Factors influencing the rooting of peach GF677 (peach almond hybrid) hardwood cuttings in a growth chamber

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1 and return this copy by mail/fax OR any corrections. New Tsipouridis Zealand et Journal al. Propagation of Crop and of Horticultural GF677 peach Science, rootstock2005, Vol. 33: XXX /05/3302 XXX The Royal Society of New Zealand Short communication Factors influencing the rooting of peach GF677 (peach almond hybrid) hardwood cuttings in a growth chamber C. TSIPOURIDIS T. THOMIDIS Z. MICHAILIDES Pomology Institute Naoussa P. C Naoussa, Greece thomi-1@otenet.gr Abstract In Greece, GF677 (peach almond hybrid) peach rootstock is widely used where replant diseases are a problem. In this study, the influence of orientation, rooting substrate, wounding, and jar environment on the rooting success of GF677 hardwood cuttings was examined. The best rooting was achieved by vertical positioning of the cuttings and the lowest by oblique and horizontal positions. The cutting lengths of 20 and 30 cm were better than 10 cm. Perlite was the best substrate for rooting, whereas cuttings in peat gave no rooting. Doublesplit wounding of cuttings increased the rooting percentage by over 29% compared with a nonwounded control. Rooting of wounded and nonwounded cuttings was achieved only in damp perlite (20% moisture) and loosely closed jars. No rooting or callusing was observed in cuttings held in the tightly closed jars. Keywords cuttings; jar environment; orientation; rooting substrate; wounding INTRODUCTION GF677 (peach almond hybrid) is a very important peach rootstock and, therefore, methods to improve its propagation are required (Stylianides et al. 1988). Hardwood cuttings are one of the least expensive and easiest methods of vegetative propagation. They are easy to prepare, are not readily perishable, may be shipped safely over long distances if necessary, and H04047 Received 7 May 2004; accepted 26 December 2004 require little or no special equipment during rooting. The choice of an appropriate substrate is crucial for rooting of cuttings (Denny & Arnold 2001; Tofanelli et al. 2003). Both rooting success and root quality are greatly affected by the substrate (Pocorny & Austin 1982). A good rooting medium should have a high water retention capacity combined with good aeration. Verdonck et al. (1983) tried to quantify these requirements for optimal rooting, recommending that an ideal substrate would have 20% of the volume as air and 20 30% of the volume as easily available water. The orientation of cuttings during placing in substrate may influence rooting success. Cristoferi et al. (1988) found that polarity reversal caused a drastic decrease in rooting percentage of hardwood cuttings of grapevine rootstock KOBER 5BB (60% less than normally orientated cuttings). Some other treatments, such as wounding of cuttings, may positively affect rooting. Howard (1984) reported increased rooting of M.26 winter cuttings in response to wounding in the presence of idole-butyric acid (IBA). Edwards & Thomas (1979) reported that wounding only affected root quality, but not rooting success of Juniperus cuttings. Air environment also affects the rooting on cuttings. It is evident that the stock plant environment exerts a strong influence on root formation in stem cuttings, and on root and bud formation in leaf cuttings (Heide 1965). The aim of these studies was to test the influence of orientation, substrate, wounding, and air environment on the rooting percentage of GF677 hardwood cuttings. MATERIAL AND METHODS Effect of the orientation of GF677 hardwood cuttings after planting on rooting percentage Hardwood cuttings, 16 cm long, were collected from 8-year-old GF677 trees on 16 November 1998 and 16 November Immediately after collection, the cuttings were treated for 15 s with 2000 ppm of IBA in a 50% ethanol solution (96% purity).

