to be removed as soon as the buds begin to break, make costs in one-quarter the time it takes to produce grafted perlite a poor choice over wax or a callus box. vines using field grafting techniques. Comparison of survival rates achieved in this study, 42% with waxes, to those achieved by experienced nursery operators in Florida of up to 50%, suggests that the tradi tionalcallus box method may be more effective than the Literature Cited

immediate transfer of waxed cuttings into soil mediaunder 1. Alley, C. J. and J. E. Peterson. 1977. Grapevine Propagation. IX. Ef greenhouse conditions. The 8% difference in survival rate fects of temperature, refrigeration, and indolebutyric acid on callus- may, however, be offset by the reduced labor costs as ing, bud push, and rooting of dormant cuttings. Amer. I. Enol. Vitic. sociated with the one-step greenhouse method. With the 28:1-6. callus box method, callused cuttings may be dipped in wax 2. Becker, H. and M. H. Hiller. 1977. Hygiene in modern bench-graft ing. Amer. J. Enol. Vitic. 28:113-118. to cover the scion and graft union, protecting both from 3. Loomis, N. H. andj. H. Weinberger. 1971. Better grafting techniques dehydration and mechanical injury. Planting the cuttings produce grapevines faster. West. Fruit Grower. 25(3):29-30. into a soilless commercially prepared potting media, to 4. Richards, M. 1976. Propagation of grapes by grafting. Plant Prop which fertilizers have been added, would allow new roots agator 22(1 ):9-10. 5. Romberger, G. A., C. W. Haeseler, and E.L. Bergman. 1979. Influ to take up nutrients quickly for fast growth. When this ence of two callusing methods on bench grafting success of 12 Vtiis fertilizer source is exhausted, application of a slow-release vinifera L. combinations in Pennsylvania. Amer. I. Enol. Vitic. 30:106- fertilizer is necessary for continued vine growth. Use of 110. citrus sleeves or milk cartons would maximize number of 6. Spiegel, P. 1955. Some internal factors affecting rooting of cuttings. plants per area. Within 4 months, vines shouldbe of salable Rep. 14th Intl. Hort. Congress 1:239-248. •7. Willhoft, F. 1983. Waxes for vine propagation. Fette Seifen size allowing the nursery operator to recoup investment Anstrichmittel 85:86-90.

Proc. Fla. State Hort. Soc. 98: 172-174. 1985.

