Plant Cell Reports

Plant Cell Reports (1989) 7:688 - 691

© Springer-Verlag 1989

Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro E. Chevreau 1, R.M. Skirvin 2, H.A. Abu-Qaoud 2, S. S. Korban 2, and J. G. Sullivan 2 1 1NRA, Station d'Arboriculture, Beaucouze, F-49000 Angers, France 2 Department of Horticulture, University of Illinois, Urbana IL 61801, USA Received October 3, 1988/Revised version received February 8, 1989 - Communicated by J. M. Widholm

ABSTRACT TO develop an adventitious regeneration system for pear cultivars, several experiments were conducted with 2 cultivars of Pyrus communis L. ('Seckel' and 'Louise Bonne') and one cultivar of P. bretschneideri Rehd. ('Crystal Pear'). Half-leaves~, taken from shoots proliferating on Lepoivre medium, were plated in petrl-dishes on medium supplemented with various combinations of cytokinins and auxins. Cultures of the above cultivars had been established from mature trees. Among the growth regulators tested, thidiazuron (TDZ), combined with naphthaleneacetic acid (NAA), was the most efficient for stimulation of adventitious shoots. The optimum level of TDZ was about 3 uM; shoot regeneration was observed over a wide range of TDZ and NAA concentrations (0.5 to 5 uM and 2.5 to 13 um, respectively). Among different macronutrient compositions, I/2 and I/4 Murashige and Skoog were the most effective. Sucrose concentrations (10 to 50 g L-I) had a linear significant effect on shoot regeneration of 'Crystal Pear'. ABBREVIATIONS BAP: 6-benzylaminopurine; IBA: indole-3-butyric acid; NAA: a-naphthaleneacetic acid; NoA: 2-naphthoxyacetic acid; 2,4-D: 2,4-dichlorophenoxyacetic acid; TDZ: thidiazuron (N-phenyl-N'-1,2,3thidiazol-5-ylurea); MS: Murashige and Skoog (1962) macroelements; L: Lepolvre (Quolrin and Lepoivre, 1977) macroelements. INTRODUCTION Adventitious regeneration occurs after cells from organized tissues dedifferentiate and then reorganize by meristem or somatic embryo formation, either directly or with a callus intermediary. This process is a very important step for the induction of variability through parasexual methods: somaclonal variation, induced mutagenesis and transformation. Although adventitious organogenesis has been achieved in several woody fruit species (Hutchinson and Zimmerman, 1987) most regenerations have been from juvenile tissues of seedling origin. Direct improvement of fruit cultivars requires the control of regeneration from mature tissues. Promising results have been obtained from apple leaf discs (James et al.~ 1984) and stem internodes (Jones et al., 1984; Evaldsson, 1985) as well as from Prunus

Offprint requests to: R.M. Skirvin

root callus (Druart, 1980; James et al., 1987). In vitro growth of Pear (Pyrus) species has been reviewed (Singha, 1986). P. communis and P. pyrifolia cultivars have been micropropagated (Singha, 1986), and stored for a long period of time (Wanas et al., 1986). Somatic embryogenesis has been reported from seedling explants (Mehra and Jaidka, 1979; 1985) and plantlets have been regenerated from protoplast cultures of a wild pear (Ochatt and Caso, 1986). Protop!asts have been isolated from 'Williams' pear, but regeneration has not been reported (Revil!a et al., 4987). The purpose of this study was to develop an efficient technique to regenerate adventitious shoots from leaf tissue of severa! pear cultivars. This is the first step in the development of an in vitro mutagenesis program. MATERIAL AND METHODS This study was conducted with 2 cultivars of P. communis ('Seckel' and 'Louise Bonne') and one cultivar of P. bretschneideri ('Crystal Pear'). Shoot cultures, which had been established from mature trees, were maintained on Lepoivre proliferation medium (Quoirin and Lepoivre, 1977), supplemented with BAP (4.4 uM), IBA (0.5) uM, GA 3 (0.3 uM), 30 g 1 -I sucrose and 6 g i -I Difco Bacto agar (Fig. I). To prevent the growth of internal bacteria on the medium, 10 mg i -I gentamycin sulfate was added prior to autoclaving. The antibiotic was necessary in all experiements. The cultures were grown at 22 ° to 24°C, under cool white fluorescent tubes (40 Em-2s-1), with a 16/8 h light/ dark photoperiod, and transferred to fresh medium every 4 weeks. For regeneration experiments, leaves were excised from the proliferating shoots, cut in halves perpendicularly to the mid rib, and randomly plated in I00x15 mm petri dishes, with the abaxial leaf surface in contact with the medium. Except in experiments 2 and 3, Lepoivre proliferation medium with 30 g I -I sucrose, 8 g 1 -I agar, and 10 mg i -I gentamycin sulfate was used with various growth regulator combinations. In all experiments, the cultures were incubated in the dark for 25 days, then transferred to a corresponding auxin-free medium in ambient light. All data were collected weekly after cultures were moved from the dark. After shoots were regenerated on an explant (Fig. 2), they spontaneously began to proliferate on the regeneration medium. The rapidly proliferating shoots often masked further differentiation. For