2 2 New Zealand Journal of Crop and Horticultural Science, 2005, Vol. 33 After surface drying, their bases were treated with Captan 75% (9:1 in talc). The cuttings were then placed in permeable polyethylene bags containing 500 cm 3 of perlite (1 5 mm) and 100 cm 3 distilled water. Bunches of 20 cuttings were inserted into each bag, with the bases of the cuttings in perlite. To avoid movement of perlite, the bags were tied in the middle, keeping the perlite at the base of the cuttings. The bags were placed in the growth room at 25 ± 10 C with a relative humidity of 80% and an 8-h photoperiod (100 µe m 2 s 1 ). The experiment was assessed after 40 days. The different treatments consisted of five orientations: vertical (control), horizontal, inverted vertical, oblique (45 ), and inverted oblique. The experimental design used was a randomised complete block (5 treatments 3 blocks) replicated in 2 years. Rooting percentage of each experimental unit was obtained from 20 cuttings. Effect of rooting substrate on rooting percentage of GF677 hardwood cuttings treated with 2000 ppm IBA Hardwood cuttings, 8 cm long, were collected from 8-year-old GF677 trees on 6 January 1998 and 6 January The bases of the cuttings (1 cm) were treated with IBA as a quick dip at 2000 ppm and Captan 75% (1:9 in talc). The cuttings were then planted in a randomised complete block (5 treatments 5 blocks replicated in 2 years). Rooting percentage of each experimental unit was obtained from 10 cuttings and for root number and root length the mean of the 10 cuttings were used. The cuttings were put in plastic cells (each plastic block consisting of 6 10 cells of 4 4 cm and 8 cm depth with a drainage hole in the bottom of each cell) containing five substrates: perlite (1 5 mm), peat, perlite 50%-peat 50%, sand, and perlite (covered cuttings were additionally enclosed in a polyethylene bag). After setting, the trays were placed on inverted wooden boxes in the growth room at 25 ± 10 C with a relative humidity of 80% and a photoperiod of 8 h (100 µe m 2 s 1 ). The cuttings were irrigated daily with 500 ml tap water per tray (60 cells). The experiment was assessed after 30 days. Effect of cutting length and double-split cutting base on rooting percentage of GF677 hardwood cuttings This experiment was established on 8 March 1998 and repeated on 8 March Three lengths of cuttings were chosen from 8-year-old GF677 trees. Double split wounds were made with secateurs and a knife, making crosscuts 1 2 cm deep, through the base of the stem towards the tip, for slice wounds (1 2-cm-long pieces of tissue were removed from the side of the cutting at the proximal end). Bases of these cuttings were then treated with 2000 ppm IBA dissolved in 50% aqueous alcohol by dipping them for 15 s to a depth of 2 cm. After surface drying, the cuttings were treated with Captan 75% (1:9 in talc) and planted in polyethylene bags containing 500 cm 3 perlite with 20% distilled water in a growth room (25 C) with a photoperiod of 8 h (100 µe m 2 s 1 ). Non-wounded cuttings were used as control. The experiment was a two-factor factorial with 2 treatments 3 cutting lengths in a randomised complete block design with 4 blocks with experimental units consisting of 10 cuttings each, replicated in 2 years. After 40 days, progress was assessed by determining the percentage rooted and the number and length of the roots. The position of roots in relation to wounding was also noted. Effect of wounding and jar environment on rooting percentage of GF677 hardwood cuttings treated with 2000 ppm IBA This experiment was established on 29 January 1998 and repeated on 29 January Cuttings, 10 cm long, kept previously in a refrigerator at 2 C for 70 days (in a big bag), were used. After incubation in the refrigerator, half of the cuttings were wounded (10 longitudinal knife cuts, 1 cm long per cutting at the proximal end) and the other half were left unwounded. All were treated with 2000 ppm of IBA as a quick dip. After surface drying, the cuttings were treated as previously with Captan 75% (9:1 in talc) and inserted (10 cuttings/jar) in glass cylinders (6 cm in diam. 11 cm in height or 312 cm 3 ) with or without perlite. The jars were tightly sealed (by using a silicone gel) or only lightly closed according to the treatment. Afterwards, all containers were placed in the growth room at 25 ± 10 C, relative hymidity 80%, with an 8 h photoperiod (100 µe m 2 s 1 ). Rooting or callusing was recorded 25 days after setting. The experiment was a three-factor factorial with 2 levels of wounding 2 levels of closure of jars 3 levels of basal treatment (air, dry perlite, wet perlite), in a randomised complete block design with 3 blocks replicated in 2 years, with experimental units the mean of 10 cuttings. Analysis of variance was carried out for data of content of lightly closed cylinder in wet perlite for wounded and nonwounded cuttings since the other example gave zero values.