IN VITRO SHOOT PROPAGATION OF GRAPE SPECIES, HYBRIDS AND CULTIVARS

D. J. Gray and L. C. Fisher feasible when the demand for a species or cultivar is great IFAS, Agricultural Research and Education Center enough to justify production costs. Expanding grape ac University of Florida reage in Florida may outstrip the existing supply of plants P.O. Box 388, Leesburg, FL 32749 produced by conventional nursery methods, thus justifying the use of in vitro propagation. Additional index words. Vitis, micropropagation. Methods to propagate grape in vitro have been de scribed previously (4,5,8) and are relatively straight for Abstract. The abilities of grape species, hybrids, and cultivars ward. Shoot apices are placed in standard tissue culture to produce shoots from cultured shoot apices approximately medium containing cytokinin-like growth regulators and 1-2 mm long in vitro were examined. Shoot propagation was are incubated under fluorescent light. Often an auxin is achieved with Vitis aestivalis, V. bourginiana, V. champini, included in the medium. The apices are induced to pro V. longii, V. munsoniana, V. rotundifolia (AD3-42, 'Carlos', duce branches with this culture regime. The apex of each 'Dixie') V. shuttleworthii, V. tiliafolia,V. vinifera ('Carig- branch can be reestablished in culture, resulting in more nane', 'Chenin Blanc', 'Flame Seedless', 'French Colombard', branches, or the branch can be rooted to produce a com 'Italia Alabastrina', 'Lambrusco', and 'Tokay'). Hybrids of V. plete plant. Thus, a continuously branching culture system rotundifolia and V. vinifera examined were NC35-4, NC39-1 results with new plants being harvested as needed. and P9-15. Complex hybrids tested were 'Carolina Blackrose', Approximately 60 grape species or cultivars have been 'Seyval Blanc', and 'Villard Blanc'. Florida hybrids included previously propagated in vitro (1,2,3,4,5,6,7,8,9,10,12), 'Daytona', 'Lake Emerald', 'Libert/, 'Norris', 'Orlando Seed however the vast majority were cultivars derived from pure less', 'Stover', and 'Suwannee'. V. vinifera parentage. Other species that have been tested The capacity for shoot proliferation in vitro was compared for propagation in vitro include: V. argentifolia, V. cinerea, for the various grapes for a total of 3 culture periods. Shoot V. labrusca, and V. riparia (3). proliferation was increased by careful dissection and selection The economically important bunch grapes grown in of apices. Because the shoots can be easily rooted and estab Florida are all complex hybrids derived from disease resis lished in soil, in vitro propagation is an available method tant native species crossed with various V. labrusca or V. for the rapid production of desirable cultivars. vinifera cultivars. Certain disease resistant Vitis (=Mus- cadinia) rotundifolia (muscadine) cultivars are also in pro duction. The abilities of these cultivars and their native parents to produce shoots in vitro has not yet been tested. In vitro propagation can be used to rapidly produce The purpose of this report is to document the in vitro shoot large numbers of disease-free clones and is economically forming capabilities of cultivars and native species that grow in Florida. These will be compared with a number of Florida Agricultural Experiment Stations Journal Series No. 6853. cultivars derived from V. labrusca and V. vinifera as well as We are grateful to C. L. Ponce for technical assistance. some native species from other regions.

