Somaclonal variation and resistance to Verticillium wilt in lucerne, Medicago sativa L., plants regenerated from callus

Plant Science, 58 (1988) 111-119

111

Elsevier Scientific Publishers Ireland Ltd.

SOMACLONAL V A R I A T I O N AND RESISTANCE TO V E R T I C I L L I U M WILT IN LUCERNE, MIcDICAGO S A T I V A L., PLANTS REGENERATED FROM CALLUS

A.O. LATUNDE-DADA* and J ~ . LUCAS**

Department of Botany, University of Nottingham, University Park, Nottingham NG7 ~ D (U..K./ (Received November 17th, 1987) (Revision received June 14th, 1988) (Accepted June 14th, 1988} Somacional variation in dkease reaction type to infection by the vascular writ pathogen VerticiUium albo-atrum Reinke & Berth was assessed in a population of lucerne plants regenerated from callus lines obtained from a susceptible cultivar. Disease severity in the regeneraut population was reduced by comparison with parental controls. Seed progeny and plants recovered via a second tissue culture cycle reverted to mainly susceptible reaction types. In a further experiment a low molecular weight toxic fraction from culture filtrates of the fungus was incorporated into the callus medium prior to regeneration. Toxin treatment reduced the regenerative capacity of callus, and there was little evidence for a higher frequency of wilt resistant plants in populations selected at low toxin concentrations. The results suggest that somacional variation as an alternative breeding strategy for disease resistance in lucerne offers no advantages over conventional recurrent selection.

Key words: Medicago satin. VerticiUium wilt; semacional variation; toxin selection; resistance breeding

Introduction Vascular wilt disease, caused by the fungus Verticillium albo-atrum Reinke & Berth., is a major problem in the cultivation of lucerne (alfalfa), Medica9o sativa L., in temperate Europe [1]. The disease is endemic and has contributed to the fall in hectarage sown to lucerne [2]. Recently the disease has spread to North America and now seriously affects the lucerne crop in several northern states and Canada [3,4]. Lucerne is an outbreeding autotetraploid, and resistance to various diseases and pests has therefore been sought through recurrent phenotypic selection. Resistance to V. alboatrum has been described as polygenic and additive [5]. A number of cultivars with *Present address: Ogun State University, College of Agricultural Sciences, Department of Crop Production, AgoIwoye, PMB 2002, Nigeria. **To whom all correspondence should be addressed. Abbreviations: BAP, 6-bensylaminopurine; IBA, 3-indole butyric acid; MS, Murashige and Skoog; UM, Uchimiya and Murashige.

enhanced resistance to VerticiUium wilt have been bred by mass selection and hybridization with M. hemicycla and M. gaetulo; however, these cultivars still contain a significant proportion of susceptible genotypes [6], and a continued search for higher levels of resistance to the disease is therefore necessary. Advances in plant cell and tissue culture have provided new methods for generating and selecting variability at the cellular level. Novel variation can be recovered in plants regenerated from non-mutagenized protoplasts and cultured cells [7,8]. In lucerne, plants regenerated from mesophyll protopiasts are morphologically variable and also show enhanced resistance to VerticiUium wilt [9]. Somaclonal variants possessing improved pest and disease resistance have been reported in other crops such as potato and sugarcane [10-13]. Significant shifts in disease reaction type may be achieved in the absence of any in vitro selection for resistance. An alternative approach is to incorporate toxins of pathogen origin in selection media to facilitate the recovery of disease

0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

112

resistant regenerants from toxin-tolerant cell lines. Recently, Fusarium wilt resistant regenerants of lucerne have been obtained via such toxin selection [14]. Culture filtrates from Verticillium species also contain toxic components [15,16] and these have been used to screen for increased resistance in lucerne seedling populations [17-19], as well as in hop tissue cultures [20]. The present study set out to assess the level of VerticiUium wilt resistance in populations of regenerants obtained from lucerne callus lines derived from the wilt susceptible cultivar Europe. Regenerants were recovered from callus maintained either on a nutrient medium, or the same medium containing a toxic fraction from culture filtrates of the fungus. The heritability of resistance identified in selected regenerants was assessed in progeny from seed, as well as in a further population of regenerants recovered from a second tissue culture cycle.