3 Tsipouridis et al. Propagation of GF677 peach rootstock 3 In all experiments, cuttings that had not rooted were checked for infections by pathogens (pieces from the bases of cuttings were transferred on cornmeal agar, hemp seed agar, potato dextrose agar, and Lutz medium). Statistical analysis In all experiments, years and blocks were taken as random effect factors and treatments as fixed effect factors. In the analysis of variance, blocks were nested within years. Treatment interactions with years were considered as random, allowing comparisons of the treatment means only within each random year. In the experiments for rooting percentages, statistical analyses of the data were carried out after angular transformation to obtain normality. Significant differences between treatments were tested by Duncan s Multiple Range Test at P = RESULTS AND DISCUSSION Effect of the orientation of GF677 hardwood cuttings after planting on rooting percentage The best rooting, as Dhru et al. (1988) also found with cuttings of Nerium oleander, was achieved by vertical positioning of the cuttings and the three lowest by oblique, oblique inverted, and horizontal positions (Table 1). Root growth was best in the vertical position; inversion did not reverse polarity and caused a marked decrease in the rooting percentage. Inverted cuttings also began rooting later and more slowly than those oriented normally. These observations clearly indicate that gravity has a major effect on rooting. Rooting of cuttings involves a gradient of growth hormone within the stem, but this hormone gradient is one of the results of the polarity of the tissues and not the cause of it (Wareing & Phillips 1975). Effect of rooting substrate on rooting percentage of GF677 hardwood cuttings treated with 2000 ppm IBA The results confirmed the importance of substrate characteristics for rooting. There were striking differences between treatments. Perlite had a significantly higher rooting percentage than other treatments, whereas cuttings in peat (very fine) gave no rooting and almost all rotted. Covering the cuttings in perlite caused a great number of sprouts and callus formation on their bases, but significantly lower rooting than perlite and peat 50%-perlite 50% (Table 2). The 50:50 peat-perlite mixture gave a reasonable amount of rooting, whereas cuttings in sand (very fine) rooted very poorly. Apart from peat, root numbers did not differ significantly among treatments. The poor performance in pure peat and sand was probably because of poor aeration of the substrate. Tofanelli et al. (2003) tested six substrate compositions (sand, carbonised rice husk, vermiculite, sand + carbonised rice husk, sand + vermiculite, and carbonised rice husk + vermiculite) and found that the highest rooting frequency was obtained with vermiculite, independent of cutting diameter. Denny & Arnold (2001) found the perlite substrate the best for the rooting of cuttings of Cotinus obovatus. Ercisli et al. (2002) investigated rooting characteristics of kiwifruit hardwood cuttings ( Hayward ) in perlite, peat, sawdust, peat + sawdust (1:1), and peat + perlite (1:1) and found that the highest rooting percentage was obtained in peat + perlite and peat + sawdust media. Effect of cutting length and double-split cutting base on rooting percentage of GF677 hardwood cuttings Wounding by a double-slit significantly improved rooting, compared with the control (Table 3). The double-split significantly increased the rooting percentage. The cutting lengths of 20 and 30 cm were significantly better than 10 cm. Callus was particularly heavy along the edges of the split stem, appearing to develop from the inner cortex, phloem, and cambium, and forming nodules overlying the xylem. Table 1 Effect of cutting orientation after setting on rooting (%) of GF677 hardwood cuttings treated with 2000 ppm of idole-butyric acid. Estimates are based on 60 cuttings. Treatment year interaction effects are significant. Year effect is not significant. Values in the same column followed by different letters are significantly different (P = 0.05) according to Duncan s Multiple Range Test. Rooting (back-transformed %) Treatment 1st year 2nd year Control (vertical) 94 a 87 a Horizontal 40 d 50 c Inverted 60 b 53 b Oblique (45) 42 d 48 cd Oblique inverted 47 c 46 c SE (angular)