172 Proc. Fla. State Hort. Soc. 98: 1985. Materials and Methods (MS) (11) medium as modified by Chee et al. (4). The medium was modified as follows: CaCl2*2H2O and KI were Plant material. Plants were grown from either green cut omitted; 3 mM Ca(NO3)2*H2O was added; MnS(V4H2O tings or dormant canes and were maintained in a soilless and myo-inositol concentrations were dropped to 5 and 55 commercial potting mix in 23-cm pots on greenhouse (xM, respectively; nicotinic acid and pyridoxine HC1 con benches. A time release fertilizer was used to promote centrations were increased to 8 and 5 |jlM, respectively; 3% rapid growth. The plants were pruned repeatedly, result sucrose and 0.7% agar were added; and the pH was ad ing in abundant production of shoot tips. The species, hyb rids and cultivars used in this study are listed in Table 1. justed to 5.7. Benzylaminopurine (BAP) at 5 fiM was used as a growth regulator to promote shoot proliferation. Establishment of cultures. Shoot tips, 5 mm long, were Naphthaleneacetic acid (NAA) at 0.5 |ljlM was tested in pre excised from the potted plants and all leaves and tendrils liminary experiments. were removed. The shoot tips were surface sterilized for 3 The cultures were incubated at 21 to 25°C with 18 hr min with agitation in 25% commercial bleach to which a (light)/6 hr (dark) cycle using cool white fluorescent lights. drop of Triton X was added and rinsed 3 times in sterile Cultures were grown for 4 weeks and the shoots produced distilled water. The terminal apex of each shoot tip (ap by each apex were counted. Apices were dissected from a proximately 1 to 2 mm long) was dissected and placed, cut sample of the resulting shoots and transferred to fresh surface down, on autoclaved solid Murashige and Skoog medium. The number of shoots produced per apex was determined for each grape over 2 to 3 culture periods. Table 1. In vitro shoot multiplication from apical meristems of Vitis species, hybrids, and cultivars. Results and Discussion Grape apices developed rapidly when cultured on mod Apices cultured Shoots/apexz ified MS medium containing 5 fiM BAP. Although auxins Vitis species and cultivars (no.) (no.) have been used in previous studies (2,3,9,12) our prelimi nary observations determined that the addition of NAA aestivalis Foxxie Lottie 7 6 did not improve shoot proliferation and its use was discon bourginiana Black Spanish 3 2 champini Dog Ridge 15 4 tinued. Two separate growth patterns were seen in culture. longii 23 3 The apices either, 1) developed as shoot tips, sparsely pro munsoniana 33 5 ducing branches in leaf axils or, 2) apical growth was shar rotundifolia ply reduced and a mass of differentiated tissue, lacking ( = Muscadinia rotundifolia) internodes, was produced from which numerous shoot tips AD3-42 39 3 Carlos 53 5 grew. The latter growth pattern produced more shoot tips Dixie 13 4 over time and, therefore, was desirable for in vitro shoot rupestris 22 4 propagation. Growth pattern 2 was identical to the re shuttleworthii 11 7 sponse reported in several previous studies (2,3,4,7,12). tiliafolia 20 4 vinifera The number of apices cultured and shoots produced Carignane 18 5 per apex for each grape tested are presented in Table 1. Chenin Blanc 28 8 Those that produced an average of only 2 to 3 shoots per Flame Seedless 5 4 apex during each 4 week culture period tended to prolifer French Colombard 27 4 ate as branching shoot tips (growth pattern 1). Those pro Italia Alabastrina 24 4 Lambrusco 35 7 ducing an average of 4 or more shoots per apex exhibited Tokay 9 5 growth pattern 2. V. rotundifolia X V. vinifera The pattern of growth could often be manipulated by NC35-4 44 4 cultural methods. We found that growth pattern 1 usually NC39-1 31 6 P9-15 11 3 resulted when apices were isolated with subtending stem Eastern U.S. hybrids tissue. Careful dissection utilizing a stereomicroscope was (primarily labrusca and necessary to isolate only thq shoot apex, thus encouraging vinifera parentage) growth pattern 2. Also, during subculture, selection of Concord 3 6 apices from highly proliferative growth pattern 2 tissue Lakemont 28 8 Remaily Seedless 21 7 encouraged similar development (Table 2). Therefore, Himrod 33 6 with careful selection and dissection, the frequency of French and U.S. hybrids shoot proliferation was increased during subsequent cul (primarily vinifera parentage) ture cycles. Similar observations were made by Chee et al. Carolina Blackrose 6 4 (4). Growth pattern expression also appeared to be at least Seyval Blanc 2 4 Villard Blanc 5 4 partially dependent on genotype. For example, V. longii Florida hybrids produced an average of 3 shoots per apex from a total of (various labrusca, vinifera 23 sample apices (Table 1) and this response was not in and native species parentages) creased despite careful cultural practices. The medium Dayton a 14 3 Lake Emerald 20 5 and cultural regime (4) used in this study was developed Liberty 21 6 primarily for hybrid cultivars grown in the eastern United N orris 15 4 States. Eastern bunch grapes such as 'Lakemont' and Re Orlando Seedless 16 5 maily Seedless' produced some of the highest numbers of Stover 23 4 Suwanee 26 6 shoots in our study suggesting that further media modifi cations may be necessary to increase shoot proliferation in 'Data combined from 2 and 3 culture periods. recalcitrant genotypes.