Leaf explants

f r o m 1 0 0 s e e d l i n g s cv. E u r o p e

1° t i s s u e c u l t u r e c y c l e

122

callicloned r e g e n e r a n t s

/ 2°tissue

culture cycle

; R1

\ Stem c u t t i n g s

produce

,,\

to

seed

/s,

I n o c u l a t e with V. a l b o - a t r u m

Assess

disease

severity

Fig. 1. Experimental design for assessment of somaclonal variation in wilt reaction type.

Methods

Plant material The commercial lucerne cultivar Europe {Elsores Seeds, Spalding, Lincolnshire) was used throughout the study. This cultivar was chosen as it is susceptible to Verticillium wilt with approximately 850/0 of individuals developing moderate to severe symptoms in growth room tests [6]. An earlier assessment of variation in protoclones was also based on this cultivar [9]. Seeds were sown in soilless compost (EFF Products Ltd) in compartmentalized plastic trays and maintained in a growth room (day, 21°C; night, 17 °C; 16-h day-length provided by 6 5 - 8 5 W fluorescent tubes at photon flux density 80 ~anol m -2 s-l). Callus induction and regeneration (Experiment 1) The overall scheme for this experiment is shown in Fig. 1. Leaves were excised from 100, 4-week old plants, surface sterilized for 10 rain in 5% Domestos bleach {Lever Bros., UK), washed in six changes of sterile distilled water, and the abaxial surface of each leaf was peeled off aseptically. The leaves were then incubated, peeled surface down, on a callus induction medium (Uchimiya and Murashige) (UM) [21] containing 0.25 mg]l kinetin and 2 mgh 2,4-D. Incubation was conducted at 25°C in continuous light for 4 weeks, during which regeneration was initiated. Green nodular regions were transferred to a regeneration medium based on that of Murashige and Skoog (MS) [22], with 0.05 mg/l IBA and 0.5 mg/l BAP {designated MSIBA). Shoots and embryoids induced on MSIBA were transferred to a hormone-free agar medium (MSO; 22). Any plantlets which developed were subsequently transferred to compost and hardened off in a humid chamber in the growth room. These regenerant plants were designated the R1 population. Prior to inoculation with V. albo-atrum, leaf explants were taken from each R1 plant and used to initiate a secondary tissue culture cycle, as described above. Regenerants from

113

this cycle were designated the R2 population. In addition, stem cuttings were taken from each R1 plant, and rooted by incubation in the growth room in a hydroponics tank containing continuously aerated water. These plants were grown on to flowering, selfed, and the seed progeny designated the $1 population. Plants from each population were then tested for reaction to inoculation with V. albo-atrum. Control plants were grown from the parental seed batch. Seed~ierived plants were tested when 4 - 5 weeks old, and regenerants once they reached a similar size and stage of development.

Fungal material The isolate of VerticiUium albo-atrum used was originally isolated from a lucerne crop at the Plant Breeding Institute, Cambridge. The fungus was maintained on Czapek Dox agar medium at 20 °C. Virulence of the isolate was checked at regular intervals by passage through lucerne seedlings. Inoculation Inoculation cultures were prepared by seeding a sterile mixture (5:1 v/v) of vermiculite and Czapek Dox liquid medium in 100 ml flasks with a spore suspension of V. albo-atrum. The flasks were maintained at 20 °C in the dark for 2 weeks. The contents of the flasks were then emptied into sterile distilled water, stirred and filtered through 4 layers of muslin. The concentration of the resulting suspension was adjusted to 4 × 106 spores ml by haemocytometer count and dilution. Routine inoculation of regenerant, seed~lerived, and control plants was carried out by cutting shoots with a sterile scalpel 8 cm above soil level and applying spore suspension to the wound. An alternative inoculation procedure, used as a check, was to uproot seedlings, wash the roots free of compost, and dip them in the spore suspension for 15 rain prior to repotting in sterile compost [6]. All inoculated plants were subsequently maintained in the growth room under the conditions described above. The development of wilt symptoms was monitored over an 8-week

period with individual plants scored weekly according to the following disease scale: 0 = no symptoms; 0.5 = symptoms limited to 1 - 2 basal leaves; I = symptoms on < 25% of foliage; 2 - symptoms on > 25% but < 50% foliage; 3 = symptoms on approx. 50% foliage; 4 = symptoms on > 50% foliage; 5 = whole plant chlorotic/necrotic except for apex; 6 = plant dead. Mean disease scores were obtained for each population. At the end of the assessment period, plants with scores 0 - 2 were classed as resistant, and the remainder susceptible.

