 'Craigella‘, a variety of the inbred tomato line 'Ailsa Craig', to provide near— isogenic lines for host-range classification of ToMV strains (Clayberg et 31.. 1960. Pelham. 1972. Hall and Bowes. 1980. Maxon Smith, 1982). Tm-l was found to be a dominant gene for suppression of mosaic symptoms. and its inhibition of virus multiplication of ToMV strain 0. the common strain of ToMV, was shown to be gene dosage dependent (Fraser and Loughlin, 1980). Though ToMV strain 1 overcame the inhibition of symptom expression by Tm-l, virus multiplication of strain 1 was reduced in plants homozygous or heterozygous for Tm-l (Fraser. Loughlin and Connor. 1980L Soost (1958. 1959)isolated the single dominant allele. Tm-2, which prevents infection by all known ToMV strains except strain 2. Tm- 22 was transferred from Lycopersicon peruvianum to L. esculentum by Alexander (1963) and was found to be allelic to Tm-2 (Schroeder et a!” 1967). Tun—22 confers resistance to nearly all ToMV isolates. Within four days of inoculation the Tm-z and Tm-22 genotype may respond hypersensitively by restricting the multiplication of virus to localized, necrotic areas. With infection at higher temperatures. a systemic necrosis may appear on plants heterozygous for Tm-2 or Tm-22 (Pelham. 1972, Hall. 1980i In their earliest sources. undesirable genes were linked to the resistance genes. Tm-2 was linked to the recessive allelen netted virescence (nv). in its original source. Plants homozygous for nv are yellow and stunted (Alexander. 1971. Soost. 1958. 1959). Tm-22 in the original source was linked to excessive vegetative growth and blotchy ripening. Probably a gamete promoter gene. linked to Tm-22. was responsible for the forked trusses and small fruits found in the early breeding lines containing Tm—22 (Hall, 1980). Unlinked sources of the genes are now available. Undesirable linkages and the time required to backcross may be limiting factors in the introduction of resistance genes to tomato lines through conventional breeding. Larkin and Scowcroft (1981) concluded that variation generated during plant cell culture, or somaclonal variation. could be of value in crop improvement. Specifically. tissue culture techniques have been reported to be instrumental in the production of disease-resistant regenerated plants (Brettell and Ingram, 1979i Sugar cane subclones propagated in vitro showed a greater degree of resistance to Fiji virus disease than the original clone (Krishnamurthi and Tlaskal. 19740. Cells from maize lines susceptible to Drechslera maydjs Race T were found to be resistant to T toxin. produced by the fungus. following subculturing of embryo-derived cells on medium containing T toxin (Gengenbach and Green. 1975). Also. pathotoxin resistance was inherited in maize plants regenerated from cell cultures (Gengenbach et a1.. 1977). Thus. where regeneration and selection methods are available, breeding objectives may be accomplished more rapidly. especially if only single loci are affected in each regenerated plant (Evans et a1.. 1984). Somaclonal variation may be epigenetic. genetic. or both. When induced. for example by the addition of hormones to a culture medium. epigenetic changes occur at a much higher rate than genetic mutation. However. epigenetic changes are reversible and are not transmitted meiotically (Meins. 1983). Epigenetic variation is most likely due to selective gene expression (Meins. 1983i An example of epigenetic variation is the induction of cytokinin habituation. or the loss of the requirement of cytokinin for growth. in tobacco pith cells. Cytokinin habituation occurs at a rate exceeding 4 x 10'3/cell generation. 100-1000 times faster than the rate of somatic mutation in Nicotiana (Meins. 1980). Sibi (1982) observed heritable epigenetic variation in four families of regenerated tomatoes. The four families had traits which differed from the control yet which did not segregate as nuclear mutations would. Significant assymetrical transmission effects observed for various traits gave evidence that epigenetic changes had occurred. Conversely. genetic variations. or mutations. are transferred meiotically. Recently. changes in chromosome number (D'amato. 1977). single gene nuclear mutations (Evans and Sharp. 1983) and cytoplasmic genetic changes (Kemble and Shepard. 1984) have been detected among regenerated plants (R) or their progeny (R1). Mutations from recessive toidominant were found among plants regenerated from immature wheat embryos (Larkin et a1.. 1984). while Lorz and Scowcroft (1983) found changes in segregation patterns ataispecific locus in one third of their protoplast-derived Nicotiana tabacum plants. Tissue culture has been shown to lead to a high rate of genetic variation among regenerated tomato plants. Sibi. Biglary and Demarly (1984) found significant increases in the cross-over or recombination rate of tomato in about half of the regenerated individuals tested. using two different sets of nuclear gene markers. Evans and Sharp (1983). using tissue culture to generate somaclonal variation. identified 13 nuclear gene mutations in progenies of 230 tomato plants regenerated from leaf discs. Buiatti et a]. (1985) selfed 88 regenerated plants and found recessive mutations which segregated out in a 3:1 ratio in 15 progenies. concluding that the 15 regenerated plants were of unicellular rather than multicellular origin. Finally. a combination of both genetic and epigenetic variation. which occurs at a high frequency yet is also transmitted in sexual crosses has been reported (Meins. 1983.‘Larkin and Scowcroft. 1981). Genetic and epigenetic changes can occur in the same cell. as was shown when seedlings from plants regenerated from tobacco cells grown on the herbicide picloram showed a higher degree of tricotyly. determined to be a physiological or epigenetic response, while some plants also showed genetic variability in resistance to picloram (Chaleff and Keil. 1981). Irradiation, another method of obtaining variation. has previously been used by plant breeders in mutation breeding. Tissue culture has been coupled with irradiation to enhance variation. For example. Van Harten et a1. (1981) found an average mutation frequency of 74h3% among adventitious sprouts produced in vitro from irradiated potato explants (rachis. petiole. leaflet-discL.whi1e the percentage of mutations among sprouts from irradiated tissue of in vivrrorigin was only 24- 38%. Murakishi and Carlson (1982) exposed 40 TMV-Flavum—inoculated Nicotiana sylvestris plants to gamma irradiation to increase the frequency of mutant events. and 40 other inoculated plants were not irradiated. Leaves of the plants were then plated on shoot-inducing medium. After four weeks of culture. green calli were selected. yellow calli were rejected. and green plantlets from selected calli