Vegetative propagation of African Mahogany : effects of auxin, node position, leaf area and cutting length

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1 New Forests 11 : , Kluwer Academic Publishers. Printed in the Netherlands. Vegetative propagation of African Mahogany : effects of auxin, node position, leaf area and cutting length Z. TCHOUNDJEU and R.R.B. LEAKEY Institute of Terrestrial Ecology Bush Estate, Penicuik, Midlothian, EH 260QB, Scotland, UK Received 15 July 1991 ; accepted 15 August 1995 Key words : rooting, mist propagation, Khaya ivorensis, tree improvement Application. This paper represents a study of the factors affecting the rooting of African Mahogany stem cuttings, a species for which little is known. It is suggested that the factors investigated here represent a good starting point for a detailed investigation. Abstract. Applied auxin, node position, leaf area and cutting length were examined to investigate the requirements for rooting stem cuttings of Khaya ivorensis. All these variables were shown to be important factors affecting rooting, confirming the hypothesis that successful rooting can be achieved if these primary variables are optimised. The best concentration of the auxin IBA was found to be 200 µg per cutting, which hastened rooting, increased the percentage of cuttings rooted and increased the number of roots per cutting. One clone (8013) was unresponsive to auxins in terms of the percentage of cuttings rooted, but was the most responsive in terms of the numbers of roots per cutting. A greater percentage of cuttings from basal nodes were rooted than from apical nodes. Cuttings cut squarely at the base produced a radially-arranged root system, whereas an oblique cut resulted in a one-sided root system. Trimming the leaf area of cuttings to 10 cm 2 gave greater rooting percentages than trimming to 100 cm2. In general, long cuttings (39 mm) rooted better than short cuttings (19 mm), however, there was an interaction between leaf area and cutting length, in which cuttings with short stems and large leaves had the lowest rooting percentage. Introduction Despite the long-term use of clonal techniques in agriculture, horticulture, and with a few temperate forest tree species, it is only in recent years that clonal techniques have received general approval for forest tree improvement and commercial forestry in the tropics (Campinos and Ikemori, 1983 ; Delwaulle et al., 1983 ; Leakey, 1987 ; Libby and Rauter, 1984). This study represents a part of a larger programme to compare the rooting requirements of two ecologically distinct groups of commercially important timber trees : light demanders like Triplochiton scleroxylon K. Schum ("Obeche") and the more shade tolerant "mature phase late succession" Current address : ICRAF, P.O. Box 30677, Nairobi, Kenya.

2 1 26 species such as Khaya ivorensis A. Chev. (African mahogany), described by Okali and Ola Adams (1987). K. ivorensis has also been classified as a "non-pioneer light demander" (Hawthorne, 1990). The timber of T scleroxylon is "Obeche". Roundwood exports of this timber have represented over 50% of the total volume from West Africa. Mahoganies contribute about 30% of this total, however, they are times more valuable than "Obeche". Thus the overall value of exports of these timbers are approximately equal. Much is known about the requirements for the vegetative propagation of T scleroxylon (Leakey, et al., 1982a; Leakey, 1983 ; Leakey and Mohammed, 1985 ; Leakey and Coutts, 1989 ; Leakey, et al., 1994), in which light and nutrients play a key role in preconditioning cuttings for easy rooting (Leakey and Storeton-West, 1992). By contrast, very little is known for K. ivorensis. In the Meliacae, vegetative propagation by stem cuttings has been previously reported for K. ivorensis and Cedrela odorata L. (Howland and Bowen, 1977 ; Leakey et al., 1982b), as well as for Swietenia species (Howard et al., 1988). Both of the studies with K. ivorensis and C. odorata also involved the screening of a range of other tree species, but few details of the methods used were presented. The present study is part of a larger investigation of macropropagation in this species and Lovoa trichiloides Harms., another late succession species of the Meliacae (Tchoundjeu, 1989). A detailed study of the in vitro propagation of K. ivorensis has been made previously (Mathias, 1988 : Newton et al., 1994). The development of vegetative propagation techniques for the mahoganies has important implications for the commercial regeneration of these species, which are seriously affected by insect pests (e.g. Mahogany shoot borer ; Hypsipyla spp.) It seems that by cloning, there may be the opportunity to capture and utilize genetic resistance to these pests (Newton 1990 ; Newton et al., 1993 a and b), and so to domesticate them (Newton, et al., 1994). The object of the present study with African mahogany was to develop vegetative propagation methods to test the hypothesis that successful rooting of leafy stem cuttings can be achieved in a previously understudied species by manipulating auxin concentration, leaf lamina area and cutting length. Materials and methods Stockplant management Plants of nine clones of K. ivorensis were propagated from stem cuttings under intermittent mist following the procedures developed and described for T scleroxylon (Leakey et al, 1982a). These clones were derived from juvenile,

