Agriculture 69, 35 ical distinetion s. Proc. Br. Insect. Fi sugar beet seed crops lication of insecticide 6. Ann. appl. Biol. 694 ws insecticides in Mysu ent. Res. 6, 191-6, Towing viruses in sugad pan opp. Bi 1968), 62, 119-128 intl in Great Britain ‘The elimination of four viruses from carnation and sweet william by meristem-tip culture By OLWEN M. STONE Glasshouse Crops Research Institute, Littlehampton, Sussex (Received 19 February 1968) SUMMARY Carnation mottle virus (CarMY), carnation ringspot virus (CRSY), carna- tion vein mottle virus (CarVMV) and carnation latent virus (CarLV) were all SHmunated from both carnation (Dianthus caryophyllus) and sweet william {D. barbatus) plants by meristem-tip culture. Camation motte virus was ore readily eliminated from D. barbatus than from carnation, Carnation vein ‘hottie and carnation latent viruses were more readily eliminated from Tarnation than from sweet william: they are rarely found in carnation but CarVMV is found frequently in sweet william. Carnation ringspot was climinated equally readily from both hosts. INTRODUCTION Meristem-tip culture is now widely used for the production of ‘virus-free’ clones ofcarnation and other crops. Itis often assumed by commercial growers that all viruses are equally readily eliminated from carnation, but this is not so. Carnation viruses differ greatly in the ease with which they can be eliminated from their host plants. This paper compares the elimination of four viruses from two host plants. The viruses were: carnation mottle (CaMV), carnation ringspot (CRSV), carnation vein mottle (CarVMV) and carnation latent (CarLV). The plants were carnation (Dianthus caryophyllus L.) and sweet william (D. barbatus L.). MATERIALS AND METHODS The viruses were cultured in four healthy clones of D. barbatus, derived from seedlings and selected to give the highest concentration of the particular virus. CarVMV and CarLV were transmitted to these plants by Mysus persicae f. dianthi (Ghrank) from infected D. barbatus, CRSV and CarMV were transmitted by mechanical inoculation with purified virus preparations. Virus-tested clones of carnation cultivars Joker and Ashington Pink, obtained by meristem-tip culture and heat treatment, were used to culture CarMV, CRSV and CarLV; purified preparations of CarMV and CRSV were inoculated mechanically, CarLV was inoculated by inarch grafting with infected D. barbatus. CarVMV was cultured in a seedling clone of carnation selected from twenty for clear symptom expression and high virus concentration; it was in- fected by inarch grafting with infected D. barbatus. ‘The presence and identity of the Viruses was checked in all plants before cutting meristem-tips and again at the end of120 Oxwen M. Stone the experiment. CRSV, CarMV and CarVMV were detected by inoculation Chenopodium amaranticolor Coste & Reyn and C. quinoa Willd., and CarLV wag] inoculated to C. quinoa; CRSV, CarMV and CarLV were also checked serologicaly The plants produced from meristem-tips were similarly tested. ‘All plants were grown in an insect-proof glasshouse and the infected plants w not heat-treated before excision of the meristem-tips. The meristem-tips were cultured as previously described (Stone, 1963), using Neergaard’s medium with the, addition of 10~ g/l of myo-inositol, and a-naphthalene acetic acid as rooting hormone, Filter-paper wicks were used throughout, in Monax tubes (75 em long, 1-5 em diameter) closed with Oxoid aluminium caps. "Approximately 100 meristem-tips were cut for each virus-host combination; ‘were not surface-sterilized before excision of the tips but all work was done in a Merle culture room. The size of each meristem-tip taken was measured and its posi- tion on the shoot noted. Tips showing no growth after 6 weeks culture were discarded. RESULTS Dianthus barbatus It proved easier to culture meristem-tips from this plant than from carnation and about half the meristem-tips survived in each of four experiments. "There were considerable differences in the ease of climinating the different viruses but no appreciable differences in the survival rate of meristem-tips cut from plants infected with them (Table 2). With CarMV, CarVMY and CarLV the proportion of ‘Table 1. Meristem-tip survival and elimination of virus from Dianthus barbatus No. No. No. No. No, Percentage Percentage ‘ot rooted — potted survived infected survived® virus-freet 109 s 4 96 7 st 103 7 2 or * Ps % + Based on number of surviving/number eut. + Based on number surviving. virus-free plants produced increased with decreasing size of tip taken, but there was ho correlation with the position of the tip on the shoot. By contrast, tips from the Iower buds on shoots of CRSV-infected plants were most likely to remain infected, regardless of size. OF the nine infected plants produced, four were from the lowest ud on the shoot, three from the second lowest and one each from the third and fourth axils; the usual number of buds per shoot was 7-9. (Of forty-one CarMV-infected plants produced from meristem-tips, thirteen con~ tained the attenuated form of the virus (Hollings & Stone, 1962), and this occurred