In vitro shoot proliferation rates of the rose cv. (hybrid tea) Dr. Verhage, as affected by apical dominance regulating substances

Transcription

1 ELSEVIER Scientia Horticulturae 6 1 ( 1995) 24 l-249 SCIENTIA HORTlCULTURA In vitro shoot proliferation rates of the rose cv. (hybrid tea) Dr. Verhage, as affected by apical dominance regulating substances Chrysothemis Voyiatzi F*, D.G. Voyiatzisb, Vasiliki Tsiakmaki Laboratory of Floriculture, Aristotelian University of Thessaloniki, GR Thessaloniki, Greece blaboratory ofbiology ofhorticulture, Aristotelian University of Thessaloniki, GR Thessaloniki, Greece Accepted 10 November 1994 Abstract The effects of three shoot tip pinching compounds incorporated into the nutrient medium and of the successive reculture technique on in vitro shoot proliferation of rose, cultivar Dr. Verhage, were studied. Of the three compounds tested, methyl ester of lauric acid (MELA) was the most effective followed, in declining order, by n-propyl-3-t-butylphenoxyacetate (M&B ) and 2,3,Wiiodobenzoic acid (TIBA). MELA at 100 mg l- nearly quadrupled the proliferation rate of untipped microshoot explants, which was comparable to the tipped controls. Successive recultures of the basal clump of tissue remaining after the initial culture, on medium free of MELA resulted in a significant increase in the total number of new shoots, compared with the tipped controls. The other two compounds also promoted branching in untipped shoots but were less effective. The results showed that manual tipping of explants can be successfully substituted by chemical tipping, thus eliminating unnecessary handling of plant material under sterile conditions. Keywords: Rose; Shoot proliferation; Pinching compounds; Tissue culture; Micropropagation * Corresponding author. Abbreviations: MELA=Methyl ester of lauric acid; TIBA = 2,3,%riiodobenzoic acid; M&B = n- propyl-3-t-butylphenoxyacetate /95/% Elsevier Science B.V. All rights reserved S.SDZO (94)00750-O

2 242 C. Voyiatzi et al. /Scientia Horticulturae 61 (1995) Introduction Highly prized rose cultivars are occasionally difficult to propagate by conventional vegetative methods owing to poor rootability of their cuttings. As an alternative, the in vitro production of microcuttings and their subsequent rooting (Debergh and Maene, 1981) can be employed to clone them. A cost-efficient production system depends, among other things, on the number of microcuttings produced. High rates of shoot proliferation can be achieved by releasing the lateral buds of microshoot explants from the repressive influence of the apex. A simple way to do this is by removing the shoot tip (tipping or pinching) of each microshoot explant. However, individual handling of microshoots under aseptic conditions should be avoided (Maene and Debergh, 1983 ), as the risk of contamination and eventually the cost per plantlet is greatly increased. In conventional cultivation practice, pinching by hand has been substituted, to a great extent, by chemical pinching. Substances that have been successfully used for this purpose, on plants grown in the field, are classified as growth inhibitors and include fatty alcohols and their methyl esters (Sill and Nelson, 1970; Vereecke, 1975; Maw, 1977 ), triiodobenzoic acid and maleic hydrazide (Baldini et al., 1973; Arora et al., 1982) and n-propyl-3-t-butylphenoxyacetate (M&B ) (Quinlan and Pakenham, 1980). They act either by destroying the apical bud or by interfering with the complex hormonal mechanism which regulates apical dominance (Bangerth, 1993). Such substances could be used in in vitro propagation to enhance shoot proliferation rates, instead of the time-consuming manual tipping of each shoot. Proliferation rates may be further enhanced by reculture of basal explants comprising the remnants of the previous culture (Xiao-Shan and Mullins, 1984; Voyiatzi and Voyiatzis, 1989 ). The purpose of this study was to evaluate the effects of three pinching compounds, namely lauric acid methyl ester (MELA), 2,3$triiodobenzoic acid (TIBA) and M&B , on the shoot proliferation rate of microshoot explants of the rose cultivar Dr. Verhage, when used in combination with the technique of reculture, during the in vitro multiplication of this cultivar. 2. Materials and methods 2.1. Establishment of stock cultures Terminal and axillary buds from young shoots of rose plants cv. Dr.Verhage grown in a greenhouse, were excised. After the removal of the protective scales the buds were surface-sterilized by immersion for 15 min in a solution of commercial laundry bleach diluted to contain 2.8% sodium hypochloride and a few drops of 0.1% Tween-20, and were then rinsed four times with sterilized distilled water. Shoot-tip explants were obtained by excising the terminal 2-4 mm of the bud axis. They were placed individually in Erlenmeyer flasks containing the in-