3 1 2 7 hedged stockplants less than two years old, originating from seeds collected in Oyo State, Nigeria by the Forestry Research Institute of Nigeria. The stockplants were grown in 13 cm pots in automatically-controlled glasshouses at the Institute of Terrestrial Ecology, Edinburgh, UK (28 C f 2 C with natural daylight supplemented by 400 W mercury vapor lamps to give 19.5 h daylength and a minimum irradiance at plant height of 150,a mol m-2 S -1 PAR). The potting compost used was made of 7 :3 :1 peat:sand :loam, on a volume basis, with 4.2 g kg -1 "Enmag" (ICI Haslemere, UK), 2.6 g kg -1 John Innes base (Bentleys, Humberside, UK) and 0.3 g kg -1 of fitted trace elements (ICI, Haslemere, UK). Plants also received weekly applications of 1 % Sangral (L & K Fertilizers Ltd., Lincoln, UK) liquid fertilizer (NPK = 20 :20 :20) in place of daily watering. White fly, red spider mite and scale insects were controlled by Diazinon (Murphy), Plictram (ICI Midox) and Vydate (Duport) respectively. These artificial conditions have been found to satisfactorily simulate natural growth conditions of T. scleroxylon (Ladipo et al, 1992) and to give propagation results that have relevance and applicability in the case of T scleroxylon in Nigeria and L. trichilioides in Cameroon. Cutting production and rooting Single-node, leafy cuttings were taken from regularly cut back (hedged : at height of 20 cm) one- or two-shoot potted stockplants. Unless otherwise stated, the leaf lamina was trimmed with scissors to 50 cm2 and auxins applied to the cutting base by a micropipette (Eppendorf Comfortpette 4700) as a 10 µl droplet, containing 40 yg per cutting of indole-3-butyric acid (IBA) dissolved in industrial methylated spirit (IMS), a mixture of ethanol and methanol. This method ensures that all cuttings regardless of size and wettability get the same treatment. Before inserting the cuttings in the gravel rooting medium, the IMS was evaporated off in a stream of cold air. In order that the node position of each cutting could be tracked, the cuttings of any one plant were set in the propagator in node order, according to a randomized experimental design (see later). The propagation beds, which have been described in detail by Leakey et al. (1982a), were composed of successive layers of stone chippings and sand, topped with a gravel (1-2 mm) rooting medium, heated to c 30 C by 100 W m -2 of insulated electric cables (Complex). Misting frequency from jets (Even products No 14, size 2) arranged 0.9 m apart and 0.56 m above the surface of the medium, was controlled by a timeclock giving 2- to 4-second bursts of mist at 2- or 16-minute intervals during the day or night, respectively. The air temperature of the propagation area was 20 C f 3 C, regulated by automatic venting and supplemented by extraction fans. Weekly assessments were made of rooting between weeks 2-12 by lifting the cuttings. Newly rooted cuttings with one or more primary roots > 1 cm

