EFFECT OF THE SUBSTRATES UPON THE REGENERATION OF THE ENGRAFTED CUTTINGS AND THE YIELD OF ENGRAFTED ROOTED VINES P. Zamanidis1, L. Maltabar2, Chr. Paschalidis3 and E.Vavoulidou4 1 NAGREF, Athens Institute of Viticulture in Likovrissi, Athens Greece 2 Kuban University of Agriculture, Russia 3 Technological Institute in Kalamata, Greece 4 NAGREF, Soil Science Institute of Athens, ,Greece *Address correspondence to: Pantelis Zamanidis National Agricultural Research Foundation (NAGREF), Athens Institute of Viniculture, 1 S. Venizelou str, 141 23, Lykovrissi, Greece Tel. :+30 210 28 16978 Fax :+30 210 2840629 e-mail: firstname.lastname@example.org ABSTRACT Engrafted cuttings of the variety Rkaciteli on stocks BxR Cober 5BB and RxR 101-14, having endured open and covered stratification on different substrates, after the post-planting preparation, were planted in a polyethylene glasshouse on four artificial substrates. The complex effect of the stock variety was examined, as well as the effect of the stratification methods and the substrates on the yield of engrafted vines. The characteristics of the regeneration of the engrafted cuttings during the stratification were established. The optimal substrates for stratification of the transplants and the growing of engrafted vines in glasshouses were defined. Key words: engrafted cuttings, stratification, regeneration, callus, substrate, engrafted vines INTRODUCTION After the mass spreading of phylloxera and the demolition of vineyards in Europe at the end of the 19th century, the grafting of European-Asian vine varieties on phylloxera-resistant stocks was the most effective and most reliable method to maintain high productivity of the vines. Currently the method for production of engrafted vines is grafting on table. During the complex process of producing vine material through the grafting on table method, the main segments of the technology are stratification of the engrafted cuttings and the growing of vines on artificial substrates in glasshouses. Two main stratification methods are used: covered, in which the engrafted cuttings are laid in layers and covered with damp peat, perlite, sawdust and, open, in which the engrafted cuttings are not covered with moisture retaining material. The covered stratification is done by covering the engrafted cuttings with moisture retaining material in special stratification crates that are placed in heated rooms (Mishurenko, 1964) or the crates with the engrafted cuttings are placed in non heated premises where the stratification is done with local electric heating (Maltabar, 1971). The open stratification is done in special rooms, in which a preset regime of light, temperature, humidity and aeration is maintained. The engrafted cuttings are covered with anti-transpirants and their lower end is placed in liquid substrate (water or fertilizing solutions), moisturized perlite, sand, porolone crumbs etc. (Constantinescu, 1964; Jukov et al. 1978), or the stratification of engrafted cuttings, placed without covering with moisture retention material in a multi-tier container by applying conditioned air (Nikolenko, 1962). After the stratification, the engrafted cutting endure pre planting preparation (sorting, removal of the shoots of the stock covering with paraffin, hardening) and are planted in a rooting place indoors or outdoors. Currently the best results are achieved when the rooted vines are grown on artificial substrates in glasshouses. For this purpose different substrates are used and their main component is peat (Deiter, 1975; Becker, 1980; Maltabar, 1983; Smirnov et al. 1998; Zamanidis, 1996). As aeration components sawdust, perlite, sand, cut straw, rice husk, etc. are used. The purpose of the research was to evaluate a set of substrates on the regeneration of engrafted cuttings during the stratification period and on the yield of standard vines in their growing on artificial substrates in polyethylene non-heated glass- houses. MATERIAL AND METHODS The research was carried out in the periodfrom 1998- to 2002 in the Crimean experimental station and Viniculture Department at the Kuban State University of agriculture. As a scion the Rkaciteli variety was used, and as stocks - BxR Cober 5BB and RxR 101-14. The preparation of the cuttings for stocks and scions, their storage, cutting, blinding of the eyes of the stock, the stratification of the tips of the stock before the transplantation etc., were carried out in accordance with the technology of Maltabar l983, 1988). The transplantation was done with the machine M.T.-7A The research included two experiments. Experiment I. Examination of the effect of the stock variety, the stratification methods and the substrates on the regeneration of the engrafted cuttings. Two stratification methods with six types of substrate were examined. 1. Covered (20 days): a) in sawdust and local electric heating (control) and b) in sawdust at common heating. 2. Open (-16 days): a) in water without replacement; b) in water without replacement and leaving an internode with length 5- 6 cm at the lower end of the stock, and removing the internode after the stratification; c) in water with periodic replacement (the lower end of the engrafted cuttings is soaked in water for 12 hours and is left without water for 12 hours ) d) in fertilization solution of mineral fertilizers (Chesnokova-Bazirina); e) in distilled water without replacement; f) in perlite; g) in sand. For each variation of stratification and each stock 2000 pieces of engrafted cuttings were produced. The electric stratification is done in crates with saw dust through the installation of ECY-M in a non-heated room. The open and covered stratification in saw dust at common heating are done in a camera with preset mode of light, temperature and humidity. For the variations with stratification in water and fertilization solution, the engrafted cuttings are tied in bundles of 100 pieces and placed in containers, in which the height of the liquid is maintained at a level of 3-5 cm. At stratification in sand and perlite, the bundles are placed in a 7-8 cm layer of moisturized substrate. The stratification begins in the middle of March. The temperature during the stratification with local electric heating is maintained at 24-26 oC , and at the other variations of the experiment at 28-30oC at specific air humidity 80-85%. Each year 36 000 pieces of engrafted cuttings were produced and stratified (2 stocks, 9 variations of stratification, and 2000 pieces per variation). Experiment 2. Vine yield in glasshouses, depending on stock, stratification method and substrate. In a non-heated polyethylene glasshouse, 4 lots with substrates were laid with a layer thickness of 50 cm. The area of each lot was 150 m2. The following substrates were used: a) soil-humus-carbonate dark brown, control; b) mix of soil, sand and peat in proportion 1:1:1; c) mix of soil, sand and rice husk 1:1:1; d) mix of soil, sand, mould (1:1:1). After hardening in water for 5-6 days, the engrafted cuttings from all variations were planted in the glasshouse in the first half of April. The planting scheme was in bands with space between the bands of 80 cm, between the rows and the bands 20 cm, and between the cuttings inside the row 5-7 cm. The depth of planting was 15-18 cm. In each substrate 300 first-grade engrafted cuttings were planted (100 pieces during the repeating of the experiments) for each variation of stratification, on two stocks. Each year 21 600 pieces of first-grade engrafted cuttings were planted in four substrates. The experiment was planned with three repetitions with random disposition of the lots. The common agrotechnical measures were executed for the plants in the glasshouses. The agrobiological reports and observations, the examination of the processes of regeneration of the engrafted cuttings during the stratification period, the control of the yield of first grade cuttings after the stratification, and the yield and the quality of the rooted vines was done in accordance with the common for the root lots methods (Maltabar et al., 1982). The statistical analysis was done with the methods of dispersion and correlation analysis. RESULTS AND DISCUSSION Regeneration of the engrafted cuttings during the stratification period The regeneration of the engrafted cuttings during the stratification period depended on the quality of the input material, the cuttings for scions and stocks, the content of nutrient amendments, and the physiological condition of the eyes of the cuttings of the scion. These parameters are not constant and they vary depending on the climate conditions through the year, the growth conditions and the care of the mother vines, the different quality of the vine shootings, the different quality of the eyes of the shoots, the presence of tendrils on the scion, the preparation period of the cuttings for stock and scion, the storage methods, etc. Forming of callus at the cohesion point of the components The yield variations with covered stratification of engrafted cuttings with a circular callus at the cohesion point stratified in saw dust and common heating, and in both stocks, was higher by 6 to 10% compared to the transplants stratified in saw dust with local electric heating (Table1). At open stratification, high yield of engrafted cuttings with a circular callus at the cohesion point on stock BxR Cober 5BB was achieved for stratification in water without replacement (90.7 %) and the engrafted cuttings on stock RxR 101-14-for stratification in distilled water without replacement and fertilization solution without replacement, where the yield was 96.3% and 93.0% respectively. At open stratification in light, the callus quickly achieved green colour and dense structure, which is a precondition for better vascular system development of the inter-component callus of the stock and the scion, whereas at covered stratification the tissues of the callus were etiolated, with loose structure, and the processes of differentiation of the elements of the vascular system were poorly manifested. The dispersion analysis (Table 2) shows that the highest effect of the components on the callus regeneration at the cohesion point was effected by factor Y at 37% (year of the observations) and at 27% by factor B (stratification’s method-substrate) and the interaction of AxB factors (stock x stratification method, substrate) . Opening of the buds of the scion and growth of the shootings The intensity of opening of the buds of the scion depended on the physiological condition of the eyes, and the quality of the eyes of the cuttings of the scion. The results of the dispersion analysis showed that the stratification method and the substrate (effect ratio -43%) had the greatest effect on the development of the eyes of the scion and the minimal effect was by the stock (effect ratio 6%). The growth of the shoots of the scion during the stratification period depended also on the stratification methods and the substrates. The maximum length of the shoots of the scion was observed in engrafted cuttings on stock BxR Cober 5BB, stratified in water without replacement, and in distilled water without replacement, and on stock RxR 101- 14 at stratification in perlite and sand (Table 1). The minimal length of the shoots was observed in engrafted cuttings on two stocks after stratification in sawdust and local electric heating. Forming of callus at the lower end of the stock No callus at the lower ends of the stock was formed or the callus was minimal at open stratification of engrafted cuttings in liquid substrates (Table 1). Intensive forming of callus was observed in engrafted cuttings on the two examined stocks, at open stratification in layered sand and perlite, and at covered stratification in sawdust at common heating. The dispersion analysis showed that factor B (stratification method, substrate), and its effect ratio of 78% had the maximum effect on the callus forming process at the base of the stock. Forming of the roots of the engrafted cuttings The rhizogenesis of the engrafted cuttings depended largely on the variety of the stock. The forming of roots on the transplants on stock RxR 101-14 was more intensive than on stock BxR Cober 5BB, which was confirmed by the dispersion analysis. The effect ratio of the stock in this process was 44%. Strong development of the roots was observed in engrafted cuttings, stratified in sawdust at common heating, in perlite and sand. The yield of engrafted cuttings with roots in these stratifications varied from 25 to 63% (Table 1). Weak root formation was observed in engrafted cuttings, stratified in fertilization solution. The regeneration of the dormant roots at the base of the stocks was active in the transplants on stock BxR Cober 5BB at stratification in water without replacement and with leaving a part of the internode at the base of the stock, and in water with periodic replacement. The yield of engrafted cuttings with dormant roots in these variations was 16% and 12% respectively in transplants on stock RxR 101-14. The maximum number of dormant roots (42%) were observed in engrafted cuttings, enduring stratification in water with periodic replacement. There were different opinions about the evaluation of the correlations between the parameters affecting the forming of callus, the regeneration of roots and the development of eyes on the scion in engrafted cuttings during the stratification period. The discussion concerns both the very existence of such correlations, and their degree and