4 4 New Zealand Journal of Crop and Horticultural Science, 2005, Vol. 33 Generally, root numbers and lengths followed the same trend. Wounding is frequently used to induce formation in responsive cuttings such as M27 apple rootstock (Pontikis et al. 1979). M26 roots easily from hardwood cuttings prepared at the basal node, but poorly if inferior quality internodal cuttings are used, unless these are wounded (Howard et at. 1984; Mackenzie et al. 1984). Shaltout et al. (1998) increased the rooting percentage of Nemaguard peach cuttings by wounding them. Kracikova (1999) reported the wounding of plum rootstocks cuttings by means of a basal split as a factor which influences the rooting of cuttings. Mackenzie et al. (1988) found that splitting the bases of M26 hardwood cuttings (followed by IBA treatment) gave a 16-fold improvement in rooting over the controls, whereas deep incisions gave only a 4-fold improvement. Effect of wounding and jar environment on rooting percentage of GF677 hardwood cuttings treated with 2000 ppm IBA Rooting of wounded and non-wounded cuttings was achieved only in the wet perlite (20% moisture) and loosely closed jars. Non-wounded cuttings showed a significantly higher rooting percentage (back-transformed 30%) than wounded (back-transformed 25%) (SE angular 2.33). A probable explanation for the low rooting percentage in this experiment compared with experiment 3 is the different time of year of cutting collection. Some quite good callusing was achieved with dry perlite or air in the other lightly closed treatments of both wounded and nonwounded cuttings. Possibly this could cause root formation if the experiment had been allowed to continue for longer. Wounding also can increase Table 2 Effect of substance on rooting (%) of GF677 hardwood cuttings treated with 2000 ppm of idole-butyric acid. Values are the mean of two experiments, each with 50 cuttings. Comparison of means within each year did not show any difference in the arrangement of significance and only the means across both years are presented. Values in the same column followed by different letters are significantly different (P = 0.05) according to Duncan s Multiple Range Test. Rooting (back-transformed %) Root no./cutting Treatment (mean) (mean) Perlite 57 a 3.6 a Peat 0 d 0.0 b Peat-perlite (50:50) 41 ab 3.9 a Sand 9 c 3.7 a Perlite covered 25 b 3.5 a SE (angular) Table 3 Effect of wounding by double-split on rooting percentage, root numbers per cutting, and root lengths of GF677 hardwood cuttings rooted in growth room after a quick dip with 2000 ppm idole-butyric acid. Values are the mean of 40 cuttings. Interaction of cuttings treatments years were not significant. For each trait, means followed by different letters within uppercase, lowercase, or Greek are significantly different by Duncan s Multiple Range Test at P = 0.05 horizontally in the table followed by the same capital letter; vertically in the same block of the table followed by the same small letter. Rooting (back-transformed %) Rooting no. Root length (cm) Cuttings (cm) Cuttings (cm) Cuttings (cm) Treatment year Control 48e 60d 60d 69b 2.0g 3.5b 3.2b 2.9b 0.5b 1.4a 1.5a 1.1a Double split 73g 88b 96a 87a 3.0b 5.0a 5.7a 4.6a 0.7b 2.0a 1.5a 1.4a SE (angular) Cuttings 60B 75A 82A B 4.3A 4.5A B 1.7A 1.5A 1.3 SE (angular)

5 Tsipouridis et al. Propagation of GF677 peach rootstock 5 survival of peach cuttings and can result in ethylene production (Fabijan et al. 1981b), which in turn can stimulate rooting, e.g., in sunflower (Fabijan et al. 1981a) and mung bean cuttings (Geneve & Heuser 1982). Wareing & Phillips (1975) draw attention to early work that wounding of plant tissues results in the release of a wound hormone which stimulates cell division. One such hormone traumatic acid HOOC.CH=CH (CH2)8 COOH, has been isolated; it is believed to cause certain cells of stem or root to become dedifferentiated and to resume meristematic activity. Another possibly quite important benefit from wounding is the alleviation of tissue tension and disruption of sclerenchyma layers. Although roots may penetrate through cells and tissues with the aid