Toxin production Shake cultures of V. albo-atrum were initiated by inoculating Czapek Dox liquid medium (300 ml in 1-1Erlenmeyer flasks) with agar blocks cut from plate cultures of the fungus. The flasks were maintained for 2 weeks in the dark at 20 °C on a rotary shaker at 120 rev./min. Cultures were harvested by filtering through 4 layers of Whatman No. 1 filter paper in a Buchner apparatus to remove the bulk of fungal spores and mycelium. The filtrate was then centrifuged at 1500 × g for 20 min, and concentrated 10-fold in a rotary evaporator at 30 °C. The concentrate was passed through a 0.25-~m Millipore membrane and stored at - 20 °C until required for chromatography. Alternatively, following centrifugation the crude filtrate was evaporated to dryness and the residue taken up in 800/0 (v/v) ethanol. The insoluble fraction was removed by centrifugation and the supernatant made ethanol-free by evaporation in vacuo. The residue was then brought up to 1/10 the original volume in distilled water, filtered through a 0.25-~m Millipore membrane, and stored at 20 °C. Fractionation of the crude culture filtrate was carried out by passing samples through a column of Sephadex G-50 (Sigma fine grade) and further separating the single low molecular weight peak eluted from this column on Sephadex G-15 (Sigma). Columns measured 25 x 2.5 cm and were eluted with deionized water at a flow rate of 8.2 ml/h. The absorbance of the eluate was monitored with a Uvicord II 8300 -

114

UV analyser with chart recorder (LKB, Sweden), and 4 ml fractions were collected on a LKB UltroRac fraction collector. All operations were carried out at 4°C. The eluate from the Sephadex G-15 column contained 4 peaks on the basis of UV absorption, and fractions under the same peak were pooled and stored at - 2 0 °C. The column was calibrated with PEG 400 (Sigma) and fusaric acid (Sigma; tool. wt., 279). Carbohydrate content of pooled fractions was determined by an anthrone method using glucose as standard [23], and fractions were also tested for reactivity to the Fofin-Ciocalteu reagent [24] using lysozyme as standard. Toxicity of pooled fractions was determined by several methods, including bioassays based on inhibition of seed germination or radicle elongation, wilting of seedlings, and effects on callus growth and the viability of suspension cultured cells [18]. The most toxic fraction was then used in the selection experiment.

Toxin selection tEzperiment 2) The overall scheme for this experiment is Wilt susceptible genotype cv. Europe

Callus lines established

/

\

Callus m e d i u m

Callus m e d i u m

plus t o x i c f r a c t i o n

(3 t r a n s f e r s )

(3 t r a n s f e r s )

Regeneration

T o x i n - s e l e c t e d regenerants

\

Regeneration

Control regenerants

/

Inoculate with V. s l b o - e t r u m

Assess disease s e v e r i t y Fig. 2.

Experimental scheme for toxin selection.

shown in Fig. 2. Three highly regenerative callus fines (coded E8, E14 and E29) derived from wilt-susceptible genotypes scoring 6 in inoculation tests were used for the experiment. Toxincontaining media were prepared by substituting the toxic fraction for distilled water in the UM callus maintenance medium. Toxic media were used at five different concentrations, 1 × (10% substitution}, 2 × (20% substitution), 3)< (30% substitution), 4 × (40% substitution), and 5 × (50°/0 substitution). Duplicate 3-week-old callus cultures (approx. 5 mm diameter} of each line were maintained on the toxic media for 3 weeks, after which surviving sectors were subcultured onto fresh toxic media of the same concentration. A total of three transfers was made, followed by regeneration on MSIBA medium. Control calfi were cultured with equivalent transfers over the same period of time on toxin-free UM medium. The viability of callus cells was estimated by a tetrazofium salt reduction method [25]. Cell suspensions were prepared by agitating small pieces of callus in 4 ml UM liquid medium in screw capped Bijou bottles. Three ml of the cell suspension was then added to 3 ml of a 1% w/v solution of tetrazofium chloride in 0.05 M phosphate buffer (pH 7.5), and the mixture incubated for 18 h in the dark at 20°C. Cells were then examined microscopically and those stained red, through reduction of tetrazolium salt to formazan, were scored as viable. Plantlets regenerating from toxin-treated and control calli were recovered and grown on as described above under Callus induction and regeneration. Toxin-selected and control plants were then inoculated with V. albo-atrum and scored for disease severity as described above.

