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

Elisabeth Chevreau, R.M. Skirvin, H.A. Abu-Qaoud, S.S. Korban, J.G. Sullivan

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Elisabeth Chevreau, R.M. Skirvin, H.A. Abu-Qaoud, S.S. Korban, J.G. Sullivan. Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro. Plant Cell Reports, 1989, 7, pp.688-691. hal-01606171

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

E. Chevreau¹, R. M. Skirvin², H. A. Abu-Qaoud², S. S. Korban², and J. G. Sullivan²

¹ INRA, 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 petri-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 naphthalene-acetic acid (NAA), was the most efficient for stimulation of adventitious shoots. The optimum level of TDZ was about 3 µM; shoot regeneration was observed over a wide range of TDZ and NAA concentrations (0.5 to 5 µM and 2.5 to 13 µM, respectively). Among different macronutrient compositions, 1/2 and 1/4 Murashige and Skoog were the most effective. Sucrose concentrations (10 to 50 g L⁻¹) had a linear significant effect on shoot regeneration of 'Crystal Pear'.

## ABBREVIATIONS

BAP: 6-benzylaminopurine; IBA: indole-3-butyric acid; NAA: α-naphthaleneacetic acid; NoA: 2-naphthoxyacetic acid; 2,4-D: 2,4-dichlorophenoxyacetic acid; TDZ: thidiazuron (N-phenyl-N'-1,2,3-thidiazol-5-ylurea); MS: Murashige and Skoog (1962) macroelements; L: Lepoivre (Quoirin 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*

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). Protoplasts have been isolated from 'Williams' pear, but regeneration has not been reported (Revilla et al., 1987).

The purpose of this study was to develop an efficient technique to regenerate adventitious shoots from leaf tissue of several 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 µM), IBA (0.5 µM), GA₃ (0.3 µM), 30 g l⁻¹ sucrose and 6 g l⁻¹ Difco Bacto agar (Fig. 1). To prevent the growth of internal bacteria on the medium, 10 mg l⁻¹ gentamycin sulfate was added prior to autoclaving. The antibiotic was necessary in all experiments. 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 100x15 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 l⁻¹ sucrose, 8 g l⁻¹ agar, and 10 mg l⁻¹ 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
this reason, when shoots developed on an explant, it
was transferred from the petri plate to prolifera-
tion medium (Quoirin and Lepovivre, 1977). There-
fore, all of our data is presented in terms of the
number of explants that differentiated shoots over
the course of 2 months.

Preliminary experiments

To evaluate the effect of different cytokinins
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 1 uM) were combined with
5 concentrations of IBA or NAA (0 to 25 uM).

Experiment 1

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 concentra-
tions (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 cultivars.
Later this experiment was repeated including a
higher level of TDZ. Four concentrations of TDZ
(0.5, 1, 2.5, 5 uM) were combined with 5 concen-
trations 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 l⁻¹) were combined
with 2 growth regulator compositions (TDZ 1 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 l⁻¹) 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 qualita-
tive modifications of the macronutrient composition
on shoot regeneration, 4 macroelement compositions
(Lepovivre, MS (Murashige and Skoog, 1962) 1/2 MS,
and 1/4 MS) were combined with 2 levels of growth
regulators (TDZ 1 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

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

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.

Preliminary experiments

The results obtained when 'Louise Bonne' leaf
pieces were incubated on regeneration media, indi-
cated 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 1 µM of TDZ.

Experiment 1

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 signifi-
cant 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 regener-
ability of the three cultivars (Fig 5). Based on
ANOVA, significant differences between cultivars and
TDZ levels were observed without significant inter-
actions. The further effect of TDZ, determined by
multiple regression analysis, showed TDZ had a quad-
ratic 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 regener-
ability of 'Louise Bonne' (data not presented).

Experiment 3

The results of macronutrient comparison experi-
ments were different for each cultivar. For 'Crys-
tal Pear', no differentiation was observed on
Lepovivre medium, but there was a significant nega-
tive linear effect of MS concentration (Fig 7). For
<figure><img src="image_2.png" /><figcaption>Figure 1. Proliferating shoots of the pear cultivar 'Seckel' in vitro.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Figure 2. Adventitious shoot regeneration from a leaf of 'Seckel' pear in vitro.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Figure 3. The effect of Thidiazuron on shoot regeneration from pear leaves in vitro (P = 0.0006) (C = Crystal pear; L = Louise Bonne; S = Seckel)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Figure 4. The effect of NAA on shoot regeneration of Seckel pear (S) in vitro (P = 0.0006)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Figure 5. The effect of Thidiazuron on shoot regeneration from pear leaves in vitro (P = 0.016) (C = Crystal pear; L = Louise Bonne; S = Seckel)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Figure 6. The effect of sucrose on shoot regeneration of Crystal pear (C) leaves in vitro with TDZ (1.0 µM); NAA (2.7 µM)</figcaption></figure>

Contrast analysis:

- CP: $R^2 = 0.44$ PR>F (Lepovivre/average MS effect) = 0.2208
PR>F (MS linear effect) = 0.0026

- LB: $R^2 = 0.63$ PR>F (Lepovivre/average MS effect) = 0.0003
PR>F (MS linear effect) = 0.0032

- S: $R^2 = 0.67$ PR>F (Lepovivre/average MS effect) = 0.0003
PR>F (MS linear effect) = 0.0004

<figure><img src="image_8.png" /><figcaption>Fig.7: The effect of macromolecule composition on shoot regeneration from pear leaves. (Experiment 3)</figcaption></figure>
'Louise Bonne' and 'Seckel', Lepovre medium was
significantly inferior to the other media. However,
the 2 cultivars showed a significant positive re-
sponse to MS salt concentration; the optimum concen-
tration was either 1/2 or 1/4 strength.

At the end of each experiment, the regenerated
shoots were transferred to ordinary Lepovivre 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 ob-
tained in vitro from leaf tissue of 3 pear cultivars
of 2 different species. There were significant dif-
ference in the ability of each cultivar to regen-
erate 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 in-
vestigation of various media components that might
affect the rate of shoot regeneration in vitro.
Growth regulators proved to be very important: re-
generation 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 pre-
dicted to be optimal for the 3 cultivars. Among the
different auxins tested, NAA seemed to be more effi-
cient 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 proved
to be important for callus induction and shoot re-
generation from apple shoot internodes (Evaldsson,
1985). In our study, we also found macroelement
composition to be an important factor for regenera-
tion. Most of our experiments used Lepovivre macro-
elements. This medium differs from MS medium by a
very low concentration of NH<sub>4</sub><sup>+</sup>, a slightly higher
concentration of Ca<sup>2+</sup>, and a total macro-salt concen-
tration equivalent to about 3/4 of MS. Lepovivre
medium has been found to be very efficient for main-
tenance of proliferating cultures of several pear
cultivars (Personal Communication, E. Chevreau).
However, the macroelements required for adventitious
regeneration are certainly different; 1/2 or 1/4 MS
proved to be optimal in our experiments.

Sucrose is the main energy source for in vitro-
grown plants and its concentration has 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' ex-
plants which differentiated improved positively as
sucrose level increased. The sucrose had no signifi-
cant effect on 'Louise Bonne'.

A particular dark/light exposure has been found
necessary for shoot regeneration from apple (per-
sonal communication, John Sanford, NY State Exp.

Stn., Geneva). Most of our experiments were con-
ducted with a dark incubation of 25 days and a trans-
fer 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 parame-
ters 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.

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