direction. The structure of the experiment allowed unbiased evaluation of the correlations between the main parameters of the regeneration, and namely, the presence of circular callus at the cohesion point between the scion and the stock, the presence of opened buds on the scion, the length of the shootings of the scion, the number of roots, the length of the roots and the presence of callus at the base of the stock. In our opinion, the optimal evaluation criterion for the degree of continuity of variation of these parameters was the grade coefficient – per Spirmen (Dospehov, 1979). The correlation analysis of the parameters was for all sources of variation (two stocks x nine variations of stratifications x three repetitions (separately for the four years of observation). As a result, correlations were repeated for most of the later time periods. They included the positive correlation between a circular callus at the cohesion point of the stock and the scion and the presence of opened buds on the scion (R= +0.44-0.66); between the number of roots, their length and the presence of callus at the base of the stock (R= +0.39 - 0.82 and R= +0.25- 0.58 respectively) and the reverse negative correlation between the circular callus at the cohesion point of the components and the presence of callus at the base of the stock (R=- 0.51), as well as the presence of in opened buds on the scion and the callus at the base of the stock (R = -0.56). The results agree with the observations of other authors (Stoev, 1973; Maltabar, 1981, 1983; Smirnov et al., 1998). The opened bud on the scion stimulates the regeneration of the callus at the cohesion point, and the callus at the base of the stock reduces callus formation at the cohesion point and the degree of development on the eyes of the scion. The conclusion is that the engrafted cuttings after stratification optimally must have circular green callus at the cohesion point of the components, short and green shooting on the scion, and only dormant roots at the base of the stock. Yield of engrafted vines grown in polyethylene glasshouses Optimal temperature and humidity in the polyethylene glasshouse and a vegetation period of 20-25 days as a result of earlier planting provide for strong above ground and root system development of the rooted plants. Regardless of the stock and the stratification methods, the engrafted vines grown in a polyethylene glasshouse are characterized by high rates of growth. They possess circular cohesion of the stock and the scion at the cohesion point, well developed growth length of the main shoots (from 110 to 225 cm), high number of roots with diameter above 2 mm (from 9 to 14), distributed evenly on the perimeter of the base of the stock. The comparative examination of the effect of the stock on the yield of standard vines shows the insignificant effect of this parameter. Thus, in all variations the average yields of vines on stock BxR Cober 5BB was 43.1 %, and of stock RxR 101-14 was 49.8% (Table 3). The yield on the second stock was higher by 8.6%. Differences were found in the yield of vines depending on the stratification method. The average yield of vines in variations with open stratification was 53.5%, and in variations with covered stratification was up to 48.8%. Overall, the stratification method effect on the yield of vines growing in different glasshouse substrates depended on the stock. Soil substrate (control) The highest yield of standard vines grown in soil with transplants on stock BxR Cober 5BB was achieved by planting the stratified engrafted cuttings in a layer of sand and distilled water at 53.5% and 54.3% respectively (Table 3). In the plants on stock RxR 101-14 none of the stratification variations with local electric heating exceeded the yield of vines of the control, which gave maximum yield of 56.6%. Substrate rice husk + soil + sand (1:1:1) High yield of vines on this substrate was achieved in growing stratified engrafted cuttings on stock BxR Cober 5BB in water without replacement and by leaving an internode length of 5-6 cm at the lower end of the stock, as well as in transplants stratified in a layer of sand at 49.1% and 53% for the two stocks. When planting the transplants of stock RxR 101-14 in a substrate of rice husk + soil + sand, the highest yield of standard vines was achieved by growing engrafted cuttings, stratified in saw dust with local electric heating (60%) and in water with periodic replacement (57.5%). Substrate peat + soil + sand (1:1:1) Regardless of the