Karyotype and guard cell measurements Chromosome numbers were determined by root-tip squashes from a sample of regenerant plants from Experiments 1 and 2. Root-tips were pretreated for 4 h in a saturated solution of 2,4-dichlorobenzene, fixed in 3.1 (v/v) ethanolglacial acetic acid, and stored in this fixative at - 20 °C. Root samples were then hydrolysed in 1 N HC1 for 6--10 min at 60 °C and stained for

115

2 h in leucobasic fuchsin (Feulgen, Ref. 9.6). Squashes were made in 45% acetic acid and chromosome counts determined for ceils in metaphase. Guard cell lengths were estimated by microscopic measurement of leaf impressions obtained by coating the abaxial surface of leaves with cellulose nitrate in amyl acetate (Humbrol, Hull, U.K.). At least 10 estimations were made from each of 3 leaflets of each plant. Results Ezperiment 1 0nly 5% of the original 100 explants from cv. Europe seedlings gave rise to regenerative callus. A total of 19.9.regenerants were recovered from these lines (Fig. 1). These R1 plants were morphologically normal and all set ample numbers of seed, in contrast to protocloned lucerne populations which contain a large proportion of protoclonal lucerene populations which contain a large proportion of aberrant and infertile individuals [9]. Measurements of guard ceil lengths suggested that 56% of R1 plants might be of increased ploidy. However, karyotype analysis of a representative sample of these plants

revealed the normal 2n = 4o: = 39. chromosome number. In the secondary tissue culture cycle (Fig. 1) approx. 30% of R1 plants gave rise to regenerative callus, confirming the feasibility of selecting for this character in lucerne [27]. Figure 3 shows the progress of VerticiUium writ in inoculated R1 plants compared with the parental seed batch control. By the end of the experiment 61% of R1 plants were scored as resistant; this compared with 15% and 20% in control populations inoculated by foot,lipping or wounding respectively (Fig. 4). A similar shift towards resistance was observed in R1 populations produced in Experiment 2 as controls without toxin selection (Fig. 6). In this case the source plants had all been rated as highly susceptible (writ score 6) in inoculation tests. In contrast, a large majority of R2 plants recovered from a secondary tissue culture cycle developed severe writ symptoms (Table I).

e

wi

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WEEKS AFTER INOCULATION

Fig. 3. Progress of writ in the regonerant (RI) population in comparison with a parental seed batch control ev. Europe. Mean disease scores + S.E.

1

2

3

4

5

6

WILT SCORE

Fig. 4. Spectrum of disease reaction types in regenerant (R1) and parental populations 8 weeks after wound inoculation (WI) or root dip fRD) treatment with VerticiUiunL Columns show number of plants in each writ score category.

116 Table I. Distribution of wilt scores in r e g e n e r a n t s derived from a secondary callus cycle. Original R1 plant writ score

R2 total recovered

11 8 4 4 1 5 9 3 3 4 1 3 1 1 2 14 5 2

Total

81

R1 w i l t score

score

N u m b e r of R2 plants with wilt score 0

6 4 4 4 3.5 3 3 3 2.5 2 2 2 2 1.5 1 1 1 1

R 1 wilt

1

1

2

3

4

5

2

2

--

1

1

1

2

--

1

2

1

6 6

55

1"5

5

1

2

--

--

4 1 1 5 5 3 3 4 1

S

1

45 1 1

I -

1

--

1

0

4

9

6

4

1

12 4 2 57

There was little correlation between R1 source plant and R2 regenerant disease scores, and the overall spectrum of reaction types appeared to have reverted to the original parental type with only 16o/o being classed as resistant. The seed progeny (S1) of 5 susceptible and 5 resistant selfed R1 plants were wound-inoculated with V. albo-atrum. Figure 5 shows the spectrum of disease scores for each of these 10 populations. In the majority of cases a large proportion of plants developed severe symptoms, suggesting segregation in favour of susceptibility. In two populations, however, both derived from resistant R1 parents, there was a preponderance of resistant individuals. All S1 plants were fertile and further selection for resistance in these lines should be feasible.