3 C. Voyiatzi et al. /Scientia Horticulturae 61 (1995) organic salts of Murashige-Skoog medium supplemented with (mg I- ): thiamine-hcl, 0.5; pyridoxine-hcl, 0.5; nicotinic acid, 0.5; glycine, 2; myo-inositol, 100; indoleacetic acid (WA), 0.3; BA, 3; sucrose, ; bacto agar, This will be referred to as the basal medium (BM). The ph of the medium was adjusted to before autoclaving at 12 1 C for 15 min. The flasks were stoppered with cotton and aluminium foil and the cultures were maintained at a constant 23 C under a light regime of 4,uE mm2 s- ( nm), provided by cool-white fluorescent tubes 16 h daily. All newly formed microshoots were harvested and those mm long were used as explants in the experiments Experimental design The explants, at their initial culture, were placed on BM supplemented with one of the above mentioned pinching compounds at various concentrations (see description of experiments, below). Each culture period lasted 4 weeks, at the end of which the newly formed microshoots were harvested and counted. The remaining basal clump of tissue (Fig. 1) was transferred to fresh BM without the pinching compound for a further 4 weeks (first reculture ). This procedure could be repeated up to two more times (second and third recultures ). In all experiments similar microshoots manually tipped and placed on BM alone served as controls. Cultures, in all experiments, were maintained at the temperature and light regime described above. All experiments were arranged according to a randomised complete-block design with 1 O-20 replicates (flasks) to each treatment. Fig. 1. New microshoots developed during the first reculture of the original explant (left) and the basal clump of tissue remaining after their harvest (right). The pinching compound was present in the medium of the initial culture only.

4 244 C. Voyiatzi et al. /Scientia Horticulturae 61(1995) I. Experiment I: The effects of MELA Intact microshoots from stock cultures were placed on BM, supplemented with MELA at 0,50,75, 100, 150 and 200 mg 1-l for their initial culture. The remaining basal clump of tissue which had been treated with 100 mg l- of MELA in its initial culture, was transferred to BM alone for 4 more weeks (first reculture ). This procedure was repeated twice (second and third recultures ) Experiment 2: The efsects of TIBA Intact microshoots were placed on BM supplemented with 0, 1, 3, 10, 30 and 100 mg 1-l of TIBA for their initial culture. After 4 weeks the remaining basal clumps of tissue from the first four treatments were transferred to BM alone for a further 4 weeks (first reculture) Experiment 3: The efses of M&B Intact microshoots were initially cultured on BM supplemented with 0, 0.0 1, 0.03,0.1,0.3 and 1.0 mgl- ofm&b At theendofthe first 4 weekperiod the remaining basal clumps of tissue from the first three treatments (0,O.O 1 and 0.03 mg 1-r) were transferred to BM alone (first reculture), for a further 4 weeks. This procedure was repeated twice (second and third recultures ). 3. Results 3. I. Experiment I: The e_dpects of MELA The number of new shoots which were produced from intact shoot explants cultured on BM supplemented with MELA at 100 mg l-, was nearly quadrupled compared with that in the absence of MELA and comparable to the tipped controls (Fig. 2). In all other treatments intermediate values were observed. Further recultures, on BM alone, of the remaining basal clumps initially cultured on 100 mg 1-l MELA, greatly enhanced shoot formation (Table 1). This potential reached its maximum in the second reculture and then declined. The total number of new shoots produced, after three recultures on medium free of MELA, was more than twice the number of new shoots derived from tipped controls without MELA in their initial culture Experiment 2: The efjects of TIBA TIBA incorporated into the BM caused an increase in the number of new shoots formed from untipped explants, which in some treatments ( 1 and 3 mg 1-l TIBA) was higher than that derived from tipped controls (Fig. 3 ). TIBA at higher concentrations was less effective but still enhanced shoot proliferation over the untipped controls. Reculturing, on TIBA-free BM, of the basal clumps after removal of the new shoots, further enhanced the potential for proliferation in all treatments so that