stratification variation and the stock, compared to the others this substrate provided higher average yield of vines (Table 3). The highest yield (60.5%) of standard vines on stock BxR Cober 5BB was achieved by growing engrafted cuttings in substrate stratified in distilled water without replacement, and in water without replacement and leaving a cut with an internode with length 5-6 cm at the base of the stock (58.2%), as well as in the control variation of stratification in sawdust and local electric heating (56.1%). In the vines on stock RxR 101-14, the best result were achieved by planting in substrate and by growing vines by transplants stratified in sawdust and with local electric heating. Substrate mould + soil + sand (1:1:1) The testing of glasshouse substrates shows that the growing of engrafted cuttings in substrate of mould + soil + sand (1:1:1), regardless of the variations of stratification, decreases the average yield of vines by 3.5 to 7.8% in comparison with the other substrates. During the rooting period, the transplants grown in this substrate were characterized by a slower forming root system. What is the reason for the disparity between the development of the above- ground part and the root system and what obstructs the early functioning of the transplants as a whole organism, what retards the rooting and finally what reduces the yield of vines? The researches show differences in the yield of vines depending on the stocks and the stratification methods. Highest yield of vines on stock BxR Cober 5BB was achieved from engrafted cuttings stratified in sand (46.2%), in distilled water without replacement (45.5%) and in saw dust and local electric heating (45.6%), and the lowest by stratification in fertilization solution (22.1%). In the plants on stock RxR 101- 14, the best yield of vines was achieved by growing transplants stratified in distilled water without replacement (52.7%) and in saw dust at common heating (52.2%). CONCLUSIONS The stratification methods and the substrates have a significant effect on the forming of callus at the cohesion point of the components. By stratification of engrafted cuttings with the base constantly immersed in water, a more circular callus was observed. The highest yield of engrafted cuttings with circular callus at the cohesion point of the components was achieved on stock BxR Cober 5BB and stratification in water (up to 90.7%), as well as on stock RxR 101-14 -in distilled water without replacement (up to 96.3%). The forming of callus was positively correlated to the opening of the buds of the eye in the shorter shooting of the scion (R = +0.66) and was negatively correlated with the forming of large quantities of callus at the base of the stock (R = -0.51). High yield of vines in polyethylene glasshouses was achieved at: a) stock BxR Cober 5BB and substrate peat + soil + sand (1:l:l) up to 52.2% of transplants stratified in water without replacement and part of an internode left at the base of the stock; b) stock RxR 101- 14 in substrate peat + soil + sand (1:1:1), and rice husk + soil + sand (1:l:l) using stratified engrafted cuttings and local electric heating (60.9% and 60.0% respectively); c) in substrate of mould + soil + sand (1:l:l) up to 45.6% from engrafted cuttings on stock BxR Cober 5BB stratified at local electric heating in distilled water without replacement, and on stock RxR 101-14 of transplants stratified in sawdust at common heating (52.2%) and distilled water without replacement (52.7%). The production of vines in polyethylene glasshouses on artificial substrates is profitable in terms of organization and revenue. It allows early production and planting of the engrafted cuttings, reduces the need of soil and water and increases the yield of vines up to 60% of the number produced by engrafted cuttings. The use of one hectare polyethylene glasshouse, even for 6 months, provides vines sufficient for planting 100 ha compared to the outdoor growing which provides plants for approximately 20 hectares. References 1. Backer, H., 1980. Modern Technology for Production of Engrafted Rooted Vines. Viniculture and Wine making, No 29, p. 18-23. 2. Constantinescu, C., 1964. Lo studio de rnovimento controllato degli ormoni nei processi di radica mento delle talles semplico ed innes tate della vitoq Estratto, dagli. Atti dell. Aa, it della e del vino, V.XVI. – p. 210-216 3. Deiter, A. , 1975. Über die Brauchbarkeit von Torfen und Torf-Erde-30 Mischungen im Weinbau.Weinbau-Wein-Wissenschafl., 30:227- 235. 4. Dospehov, B. A., 1979. Field Experiments Methods. Kolos, Moscow, p.376 Formatted: English (United States) 5. Jukov, A. I., Perov N. N. , 1978. Application of the Perlite in Vine Rooting Lots. Recommendations of MOSVIVIV, Anapa, p. 28 6. Maltabar, L. M. , 1971. Production on engrafted grafted rooted Vines in Moldova. Kartja Moldovenjaske, Kishinew, p. 284. 7. Maltabar, L. M., 1981. Theoretical Principles of the Vegetative Propagation of the Vine. Scientific works Kuban, p. 72. 8. Maltabar, L. M., 1983. Technology for Production of Engrafted Vine Material for Planting. Krasnodar, p. 128. 9. Maltabar, L. M., Jdarnarova, A. G., 1982.Methods for Carrying out Agrobioiogical Readings and Observations in the Viniculture. Krasnodar, p.28. 10. Mishurenko, A. G., 1964 .Vine Rooting Lots. Kolos, Moscow, 343 C. 11. Nikolenko, V. G., 1962. New Method for Stratification of vine scions. Gardening, Viniculture and Vine Making in Moldavia, 11:4-8 12. Stoev, K. , 1973. Physiological Principles of the Viniculture. BAS, Sofia, Part 2, p. 168-193. 13. Smirnov, K. V., Maltabar, L. M., Radjabov,A. K., Matuzok, N. V., 1988. Viniculture. Formatted: English (United States) Formatted: English (United States) MAA, Moscow, p.160-254 Formatted: English (United States) 14. Zamanidis, P. , 1996. Propagation of the vine through Grafting on Table. Athens. Formatted: English (United States) Magazine for Agriculture and Farming, 6:10-13; 7:39-43; and 8:12-1 3. Formatted: English (United States) Formatted: English (United States) Formatted: English (United States) Formatted: Centered Table 1. Regeneration of engrafted cuttings of the variety Rkaciteli depending on the stock, the stratification method and the substrate (1998-2002) Stratification method Substrate Yield of engrafted cuttings, % C Circular callus at the Dormant roots at the Opened buds on the Callus at the base of Roots at the base of base of the stock cohesion point Stock the stock the stock scion Covered (20 days) 1 78 49 28 20.6 2.2 Saw-dust and local electric heating KanHO 2 85.2 49 32 25.6 15.2 (control) Saw-dust and common heating 1 88 52 42 40.1 4 2 91.1 42.1 45.1 59.7 6.1 Open, (16 days) Water, without replacement 1 90.7 58.2 0 3.8 9.4 2 90.5 53.7 0 30.09 30.8 Water, without replacement and leaving a part 1 79.3 44 0 10 16.5 with intemode at the base of the stock 2 92.3 48.3 0 15 32 Water, with periodic replacement 1 86.5 65.2 6 18.8 11.8 2 87.5 58.7 3 39 41.6 Distilled water, without replacement 1 82 51 0 4.6 9.8 2 96.3 55.3 0 38 33.6 Fertilizing solution, without replacement 1 74.6 33.3 0 3.4 0 2 93 44.3 0 10.4 12.8 1 75 65 37 27 5.7 Perlite 2 86.7 57 33 43.5 15.6 Sand 1 70.7 42.0 40.0 | 25.1 6.0 2 78.7 39.0 49.0 39 49 63.3 27.5 HCP05 6.3% 3.5% 6.2% 13.1 14.5% % Table 2. Effect ratio of the factors (%) on the regeneration of engrafted cuttings during the Formatted: Centered stratification period (1998-2002) Factor Effect ratio of the factors, % on the length of the roots callus at the stock base on the development of on the number of roots on the formation of circular callus at the on the length of the on the formation of shoots of the scion eyes of the cutting cohesion point A 9 6 17 0 44 10 Β 27 43 18 78 15 39 AxB 27 21 21 0 0 0 Υ 37 30 44 22 41 51 100 100 100 100 100 100 A - stock; Β - stratification method, substrate; AxB - interaction of the factors A and Β; Υ- year of the observations. Formatted: Centered Table 3. Yield of one-year old vines of variety Rkaciteli in spring glasshouses, % on the carried out grafts (1998-2002) Stratification method Substrate Substartum Rice husk +soil +sand Mould +soil +sand Peat+soil (1:1:1) Soil (control) Stock (1:1:1) (1:1:1) Covered (20 days) 1 45.1 56.1 40.8 45.6 Saw-dust and local electric heating KanHO 2 56.6 60.9 60 49.5 (control) Saw-dust and common heating 1 37.8 47.4 41.1 52.2 2 37.8 47.4 41.1 52.2 Open, (16 days) Water, without replacement 1 37.6 44.4 40.7 31.5 2 55.8 54.7 48.1 37.8 Water, without replacement and leaving a part 1 45.2 58.2 49.1 39.1 with intemode at the base of the stock 2 54 53.9 53.1 48.4 Water, with periodic replacement 1 44.7 37.8 43.8 40.2 2 54.2 53.9 57.5 47.3 Distilled water, without replacement 1 54.3 60.5 46.4 45.5 2 53.3 55.7 54.6 52.7 Fertilizing solution, without replacement 1 24.8 25.5 24.5 22.1 2 48.3 47.6 33.6 41.8 1 38.4 45.9 44.9 42.7 Perlite 2 55.4 52.6 44.3 34.3 1 53.5 49.1 53 46.2 Sand 2 48.9 46.6 44.5 45.2 HCP05 4 4.5 3.1 4.9 Note. 1 - Stock BxR Cober 5BB ; 2 - Stock RxR 101-14.
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