689 this reason, when shoots developed on an explant, it was transferred from the petri plate to proliferation medium (Quoirin and Lepoivre, 1977). Therefore, all of our data is presented in terms of the number of explants that differentiated shoots over the course of 2 months.

investigated by multiple regression analysis. All computations were made using the computer program SAS. Each factor was tested individually and in all possible combinations. In the results, only the significant factors are discussed. RESULTS

Preliminary

experiments

To evaluate the effect of different eytokinins and auxins on shoot regeneration, 2 preliminary experiments were conducted with limited numbers of explants per treatment. In the first experiment, 5 concentrations of BAP (0 to 22 uM) were combined with 5 concentrations of the following auxins: IBA, NAA, 2,4-D or NoA (0 to 22 uM, except for 2,4-D: 0 to 9 uM). In the second preliminary experiment, 5 concentrations of TDZ (0 to I uM) were combined with 5 concentrations of IBA or NAA (0 to 25 uM).

Shoot differentiation began about 3 weeks after plating on the regeneration medium. Adventitious shoots continued to develop for another 2 months. The frequency of regeneration from the distal or proximal half of the leaves was identical. Most regenerants developed on the wounded edges, on the mid-rib or on the petiole, after the formation of a small callus. Occasionally, shoot regeneration was observed directly from the leaf mesophyll, without callus. One to 3 independent sites of regeneration were usually found per regenerating half-leaf.

Experiment

Preliminary

I

On the basis of the preliminary studies, 2 experiments were designed to test the effect of higher concentrations of TDZ combined with NAA or IBA. In the first experiment, TDZ, at 4 concentrations (0.5, 1.5, 2.5, 3.5 uM), was combined with the following auxin levels: IBA (2.5, 7.5, 12.5 uM) or NAA (2.7, 8.1, 13.5 uM). Each treatment consisted of 4 petri dishes with 6 to 8 explants each. The experiment was conducted with all 3 eultivars. Later this experiment was repeated including a higher level of TDZ. Four concentrations of TDZ (0.5, I, 2.5, 5 uM) were combined with 5 concentrations of NAA (0, 2.7, 5.4, 13, 27 uM). Each treatment consisted of 2 petri dishes with 6 or 7 explants each; the experiment was conducted with the 3 cultivars. Experiment

2

Two cultivars were used to test the effect of various sucrose concentrations on the rate of shoot regeneration. For 'Crystal Pear', 5 levels of sucrose (10, 20, 30, 40, 50 g 1 -I) were combined with 2 growth regulator compositions (TDZ I uM with or without NAA at 2.7 uM). (These levels of growth regulators were selected because they were observed to give satisfactory regeneration in our first experiments, before the data had been statistically analyzed). Three petri dishes containing 10 explants each were used for each treatment. For 'Louise Bonne', 6 levels of sucrose (10, 20, 30, 40, 50, 60 g 1 -I) were combined with TDZ (2.5 uM) and NAA (5.4 uM); 4 petri dishes with 10 explants each were used in all treatments. Experiment

3

To test the effect of quantitative and qualitative modifications of the macronutrient composition on shoot regeneration, 4 macroelement compositions (Lepoivre, MS (Murashige and Skoog, 1962) I/2 MS, and I/4 MS) were combined with 2 levels of growth regulators (TDZ I uM and NAA 2.7 uM, or TDZ 2.5 uM and NAA 5.4 uM). These levels of growth regulators were selected because they had been observed to give satisfactory regeneration in our first experiments, before the data had been statistically analyzed. The experiment was conducted with all 3 cultivars, using 4 petri dishes containing 8 explants for each treatment. This experiment was interpreted by contrast analysis (Steel and Torrie, 1980). All experiments were first analyzed by simple analysis of variance (ANOVA). The significant quantitative factors (e.g. TDZ, NAA) were further

experiments

The results obtained when 'Louise Bonne' leaf pieces were incubated on regeneration media, indicated that BAP was necessary for shoot regeneration, but there was no significant difference in the rate of regeneration among the different concentrations of BAP (data not shown). Among the auxins, NAA seemed to be the most efficient for the induction of shoot regeneration, followed by IBA, then 2,4-D. However, some shoots were obtained without auxins, in the presence of BAP. In the second preliminary experiment, shoot regeneration of 'Crystal Pear' and 'Seckel' was obtained only at I ~M of TDZ. Experiment

I

The investigation of TDZ and auxin effects on differentiation, showed that there was a significant difference in the percentage regeneration among the three cultivars (Fig 3). TDZ had a highly significant linear effect on regeneration of the three cultivars. There was no significant interaction between TDZ and cultivars. NAA exhibited a highly positive linear effect on regeneration of 'Seckel' explants only (Fig 4). IBA did not significantly affect regeneration. There was no significant interaction between TDZ and either auxin. When higher concentrations of TDZ were used, there was a significant difference in the regenerability of the three cultivars (Fig 5). Based on ANOVA, significant differences between cultivars and TDZ levels were observed without significant interactions. The further effect of TDZ, determined by multiple regression analysis, showed TDZ had a quadratic effect on regeneration, which was similar for the three cultivars. In this experiment, NAA had no significant effect on the percentage of regenerating explants for the three cultivars. Experiment 2 Sucrose, in the presence of both TDZ and NAA, significantly affected regeneration of 'Crystal Pear'. There was a positive linear effect (Fig. 6). Sucrose did not significantly affect the regenerability of 'Louise Bonne' (data not presented). Experiment 3 The results of macronutrient comparison experiments were different for each cultivar. For 'Crystal Pear', no differentiation was observed on Lepoivre medium, but there was a significant negative linear effect of MS concentration (Fig 7). For

690

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691 'Louise Bonne' and 'Seckel', Lepoivre medium was significantly inferior to the other media. However, the 2 cultivars showed a significant positive response to MS salt concentration; the optimum concentration was either I/2 or I/4 strength. At the end of each experiment, the regenerated shoots were transferred to ordinary Lepoivre medium in 25x150 mm culture tubes. About 80% of the shoots survived and most of them proliferated very well. More than 50% rooting of some cultivars has been observed (unpublished). DISCUSSION Adventitious shoot regeneration has been obtained in vitro from leaf tissue of 3 pear cultivars of 2 different species. There were significant difference in the ability of each cultivar to regenerate on similar media. These results and those of Ochatt and Caso (1986) and Revilla et al (1987) indicate the potential for organogenesis is probably widespread within the genus Pyrus. This study was designed as a preliminary investigation of various media components that might affect the rate of shoot regeneration in vitro. Growth regulators proved to be very important: regeneration could be obtained without auxin, but a cytokinin was always required. TDZ, which has been reported to be very efficient for adventitious shoot production in woody plants (van Nieuwkerk et al., 1986; Kerns and Meyer, 1986) gave very good results in this study. From the quadratic equation of Fig. 5, an approximate concentration of 3 uM TDZ is predicted to be optimal for the 3 cultivars. Among the different auxins tested, NAA seemed to be more efficient than IBA. However, because of the variability among experiments, and lack of sufficient data we were unable to recommend an optimal concentration of NAA for the 3 cultivars. The concentration of macronutrients has preyed to be important for callus induction and shoot regeneration from apple shoot internodes (Evaldsson, 1985). In our study, we also found macroelement composition to be an important factor for regeneration. Most of our experiments used Lepoivre macroelements. This medium differs from MS medium by a very low concentration of NH4 +, a slightly higher concentration of Ca 2+, and a total macro-salt concentration equivalent to about 3/4 of MS. Lepoivre medium has been found to be very efficient for maintenance of proliferating cultures of several pear cultivars (Personal Communication, E. Chevreau). However, the macroelements required for adventitious regeneration are certainly different; I/2 or I/4 MS proved to be optimal in our experiments. Sucrose is the main energy source for in vitrogrown plants and its concentration ~ims proved to be critical for adventitious rooting (Hutchinson and Zimmerman, 1987). We found that, in the presence of TDZ and NAA, the percentage of 'Crystal Pear' explants which differentiated improved positively as sucrose level increased. The sucrose had no significant effect on 'Louise Bonne'. A particular dark/light exposure has been found necessary for shoot regeneration from apple (personal communication, John Sanford, NY State Exp.