Experiment 2: Tozin selection Fraetionation of crude culture filtrate on Sephadex G-50 yielded two main peaks when

0"5 O

6

0

6

Spectrum of disease reaction t y p e s in seed progeny derived from individual r e s i s t a n t and susceptible r e g e n e r a n t (R1) plants. The original wilt scores of each r e g e n e r a n t p a r e n t are indicated.

absorbance was monitored at 280 nm; a high molecular weight peak > 30 000 Da eluting at the end of the void volume, and a low molecular weight peak eluting in the inclusion volume of the column. When further separated on Sephadex G-15, the latter was resolved into 4 peaks with approximate molecular weights as follows: Peak I 1500, Peak II 400, Peak III 200, Peak IV 100. Pooled fractions under each peak were tested in bioassays for inhibition of seed germination on soaked filter paper, inhibition of radicle elongation, and yellowing and wilting of excised shoots [18]. Only Peaks II and III showed toxic activity, with Peak II being the more potent. This fraction also showed lethal activity to suspension cultured lucerne cells, and was used thereafter for the toxin selection experiment. Peak II contained carbohydrate, as determined by the anthrone method, and was also Folin positive, indicating the presence of aromatic amino acids. This latter property was

117

Table II. Effect of different toxin concentrations on growthand viabilityof lucerne callus. Medium

Initial callus diameter

Final callus diameter"

Viabilityb

20.0 ± 0 21.7 ± 1.7 13.0± 1 5 5

+ + + + --

30"

Control

X

3X n:34

(ram) ~ 10'

UM(control) UM + 1 x UM + 2 x UM+4x UM + 5 x

5 5 5 5 5

•Measurementstaken 4 weeks after exposure using3 replicate calli. ~Cellviabilitydeterminedby the tetrazoliumsalt reduction method[25].

used as an indirect means of quantifying the toxic fraction, although in the absence of any definitive chemical characterization the relationship between Folin reactivity and toxin content is not known. Based on calibration curves with lysozyme as the standard the 1 x , 2 × and 3 × levels of toxin were equivalent to 65, 132, and 165 ~g 'protein' per m1-1. The toxic fraction reduced callus growth at 2 x concentration, and inhibited growth completely at 4 x concentration, although viability tests showed that surviving sectors could be recovered (Table II). The toxin also reduced regenerative capacity to the extent that in two of the three callus lines used no regenerants were recovered even at the lowest concentration used for toxin selection. In the third line 44 plants were recovered from callus exposed to I x concentration, 48 from 2 x , 34 from 3 x and a single regenerant from 4 x . In addition a total of 41 unselected plants were regenerated from callus on toxin-free control medium. Results of inoculation tests on these populations are shown in Fig. 6. The proportion of susceptible reaction types was again reduced in regenerant versus parental control populations, but the majority of plants recovered from the 1 x and 2 x selection proved to be susceptible. The only evidence for enhanced writ resistance in toxin-selected populations was observed in the 3 x sample where some plants scored 0. However, sample sizes in

/ 30 t U n s e l e c t e d

i

20

2X

4X

n,41

; I°I_~ 0

Fig. ~

n=l

mm

6

0

6

0

6

Spectrum of d~e--e rea~ion types i . an Um.~

lected regenerant population, and populations selected at different toxin concentration (1 x--4 x ), compared with a parental seed batch control.

this experiment were too small to draw valid conclusions on the benefits, if any, of toxin selection at the callus stage. Discussion Regenerant populations obtained from callus differed markedly from protocloned lucerne populations in the low frequency of morphological variants. A majority of protoclones from cv. Europe possessed abnormalities such as multifollate leaves and stunting, correlated in most cases with increased ploidy [9]. Estimates of guard cell measurements, a parameter often linked with ploidy [28], suggested that approximately half of the callicloned plants recovered were of higher ploidy, but this was not confirmed by chromosome counts. The preponderance of hexaploid and octaploid regenerants (or near aneuploids) in protoclones may reflect the more extended period spent in culture; Hartmann et al. [14] found that lucerne regenerants from long term culture had elevated ploidy levels, whereas a

