Scientia Horticulturae, 28 (1986) 315--321 315 Elsevier Science Publishers B.V., Amsterdam --Pr in ted in The Netherlands
IN VITRO MICROPROPAGATION OF QUINCE (CYDONIA OBLONGA MILL.)
KHALIL AL MAARRI, YOLANDE ARNAUD and EMILE MIGINIAC
Laboratoire de Physiologie Vdgdtale, Ontogen~se Expdrimentale, Universitd P. et M. Curie, Tour 53-5~me dtage 4, Place Jussieu, 75230 Paris Cddex 05 (France)
(Accepted for publication 12 November 1985)
ABSTRACT
AI Maarri, K., Arnaud, Y. and Miginiac, E., 1986. In vitro micropropagation of quince (Cydonia oblonga Mill.). Scientia Hortic., 28: 315--321.
Methods are described for the in vitro propagation of Provence quince for pear root- stock, making possible the production of a large number of plants from micro-cuttings. The influence of 6-benzylaminopurine concentrations on shoot multiplication and elongation was determined. The best rate of shoot multiplication was obtained with 2 mg l -z 6-benzylaminopurine (more than 5 shoots per culture).
Rooting was obtained on media containing 0.1--1 mg I -I naphthaleneacetic acid; the best combination of percentage and quality of roots was with 0.1 mg l -I naphthalene- acetic acid. The effect of transferring to a hormone-free medium is discussed.
Keywords: in vitro propagation; pear rootstock; Provence quince.
Abbreviations: BAP = 6-benzylaminopurine; GA 3 = gibbereUic acid; IBA = indole-3- butyric acid; MSJ = mineral salts of Murashige and Skoog (1962) modified by Jones et al. (1977); NAA = naphthaleneacetic acid.
INTRODUCTION
Pear is usually propagated by grafting onto quince (Cydonia oblonga Mill.) or pear seedlings (Pyrus communis 'Franc') (Brian and Duron, 1971). Quince as rootstocks produce dwarf trees, suitable for high-density planting systems. Two French sources of quince are Provence and Angers (Broissier, 1978); these rootstocks are usually propagated by rooting cuttings or by layering.
It would be valuable to be able to propagate fruit trees by tissue-culture techniques because the propagation rate is much higher than with traditional methods. The first in vitro propagation of apple rootstock M26 (Quoirin, 1974) was about 11 years ago. Since then, many other fruit-tree rootstocks have been propagated by in vitro techniques, e.g. apple (Jones et al., 1977; James and Thurbon, 1979; Navatel, 1980; Welander, 1983) and peach
(Mosella et al., 1980; Hammerschlag, 1982). Some fruit tree cultivars have also been propagated in vitro, e.g. apple (Lane, 1978; Zimmerman and Broome, 1981; Sriskandarajah and Mullins, 1981), pear (Lane, 1979; Singha, 1980), plum and quince (Boxus and Quoirin, 1977; N~meth, 1979). This paper describes methods for the in vitro propagation of Provence quince, an important pear rootstock.
M A T E R I A L AND METHODS
Scions of 'Passe-Crassane' pear cultivar grafted onto Provence quince were placed in a greenhouse under the following conditions: 22°C day-- 12°C night temperatures, with 16 h light per day and 65% humidity. Micro- cuttings (10--15 mm), containing one terminal or axillary bud, were taken from axillary shoots of rootstocks. These initial explants were surface- sterilized by dipping them in 95% ethanol for 30 s and then immersing them for 20 min in 10% Domestos (a commercial preparation of sodium hypo- chlorite, with 7% active chlorine; made in Warrington (Great Britain) by Lever), followed by three washings in sterile water.
Micro-cuttings were inoculated into test tubes (200 X 24 mm) with 15 ml culture medium containing the mineral salts of Lepoivre (Quoirin et al., 1977) with the addition of 3% sucrose, 0.6% bacto agar (Difco), 0.5 mg 1-1 BAP, 0.1 mg 1-1 IBA, 0.2 mg 1-1 GA3, 0.4 mg 1-1 thiamine hydro- chloride and 100 mg 1-1 inositol. This medium is used for the initial culture and for shoot multiplication and elongation.
Three sub-cultures were made at monthly intervals. When there was a sufficient quanti ty of material, we studied the influence of different BAP concentrations (0, 0.5, 1, 2 and 4 mg 1-1) on shoot multiplication and elongation. For this experiment, 24 shoot tips {10--15 mm) from in vitro explants were used per treatment.
For rooting, 24 shoot tips (15--20 mm) per t reatment were inoculated vertically on media consisting of 1/3 X MSJ (mineral salts of Murashige and Skoog, but with the NaEDTA and FeNaSO4 replaced by FeNaEDTA at 20 mg 1-1) plus 2% sucrose, 0.6% agar, 100 mg 1-1 meso-inositol, 0.4 mg 1-1 thiamine and NAA at different concentrations (0, 0.1, 0.5 or 1 mg 1-1). Some explants were left on the rooting media for 1 month; others were transferred after 1 week to a hormone-free medium. All media were adjusted to pH 5.5--5.7 and then sterilized by autoclaving at 115°C for 30 min. All cultures were kept at 22 -+ I°C with a 16-h photoper iod sup- plied by fluorescent tubes at 25 W m -2.
Roo ted micro-cuttings were transferred to plastic containers (45 X 30 X 14 cm) holding a mixture of sterilized vermiculite and perlite (1/1); each week this substrate was sprayed twice with water and once with 1/3 X MSJ mineral salts.
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R E S U L T S
I n i t i a l c u l t u r e . - - After 2 months in culture, the bud of each micro-cutting burst and elongated to give a single shoot averaging 2.6 cm in length.