5 C. Voyiatzi et al. /Scientia Horticulturae 61(1995) Tipped MELA mg I - control s Fig. 2. The effect of MELA on the number of new shoots formed from untipped shoot explants. Each value is the mean of 16 replicates. Vertical bars represent the standard error. Table 1 In vitro formation of new shoots from untipped and tipped shoot explants, as affected by MELA and the number of successive recultures of the basal clump. Tipped shoots were cultured on medium free of MELA. Untipped shoots were treated with 100 mg 1-i of MELA in their initial culture only No. of new shootsa Tipped shoot explants (controls without MELA) Untipped shoot explants (with MELA) Initial culture 3.4d 4.5c 1 st reculture 4.9c 13.lb 2nd reculture?.2b 18.8a 3rd reculture 8.3a 15.5b Total no. of new shoots from four cultures BMeans of 20 replicates (flasks) for each treatment. Means within each column followed by different letters differ significantly at P=O.O5. Mean separation with Duncan s multiple range test. the number of new shoots formed was higher than that of the respective treatments in the initial culture (Table 2). In certain cases (clumps derived from 3 and 10 mg l- TIBA) there was a 67% and 100% increase in the number of new shoots per explant over that of the initial culture in the presence of TIBA, and it was, certainly, higher than the number of new shoots derived from tipped controls Experiment 3: The eflects of M&B The addition of M&B into the BM increased the proliferation capacity of untipped explants over their controls (Table 3). The number of new shoots

6 246 C. Voyiatzi et al. /Scientia Horticulturae 61 (1995) TIBA mg I Tipped controls Fig. 3. The effect of TIBA on the number of new shoots formed from untipped shoot explants. Each value is the mean of 16 replicates. Vertical bars represent the standard error. Table 2 The effect of TIBA on the number of new shoots derived from the initial culture of untipped shoot explants and from the first reculture of the remaining basal clump. TIBA was present in the medium of the initial culture only TIBA (mgl- ) No. of new shoots per flask Initial culture 1 st reculture (with TIBA) (without TIBA ) Untipped shoot explants 0 0.5fO.l f kO.2 OS+_O.l 4.0f f f0.3 Tipped controls 0 3.0f kO.3 Means ( + SE) of ten replicates (flasks) in each treatment. per explant at and 0.03 mg 1-l exceeded that of tipped controls, though not significantly. However, these shoots, on average, were significantly longer than shoots from either the untipped or tipped controls, an effect of this inhibitor that was not observed in the case of MELA and TIBA. Subsequent transfers to a medium free of M&B 25-l 05, of the remaining clumps of tissue after their initial culture, further induced their ability to form new shoots, up to the second reculture. An average of 19 new shoots per clump were formed on clumps that had been subjected to the influence of 0.03 mg 1-r M&B only in their initial culture. The respective numbers for the untipped and tipped controls were 6.5 and 1.5 new shoots per clump (Fig. 4).

7 C. Voyiatzi et al. /Scientia Horticulturae 61(1995) Table 3 The effect of M&B on the in vitro shoot proliferation of the rose cv. Dr. Verhage M&B No. of new Mean length concentration shoots per flask per shoot (mgl- ) (cm) Untippedshoot explants 0 1.3f f kO f ko f f0.3 Tipped controls f kO.5 Mean ( &SE) of ten replicates (flasks) in each treatment M&B Lz 2- _ _+-- ~ * Initial culture No. OF RECULTURES Fig. 4. The effect of M&B and the number of recultures on the formation of new shoots from untipped shoot explants. M&B was present in the medium of the initial culture only. Means of ten replicates. Vertical bars represent the standard error. 4. Discussion In vitro shoot proliferation comprises a manifestation of apical dominance and as such it is regulated by the correlative action of the apical bud upon axillary bud growth (Tamas, 1987). An extremely complex hormonal mechanism is involved where the polar transport of auxin plays a predominant role (Bangerth, ). Rose shoots exhibit strong apical dominance (Zieslin and Halevy, 1976 ), which is diffkult to overcome completely in in vitro culture even with the addition of cytokinins in the medium (Hasegawa, 1979). All three chemicals used in this

