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Double-phase in vitro culture using sorbitol increases shoot proliferation and reduces hyperhydricity in Japanese pear
Scientia Horticulturae 89 (2001) 207±215
Double-phase in vitro culture using sorbitol increases shoot proliferation and reduces hyperhydricity in Japanese pear M. Kadota*, K. Imizu, T. Hirano Toyama City Agricultural Center, 3-101 Tsukioka-cho, Toyama 939-8132, Japan Accepted 11 October 2000
Abstract This study aimed to improve in vitro shoot proliferation ef®ciency without inducing hyperhydricity in Japanese pear. The shoot number increased at 2.5±10.0 mg l 1 benzyladenine (BA), while shoot fresh mass increased at 1.0 and 2.5 mg l 1 BA. Different macroelement formulation did not affect shoot proliferation, but adding activated charcoal (AC) to the medium inhibited markedly the production of axillary shoots and biomass and many shoots were hyperhydric. Different carbon sources (CS) signi®cantly increased the shoot number and fresh mass, with the best results for shoot proliferation at 20±30 g l 1 sorbitol. With gelling agents, the shoot number increased at 0.4 and 0.6% agar and 0.3% gellan gum, while fresh mass increased at 0.4% agar. The hyperhydric explants were more than 30% at 0.4±0.6% agar and at any concentration of gellan gum. The improved culture (woody plant (WP) supplemented with 20 g l 1 sorbitol, 0.1 mg l 1 3-indolyl-butyric acid (IBA), 2.5 mg l 1 BA and 0.8% agar) and double-phase culture (the same medium using a double-phase liquid-gelling agent solidi®ed culture system) produced a higher number of axillary shoots than the conventional culture (1/2MS supplemented with 0.1 mg l 1 IBA, 1.0 mg l 1 BA, 30 g l 1 sucrose and 0.8% agar), moreover, double-phase culture had a higher fresh mass than the other cultures. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Carbon source; Double-phase culture; Gelling agent; Hyperhydricity; Japanese pear; Pyrus pyrifolia N.; Shoot proliferation; Sorbitol
* Corresponding author. Tel.: 81-76-429-4504; fax: 81-76-429-2449. E-mail address: [email protected] (M. Kadota).
0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 3 4 - X
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M. Kadota et al. / Scientia Horticulturae 89 (2001) 207±215
1. Introduction Japanese pear (Pyrus pyrifolia N.) is one of the most important deciduous fruit trees in Japan, China, South Korea and New Zealand. Several studies on the micropropagation of European pear (Pyrus communis L.) have been carried out and factors in¯uencing shoot proliferation and rooting (Singha, 1982; Shen and Mulling, 1984; Viseur, 1987; Moretti et al., 1991; RodrõÂguez et al., 1991; Wang, 1991, 1992; Bertazza et al., 1995; Reed, 1995; Yeo and Reed, 1995) have been identi®ed. Relatively, the in vitro behavior of the Japanese pear has been less investigated. In the genus Pyrus, adventitious shoots obtained from leaf segments (Chevreau et al., 1989; Predieri et al., 1989; Lane et al., 1998), protoplasts derived calli (Ochatt and Power, 1988; Ochatt et al., 1992) or anthers (unpublished data) are often stunted and do not elongate satisfactorily until after several subcultures in proliferation phase. Therefore, establishing a reliable and rapid micropropagation of the adventitious shoots derived from various organs or tissues is important when breeding by applying biotechnological techniques and propagating virusfree plants. In Japanese pear, micropropagation by apical meristem culture has been developed (Banno et al., 1989), but the ef®ciency of this propagation system is very low. Therefore, this study aimed to improve in vitro shoot proliferation ef®ciency without inducing hyperhydricity in Japanese pear. 2. Materials and methods 2.1. Plant materials and culture conditions Shoots from Japanese pear cultivar Hosui that had been maintained in vitro by monthly subcultures for 12 months were used for this study. Hosui is one of the most important cultivars in Japan. Shoot cultures were kept in a proliferation medium consisting of half strength MS (Murashige and Skoog, 1962) (1/2MS) with 0.1 mg l 1 3-indolyl-butyric acid (IBA), 1.0 mg l 1 benzyladenine (BA), 30 g l 1 sucrose and 0.8% powder agar (Wako Co. Ltd., Japan). All media were adjusted with 0.1 N NaOH to pH 5.7 and were autoclaved at 1.1 kg cm 2 (1218C) for 20 min. All cultures were incubated under a 16 h photoperiod illuminated by a cool-white ¯uorescent light (50 mmol m 2 s 1) at 25 1 C in a plastic pot (5 15 cm) containing 30 ml of gelled medium (unless otherwise speci®ed). 2.2. Shoot proliferation experiments First, the effect of cytokinins and their concentrations on shoot proliferation and hyperhydricity were studied. The shoots were transferred to 1/2MS medium containing 30 g l 1 sucrose, 0.1 mg l 1 IBA, 0.8% powder agar and 0.1, 1.0, 2.5,