S h o o t m u l t i p l i c a t i o n a n d e l o n g a t i o n . - - Shoots provided from the first phase were cut into 1--2 node segments and transferred to fresh medium, where further axillary shoots developed. Shoots were sub-cultured every 4 weeks. With each sub-culture there was a 2.8-fold increase in shoot num- ber, with a mean shoot length of 3.6 cm.
After 1 month of culture on media at different BAP concentrations, we made the following observations: cytokinin-free medium was unfavour- able for shoot multiplication and elongation (Fig. 1). The rate of shoot multiplication increased with increasing BAP concentrations, but their elongation decreased (Fig. 1). Shoots were often fasciated in media con- taining 4 mg 1-1 BAP. The best results were obtained with 2 mg 1-1 BAP; the rate of multiplication was high, and shoots were well elongated and
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2
o 0'.51 ~ g ' ,:si 2 BAP CONCENTRATIONS(mgb 1)
Fig. 1. In v i t ro mu l t i p l i ca t i on and e longat ion o f quince shoots: effect o f d i f fe rent BAP concentrat ions. Twen ty - fou r explants per treatment, observed af ter 1 mon th of cul ture. A. Shoot mu l t i p l i ca t ion rate (number o f shoots fo rmed f rom one shoot). B. Shoot e longat ion (era). Bars = s.d.
Fig. 2. Ef fec t of d i f f e ren t N A A c o n c e n t r a t i o n s o n roo t ing of qu ince cu t t ings (24 ex- p lants ) : , w i t h o u t t ransfer ; . . . . , w i th t ransfer . A. Roo t i ng percentage . B. Average e longa t ion of the longes t r o o t s (cm). C. Average r o o t n u m b e r per r o o t e d cu t t ing . Bars = s.d. The same resul t s were o b t a i n e d in t w o r epe t i t i ons of these roo t ing expe r imen t s .
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Fig. 3. Rooting quince in the presence of different NAA concentrations. Top. a, b, c and d correspond to the following NAA concentrations, respectively: 0, 0.1, 0.5 and 1 mg |-, applied for 1 month. Bottom. e, f, g and h correspond to the following NAA concentrations, respectively 0, 0.1, 0.5 and I mg l-' applied for the first week of the experiment, with the explants then being transferred to a hormone-free medium.
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normally developed. After this experiment, the apical port ions of these shoots were used for rooting and the basal port ions were cultured on Le- poivre's medium with 2 mg 1-1 BAP for shoot multiplication (in preliminary experiments, basal explants multiplied better than apical explants).
Shoots were sub-cultured at monthly intervals for 1 year on the same medium; we obtained a good rate of shoot multiplication (more than 5-fold per sub-culture).
R o o t i n g . - - Hormone-free medium was unfavourable for rooting. The rooting percentage, the number of roots per rooted cutting, the callus formation and the root thickness increased with increasing NAA concentrations ap- plied for 1 month, but the average root elongation decreased (Figs. 2 and 3). The rooting percentage was highest with 0.5 mg 1-1 NAA, but the quality of root formation was best with 0.1 mg 1-1 NAA (Fig. 3b).
In another experiment, the same concentrations of NAA were used, but after 1 week the micro-cuttings were transferred to a hormone-free medium for 1 month. The rooting percentage, the number of roots per rooted cutting and the callus formation rose with increasing NAA con- centrations (Figs. 2 and 3). With 0.5 or 1 mg 1-1 NAA applied for 1 week, the rooting percentage and the root quality were better than with 0.1 mg 1-1 NAA also applied for 1 week; at the same time, the length of roots increased and the callus size decreased with 0.5 or 1 mg 1-' NAA applied for 1 week compared to the same concentrations applied for 1 month (Fig. 3c, d, g and h).
T r a n s p l a n t a t i o n . - - After 3 months, rooted shoots had grown to a height of more than 30 cm. All these plants were identical to Provence quince and did not show morphological abnormalities.
DISCUSSION AND CONCLUSIONS
There are many applications of aseptic methods in the propagation and breeding of fruit plants. Our experiments show for the first t ime (as far as is known) that in vitro culture can be used to rapidly propagate large numbers of Provence quince plants.
The rate of propagation in vitro is much higer than traditional methods (Jones et al., 1977; Martin et al., 1981). In our case, more than 5 shoots per month could be produced from the basal port ion of a shoot cultivated horizontally on Lepoivre's medium with 2 mg 1-1 BAP. Roo t formation of the quince BA 29 was obtained by N~meth (1979) on media containing cytokinin with or wi thout exogenously applied auxin. In our case, rooting of the Provence quince could not be induced in the presence of BAP or IBA, but cultivars and culture conditions were not similar.
Two requirements for successful propagation are a high rooting percentage and good quality (long roots, small callus). The best results were obtained
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from apical cuttings cultured on a medium containing mineral salts of 1/3 X MSJ with 0.1 mg 1-1 NAA applied for 1 month. Satisfactory rooting percentage combined with good root quality without callus formation were observed. This method has been conducted over 10 times, repeatedly giving good-quality rooting. When a high concentration of NAA (0.5 or 1 mg 1-1) was used, it was imperative to transfer explants to hormone-free medium after 1 week. Cuttings left in continuous contact with 0.5 or 1 mg 1-1 NAA formed abundant callus at the cut surface and root elonga- tion was inhibited. James and Thurbon (1979) made the same observation on apple rootstock M 9.
In conclusion, these methods can be used commercially for the rapid multiplication of Provence quince to produce a large number of plants in a short time.
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