8 248 C. Voyiatzi et al. /Scientia Horticulturae 61(199S) study promoted the formation of new shoots from untipped microshoot explants. They act by interfering with the mechanism of apical dominance either by destroying or inhibiting terminal bud growth, as fatty alcohols and their esters do (Vereecke, 1975), or by inhibiting basipetal auxin transport, as TIBA (Rubery, 1987) and M&B (Quinlan, 1985) do. In both cases axillary buds were released of the repressive influence of the apex and sprouted into new shoots in rates that matched or even exceeded those of tipped controls. In a previous attempt where TIBA was applied on the apex of each microshoot individually, proliferation rates were not improved compared with those obtained by tipped controls (Bressan et al., 1982). All three substances used exhibited a marked after-effect, which in the case of MELA and M&B persisted long after their application. A carry-over effect of these substances could not be excluded, though in this case it could not be considered as a problem. Although the mechanism of action of this phenomenon is not clear, it may have a significant implication in practice as a large number of new shoots can be formed during successive cultures after the pinching chemical has been applied once only in the initial culture. The question arises whether these newly formed shoots are adventitious or derived from pre-existing lateral buds which occasionally are formed, especially after prolonged maintenance of tissue in in vitro conditions (Jones and Murashige, 1974). According to Hasegawa ( 1980) the development of new shoots in rose explants, during the proliferation stage, may be attributed to numerous pre-existing lateral buds. Our observations on more than a hundred plantlets, acquired by this method and transplanted in the field, did not reveal any abberations concerning growth habit and flower characteristics. One can assume that in rose, given adequate time (in fact time equal to the duration of three successive recultures, i.e. 12 weeks) the majority of lateral buds in the recultured tissue are able to grow into shoots, hence the rise and subsequent decline in the number of new shoots after three recultures. In conclusion, it was shown that the proliferation rate of rose microshoots could be significantly increased when pinching of individual microshoot explants was substituted by the incorporation into the nutrient medium of one of the tested pinching chemicals, in combination with the technique of repeated recultures. This offered the added advantage that by eliminating all unnecessary manual handling of sterilized plant material, the possibility of loses which usually occur through contamination and mishandling was greatly diminished. This in turn, suppresses the cost per plantlet produced and renders the in vitro technique more attractive as a means of propagating difficult but desirable rose cultivars. Acknowledgements The donation of M&B by May&Baker Ltd., Dagenham, England, is gratefully acknowledged by the authors.

9 C. Voyiatzi et al. /Scientia Horticulturae 61(1995) References Arora, S.K., Pandita, M.L. and Sidhu, A.S., Effect of maleic hydrazide on vegetative growth, flowering and fruiting of bottle gourd. Sci. Hortic., 17: 2 1 l Baldini, E., Sansavini, S. and Zocca, A., Induction of feathers by growth regulators on maiden trees of apple and pear. J. Hortic. Sci., 48: Bangerth, F., Polar auxin transport as a signal in the regulation of tree and fruit development. Acta Hot-tic., 329: Bressan, P.H., Kim, Y-J., Hyndman, S.E., Hasegawa, P.M. and Bressan, R.A., Factors affecting in vitro propagation of rose. J. Am. Sot. Hortic. Sci., 107: Debergh, P.C. and Maene, L.J., A scheme for commercial propagation of ornamental plants by tissue culture. Sci. Hortic., 14: Hasegawa, P.M., In vitro propagation of rose. HortScience, 14: Hasegawa, P.M., Factors affecting shoot and root initiation from cultured rose shoot tips. J. Am. Sot. Hortic. Sci., 105: Jones, B.J. and Murashige, T., Tissue culture propagation of Aechmea fksciata Baker and other Bromeliads. Proc. Int. Prop. Sot., 24: Maene, L.M. and Debergh, P.C., Rooting of tissue cultured plants under in vivo conditions. Acta Hortic., 131: Maw, G.A., Aliphatic alcohols as pruning-agents for tomato side shoots. Sci. Hortic., 7: Quinlan, J.D., Chemical regulation of fmit tree growth in the development of new production systems. In: R. Menhenett and M.B. Jackson (Editors), Growth Regulators in Horticulture, Monogr. 13. British Plant Growth Regulators Group, Bristol, pp Quinlan, J.D. and Pakenham, E.M., Chemical induction of branching and translocation of M&B East Malling Research Station Report for 1979, p. 36. Rubery, P.H., Auxin transport. In: P.J. Davies (Editor), Plant Hormones and their Role in Plant Growth and Development. Kluwer, Dordrecht, pp Sill, A.Z. and Nelson, P.V., Relationship between azalea bud morphology and effectiveness of methyl decanoate, a chemical pinching agent. J. Am. Sot. Hortic. Sci., 95: Tamas, LA., Hormonal regulation of apical dominance. In: P.J. Davies (Editor), Plant Hormones and their Role in Plant Growth and Development. Kluwer, Dordrecht, pp Vereecke, M., Response of brussel sprouts to chemical pinching with fatty alcohols and methyl esters of fatty acids. HortScience, 10: Voyiatzi, C. and Voyiatzis, D.G., In vitro shoot proliferation rate of Dieffenbachia exotica cultivar Marianna as affected by cytokinins, the number of recultures and the temperature. Sci. Hortic., 40: Xiao-Shan Shen and Mullins, M.G., Propagation in vitro of pear, Pyrus communis L. cultivars William s Bon Chretien, Packham s Triumph and Beurre Bose. Sci. Hortic., 23: 5 l-57. Zieslin, N. and Halevy, A.H., Components of axillary bud inhibition in rose plants. I. The effect of different plant parts correlative inhibition. Bot. Gaz., 137: