The Inactivation of Virus in Cultured Shoot Tips of Nicotiana rustica L.

Transcription

1 J. gen. ViroL (1969), 5, I With I plate Printed in Great Britain 237 The Inactivation of Virus in Cultured Shoot Tips of Nicotiana rustica L. By D. G. A. WALKEY, JANET FITZPATRICK JUDITH M. G. WOOLFITT National Vegetable Research Station, Wellesbourne, Warwickshire AND (Accepted 21 March 1969 ) SUMMARY Three weeks after inoculation, cherry leaf roll virus and arabis mosaic virus were each detected in the apices of at least seven axillary buds above the inoculated leaves of Nieotiana rustica plants. Such infected apices were grown into young plants on Linsmaier and Skoog's (1965) medium, and their virus content tested after 14 to I35 days; 5 to 78 % were apparently free from cherry leaf roll virus and 69 % from arabis mosaic virus. In a more detailed test with cherry leaf roll virus, the proportion of virus-free plants increased from 50% after 16 days to IOO% after io8 days. Similar plants grown for 3o weeks remained free of symptoms and detectable virus. INTRODUCTION Healthy rhubarb plants can be grown from the shoot-tips of buds taken from plants infected with one or more of five viruses (Walkey, I968a). Some of the viruses are, however, detectable initially in some of the excised tips. Thus production of healthy plants may involve elimination of virus from infected tips during their development, a mechanism similar to that postulated by HoUings & Stone (1964) for the loss of carnation mottle virus from carnation. This differs from that suggested in potato by Kassanis (1957) and Kassanis & Varma (1967) in which the young plantlets were free from infection because the tips from which they were grown were uninfected-- presumably because the virus in the parent source plants had not entered the meristematic region of the apical-buds. Attempts were made to study the possible in vivo inactivation of virus in rhubarb tip cultures but it proved difficult either to infect rhubarb experimentally, or to detect viruses in its shoot-tips because of the need to counteract the acidity of the sap to be used as inoeulum for indicator plants. A search for other more suitable plants, and the results of detailed tests on one species, are described here. METHODS Culture of shoot-tips. The healthy plants were grown in pots in the glasshouse and when of suitable size, shoot-tips, consisting of the meristematic dome and one or two pairs of leaf primordia, were dissected from axillary buds. This was done under a microscope in a sterile room, using scalpels made from pieces of razor blade (Stone, 1963). The tips were cultured on filter paper bridges in 3 x I in. Monax tubes, each

2 238 D. G. A. WALKEY AND OTHERS containing 8 ml. of Linsmaier & Skoog's (1965) nutrient solution, excluding the optional constituents but including indole acetic acid (8 mg./1.) and kinetin (2.56 mg./l.). After covering with sterile polypropylene film (Blake, I966) the tubes were incubated at about 18 in an illuminated cabinet (Walkey, I968a). Tips from infected plants were excised and cultured in the same way. Viruses. The cherry leaf roll virus (CLRV) isolate was obtained from rhubarb (Tomlinson & Walkey, 1967) and the arabis mosaic virus (AMV) from celery (Walkey, I968b). Both viruses were cultured in Nicotiana rustica L. Inoculation of test plants and virus assay. The plants under test were mechanically inoculated when 4 to 6 in. high by rubbing the 2 to 3 lowest leaves with infected sap of N. rustics, obtained by grinding systemically infected leaves in o.i M-potassium phosphate buffer (ph 7"5). Tips were removed from axillary buds on the test plants some 3 to 6 weeks after inoculation and assayed for virus content by placing each tip in a small drop of o.t M-potassium phosphate buffer on a glass slide together with Celite (Johns-Manville Co.). The tip was then ground in this mixture using the end of a glass rod and the resultant homogenate inoculated on to the leaves of 2 to 3 in. tall plants of Chenopodium amaranticolor Coste & Reyn. The presence of CLRV or AMV was indicated by the development of systemic symptoms typical of infection by these viruses. Although the tips were very small (Table 2) the method of assay was reliable. In later tests the fact that virus particles could be detected in infected tips (in characteristic 'tubules') by electron microscopy of tip squashes (Walkey & Webb, 1968) was used to confirm the results obtained from mechanical inoculations. RESULTS Test of six plant species Survival of shoot-tips. Tips taken from six species were measured and their survival tested (Table I). Three Nicotiana species showed the highest survival rate. Infection of shoot-tips of the six plant species. For the study of in vivo inactivation it was necessary that a high proportion of the tips used in the culture work should become infected; this was tested. All the tips of Nicotiana rustica plants were infected with both CLRV and AMV (Table 2). Since tips from virus-free N. rustics plants had a high survival rate the species was chosen for further experimental work. Studies on Nicotiana rustica The occurrence of virus was followed in plantlets of N. rustica grown from infected shoot-tips. Since the test for virus in the shoot-tips was destructive, comparable tips were chosen at random from infected plants and used for further studies. The immediate tests showed that, within 3 weeks after inoculation, virus was present in the tips of buds in the axils of the inoculated leaves and also in those of at least a further seven buds up the plant (in the axils of systemically infected leaves). The lowest six to eight axillary buds were assumed to be infected and were used in the culture tests. As a further check the 6th bud on every plant used in Expts 2 and 3, and the ISt and 8th buds in Expt 5 (Table 4) were tested for virus. All were infected. After 14 to I35 days young plantlets were removed from the growth medium for virus assay. In the early stages of growth the cultures consisted of a basal ball of callus with terminal leaves (P1 I a), so the entire culture was homogenized in buffer and the