mainly in plants raised from the smaller meristem-tips, Thus, twelve of twenty-two tips of size 02-0-4 mm, but only one of fifteen of size o-4-0'6 mm had the attenuated type of virus, whereas all four of size o-6-0'8 mm had typical CarMV.by inoculation and CarLV ecked serologically ected plants werd neristem-tips s medium with th \s rooting hormone sem long, 15 em ombination; shootg ork was done in vsured and its posi ure were discarded from carnation and he different virusea ips cut from plant Percentage Percentage survived” virusfret st 4 st % ” 36 6 5 aken, but there was) trast, tips from th to remain infect sre from the lowes the third and fo tips, thirteen o and this oc¢ Virus elimination by meristem-tip culture Carnation Fewer meristem-tips survived from carnation than from sweet william, the greatest tox occurring after transfer ofthe rooted tips to sol. ‘The numbers surviving wore ss a to those obtained in previous experiments from seedling infected with CarMWV ‘Stone, 1963); survival then was 26% and 27% for two seedling clones, ‘The results ire summarized in Table 2. ‘Table 2. Meristem-tip survival and elimination of virus from Dianthus caryophyllus No, No. No. -—'No,__No._ Percentage Percentage Virus No jauted potted survived infected survived® virus-frect canal 10 & 6 2 6 26 10 RSV “7 no 0 25 4 6 % GavMV 100 8 7% 6 4 6 88 Cork 108 o 8 4 7 2 7 + Based on number survivinginumber cut. Based on number surviving The postion of the bud on the carnation stem appeared to have no effect on the proportion of surviving explants that were still infected, with any ofthe four viruses, he proportion of virus-free plants increased with decreasing size of meristem-tip for hoth CarMV and CarLV, but the number of CRSV- and CarVMV-infected plants tas too small for any inference to be drawn. Unlike the result from D. barbatus, Veenuated CarMV was found in only one carnation plant, which derived from one of the smallest tips (o-2-'4 mm) and was one of eighteen infected plants inthis category “Thus, of the four viruses tested, CRSV was equally readily eliminated from D. barbatus and from carnation by meristem-tip culture, CarMV was more readily eliminated from D. barbatus than from carnation, and CarVMV and CarLV were ‘nore readily eliminated from carnation than from D. barbatus. Plants of both species can be freed from all four viruses provided that enough meristems are cut. Thorough testing of the plants is also necessary to ensure detection of the attenuated form of CaMV DIscUssION “The differences in ease of elimination of the viruses in the two hosts are interesting. Not all clones of D. barbatus can be infected with CarMV (Hollings & Stone, 1964); the clone used in this work was the best of twelve tested for susceptibility and high virus concentration, and at the end of the experiment the mother plant was still infected. Yet the percentage of virus-free plants obtained was more than twice that ion and, of those infected, one-third contained only the attenuated form of CarMV in very low concentration. CarMV is widely distributed in commercial carnation crops but is not recorded as being common in D. barbatus; it may therefore be primarily a carnation virus which can also infect other hosts in the Caryophyllaceae. ‘The status of CRSV is less clear; it is less infectious to D. barbatus and to other species of Dianthus than to carnation, although equally readily eliminated from the two species tested. Its conspicuous symptoms in carnation ensure that it is easilyOwen M. Stone ‘identified and rogued from commercial stocks and therefore not spread by intensive ‘vegetative propagation of these stocks. By contrast, both CarVMV and CarLV were much more readily eliminated from ‘carnation than from D. barbatus. CarVMV occurs commonly every year in D. barbatud in gardens and it is possible that CarLV may also occur in this host, for alone it ig symptomless. When present in mixed infections with CarVMV it merely intensifies the symptoms of the latter and is probably overlooked. Both viruses are seldom recorded in commercial crops of carnation. We have found CarLV once in 8 years at this Institute and have not so far found CarVMV. CarVMV causes mild symptoms in carnation and CarLV is symptomless, so that they are unlikely to be rogued from! commercial stocks. In view of the relatively small number of cultivars grown and the intensive vegetative propagation of them it is surprising that these two viruses are not, now widely distributed in carnation crops. Possibly they are primarily pathogens of D. barbatus and infect carnation only when aphid vectors are numerous and infected D. barbatus is growing close to carnation glasshouses. I wish to thank Miss G. C. Bouttell and Mr R. R. Pawley for technical assistance, REFERENCES Hotumos, M. & Sroxe, O. M. (1962). The attenuation of carnation mottle virus in plants, Rep. Glasshouse Crops Ret. Inst. for 1961, p. 90. Houtinos, M. & Stove, O. M. (1964). Investigation of carnation viruses I. Carnation motte. Ann. appl. Biol. $3, 103 Srowt, O. M. (1963). Factors affecting the growth of carnation plants from shoot apices. Am. ‘appl. Biol. 52. 199,