of cellulases, heavily lignified tissues may resist, and here a shallow incision may be essential. No rooting or callusing was observed in cuttings held in the tightly closed jars. A somewhat similar appearance of cuttings was described by Jackson (1990) and explained as ethylene effects. Other possible causes could be the increased CO 2 concentration in contrast with the reduced O 2 concentration. Fungi of the genus Sclerotinia spp. were isolated only from four non-rooted cuttings placed for rooting in sand (experiment 2). CONCLUSION The experiments show that the usual vertical orientation of hardwood cuttings after planting is the best way of planting the GF677 hardwood cuttings. Rooting could be improved by using the appropriate cutting length (20 30 cm), suitable substrate with adequate water-air capacity, and by applying splitting at the base of the hardwood cuttings, possibly with a mechanical device. REFERENCES Cristoferi G, Filiti N, Rossi F The effects of reversed polarity and acropetal centrifugation on the rooting of hardwood cuttings of grapevine rootstock Kober 5BB. Acta Horticulturae 227: Denny C, Arnold A Interactions among rooting substrate, phenological stage of cuttings and auxin concentration on the rooting of Cotinus obovatus. Journal of Applied Horticulture Lucknow 1: Dhru P, Gupta S, Nursat A, Charam S Regeneration of stemcuttings of Nerium oleander L. as influenced by indoloacetic acid and planting posture. Advances of Plant Sciences 1: Edwards R, Thomas M Influence of wounding and IBA treatments on the rooting of cuttings of several woody perennial species. The Plant Propagator 24: Ercisli S, Anapali O, Esitken A, Sahin U The effects of IBA, rooting media and cutting collection time on rooting of kiwifruit. Gartenbauwissenschaft 67: Fabijan D, Taylor J, Reid D 1981a. Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. II. Action of gibberellins, cytokinins, auxins and ethylene. Physiologia Plantarum 53: Fabijan D, Yeung E, Mukherjee I, Reid D 1981b. Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. I. Correlative influences and developmental sequence. Physiologia Plantarum 53: Geneve R, Heuser C The effect of IAA, IBA, NAA and 2,4-D on root promotion and ethylene evolution in Vigna radiate cuttings. Journal of the American Society for Horticultural Science 107: Heide O Photoperiodic effects on the regeneration ability of Begonia leaf cuttings. Physiologia Plantarum 18: Howard B Plant propagation. Report of East Malling Research Station Pp Howard B Plant propagation. Report of East Malling Research Station Pp Jackson M Communication between the roots and shoots of flooded plants. Monograph of the British Society for Plant Growth Regulation No. 21: Kracikova M Determining the influence of several factors on propagation of plum rootstocks by hardwood cuttings. Vedecke Prace Ovocnarske 16: Mackenzie K, Howard B, Harrison-Murray R The anatomical relationship between cambial regeneration and root initiation in wounded winter cuttings of apple rootstock M.26. Annals of Botany 58: Mackenzie K, Howard B, Harrison-Murray R Anatomical features or rooting in wounded winter of the apple rootstock M.26. Acta Horticulturae 227: Pocorny F, Austin M Propagation of blueberry by softwood terminal cuttings in pine bark and peat media. HortScience 16:

6 6 New Zealand Journal of Crop and Horticultural Science, 2005, Vol. 33 Pontikis C, Mackenzie K, Howard B Establishment of initially unrooted stool shoots of M.27 apple rootstock. Journal of Horticultural Science 54: Shaltout A, Salama M, El-Wakeel F, Aziz M, Ismail O Propagation of Nemaguard peach by stem hardwood cuttings and layerings. Annals of Agricultural Science Cairo, Special Issue 3: Stylianides D, Syrgianidis G, Almaliotis D The peach rootstocks: a review of bibliography with relative observations in Greece. Agriculture Technology 12: Tofanelli M, Rodrigues J, Ono E Rooting of peach cv. Okinawa hardwood cuttings at different stem diameters, substrates, and pots. Ciencia Rural 33: Verdonck O, Penninck R, De Boodt M The physical properties of different horticultural substrates. Acta Horticulturae 150: Wareing P, Phillips I The control of growth and differentiation in plants. Oxford, Pergamon Press. P. 96.