4 1 28 were potted into the same compost as previously described for stockplants, and removed from the experiment. Cutting length was measured at the first rooting assessment (week 2). Statistical analysis (ANOVA or t-test, as appropriate) was done using the Statview 512 (Abacus Concepts, Inc., Venture Blvd., Calabasas, California, USA) computer software package and, unless otherwise stated, statistical significance is given at the 5% level (P < 0.05). Standard errors for percentages of cuttings rooted were calculated using the procedures of Snedecor and Cochrane (1989) for data with binomial distribution. Effects of auxin concentration and angle of basal cut (Experiment 1) Cuttings from eight plants each of clones 8002, 8004 and 8013 and 16 plants of clone 8012, were allocated equally to each of four auxin treatments : 0, 8, 40 or 200 pg IBA per cutting (n = 100 cuttings per treatment). The control treatment received solvent alone. Ten single-node cuttings were harvested sequentially from the top shoot of each plant and the base of each was cut alternately, either obliquely (^_J 45 ) or squarely (90 ). Equal numbers of cuttings of each clone/treatment were allocated randomly, but in node order, to each of 10 randomized blocks on the mist bed. The length of each cutting (mm) was assessed at week 2. The propagation period was 12 weeks. Effects of auxin concentration (Experiment 2) One plant of each of nine clones (8002, 8003, 8004, 8007, 8009, 8011, 8012, 8013 and 8023) was allocated to each of 4 auxin treatments : 0, 100, 200 or 300 Fag IBA per cutting. The control (solvent only) was applied to cuttings from 5-6 plants of clones 8002 and Ten single-node cuttings were taken sequentially from the top shoot of each plant (n = 90). Equal numbers of cuttings of each clone/treatment were allocated at random to each of 9 randomized blocks in the mist bed. The propagation period was 8 weeks. Leaf area and cutting length (Experiment 3) One plant of each of the 9 clones used in experiment 2 was randomly allocated to each of 3 leaf lamina area treatments : 10, 50 and 100 cm2. A second plant of clone 8002 was also allocated to each treatment. Templates cut from graph paper were used to impose leaf area. After trimming leaves to the correct size, 10 sequentially-harvested cuttings were alternately cut from the top shoot of each stockplant to stem lengths of 19.0 ± 0.2 or 39.0 f 0.2 mm (n = 100 cuttings for each leaf area, n = 50 for leaf area x length combination). Equal numbers of cuttings of each clone/treatment were allocated in node order to

5 each of 10 randomised blocks on the mist bed. The cuttings were set to the same depth. The propagation period was 9 weeks. 129 Results Effects of auxin concentration, angle of basal cut and node order (Experiments 1-2) In the first experiment, IBA application above the 8 pg level hastened rooting. Compared with controls, 40 pg IBA per cutting reduced the time to achieve 40% rooting from 10 to 6 weeks (Fig. la). 200 Mg IBA reduced this period to 3 weeks. Compared with the control, 40 and 200 pg IBA per cutting significantly (P = 0.01) increased the percentage of cuttings rooted. However, the final difference (week 12) in rooting percentage between the 40 and 200 pg IBA treatments was not significant. The highest level of IBA significantly (P = 0.01) increased the mean number of roots formed per rooted cutting for the 4 clones (6.5 f 0.5 for 200 pg IBA per cutting, f 0.3 for 0, 8 and 40 pg per cutting) at week 10 (Fig. 2b). Over 60% of the cuttings of clone 8013 rooted in all treatments, with 80-90% rooting with 0, 40 and 200 pg IBA per cutting. Clones 8002, 8004 and 8012 were very responsive to increasing levels of auxin, with 200 jig IBA giving the greatest rooting percentages in clones 8004 and 8012 (Fig. 2a). In terms of the stimulation of root numbers after 10 weeks, the effects of 200 pg IBA per cutting were significant in all clones, but clones 8002 and 8004 were relatively less responsive than clones 8012 and 8013 (Fig. 2b). Averaged across treatments and clones, cuttings from basal nodes had greater rooting percentages than did those from apical nodes (apical nodes 1-5 = 59.6% f 10.0 ; basal nodes 6-10 = 84.2% f 4.0). There was a weak, but significant, positive relationship between cutting length and rooting ability (y = x, r = 0.53 ; p = 0.01). In an assessment of cuttings producing more than 3 roots, those whose basal ends had been cut squarely tended to produce roots radially around their circumference (68% f 6.3 radial v 32% f 6.3 one-sided), while those cut obliquely tended to produce one-sided root systems (30% f 6.0 radial ; v 70% f 6.0 one-sided). There were no effects of the angle of the cut on rooting percentage or number of roots per cutting. In the second experiment, applied auxins again greatly enhanced rooting (P = 0.001) with 100 and 200 pg IBA cutting -1 resulting in faster rooting than 300 pg IBA cutting -1. The final percentage of cuttings rooted (week 8) was significantly greater with 200 pg than with 300 pg IBA cutting -1 (Fig. lb). However, by week 8, 200 and 300 pg IBA cutting -1 resulted in

6 1 30 A Weeks after treatment B Weeks after treatment Fig. 1. Effects of IBA concentration on rooting percentages for single-node, leafy stem cuttings of K. ivorensis. (a) Experiment 1 : (/ = 0 pg IBA ; = 8 pg IBA ; A = 40 pg IBA and V = 200 pg IBA ; (b) Experiment 2 : (/ = 0 pg IBA; 0 = 100 pg IBA ; 1 = 200 pg IBA and = 300 pg IBA). Error bars = t one SE.