118

shorter selection cycle gave regenerants with the normal tetraploid chromosome number. Inoculation tests with V. albo-atrum showed that the proportion of plants developing severe wilt symptoms was substantially reduced among regenerants from callus by comparison with original seed~terived controls. This result was also confirmed in the toxin-selection experiment where the original source plant was highly susceptible. It is not known whether the reduced symptom severity reflects resistance per se or increased tolerance to invasion by the fungus. A similar shift in the overall disease resistance spectrum of a regenerant population has been observed in potatoes infected by common scab [29]. The enhanced wilt resistance of R1 plants appeared to be lost in regenerants recovered from a second culture cycle. Other authors [12] have suggested that a 'stacking' of desirable characteristics might be achieved through further cycles of culture. Our results indicate a reversion to parental type, and suggest that the initial change in resistance might in part be epigenetic. Numerous genetic mechanisms for somaclonal variation have been advanced, including gene amplification and unstable mutations, as well as heritable genetic changes [13]. As the genetic basis of VerticiUium wilt resistance in lucerne is only partly understood [5], the mechanisms of enhanced resistance in somacloned populations remains speculative. To determine the heritability of useful traits in somaclones, sexual progeny need to be examined [13]. Assessment of seifed progeny from R1 plants exhibiting a representative range of wilt reaction types indicated that the majority were susceptible to V. albo-atrurn. This result is to some extent predictable in view of the heterozygosity of an autotetraploid outbreeding crop such as lucerne, particularly when the source cultivar for the study was itself highly susceptible. However, a few progeny populations contained an increased proportion of resistant individuals and seifed seed from these has been collected to further analyse segregation for wilt reaction type in the next generation.

Previous studies [17--19] have suggested that exposure of lucerne seedlings to culture filtrates from V. albo-atrum may select plants with improved resistance to the pathogen in glasshouse and field tests. Our programme of toxin selection at the cell level has confirmed the existence of biologically active low molecular weight fractions from V. albo-atrum, but the potential value of such selection remains unresolved. The inhibitory effects of the toxin(s) on regenerative capacity, coupled with the shift towards resistance in unselected control regenerants, may have masked any beneficial effects of the selection regime. Overall, however, there was little evidence for improved resistance in the selected population. Further progress may rely on characterization of the toxins involved and clarification of their role in pathogenesis. Other workers now claim to have identified specific low molecular weight toxins from VerticiUium species [15,16,20]. The appeal of somaclonal variation as a breeding strategy is that it can be used to recover at high frequency natural genetic variability from existing crop varieties [13]. The present study suggests that this also applies to lucerne. However, the potential benefits are hampered by the complex genetics and outbreeding characteristics of this seed-propagated legume. Improvements in writ-resistance noted in somaclonal regenerants were lost in sexual progeny from these plants. It is questionable, therefore, whether somarloning offers any advantages over recurrent phenotypic selection as a procedure for resistance breeding in this crop. Aclmowl~lgemento The authors thank the Agricultural and Food Research Council for a grant in support of this work. References J.B. Heale, Vertiefilium writ of alfalfa, background and current research. Can. J. Plant Pathol., 7 (1985) 191198.

119 2

8 4

5

6

7

8

GAt.Dixon, Lucerne diseases - with particular reference to varietal centrol of writ. NIAB Fellows Conf. Pep., 1 (1977)4 - 1 1 . A~,. Christen and Ra'q. Peaden, Vertieillium writ in alfalfa. Plant Dis., 65 (1981) 319-321. D.C. Amy and CAt. Gran, Importance of Vertieillium writ of alfalfa in North America. Can. J. Plant Pathol., 7 (1985)187-190. C~.. Panton, The breeding of lucerne, Medicago sat/m L. for resistance to Vert/ciUium aibo-atrnm Rke. et Berth 1I. The quantitative nature of the genetic mechanism controllingresistance in inbred and hybrid generations. Aeta Agrie. Seand,, 17 (1967)4 8 - 52. A.O.Latunde-Dada and J.A. Lueas, Variation in resistance to Vert/ei~vm wilt within seedling populations of some varieties of lucerne (Medicago eat/m). Plant Pathol., 31 (1982)179-186. R.I.S. Brettoll and D.S. Ingrain, Tissue culture in the production of novel disease resistant erep plants. Biol. Rev., 54 (1979)3 2 9 - 848. P3. Larkin and WAt. Sceweroft, Somaelonal variation a novel source of variability from cell cultures for plant improvement. Theer. Appl. Genet., 60 (1981) 197 214. A.O. Latundo-Dada and J.A. Lueas, Somac]onal variation and ruction to Ve~-//B#m w/It in M e d i c o sat/m L. plants regenerated from protopluts. Plant Sei. Lott., 32 (1988}205--211. JAt. Shepard, Protopluts as sources of disease resistance in plants. Annu. Rev. Phytopathol., 19 (1981) 145 161. D•. Heinz, M. Krishnamurthi, L.G. Niekell and A. Maretzaki, Cell tissue and organ culture in sugarcane improvement, in: J. Peinert and Y.P.S. Bajaj (Eds.), Applied and Fundamental Aspects of Plant Cell Tissue, and Organ Culture, Springer-Verlag, Berlin, 1977, pp. 1-17. P J . Lark,in and WAt. Scowcroft, Somaclonal variation and eyespot toxin tolerance in sugarcane. Plant Cell Tissue Organ. Culture, 2 (1988) U l - 121. D~,. Evans and WAt. Sharp, Applications of somaelonal variation. Bioteehnology, 4 (1986)526- 532. C.L. Hartman, T.J. McCoy and TAt. Knous, Selection of alfalfa (Medicago sat/m) cell Lines and regeneration of plants resistant to the toxin(s) produced by Fugari#m ozyspornm Lsp. medicagi~tie. Plant Sei. Lett., 24 (1984) 183-194. A. Naehmins, V. Buchnor and Y. Burstoin, Biological and immunoehemieal characterization of a low molecular weight phytotoxin isolated from a protein-lipopolyseecharide complex produced by a potato isolate of Vert/ciliiv~m daAliae Kleb. Physiol. Plant Pathol., 26 (1985) 48-- 55. -