Proc. Fla. State Hort. Soc. 98: 1985. 173 Table 2. Effect of subculture on shoot multiplication in selected Vitis experiments, individual apices from several cultivars ex species and cultivars. hibited shoot numbers of 20 or more per apex, suggesting that careful selection and dissection procedures will lead Species or cultivars Apices to increased average numbers of shoots. and cultured Shoots/apex Shoots produced in vitro are easily induced to produce culture period (no.) (no.) roots either by the use of an auxin-containing rooting Daytona medium or with no growth regulator at all. Studies of 1st culture period7 4 2 plants produced by in vitro shoot propagation have de 2 2nd culture period 5 monstrated that phenotype is faithfully maintained 3rd culture period 5 3 (1,2,12). To date, we have established rooted shoots of sev Dog Ridge 1st culture period 3 2 eral species and cultivars in soil. The rooting capacity and 2nd culture period 7 3 performance of various grapes will be presented in sub 3rd culture period 5 4 sequent reports. Lake Emerald 1st culture period 4 3 Literature Cited 2nd culture period 10 5 3rd culture period 6 7 1. Barlass, M. and K. G. M. Skene. 1983. In vitro adventitious bud for V. munsoniana mation from the grapevine shoot apex. Proc. Aus. Plant Breed. Conf. 1st culture period 5 3 2nd culture period 19 4 :310-311. 2. Chee, R. and R. M. Pool. 1982. The effects of growth substances and 3rd culture period 9 9 photoperiod on the development of shoot apices of Vitis cultured in 'Each culture period was 4 weeks. vitro. Sci. Hort. 16:17-27. 3. Chee, R. and R. M. Pool. 1983. In vitro vegetative propagation of Vitis: Application of previously defined culture conditions to a selec tion of genotypes. Vitis 22:363-374. Studies of in vitro propagation of grape have dealt pre 4. Chee, R., R. M. Pool, and D. Bucher. 1984. A method for large scale dominantly with cultivars of V. vinifera, V. labrusca and in vitro propagation of Vitis. New York Food and Life Sci. Bui. 109:1- their hybrids (4,5,6,7,8,12). There is little information on 9. the culture of other Vitis species. Chee and Pool (3) success 5. Ciccotti, A. M. 1982. Micropropagation of Vitis vinifera L. cvs. 4Mos- fully cultured V. argentifolia and V. riparia but found that cato D'Amburgo' and 'Pinot Bianco'. Stazione Sperimentale Agraria Forestale S. Michele All'Adige. Estratto da Esperienze e Richerche— V. cinerea would not respond. In the present study, a Nuova ser. vol XL Grafiche Instituto Artigianelli, Trento, Italy. number of additional species have been tested (Table 1) 6. Fanizza, G., O. A. Tanzarella, and G. Carrozzo. 1984. Influence of including V. champini 'Dog Ridge', an important rootstock, Vitis source on in vitro shoot apex culture. Ann. Appl. Biol. 105:577- and several V. rotundifolia (muscadine) cultivars. Because 578. 7. Goussard, P. G. 1981. Effects of cytokinins on elongation, prolifera muscadine grapes are grown commercially throughout the tion and total mass of shoots derived from shoot apices of grapevine Southeastern United States, in vitro propagation may be cultured in vitro. Vitis 20:228-234. useful for rapid production of plants from new cultivars. 8. Harris, R. E. and J. H. Stevenson. 1982. In vitro propagation of Vitis. With the exception of 'Daytona', Florida hybrids exhib Vitis 21:22-32. ited average to high shoot proliferation numbers. This 9. Jona, R. and K. J. Webb. 1978. Callus and axillary-bud culture of Vitis vinifera 'Sylvaner Riesling'. Sci. Hort. 9:55-60. suggests that in vitro propagation may be a feasible method 10. Li, J. R. and G. W. Eaton. 1984. Growth and rooting of grape shoot of economically supplying vines. For example, considering apices in vitro. HortScience 19:64-66. an initial plating of 20 'Liberty' apices with a proliferation 11. Murashige, T. and F. Skoog. 1962. A revised medium for rapid rate of 6 shoots each per month, over 5,590,000 shoots growth and bioassays with Tobacco tissue culture. Physiol. Plant. would be produced in 7 months. Our average proliferation 15:473-497. 12. Rosu, A., A. Brezeanu, and M.Jordan. 1983. Micropropagation in rates were below those reported by Chee et al. (4), who Vitis vinifera L.: III. Studies regarding 'in vitro' stimulation of multi obtained an average of 17 'Remaily Seedless' shoots per ple axillary shoot development in some grapevine cultivars for clonal apex during 6 to 8 week culture periods. However, in our multiplication. Rev. Roum. Biol.—Biol. Veget. 28:115-122.

174 Proc. Fla. State Hort. Soc. 98: 1985.