7 1 3 1 A IBA concentration (pg per cutting) B IBA concentration (pg per cutting) Fig. 2. Effects of IBA concentration (Experiment 1) on: (a) rooting percentages of single-node, leafy stem cuttings (week 12) and (b) the mean number of roots per cutting (week 10) of 4 K. ivorensis clones. Error bars = ± one SE. more roots per cutting (3.5 f 0.2) than 100 pg cutting -1 (2.5 f 0.1) or the solvent control (1.5 ± 0.1). Again cuttings from basal nodes rooted best. Effects of leaf area and cutting length (Experiment 3) Trimming leaf laminas to different areas significantly affected rooting percentage after 9 weeks, such that greater percentages of cuttings with leaves reduced to 10 cm 2 rooted, than those with leaves reduced to 100 cm 2 (Fig. 3). Overall, short cuttings rooted less frequently than long cuttings (45% vs 60%

8 c 60-0 Ame a) 100~ M 30 - c a Weeks after treatment 8 10 Fig. 3. Effects of leaf area (Experiment 3 : = 10 cm2 ; A = 50 cm2 ; = 100 cm2) on rooting percentages of single-node, leafy stem cuttings of K. ivorensis. Error bars = ± one SE. f 4.1 % respectively), but there was a significant interaction between leaf area and cutting length. Cutting length did not significantly affect rooting ability in cuttings with small or medium leaf areas, but there was a significant reduction in the rooting ability of short cuttings with a large leaf area (Fig. 4). As in Experiment 1, there was a significant propensity (P = 0.001) for cuttings from basal nodes to root more frequently after 8-10 weeks (Fig. 5) and to produce more roots (data not shown) than those from apical nodes. Discussion The results of the present study appear to confirm the hypothesis that high rooting percentages can be achieved in a previously understudied species, by manipulating auxin concentration, leaf lamina area, and cutting length. Individually, the optimum treatment for each of the factors studied increased rooting success significantly, with indications that interactions can occur at least between leaf area and cutting length. A better understanding of these factors appears necessary to maximize the success and cost effectiveness of a vegetative propagation program. It is also evident from the present study

9 a) 70- m U) c m 30-2 c 20- CD CL 10- S L 10 cm2 S L S L S L 50cm2 100cm2 Mean Fig. 4. Effects of leaf area and cutting length (Experiment 3 : Short = 19 mm, Long = 39 mm) on rooting percentages of single-node, leafy stem cuttings of K. ivorensis (week 9). Error bars = ± one SE. Weeks after treatment Fig. 5. Effects of node position (Experiment 3 : 0 = 1 ; 0 = 3; = 5 ; D = 7; A = 10) on rooting percentages of single-node, leafy stem cuttings of K. ivorensis (mean of 3 leaf areas). Error bars = ± one SE. Nodes 2, 4, 6, 8 and 9 omitted for clarity.

10 1 34 that, even from juvenile stockplants, a cuttings' node position of origin is very important. This result concurs with studies on T. scleroxylon (Leakey, 1983 ; Leakey and Mohammed, 1985 ; Leakey and Coutts, 1989). However, these species differ in that in T scleroxylon apical cuttings root best, while in K. ivorensis the basal ones root best. The need to determine the best treatment for each of the factors that affect the rooting of cuttings of each species studied can be shown by contrasting other tree species with K. ivorensis : (i) The ideal auxin (IBA + NAA) concentration for a range of T scleroxylon clones was found to be around 40 yg cutting' (Leakey et al, 1982a), while that for K. ivorensis seems to be considerably greater, around 200 µg cutting -1. The close similarity between the percentage of cuttings rooted with 200 yg IBA cutting - ' in Experiments 1 and 2 (Figs. 1 and 3) gives confidence in the comparability of these two experiments. The reason for the difference between T. scleroxylon and K. ivorensis is unclear, but could perhaps relate to differences in their modes of shoot elongation. Shoots of T. scleroxylon elongate by free growth, whereas in K. ivorensis shoot growth is by recurrent flushing. (ii) The best leaf area for K. ivorensis (10 cm2 ) contrasts with the greater optima of other species (Leakey, 1985). In T scleroxylon the optimum of about 50 cm2 appears to be due to the balance between the effects of leaf area on current assimilate production and the effects of water loss through transpiration (Leakey and Coutts, 1989). It seems possible that the reported adaptations of Khaya spp to shade via changes in specific leaf area and quantum flux efficiency (Kwesiga and Grace, 1986), could influence both rates of photosynthesis and transpiration. This might explain the observed differences for leaf area influences on rooting between cuttings of K. ivorensis and T scleroxylon. The interaction between leaf area and cutting length on rooting ability in K. ivorensis is of interest because little attention has been paid to such interactions (Newton et al., 1993). In T scleroxylon, as in K. ivorensis, long cuttings rooted in higher percentages than short cuttings (Leakey, 1983), regardless of their position of origin (Leakey and Mohammed, 1985). However, for T scleroxylon cuttings of the same length, the basal ones with the largest diameter had the greatest rooting percentages, indicating that cutting stem volume may in fact be more critical than length (Leakey et al, 1993), perhaps as a storage sink for current assimilates prior to the formation of the new roots. Further evidence that a cutting's storage capacity is an important determinant of rooting has been found in stem cuttings of Eucalyptus grandis (Hoad and Leakey, 1993). The interaction between leaf area and cutting length in