3 Journal of General Virology, Vol. 5, No. 2 Plate (a) Three-week-old Nicotiana rustica culture showing basal mass of callus. (b) Ten-week-old plantlet with well developed roots and leaves. D. G. A. WALKEY, J. FITZPATRICK AND J. M. G. WOOLFITT (Facing p. 238)

4 In vivo virus inactivation in cultured shoots 239 homogenate used to inoculate tests plants of Chenopodium amaranticolor. The older cultures, however, often consisted of well developed plantlets with roots and numerous leaves (P1. I b) and in tests of such plantlets one or two leaves were removed for assay. This allowed the plantlets to be transferred to pots so that further virus tests could be made on them. To increase the chances of detecting virus when the older cultures were Table i. The survival in culture of shoot-tips from six species Mean diam. of No. tips (%) Species excised tip (/zm.) tested Survival Nicotiana rustica N. glutinosa N. tabacum var. 'White Burley' N. clevelandii 3oi 35 5 Atriplex hortensis 3zo 4I IO Chenopodium amaranticolor o Table 2. Virus infection of shoot-tips in six species* Mean Mean Infection diam. of diam. of (%) meristem excised No. tips r ~ - - ~, Species dome (/~m.) tip (/~m.) tested CLRV AMV Nicotiana rustiea IOO loo N. glutinosa N. tabacum vat. 'White Burley' oo N. clevelandii I A triplex hortensis t t Chenopodium amaranticolor * The excised tip included the meristem dome+ one to three pairs of primordial leaves. Table 3. The elimination of virus from Nicotiana rustica shoot-tips in culture No. tips Age of culture No. tips containing Infected with Expt no. Virus (days) tested virus virus (%) I CLRV o IOO 29t--o3 55 2t 38 2 CLRV o ioo CLRV o lo io ioo I4-I35 I2I AMV o Ioo J4-7I I assayed, six of the larger plantlets from each batch were homogenized and their sap subjected to differential centrifugation, to concentrate any virus that might be present, before its inoculation to test plants. During the period when the tips were in culture, cherry leaf roll virus was eliminated from 5o to 78 % of the cultures and arabis mosaic virus from 69 % (Tables 3, 4). Differential centrifugation of homogenized plantlets did not increase the detection of virus infection and repeated tests on initially virusfree plantlets which were grown on to maturity failed to detect virus.

5 240 D.G.A. WALKEY AND OTHERS The time required for the elimination of CLRV from N. rustica cultures was studied in detail in Expt 5, in which batches of tips were assayed for virus at approximately fortnightly intervals from the beginning of the experiment. In these tests there was a fairly rapid loss of virus from some of the plants, only 5 % remaining infected after I6 days in culture (Table 4)- This proportion remained fairly constant for a further 4 weeks and then diminished, until after 15 weeks none or only very few of the plants were infected. Table 4. Relation between inactivation of cherty leaf roll virus and time in culture Plants with no No. days in No. plants No. plants detectable virus culture tested infected (~) o 6o 60 o i6 2o lo io I I I7 z 88 io8 ~6 o ~oo DISCUSSION We have shown dearly that the viruses, although initially present in excised shoottips of N. rustica, and capable of inducing symptoms on indicator plants, were inactivated during subsequent growth on culture medium. Inactivation occurred within 3 weeks in about 5o % of the plants but more slowly in others, and occasionally virus survived for I3 weeks. Neither the reasons for these differences between plants nor the mechanism of inactivation are known. No evidence was obtained that virus survived in plantlets below the limits of detection. Plants kept for up to 3o weeks never developed any symptoms nor could virus be obtained from them, either by inoculation of crude sap, or of preparations obtained by ultracentrifugation of plant extracts. The experimental system used appears very suitable for in vivo virus inactivation studies. Each of the viruses infected all the shoot tips of lateral buds of N. rustica and each was eliminated during culture of the tips. It is probable that the relatively large size of the meristems (and consequently the shoot-tips) of N. rustica contributed towards both their high rate of survival and their high initial virus content. We wish to thank Misses R. Jones and S. Whittingham-Jones for technical assistance. REFERENCES BLAKE, J. (I966). Flower apices cultured in vitro. Nature, Lond. zxx, 990. HOLHNGS, M. & STO~E, O. M. (1964). Investigation of carnation viruses. L Carnation mottle. Ann. appl. Biol. 53, Io3. KASSANIS, B. (I957). The use of tissue culture to produce virus-free clones from infected potato varieties. Ann. appl Biol. 45, 422. KASSANIS, B. & VARMA, A. (1967). The production of virus-free clones of some British potato varieties. Ann. appl Biol. 59, 447-

6 In vivo virus inactivation in cultured shoots LINSMAmR, E. M. & SKOOG, F. (I965). Organic growth factor requirements of tobacco tissue cultures. Physiologia Pl. xs, Ioo. STONE, O. M. (1963). Factors affecting the growth of carnation plants from shoot apices. Ann. appl. Biol. 5z, I99. TOMLINSON, J. A. & WALKEY, D. G. A. (1967). The isolation and identification of rhubarb viruses occurring in Britain. Ann. appl. BioL 59, 415. WALKEY, D. G. A. (I968a). The production of virus-free rhubarb by apical tip culture. J. hort. ScL 43, Z83. WALKEY, D. G. A. (I968b). Celery viruses. Rep. hath. Veg. Res. Stnfor 1967, P. 78. WALr, EY, D. G. A. & WEaB, M. J. W (1968). Virus in plant apical meristems. J. gen. Virol. 3, 3 II. (Received 26 November I968 ) 24I x6 J. Virol. 5