were potted and tested for the presence of virus. Both irradiated and control calli produced virus-free plantlets (168 and 33 plantlets. respectively). When cuttings of these plants were inoculated with TMV? Flavum. symptom expression was delayed in cuttings of sevenlof the irradiated plants which were originally virus-free. Perhaps mutagenesis by irradiation was responsible for this ephemeral type of resistance. The present study was conducted to determine whether variation in the reaction of susceptible lines of tomato to inoculation with ToMV could be generated by tissue culture of tomato leaf and stem sections. gamma irradiation of tomato seeds or tissue. or a combination of tissue culture and irradiation. Secondly, protoplasts of a ToMV-susceptible line of tomato were cultured to evaluate the possibility of regenerating plants. cn~'protoclones'. and screening these plants for variation in disease reaction. Protoplasts of the GCRI line 26 formed calli. but no regeneration was obtained, so all regenerated plants. or somaclones. used in screening were from leaf or stem explants. Also, nearly all screening was done at the level of the regenerated plant. as difficulty was encountered in regenerating plants from leaves of virus—infected plants. The first group of seedlings and somaclones screened were commercial tomato lines (Asgrow Seed Company. San Juan Bautista. CA) presumed to be susceptible to a strain of virus, ToMV-2A, isolated by the Asgrow Seed Company. This virus strain was first characterized by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis of virus infected. near-isogenic lines in 'Craigellah Secondly. a group of somaclones obtained from leaf discs of GCRI— 26. known to be susceptible to all strains of ToMV. were screened with the yellow aucuba variant of ToMV—0. designated TMV—YA. and with TMV- Flavum. The host range of TMV-Flavum was determined to insure that the two viruses could be used interchangeably in screening. The screening of somaclones of a known susceptible tomato line with a virus having a host range similar to the common strain could provide a model system for other studies in the use of tissue culture to generate variability in response to virus infection. MATERIALS AND METHODS Sourcesfl purification of viruses. The differential centrifugation method (Knight. 1962. Stanley and Wyckoff. 1937) was used to purify ToMV-2A from a plant homogenate supplied by the Asgrow Seed Company, San Juan Bautista. CA. The frozen homogenate was thawed. then centrifuged at 10,000 rpm for 20 minutes (Sorvall. SS-34 rotor) to pellet cell debris. Low speed centrifugation was followed by a high speed centrifugation of the supernatant. 2 hours at 22.5 kRPM (Beckman Model L ultracentrifuge). The virus pellet was kept overnight at 4C in 0.01M phosphate buffer. pH 7.0. of sufficient amount to submerge the pellet. The following day. the round of low and high speed centrifugations was repeated and the concentration of the partially purified virus was estimated (Takahashi, 1951) by absorbance readings at 265 nm using a Beckman DB spectrophotometer. The virus was adjusted to a concentration of 1.0 mg/ml in 0.01M Na.K phosphate buffer and stored at -20 C. TMV-Flavum (Murakishi and Carlson. 1982). TMV-YA (litter and Murakishi, 1969), and ToMV-0 (Hall and Bowes. 1980) were also purified by the same method. Potato virus X (PVX) was furnished by Dr. W. J. Hooker. Michigan State University. and was maintained in Nicotiana glutinosa. 19.91119 lines and seed irradiation. Commercial tomato breeding lines 541. 569. 574. 576. and 577. presumed to be susceptible to the common strain of ToMV. ToMV-0. were obtained from the Asgrow Seed Company, San Juan Bautista. CA. To screen for variation in the reaction or commercial lines to inoculation with virus strain ToMV-2A. 20 seeds 01' each of the lines were irradiated at 20. 30 or 40 kR gamma- irradiation. By irradiating seeds. a comparison could be made of reactions due to irradiation alone versus irradiation in combination with tissue culture plantlet regeneration. A variable flux gamma irradiator with a 60C0 source (Chemistry Department. Michigan State University) was used for irradiation. Shoots of six. near-isogenic lines of tomato (Glasshouse Crops Research Institute (GCRI). Littlehampton, UK). were regenerated from leaf discs. The following virus resistance genotypes in lines of near- isogenic 'Craigella' were used: 26 (+/+). 236 (Tm-2/Tm-2), 237 (Tm- 1/Tm-1). 267 (Tm-22/Tm-22). 488 (Tm-1/-.Tm-2/*), and 490 (Tm-1/+.Tm- 2/Tm-22). Lines 26. 236. 237 and 267 were used to determine the host range of ToMV-2A and TMV-Flavum. and leaf-regenerated plants of the susceptible line GCR126 were screened for variability in disease reaction to inoculation with TMV-Flavum or TMV-YA. Somaclones. To regenerate plants from stem explants. tomato seeds were surface sterilized one minute in 95% ethanol and five minutes in 10: commercial bleach (0.5255 NaClO.5HZO). followed by three rinses of five minutes each in sterile water. After germination on moistened filter paper or potato dextrose agar (Difco Laboratories. Detroit. Michigan) in petri plates. the tap roots of seedlings were removed and seedlings placed individually in tubes onto EL maintenance medium (Table 1) under light of 60-90 uEm’zs'l. a 16 hour daylength. and at 24-26 C for 3-4 weeks. Once plants grew nearly to the tops of the tubes. 5-6 stem explants (excluding nodal tissue) of approximately 5 mm in length were plated on 25 ml of shoot inducing medium (Table 2) in 100 x 15 mm petri plates (Miles Laboratories. Inc.. Naperville. IL 60540). 560 tomato somaclones regenerated from stem were obtained from Table 1. Composition of EL rooting medium a Murashige and Skoog NH4N03 KNO CaCT2 MgSO4 KH2P04 FeNazEDTA H3803 MnSO4.H20 ZnSO4.7H20 KI Na2M004.2H20 CuSO4.5H20 85 vitaminsC i-inositol nicotinic acid thiamine.HCl pyridoxine.HCl sucrose agar pH 6.0 salts weight/liter 333 181 170 36. .2 mg .9 mg .6 mg .83 mg .25 mg .025 mg .025 mg H OCOOQGG .65 g .90 g mg mg mg 7 mg 100 mg 10 mg 30 g mM 20 18. 0000 88. OOCOOOOOHHOD meooo H .03 .005 .001 .0001 .0001 .56 .008 .03 .004 aEL rooting medium devised by R. Smith (International Plant Research Institute. San Carlos, CAL bMurashige and Skoog. 1962 (KC Biological. Inc.. Lenexa. KS 66215L CGamborg et 81.. 1968. 10 Table 2. Composition of shoot-inducing medium. a MS saltsb weight/liter NH4N03 1.65 g KNO 1.90 g MgSO47H20 370 mg MnSO4.H20 16.9 mg ZnSO4.7H20 8.6 mg CuSO4.5H20 0.025 mg CaC12.2H20 440 mg KI 0.83 mg CoC12.6H20 0.025 mg KH2P04 170 mg H3BO3 6.2 mg Na2M004.2H20 0.25 mg FeSO4.7H20 27.84 mg NazEDTA 37.24 mg NaH2P04.H20 0.17 g thiamine.HCl 0.5 mg m-inositol 100 mg nicotinic acid 0.5 mg pyridoxine-HCI 0.1 mg sucrose 30 g indole-3-acetic acid (IAA) 0.3 mg benzyladenine (BA) 3.0 mg glycine 3.0 mg N O HOIQO H CHOOHOOOHQ mM .03 .0001 .005 .0001 .1 0.001 88. 