11 135 the present study suggests that larger-leaved cuttings require a greater stem volume for the storage of current assimilates. On a more practical theme, the formation of a one-sided root system on obliquely severed cuttings poses a potential hazard for tree stability. It is therefore important, in this species at least, to promote a radial arrangement of roots by trimming the cutting base squarely. Acknowledgements We wish to thank the Commission of European Communities (DG XII) and UK Overseas Development Administration for funding this research. We also gratefully acknowledge the assistance of Messrs R. Ottley and F Harvey. References Campinos, E.J. and Ikemori, Y.K Mass production of Eucalyptus species by rooting cuttings. Silvicultura. 8 : Delwaulle, J.C., Laplace, Y and Quillet, G Production massive de boutures d'eucalyptus en Republique Populaire du Congo. Silvicultura. 8 : Hawthorne, W Field Guide to the Forest Trees of Ghana, Natural Resources Institute, Chatham, England. Ghana Forestry Series 1 : 278 p. Hoad, S.P. and Leakey, R.R.B Morphological and physiological factors induced by light quality and affecting rooting in Eucalyptus grandis. pp In: Mass Production Technology for Genetically Improved Fast Growing Forest Tree Species Vol. 1. AFO- CEL/IUFRO Conference, September 1992, Bordeaux, France. Howard, F.W., Verdace, S.D. and de Filippis, J.D Propagation of West Indies, Honduran and hybrid Mahoganies by cuttings, compared with seed propagation. Proc. Fla. State Hort. Soc. 101 : Howland, P. and Bowen, M.R Triplochiton scleroxylon (K. Schum) and other West African tropical hardwoods. West African Hardwoods Improvement Project. Research Report , Forest Research Institute of Nigeria, PMB 5059, Ibadan, Nigeria : 154 p. Kwesiga, F.R. and Grace, J The role of red :far red ratio in the response of tropical tree seedlings to shade. Ann. Bot. 57 : Ladipo, D.O., Leakey, R.R.B. and Grace, J Bud activity of decapitated, nursery-grown plants of Triplochiton scleroxylon in Nigeria : effects of light, temperature and humidity. For. Ecol. Manage. 50 : Leakey, R.R.B Stockplant factors affecting root initiation in cuttings of Triplochiton scleroxylon K. Schum., an indigenous hardwood of West Africa. J. Hort. Sci. 58 : Leakey, R.R.B The capacity for vegetative propagation in trees, pp In : Cannell, M.G.R. and Jackson, J.E. (Eds.) Attributes of Trees as Crop Plants. Proc. IUFRO Meeting, Edinburgh, July Institute of Terrestrial Ecology, Monks Wood, England. Leakey, R.R.B Clonal forestry in the tropics - a review of developments, strategies and opportunities. Commonw. For. Rev. 66 : Leakey, R.R.B. and Courts, M.P The dynamics of rooting in Triplochiton scleroxylon cuttings : their relation to leaf area, node position, dry weight accumulation, leaf water potential and carbohydrate composition. Tree Physiology. 5 : Leakey, R.R.B. and Mohammed, H.R.S Effects of stem length on root initiation in sequential single-node cuttings of Triplochiton scleroxylon K. Schum., J. Hort. Sci. 60:

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