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13 14

15

16 A. Naehmias, V. Buchnor, L. Tsror, Y. Burstein and N. Keen, DiHorential phytotoxieity of poptides from culture fluids of VevticiU~#m d~/~ns races 1 and 2 and their relationsh/p to pathogenic/ty of the fungi on tomato. Phytopatbology, 7"/(1987)506- 510. 17 S~I. Michall and A,J.H. Cart, Use of culture filtrates as a rapid technique for screening lucerne for resistsnce to VerticiUi#m albo-atrum. Trans. Br. Mycol. Soe., 49 (1966) 135-138. 18 A.O. LatundwDada, The interaction of Vert/ciUium albo-atrum Reinke & Berthold with plants and tissue cultures of lucerne (Medicago sat/m L.), Ph.D. Thesis, University of Nottingham, 1988. 19 K~. Ireland and K.T. Leath, Potential of using culture filtrates from Vert/cillium aZbo-atrum to evaluate alfalfa germ p l u m for resistance to Vertieillium writ. Piant Dis., 71 (1987)900-903. 20 S.A. Connell and J.B. Heale, Development of an in v/fro selection system for novel sources of resistance to Vert/ciUi#m writ in hops, in: L. Withers and P.G. Anderson (Eds.), Tissue Culture and Agriculture, Butterworths, 1986, pp. 451 - 459. 21 H. Uchimiya and T. Murashige, Evaluation of parameters in the isolation of viable protopluts from cultured tobacco cells. Plant Physiol., 54 (1974)936 - 944. 22 T. Murashige and F. Skoog, A revised medium for rapid growth and bioasaays with tobacco cultures. Physiol. Plant, 15 (1982)478-497. 23 G.E. Hodge and B.T. Hofreiter, Determination of reducing sugars and carbohydrates, in: R.L. Whistler and M.L. Wolfrom (Eds.), Methods in Carbohydrate Chemistry, VUl. I. Academic Press, New York, 1962, pp. 380-394. 24 O.H.Lowry, NJ. Rosebrough, A.L. Farr and R.J. Randell, Protein measurement with the Folin phenol reagent. J. Biol. Chem., 198 (1651)265- 275. 25 L.E. Towill and P. Mazur, Studies on the reduction of 2,3,5-triphenyltetrasolium chloride as a viability assay for plant tissue cultures. Can. J. Bot., 53 (1975) 10971102. 26 L.C. Coleman, Preparation of leueobnsic fuchsin for use in the Feulgen reaction. Stain Teehnol., 13 (1988) 123-124. 27 E.T. Bingham, L.V. Hurley, D.M. Kaatz and J.W. Saunders, Breeding alfalfa which regenerates from callus tissue culture. Crop Sei., 15 (1975) 719-721. 26 C. North, Plant Breeding and Genetics in Horticulture, Macmillan, London, 1979, pp. 1 - 150. 29 N.E. Evans, D. Foulger, L. Farrar and S.W.J. Bright, Somaelonal variation in explant-derived potato clones over three tuber generations. Euphytiea, 35 (1986) 353 --381.