13. .1 .l .0012 .0015 .56 .004 .0004 .04 aReynolds. Upjohn Company bMurashige and Skoog. 1962 11 the Asgrow Seed Company (San Juan Bautista. CA) to be screened for resistance or tolerance to ToMV-2A. Adventitious shoots appeared from explants of the commercial lines within approximately four weeks. Leaf explants were taken from four week old plants grown in soil under the same lighting conditions as the axenically grown plants used for stem explants. The fully expanded leaves of each plant were sterilized in 7’6 commercial chlorox and 0.1: Tween 20. and rinsed 3 times with sterile water. Six mm leaf discs were obtained (Kartha et a1.. 1976). and 5-8 leaf discs were plated on 25 ml shoot inducing medium (Table 2) in 100 x 15 mm petri plates. Various concentrations of benzyladenine (BA) and indole-3-acetic acid (IAA) were tested in the shoot-inducing medium to determine the best combination for shoot regeneration. Leaf explants were usually subcultured individually in tubes of the same medium at approximately 3 weeks. Shoots began to appear approximately four weeks after initial plating of explants from the five commercial lines (541. 569, 574. 576. 577) as well as from the six GCRI lines (26. 236. 237. 267. 488 and 490). . Virus characterization by ggnotype interaction. Visualization of ToMV colt protein buel electrophoresis. Near- isogenic lines of tomato (GCRI. Littlehampton, UR). differing in the genes for resistance to ToMV. were used to determine the host range of ToMV-2A and TMV-Flavum. Samples of the inoculated host range tomato plants were prepared for visualization of viral protein by gel electrophoresis as described by Fraser and Loughlin (1980). The buffer used to grind tissues and extract proteins at a 1:10 dilution consisted of 40 mM boric acid. 41 mM Tris (pH 8.6). 4: v/v 2-mercaptoethanol. 10$ w/v sucrose. and 0.758 w/v sodium dodecyl sulfate (SDS). The plant 12 homogenates were placed in small tubes. heated to 100 C for 3 minutes and then centrifuged 3 minutes at 12,000 g. Protein extract was stored at -20(3and thawed at room temperature before running samples on a SDS-polyacrylamide slab gel (Laemmli, 1970). A stock solution of 30% (w/v) acrylamide and 0.8% (w/v) N.N'-bis-methylene acrylamide (Bio-Rad. Richmond. CA) was used in preparing the gel. The running gel was 12% acrylamide (Bio-Rad. Richmond, CA). 0.375 M Tris.HCl. pH 8.8. and the stacking gel was 3.75% acrylamide, 0.125 M Tris.HCl. pH 6.8. Both gels were 10% SDS and were polymerized with 0.05-0.06% (w/v) ammonium persulfate and 0.05-0.07% (v/v) N.N.NHJP-tetramethylethylenediamine (Bio-Rad). The samples were run with bromphenol blue tracking dye 9 hours at 40 milliamps. A sample composed of bovine serum albumin (MW 66.000L alcohol dehydrogenase (MW 29,000) and egg albumin (45,000) at 1 mg/ml was used as a marker lane. Gels were stained one hour at room temperature with 0.25% Coomassie brilliant blue in 4.6% glacial acetic acid and 45.4% methanol, then gels were destained in 7% glacial acetic acid and 10% methanol for several hours. Enzyme—linked immunosorbent assay (ELISAL The enzyme-linked immunosorbent assay (ELISA) and the presence or absence of symptoms were recorded for inoculated plants. Virus multiplication was determined by ELISA when symptoms were observed on susceptible plants. All ELISA procedures were based on those of Clark and Adams (1977). TMV-0 antiserum from rabbit was obtained from Dr. M. Nishiguchi (Institute for Plant Virus Research, Tsukuba. Japan). The protein of 1 ml crude ToMV-0 antiserum diluted to 10 ml with distilled water was precipitated by the addition of 10 ml saturated ammonium sulfate 13 solution. After 1 hour of precipitation at room temperature. the antiserum was centrifuged at 6000 rpm for 5 min (Sorvall type SS-34). Upon dissolving the pellet in 2 ml 1/2-strength phosphate buffered saline (PBS)(Clark and Adams, 1977). the solution was desalted by dialyzing the preparation 3 times against 500 ml 1/2-strength PBS including once overnight. The gamma—globulin was further purified by washing through a DE 22 cellulose column with 1/2-strength PBS. A fraction was obtained with an absorbance of 1.0 at 280nm which corresponds to a gamma-globulin concentration of approximately 0.7 mg/ml. In the conjugate preparation. 0.2 ml (1mg) alkaline phosphatase (Sigma) was added to 0.5 ml gamma-globulin (0.35 mg). and glutaraldehyde was added to 0.05% (v/v). Crosslinking was allowed to proceed for 4 hours at room temperature. then glutaraldehyde was removed by dialyzing 3 times against 500 ml PBS + 0.01% sodium azide. Bovine serum albumin was added to 5 mg/ml for stabilization and the gamma-globulin conjugate was stored at +4 C. For the assay, wells of microtiter plates (Dynatech Labs, Inc.. Alexandria, VA) were coated with a 1:1000 dilution of purified rabbit anti-TMV-L gamma-globulin in 0.05 M sodium carbonate buffer. pH 9.6. Plates were incubated at 37 C 2-4 hours or overnight at 4 C. rinsed 3 times with PBS + 0.05% Tween 20 (PBS-Tween), then a 1:10 dilution of ground plant samples in virus buffer (2% polyvinylpyrrolidone. 0.2% ovalbumin) was added and plates incubated 3-4 hours at 37 C or overnight at 4 C. A healthy control and a known diseased (symptomatic) control sample were included in each plate. Plates were again rinsed 3 times with PBS-Tween, the gamma- globulin conjugate was added at a 1:1000 dilution in virus buffer. plates were incubated 3-4 hours at 37 C. were rinsed again 3 times with 14 PBS-Tween. and p-nitrophenyl phosphate , substrate of the phosphatase enzyme. (Sigma Company) was added. Within 30 minutes following the addition of substrate in 10% diethanolamine. pH 9.8, absorbance readings (405 nm) were taken and recorded with a Microelisa minireader II (Dynatech Labs) coupled to a printer (COEX 80F/T. Components Express Incorporated. Santa Ana, CA 92705L If diseased control absorbance readings had not reached 0.5 30 minutes after substrate addition. readings were taken at one hour. Inoculation of seedlings or regenerated plants. Seedlings were inoculated after approximately two weeks of growth at 24-26 C and 60- 90 uEm'Zs’1 fluorescent light. For screening at the level of stem or leaf regenerated plants. shoots were first rooted in EL or TM-1 media (Table 3). or the cut ends were covered with indole butyric acidztalc (200mg/100cc). and then planted in a commercial peat mixture (Sunshine Mix. No. 1, Fisons Western Corp..‘Vancouver. B11). Plants were allowed to harden in a cellophane-covered plastic box approximately one week under fluorescent light of 60-90 uEm'zs'l with a 16 hour daylength at 24 C. and were then dusted with carborundum and rub-inoculated using a cotton swab. Approximately 24 nodes of one GCRI-26 seedling (obtained by successive subcultures) were cultured cui'TM-i medium (Table 3) to obtain plants which were inoculated in vitro using filter-sterilized TMV-Flavum at 20 ug/ml. Commercial tomato breeding lines (Asgrow Seed Company) were inoculated with ToMV-2A. Symptom detection was facilitated by using ToMV-2A which is a yellow strain of ToMV. Although concentrations of ToMV-2A as low asiL7 ug/ml gave disease symptoms. the majority of plants regenerated from commercial lines were inoculated at 33 ug/ml. 15 Table 3. Composition of TM-1 rooting medium.a mg/ml mM CaC12.2H20 150 1.0 KNO3 2530 25.0 NH4N03 320 3.9 NH4H P04 230 2.0 (NH4 2SO4 134 1.0 MgSO4.7H20 250 1.0 KI 0.83 0.005 H3BO3 6.20 0.1 MnSO4.7H20 22.30 0.1 ZnSO4.7H20 8.60 0.03 Na2M004.2H20 0.25 0.001 CuSO4.5H20 0 025 0.0001 CoC12.6H20 0 025 0.0001 FeSO4.7H20 18.50 0 0001 Na2.EDTA 18.50 0.055 Nicotinic acid 2.50 0.02 Thiamine.HCl 10 0.03 Pyridoxine.HCl 1 0.005 Folic acid 0.50 0.001 Biotin 0.05 0.0002 D-Ca-pantothenate 0.50 0.002 Choline chloride 0.10 0.0007 Glycine 0.50 0.0067 Casein hydrolysate 50 L-Cysteine 1 0.008 Malic acid 10 0.074 Ascorbic acid 0.50 0.003 Myo-inositol 100 0.56 Riboflavin 0.25 0.00066 Sucrose 30.0 g 88.0 agar 6.0 g pH 5.8 aShahin.1984. 16 The yellow strains TMV-Flavum or TMV-YA were used to look for variability'in disease reaction among regenerated plants of GCR126. Either the purified viruses or 1:20 dilutions of virus-infected plant samples ground in 0.01 M Na, K phosphate buffer pH 7.0 were used in inoculations. Also, double inoculations using a dilution of crude sap of either TMV-Flavum or TMV-YA in combination with potato virus X (PVX) were conducted (”1 a group of GCRI—26 seedlings. These double inoculations caused plants susceptible to ToMV to become necrotic. providing a more sensitive assay (Murakishi and Honma, 1963i However, PVX is carried on the seed coat to the progeny. so to avoid the necessity to eliminate PVX from a putatively ToMV-resistant somaclone. inoculations of ToMV alone were used in these experiments. After inoculation. plants were transferred to a growth chamber as“1 light at 24-26 c. under a 16 hour photoperiod with 150-200 uEm’ Symptoms usually appeared within 10 days. but if symptoms were latent or did not appear after approximately two weeks. a sample of uppermost leaves near the growing tip was ground in virus buffer at a 1:100 dilution and used in ELISA tests to determine virus multiplication. Plants with ELISA readings less than 0.5 were maintained. reinoculated, and after a second ELISA test infected plants were discarded and virus- free plants were placed in the greenhouse to obtain cuttings from nodes and to allow plants to set fruit for testing of the progeny. One or two leaves of these virus-free plants were detached. placed in petri plates of 0.8% water agar. and inoculated to test for virus multiplication. As a final test for virus multiplication in several selected test plants. 1:10 dilutions of the test plant samples in ELISA virus buffer were 17 rub-inoculated onto seedlings of Nicotiana glutinosa. In vitro screening. For the in vitro screen. four 10 day old seedlings of line 577 growing in soil were inoculated with either purified TMV— Flavum (20ug/ml) or TMV-2A (33 ug/ml) and were irradiated with 650 RR gamma-irradiation one month later. when symptoms were obvious. The day following irradiation, leaves of the plants were sterilized and leaf discs were plated on shooting media as described previously. Shoots were sampled directly from the culture tube for ELISA testing of virus multiplication. Protoplast isolation and culture: GCRI-26. A modification of the method for isolation and dqq— u d uqdq-d— d‘ d qua-dq— 1 1441414 no Loo .ooo .oooo <._.cm 003833303. no DE. in: 50000 25 Table 7. Comparison of host ranges. Symptoms and ELISA readings were used to compare the host ranges of ToMV-2A and TMV-Flavum with that of the common strain, ToMV-0. The fol lowing genotypes were examined: 26 (+/+). 236 (Tm—2/Tm-2), 237 (Tm-1/Tm-1) and 267 (Tm-22/Tm-22). ELISA readings represent one replication. and no symptoms were observed in lines 236. 237 or 267 inoculated with either of these strains. Table 7. 26 Description ToMV-0a (17 day ELISA) ToMV-2Aa TMV-Flavuma (13 day ELISA) (13 day ELISA) Healthy plant line 26 (+/+) line 236 (Tm-2/Tm-2) line 237 (Tm-l/Tm-I) line 267 (Tm-22/Tm-22) 0.08 2.54b 2.54 0.09 0.19 0.02 2.52C 2.52 0.02 0.01 0.00 0.00 0.01 0.00 0.02 1.74d 1.74 0.03 0.03 0.21 0.19 0.09 0.09 aELISA absorbance readings at 405 nm were taken the indicated number of days after inoculation of plants. bMosaic symptoms observed 16 days after inoculation. cWrinkled leaves observed 13 days after inoculation. dMosaic symptoms observed 9 days after inoculation. 27 in these plants. Also. the ELISA readings of leaves from 12 leaf explant somaclones of line 488. 9 of which were inoculated with TMV-YA and 3 with TMV-Flavum. never exceeded 0.LL An ELISA absorbance reading of(l5 was used as a threshold value below which plants were defined as having a 'low' virus titer. Results of SDS-polyacrylamide gel electrophoresis of GCRI near- isogenic lines inoculated with ToMV-2A and TMV-Flavum are shown in figures 2 and 3, respectively. For both viruses. a relatively large band of protein appears near the gel front exclusively in the lane of the inoculated GCRI-26 sample. Mobilities of 2 of the 3 molecular weight (MW) markers were 01%) for bovine serum albumin and 0.47 for egg albumin. There was no obvious alcohol dehydrogenase band. Based on a linear extrapolation of a graph of log MW of markers versus mobility. the MW of the rapidly migrating band of mobility 0.95 (ToMV-2A) or 0.98 (TMV-Flavum) found in the lane containing inoculated GCRI-26 was calculated to be approximately 17,000. Commercial lines: disease reactions to inoculation with ToMV-2A. _Selection of putatively resistant plants. 20 seeds of each of five Asgrow lines (San Juan Bautista. CA). designated 541, 569, 574. 576. and 577. were irradiated at 20, 30 or 40 kilorads (kR) 60Co gamma- irradiation. Seeds were then divided into three groups. About five seeds were allowed to grow into various numbers of seedlings (S) and were inoculated with ToMV-2A. Non-irradiated seeds were inoculated as controls to determine variation in disease reaction due to irradiation alone. Another group of irradiated seeds were grown and selfed to PPOduce progeny (S1) which were inoculated. The third group of seeds 28 Figure 2. SDS-polyacrylamide gel electrophoresis of GCRI near- isogenic lines inoculated with ToMV-2A. 10 day old plants were inoculated with ToMV-2A, and leaf samples for electrophoresis were taken 10 days later. Lanes left to right are: MW marker (bovine serum albumin. 66.000; alcohol dehydrogenase. 29.000; egg albumin. 45.000), non-inoculated GCRI-26. and lines 26. 236. 237 and 267 inoculated with ToMV-2A. The front is at lower edge of figure. 