Stn., Geneva). Most of our experiments were conducted with a dark incubation of 25 days and a transfer to auxin-free medium after this period. The importance of this treatment should be investigated further. Although pear seedlings and protoplast-derived callus from seedlings have produced adventitious shoots in vitro, (Ochatt and Caso, 1986; Revilla et al., 1987) this is the first time that whole plants have been regenerated from pear cultivars. Regeneration of up to 50% of the explants has been obtained by adjusting some of the in vitro parameters such as growth regulators, sucrose level, and macrosalt concentration. Further improvement can certainly be achieved. Our simple regeneration system has promising applications for improving pear cultivars through somaclonal Variation, induced mutagenesis or genetic engineering. ACKNOWLEDGEMENT This research was supported in part by the Illinois Agricultural Experiment Station, INRA, FRANCE, and NATO. The authors would like to thank M. C. Chu and S. Singha for plant material. REFERENCES Druart P (1980) Sci Hort 12:339-342 Evaldsson I (1985) Swedish J Agric Res 15:119-122 Hutchinson JF, Zimmerman RH (1987) Hort Review 273-349 James DJ, Mackenzie KAD, Mahlotra SB (1987) Theor Appl Genet 73:589-594 James DJ, Passey AJ, Malhotra SB (1984) Plant Cell Tissue Organ Culture 3(4):333-341 Jones OP, Gayner JA, Watkins R (1984) J Hort Sci 59(4):463-467 Kerns HR, Meyer MM (1986) HortSci 21(5):12091210 Mehra PN, Jaidka K (1979) Phytomorphology 29:286-298 Mehra PN, Jaidka K (1985) Phytomorphology 35:1-9 Murashige T, Skoog F (1962) Physiologia PI 15:473-497 Ochatt SJ, Caso OH (1986) J Plant Physiol 122:243-249 Quoirin M, Lepoivre P (1977) Acta Hortic 78:437 Revilla MA, Ochatt SJ, Doughty S, Power JB (1987) Plant Science, 50:133-137. Singha S (1986) Pear (Pyrus communis). In YPS Bajaj (ed.) Biotechnology in Agricul~ure and Forestry Vol.1:Trees, Springer-Verlag Berlin, pp 198-206 Steel RGD, Torrie J (1980) Principles and proeedures of statistics. McGraw-Hill Book Company, pp 177-181 van Nieuwkerk JP, Zimmerman RH, Fordham I (1986) HortScience 21(3):516-518 Wanas WH, Callow JA, Withers (1986) In Withers LA and Alderson PG (eds.), Plant tissue culture and its agricultural applications, Butterworths, London, pp 285-290

Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro.

To develop an adventitious regeneration system for pear cultivars, several experiments were conducted with 2 cultivars of Pyrus communis L. ('Seckel' ...
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