29 30 Figure 3.8DS-polyacrylamide gel electrophoresiscfl’GCRI near- isogenic lines inoculated with TMV-Flavum. 10 day old plants were inoculated with TMV-Flavum and leaf samples for electrophoresis were taken 9 days later. Lanes left to right are: MW marker (bovine serum albumin, 66.000; alcohol dehydrogenase. 29.000; egg albumin. 45.000), non-inoculated GCRI-26. and lines 26. 236. 237 and 267 inoculated with TMV—Flavum.‘The front is at lower edge of figure. 31 32 were grown and used to regenerate plants from either stem or leaf. Regenerated plants, designatedI2(Chaleff, 1981).of seedlings from both irradiated and non-irradiated seed were inoculated with ToMV-2A. Irradiated plants grew more slowly and regeneration of shoots from leaves and stems of these plants was difficult. Asgrow seedlings and regenerated plants (Table 8) were screened by inoculation with ToMV-2A at concentrations ranging from 305100 ug/ml. Eight of these plants were maintained in the greenhouse after screening because they had no symptoms and had low virus titers longer than one month after inoculation. Three of the plants were non-irradiated control seedlings (S) of Asgrow line 574. Table 9 shows that these plants were still resistant 40 days following the initial inoculation. Virus multiplication was evident eight days after inoculation in the two susceptible control seedlings shown. ELISA tests and descriptions of the other five plants are shown in Table 10. Two of these plants were from line 541 and three were of line 574.‘The virus titer as detected by ELISA had increased in plants #1 (line 541) and #3 (line 574) by 112 and 91 days post-inoculation. respectively. 2152s inoculation of detached leaves. cuttings and progeny of selected plants. One leaf was taken from plants having low virus titers in ELISA tests described in Table 10. 13 days following inoculation of the detached leaf with ToMV-2A. virus multiplication could be detected in the leaf from #2 but no virus multiplication was detected in the leaves from #4 and #5 (Table 11). In another assay, cuttings were taken from the nodes of plants *2. ‘34 and #5 and inoculated with ToMV-2A. Virus multiplication was not 33 Table 8. Number of seedlings and regenerated plants of five commercial lines screened for variation in disease reaction. Following gamma irradiation of a portion of the seeds. seedlings (S). progeny of seedlings (SI) and stem and leaf regenerated plants (R) were inoculated with purified ToMV—2A. 34 Table 8. line kRa S S1 (fruits)b stemc leafC R R 541 0 17 0 31 1 20 2 0 14 0 30 3 47 (4) 3 0 40 2 39 (3) 4 0 569 0 15 0 11 1 20 2 0 12 0 30 0 8 (l) 0 0 40 1 0 2 0 574 0 43 0 19 19 20 0 O 5 0 30 3 39 (3) 6 1 40 l 47 (3) 3 0 576 0 16 0 1 5 20 2 0 1 0 30 0 35 (2) 0 0 40 1 9 (1) 1 0 577 o 20 o 603d 69 20 3 0 1 0 30 2 44 (3) 8 0 40 1 7 (1) 4 0 aGermination of 30 and 40 kR-irradiated seeds was relatively poor (S). and explants from these plants were recalcitrant in regeneration (R). bProgeny (81) of one fruit were tested per plant (S). cStem and leaf R were regenerated from an undetermined number of S. d560 of the 603 line 577 stem-regenerated plants were obtained from the Asgrow Seed Company. 35 Table 9. Putatively resistant control (non-irradiated seedlings) plants of line 574. All but 3 of the 43 seedlings (S) of line 574 inoculated with purified ToMV-2A (Table 8) had symptoms or virus multiplication as detected by ELISA within approximately 40 days. ELISA absorbance readings of these seedlings are shown. together with those of susceptible seedlings inoculated under the same conditions. 36 Table 9. Days following initial inoculation: Description 6 8 30 40 50 574-1a 0.00 0.00 0 00 0.00 574—5 2.48 2.30 (susceptible) 2.48 2.14 574-7b 0.00 0.02 0.04 0.00 0.01 0.10 574—16b 0.00 0.00 0.06 0.01 0.01 0.08- 574-20 2.02 - - (susceptible) 1.93 — — aReinoculated 9 days following initial inoculation. bReinoculated at 11, 21 and 33 days 37 Table 10. ELISA readings of samples from selected plants following initial virus screen. 5 symptomless Table 8 which had low virus titers for over 3 maintained in the greenhouse and monitored multiplication. Samples were taken from leaves near tip unless otherwise noted. commercial plants from weeks were for virus the growing 38 Table 10. Description days post— Inoculateda Healthy inoculation plant plant #1 stem regenerate 541-OkR 23 0.04 (106 (R) inoc. 3.7 ug/ml (L03 0.05 112 1.22 0.00 1.39 0.00 #2 stem regenerate 541-30kR 123 0 00/0 08 0.02 (R) inoc. 11 ug/ml 0.00/0.02 0.00 #3 stem regenerate 574-0kR 42 0.11 0.04 (R) inoc. 33 ug/ml 0.06 0.04 91 1.84 0.06 1.84 0.05 #4 Leaf regenerate 574-0kR 135 0 00/0 22 0.02 (R) inoc. 33 ug/ml 0.01/0.21 0.00 #5 Progeny 574-40kR 139 0.04/0.05 0.02 (S1) inoc. 33 ug/ml 0.04/0.06 0.00 aSecond numbers represent samples from older leaves. 39 Table LL Virus multiplication:hidetached leaves of selected commercial plants. One leaf from each plant (#2, 4, 5) in Table 6 which had a low virus titer as determined by ELISA 123 days after inoculation. as well as one leaf from each of 2 control plants. was placed in a petri plate containing 0.8% water agar. At 6 and 13 days. 1:100 dilutions of samples of each leaf were used in ELISA tests. 40 Table 11.a Source of detached 6 days 13 days leaves 541 control (S) 2.41 2.09 2.44 2.17 574 control (S) 1.92 1.94 2.43 2.15 #2 541-30kR (R) 0.09 1.91 0.00 2.20 #4 574-30kR (R) 0.00 0.00 0.00 0.00 #5 574-40kR (81) 0.00 0.00 0.00 0.00 8|ELISA absorbance readings at 405nm. Uninoculated control reading was 0.00. 41 detected in the cutting from plant #4 but the other cuttings were susceptible (Table 12). When plants #2. #3 and #5 were selfed. all 11 progeny of #4 tested gave low ELISA readings. indicating that virus multiplication had not occurred. whereas the progeny of #2. #3 and #5 either showed symptoms or the presence of virus was detected by ELISA in these plants within 9 days. One exception was that of progeny #1 of somaclone #2. in which no virus multiplication was detected 33 days after inoculation (Table 13). No virus multiplication was detected in plant #4, a somaclone regenerated from a leaf disc of line 574. nor in the cutting. detached leaf or progeny experiments involving #4. Table 14 shows the percentages of symptomless plants from each commercial line having low ELISA titers at the time that testing was concluded. Line 574 was the only commercial line in which putatively resistant plants remained. Somaclones of GCRI-26: Disease reaction to inoculation with ToMV. Method of screening. 370 somaclones from 327 separate leaf disc explants of GCRI-26 were inoculated with either purified TMV-Flavum or TMV-YA, or TMV-Flavum- or TMV-YA-infected plant sap. TMV-Flavum infected plants more consistently than did TMV-YAH.Also, in one experiment.10 GCRI-26 seedlings were inoculated with TMV—Flavum— infected plant sap. and 10 seedlings were inoculated with 20 ug/ml purified TMV-Flavum. Inoculation with infected plant sap gave a 90 percent show of symptoms within six days compared to a 10 percent show of symptoms with inoculation with purified virus. so the primary method of inoculation of somaclones of GCRI-26 was to use'TMV-Flavum-infected plant sap. 42 Table 12. Virus multiplication in cuttings of selected commercial plants. Cuttings from nodes of plants which had low virus titers in Table 8 were inoculated with 33 ug/ml ToMV-2A and reinoculated 9 days later. ELISA readings were made in the indicated number of days following inoculation. 43 Table 12.a Source of cuttings 8 days 13 days 17 days 40 days #2 541-30kR 0.02 1.52 2.36 1.89 stem (R) 0.05 1.79 2.44 2.23 #4 574-ORR 0.06 0.00 0.00 0.00 leaf (R) 0.03 0.00 0.01 0.00 #58 574-40kR 0.02 0.16 2.37 1.71 progeny (31) 0.03 0.05 2.25 1.91 #Sb 574-40kR 2.48 2.19 2.44 2.08 progeny (S1) 2.48 2.07 2.47 2.09 aELISA absorbance readings at 405nm. Uninoculated control readings were 0.01. 0.03. 44 Table 13. Progeny tests of selected commercial plants. Selfed progeny of plants #2. 3. 4 and 5 of Table 9 were inoculated with ToMV at 33 ug/ml and assayed for symptom expression or virus multiplication at the indicated number of days following inoculation. Uninoculated control reading was 0.00. 45 Table 13.a seedling #2 (R1) #3(R1) #4(R ) #5(82) 541-30kR 574-ORR 574-0 R 574-30kR 33 days 6 days 6 days 23 days 6 days 1 0.00 1.17 0.00 0.00 1.01 2 b 1.26 0.00 0.05 0.99 3 b 1.09 0.00 0.00 0.90 4 b 0.74 0.00 0.00 0.95 5 b 0.82 0.00 0.02 1.02 6 b 0.80 0.00 0.00 1.00 7 b 1.00 0.00 0.00 1.00 8 b 1.00 0.00 0.00 0.53 9 b 0.93 0.00 0.00 0.97 10 b 1.10 0.00 0.00 1.28 11 b 1.07 0.00 0.00 0.78 12 b 0.70 0.80 13 b 0.91 1.13 14 b 0.84 0.86 15 b 0.78 1.15 16 b 0.65 aELISA absorbance readings at 405nm the indicated number of days after inoculation. bSymptoms observed 9 days following inoculation. 46 Table 14. Percentages of plants from commercial lines with low ELISA values upon completion of screening. Commercial line Irradiated and non- Plants regenerated from irradiated control stem or leaf of seedlings irradiated and control seedlings 541 0 O 569 0 0 574 7a 2 6b 576 0 0 577 0 0 aPercentage represents 3 non-irradiated seedlings (S)cn?43 total; only 4 of the 43 seedlings were irradiated. bPercentage represents one of 38 regenerated plants (R). 47 Selection of somaclones. Table 15 is a list of ELISA readings of symptomless somaclones in which virus titers (as determined by ELISA) remained low for one month after inoculation. All somaclones were reinoculated with ground tissue of Flavum-infected plants (Flavum sapL Symptoms were later observed on somaclones #53C, 343 and 357, and virus multiplication was detected by ELISA, as well as by rub-inoculation of an indicator plant. in the ground plant sap of somaclone #264. Virus inoculation of detached leaves. cuttings and progeny of selected sggaclones. Detached leaves from the 18 somaclones listed in Table 15 were inoculated with ToMV and ELISA readings of these leaves 10 and 19 days post-inoculation are shown in Table 16. Also shown are the results of cutting experiments in which inoculated cuttings were observed for symptom expression and were assayed by ELISA. Virus multiplication was detected in detached leaves of somaclones #53C. 233B and 251. Symptom expression or virus multiplication as determined by ELISA occurred in cuttings of somaclones #12. 27. 44. 53B. 53C, 2338. 257. 324. 330. 331. 343 and 357. Neither symptom expression nor virus multiplication was detected in inoculated detached leaves or cuttings of somaclones #215. 219. 247. 322 and 330. 1:10 dilutions of leaves of these somaclones irl(L01M phosphate buffer caused no necrotic lesions when rub-inoculated onto Nicotiana glutinosa plants. The results of inoculations of progeny (R1) of selfed somaclones #12 and 247 are shown in Table 17. Virus multiplication was not detected in these two somaclones (R). and ELISA readings of inoculated. detached leaves were low or zero for each. However. ELISA readings of an inoculated. rooted cutting of somaclone #12 exceeded 0.50. 15 days after inoculation (Table 16). 30 days following inoculation with TMV— 48 Table 1H1 ELISA readings of selected GCRI-26 somaclones. Conditions of inoculation and ELISA readings of 18 somaclones. selected from a total of 370 inoculated somaclones. are shown. These plants maintained low ELISA readings over a period of one month and through a second inoculation. A 1:20 dilution of TMV- Flavum-infected tomato leaves in CLO1M phosphate buffer was used for reinoculations. Table 15 49 Somaclone 1st inoculation Days post- Day of re- Days post- inoculation: inoculation: inoculation: ELISA ELISA 12 50 ug/ml TMV-YA 37: 0.00 41 51: 0.00 0.00 27 50 ug/ml TMV-YA 37: 0.00 41 49: 0.02 44 20 ug/ml TMV-Flavum 17: 0.00 21 40: 0.00 0.00 538 20 ug/ml TMV-Flavum 17: 0.00 21 31: 0.00 0.00 53c8 20 ug/ml TMV-Flavum 17 (100 21 31:(100 0.00 215 purified TMV-YA 47: 0.00 51 161: 0.03 0.04 219 purified TMV-YA 59: 0.00 63 173: 0.03 0.02 2338 20 ug/ml TMV-Flavum 17: 0.00 21 40: 0.00 0.00 247 20 ug/ml TMV-Flavum 23:0.00 27 35: 0.31 101: 0.03 251 TMV-Flavum sap 15: 0.00 74 93: 0.00 257 50 ug/ml TMV-YA 37: 0.00 41 51: 0.00 0.00 264b 20 ug/ml TMV-Flavum 27: 0.00 31 105: 0.03 0.05 322 50 ug/ml TMV-Flavum 59: 0.00 63 137: 0.04 0.04 324 20 ug/ml TMV-Flavum 23: 0.00 27 35: 0.00 330 20 ug/ml TMV-Flavum 17: 0.00 21 95: 0.04 0.04 331 TMV-Flavum sap 7: 0.00 40 80: 0.03 0.01 0.03 343C TMV-Flavum sap 7: 0.00 40 59: 0.03 0.00 357d TMV-Flavum sap 26: 0.01 40 59: 0.02 0.03 aMosaic symptoms observed 109 days after initial inoculation. bA 1:10 dilution of a plant sample gave an ELISA reading of (L77 and caused necrotic lesions on NTcotiana glutinosa 107 after inoculation of this plant. days CMosaic symptoms observed 82 days after initial inoculation. dMosaic symptoms observed 62 days after initial inoculation. 50 Table 16. Virus multiplication in cuttings and detached leaves of selected GCRI-26 somaclones. Rooted cuttings and detached leaves of somaclones listed in Table 15 were inoculated with a 1:20 dilution of TMV-Flavum-infected tomato leaves in 0.01M phosphate buffer. 51 Table 16. Somaclone ELISA readings of Symptoms (3) observed or designathni detachedleaves ELISA readings of cuttings: 10 and 19 days post- days post-inoculation inoculationa 12 0.00 0.02 2.55: 15 0.00 0.02 2.55 27 0.19 0.19 1.78: 15 0.15 0.14 1.78 44 0.10 0.04 s: 8 0.10 0.02 53B 0.15 0.00 S: 8 0.14 0.00 53C 1.42 0.96 2.55: 15 1.44 0.81 2.55 215 0.00 0.00 0.13: 15 0.00 0.01 0.10 219 0.03 0.05 0.06: 15 0.05 0.04 0.14 233B 2.12 0.73 s: 8 2.22 0.43 247 0.02 0.16 0.08: 15 0.04 0.11 0.10 251 0.15 0.67 0.18: 12 0.12 0.84 257 0.24 0.22 2.55: 15 0.22 0.12 2.55 264 0.20 0.13 0.30: 15 0.23 0.31 0.25 322 0.00 0.00 0.23: 15 0.00 0.01 0.32 324 0.16 0.16 S: 8 0.19 0.15 330 0.00 0.01 0.05: 15 0.01 0.01 0.06 331 0.28 2.52 1.05: 12 0.20 343 0.26 0.18 0.98: 12 0.23 0.30 357 0.09 0.70 0.87: 12 0.10 aHealthy control readings were 0.00. 52 Table 17. Progeny tests of somaclones 0f GCRI-26. 14 day old progeny of one fruit of the selfed somaclone #12 (virus multiplication detected 1T) cutting). and #247 (putatively resistant throughout testing), were inoculated with a 1:20 dilution of TMV-Flavum-infected plant leaves in 0.01M phosphate buffer. 7 day old progeny of the same fruit of #257 were also inoculated with 33ug/ml ToMV-O. Progeny of a known susceptible GCRI-26 somaclone were included. ELISA was performed the indicated number of days following inoculation. 53 Table 17.a ToMV-0 TMV-Flavum sap seedling 26 247 26 12c 247C 1 0.69 0.06 1.43 0.00 0.10 2 0.81 0.13 1.51 0.00 0.00 3 0.88 0.23 1.68 0.00 0.00 4 0.74 0.34 1.25 0.00 0.00 5 0.01 0.77b 0.00 0.00 6 0.11 1.22 0.00 0.00 7 0.03 1.45 0.00 0.00 8 1.33 0.00 0.00 9 0.53b 0.00 0.00 10 1.41 0.00 0.00 11 0.04C 0.00 0.00 12 0.48C 0.00 0.00 13 1.36 0.00 0.56 14 1.11 0.00 0.00 15 0.26b 0.00 16 1.32 17 0.42b 18 1.17 19 1.40 20 1.63 21 1.85 22 1.30 23 1.36 24 1.37 25 1.27 26 0.66b 27 1.42 aProgeny with low readings (0.00-0.10).at 13 days post- reinoculated at 14 days. of the susceptible somaclone inoculation were readings of all progeny (#26). and of progeny were taken at 13 days indicated. of #247 post-inoculation bELISA readings at 20 days post-inoculation. cELISA readings at 30 days post-inoculation. ELISA absorbance inoculated with ToMV-0. unless otherwise 54 Flavum sap, virus multiplication was detected in only one progeny of #247 and in none of the progeny of #12. Readings of 0.23 and 0.34 for samples of #247 progeny inoculated with ToMV-0 suggest that multiplication of the common strain may not be inhibited by these p]ants.‘Virus strain TMV-0 multiplied in the four inoculated progeny of the susceptible somaclone control, and readings exceeding(140 were obtained for 25 of the 27 progeny of a susceptible somaclone control inoculated with TMV-Flavum. Pending further inoculations of progeny of somaclones #12 and 247, and tests of progeny of other plants, somaclones #12. 215. 219. 247, 322 and 330 may possibly have a transmissible type of resistance to multiplication of TMV-Flavum. In vitro screening. Bacterial contamination and browning of leaf discs was encountered in the regeneration of tomatoes from explants of previously-inoculated plants. Thus. only 12 somaclones were regenerated from leaf discs of four seedlings of the commercial line 577 which had been preinoculated with ToMV-2A at 33 ug/ml. The virus had multiplied in all 12 somaclones. as determined by ELISA readings of samples taken directly from culture tubes. Of 24 plants derived from nodes of a single GCRI-26 seedling inoculated in vitro. only 11 had ELISA readings exceeding 0.50. and mosaic symptoms were observed in only one of the clones. Protoplast isolation and culture: GCRI-26. Though many plates of freshly isolated protoplasts in TM-2 medium became contaminated with bacteria. groups of cells presumed to be dividing were observed in two plates approximately 16 days after plating. Aliquots of the media and cell groups were placed on TM-3 medium. where tiny white mini-cal ] i 55 formed. These calli were transferred to TM-4 after 15 days. The calli were subdivided. but all but one piece were lost to contamination. That piece. transferred to TM-4 with 20 g glucose substituted for 30 g glucose (Shahin. 1984). continued to grow, and was placed with leaf discs on shoot-inducing medium (Table 2) after 46 days on TM-4. DISCUSSION The search for disease resistance among somaclones has been most successful with crops which are polyploid and asexual 1y propagated. Sugarcane clones were recovered which had resistance to Fiji virus disease. downy mildew. eyespot disease and culmicolous smut (Evans et a!” 1984). Variations in response to Phytophthora infestans. the fungus causing late blightq and tozltoxin from culture filtrates of Alternaria solani. causing early blight, were found among protoplast— derived clones of potatoes (Shepard. 1980). Unlike the sexually propagated. diploid tomato, asexually propagated polyploid plants can tolerate more chromosomal variation (Evans et 81.. 1984). Carlson (1983) explains that some superior genotypes arising through tissue culture are heterozygous for mutations which would be deleterious in the homozygous condition which would segregate in the progeny of a sexually propogated crop. Nevertheless, in this study, tomato seedlings or somaclones (R) regenerated from leaf discs or stem sections from non-irradiated and a few irradiated donor plants were inoculated with ToMV to evaluate the possibility of finding resistant variants among somaclones of a susceptible cultivar. In screening the R generation. rather than the R1. for the absence of symptoms or virus multiplication. only a dominant mutation to resistance in the chromosomes. a cytoplasmic change or an epigenetic effect preventing virus multiplication would be detected. The chances of obtaining a true-breeding mutation for virus resistance in a particular gene within a large genome such as the tomato would seem to require screening an enormous number of 56 57 regenerated or irradiated plants, therefore the small numbers of irradiated plants and somaclones in this study'may'have limited the search for disease resistance. Somaclones of commercial lines were screened with the ToMV strain ToMV-2A. and somaclones of a known susceptible line were screened with TMV-YA or TMV-Flavum. which provided a model system eliminating the possibility of pre-existing resistance that could be present in commercial lines. The progeny of any putatively resistant somaclone were inoculated to determine whether or not a stable dominant mutation. which would be transmitted, had occurred. Virus chaLacterization by genotype interaction. Based on the linear relationship between log MW and mobilities of proteins in a polyacrylamide gel. the protein seen exclusively in the lane of TMV- Flavum or ToMV-2A-inoculated GCRI-26 (+/+) following polyacrylamide gel electrophoresis was probably the 17,500 MW coat protein described by Matthews (1981). Coat protein was detected in GCRI-26 by ELISA as well. and values for inoculated plants of the other near-isogenic lines were considerably lower. Thus. gel electrophoresis of proteins and ELISA host range using GCRI lines 26. 236. 237 and 267 demonstrated that the resistance genes Tm-i. Tm-2 and Tm-22 prevent multiplication of TMV- Flavum and ToMV-2A. and these strains can be considered to have a similar host range as that of the common strain. ToMV-0 (Hall and Bowes. 1980, Fraser and Loughlin. 1980). Variation in disease reactions amongiseedlings and somaclones of pglngrgial_ljn§§, Overall. because of the limiting number of somaclones and irradiated plants in this study, tomato genotype seemed to have the largest effect on disease response. The 603 stem- 58 regenerated plants of line 577 showed very little variation in disease symptoms following inoculation with ToMV-2A. Nearly all showed mosaic symptoms or virus multiplication was detected within one month. Conversely, among fewer somaclones of line 541 and 574 were found several plants which had low ELISA titers for over one month. Because the plants may have simply 'escaped' infection. control seedlings were inoculated.and cutting and detached leaf inoculations were conducted on the five somaclones in question. Only a leaf-regenerated plant of line 574 seemed to be resistant following these tests. ToMV-2A multiplication seemed to be inhibited in three of 43 non- irradiated 574 seedlings inoculated. though progeny tests were not yet conducted Because the somaclcuue(#4) proving tolerant or resistant through cutting and detached leaf and progeny tests was also of line 574. pre-existing resistance in this line was suspected.4All progeny of #4 tested seemed to be resistant. indicating that some seeds within the seed lot may have been homozygous for a type of resistance. Another possibility is that a cytoplasmically inherited resistance could be present. giving a uniform display of resistance in the selfed seed. Virus multiplication was not detected in one out of 16 progeny of #2. but because somaclone #2 itself was susceptible to ToMV-2A. the R1 seedling was probably not infected due to environmental factors (Fletcher and Macneill. 1971). Though conditions for screening were 25-27 C with 150-200 uEm'zs’1 light. higher temperatures and light intensities may have led to greater virus multiplication and more obvious disease symptoms in susceptible plants. Also. it was apparent that plants must be growing rather rapidly for the most efficient virus screening. 59 If further screening for disease resistance in commercial lines of unknown parentage were to be conducted. disease resistance homogeneity within the original seed selection should not be assumed. Segregation of desirable characteristics may still be occurring; Secondly. if plants of unknown disease-resistance genotype were used for generating somaclones through tissue culture~and screening for resistance. the donor plants should be inoculated after taking the explants to insure that the donor plants were not resistant. Variation in disease reactions among somaclones of GCRI-26. Inoculation of GCRI-26 with TMV-Flavum caused more rapid symptom expression and was a more sensitive screen of susceptible plants than was inoculation with TMV-YA. Also. the virus titer in previously inoculated. symptomatic plants is probably much higher than the concentrations (20 ug/ml) of TMV-Flavum used in inoculations with purified virus. After an initial screening of 370 somaclones of the known susceptible line. GCRI-26. 18 appeared resistant. Once again. the conditions may not have been optimal for symptom expression. Also the ELISA sandwich method using antibody of ToMV-L was not as sensitive to TMV-Flavum as it was to ToMV-2A. Therefore, it was necessary to reinoculate the plants and to carefully check cuttings. detached leaves and progeny for virus susceptibility. Once this had been done. the number of putatively resistant plants was reduced to five. #215, 219. 247. 322 and 330. Progeny tests were not completed on all five plants. but tests for virus multiplication in the progeny of one of the five (#247) were negative in 13 of 14 plants 30 days after inoculation of the seedlings with TMV-Flavum sap. However. ToMV-0 appeared to be multiplying in a few progeny of this somaclone. 60 Surprisingly, TMV-Flavum virus multiplication was not detected among progeny of a somaclone (#12) which tested positive for virus multiplication in a rooted cutting inoculation. Thus. further testing should involve reinoculations. and future work could include the characterization of the type of resistance. Breeding should be conducted to determine the genetic location of the resistance and to determine whether this resistance is novel or whether it is seed of a previously known type of resistance. In vitro screening. Bacterial contamination and browning of virus— inoculated leaf explants occurred during culture. Though this method of tissue culture has succeeded previously to generate virus-free tobacco plants (Murakishi and Carlson. 1982), the frequency of tomato regenerates was very low. and virus was detected in each shoot. Contamination could be reduced by growing donor plants in vitro. but often leaves were thin and easily damaged in the cutting of explants. Inoculation at the level of the somaclones (R) in vitro‘would eliminate the transplanting step in screening. However, the virus did not replicate quickly when plants were inoculated in the culture conditions. and symptoms did not readily appear. making this a poor screen for resistance. Perhaps the lack of disease symptoms was due to low vigor of the plants in culture. which prevents rapid utilization of the hostfs replication mechanisms by the virus inoculum. gglture of GCRI-26 protoplasts. Regeneration of protoplasts from GCRI- 26. which may have provided a large number of 'protoclones' from which to screen for variability. was not successful. Probably the protoplasts had undergone deleterious changes in culture and had lost their totipotency. Protoplast regeneration of this line in the future may be 61 beneficial with the advent of transformation of protoplasts with'naked DNA' (Hein et a1.. 1985). When more is known about virus disease resistance on a molecular level, perhaps the transfer of genes by recombinant techniques may provide an alternative to both breeding and generation of variability through tissue culture in the search for disease resistant plants. For convenience, throughout this work plants regenerated from stem or leaf sections in vitro were called somaclones. Evans et a1. (1984) use 'somaclone' to refer to plants regenerated from cell culture derived from somatic tissue. Larkin and Scowcroft (1981) used the term 'somaclonal variation' to describe variation among plants regenerated at the end of a tissue culture cycle which involved proliferation of a dedifferentiated cell or tissue culture for several generations. Somaclonal variation has been observed in many species. However. in this study, using leaf and stem explants to obtain regenerated plants. could more variation be expected than that which would be found upon simple vegetative propagation? Did the culture conditions lead to the genetic changes(mitotic crossingzoverg organelle variation and sorting) suspected to lead to somaclonal variation? 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