M. Kadota et al. / Scientia Horticulturae 89 (2001) 207±215
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5.0 and 10 mg l 1 BA or 0.1 and 1.0 mg l 1 thidiazuron (TDZ). To determine the effect of three different basal salts mixtures (macro and micro) and activated charcoal (AC), the shoots were transferred to MS, 1/2MS and woody plant (WP) (Lloyd and McCown, 1981) medium supplemented with 30 g l 1 sucrose, 0.1 mg l 1 IBA, 2.5 mg l 1 BA and 0.8% powder agar with or without 0.3% AC. To investigate the effect of carbon sources (CS) at different concentrations, the shoots were transferred to WP supplemented with 0.1 mg l 1 IBA, 2.5 mg l 1 BA, 0.8% powder agar and one of the following CS, sucrose, sorbitol, or glucose. Each CS was provided at three levels: 20, 30 and 40 g l 1. To compare the effect of gelling agents and their concentrations, the shoots were transferred to WP supplemented with 20 g l 1 sorbitol, 0.1 mg l 1 IBA, 2.5 mg l 1 BA solidi®ed by 0.1, 0.15, 0.2, 0.25 and 0.3% gellan gum (Wako Co. Ltd.) or 0.4, 0.6, 0.8, 1.0 and 1.2% powder agar. Finally, the effect of a double-phase culture system was evaluated. The shoots were transferred to WP supplemented with 20 g l 1 sorbitol, 0.1 mg l 1 IBA, 2.5 mg l 1 BA and 0.8% powder agar (the improved culture), or the same medium using a double-phase liquid-gelling agent solidi®ed culture system. For double-phase culture, 10 ml of liquid medium composed of the same as solid phase minus agar was added after horizontally positioning the primary explants. At the same time, the conventional culture of Banno et al. (1989), which is consisting of 1/2MS with 0.1 mg l 1 IBA, 1.0 mg l 1 BA, 30 g l 1 sucrose and 0.8% powder agar, was compared. 2.3. Data analysis All experiments were carried out completely randomly and were repeated three times with three shoots within ®ve pots per treatment. After 35 days from the start of the experiment, the number of shoots formed per explant, percentage of fresh mass increase and percentage of hyperhydric shoot were recorded and the former two were analyzed using Duncan's multiple range test at P 0:05. Also, for the experiment on the effect of macroelements and AC, the number of shoots formed per explant and rate of fresh mass increase were subjected to a two-way ANOVA with macroelements and AC. For the experiment on the effect of CS at different concentrations, the two indicators of shoot proliferation were subjected to a twoway ANOVA with CS and CS concentrations. 3. Results Hyperhydric shoots were observed in the experiment on the effect of macroelements and the experiment on the effect of gelling agents, but they were subcultured in the same fresh medium, they stopped growing within 2±3 months and then died. The number of shoots increased at 2.5±10.0 mg l 1 BA, and the
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Table 1 Effect of cytokinins on shoot proliferation and hyperhydricitya Cytokinin
Concentration (mg l 1)
Number of shoots formed per explant
±b
Fresh mass increase (%)
1.1 cc
BA
0.1 1.0 2.5 5.0 10.0
TDZ
0.1 1.0
1.1 2.2 4.2 4.0 5.6
66.9 c
c b a a a
294.7 640.6 559.6 374.0 204.1
1.5 bc 1.1 c
bc a a b bc
301.8 bc 96.4 c
a
Fresh mass of shoot tips at the start of the experiment were 80:1 5:7 mg. Proliferation medium with no cytokinin. c Mean separation by Duncan's multiple range test at P 0:05. b
percentage of shoot fresh mass increased at 1.0 and 2.5 mg l 1 BA (Table 1). TDZ had no stimulatory effect on the two indicators of proliferation. Hyperhydric shoots were not observed. The different macroelements did not affect shoot proliferation, but adding AC to the medium markedly inhibited the production of axillary shoots and biomass and many normal shoots were hyperhydric (Table 2). The different CS concentrations increased signi®cantly the shoot number and the percentage of fresh mass increase, with sorbitol producing a greater increase than sucrose and Table 2 Effect of macroelements and AC on shoot proliferation and hyperhydricitya Macroelement MS MS 1/2MS 1/2MS WP WP
d
Significant macroelement Significant AC Significant macroelement AC a
ACb
Number of shoots formed per explant 4.0 1.3 4.7 1.5 5.0 1.1 NS ** NS
ac b a b a b
Fresh mass increase (%)
Hyperhydric individuals (%)
499.0 152.5 628.9 226.1 591.2 130.0
0 73.3 0 66.7 0 60.0
a b a b a b
NS ** NS
Fresh mass of shoot tips at the start of the experiment were 78:5 4:8 mg. Proliferation medium with () or without ( ) 0.3% AC. c Mean separation by Duncan's multiple range test at P 0:05. d NS and ** indicate not signi®cant, signi®cant at P 0:01, respectively. b
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Table 3 Effect of CS and their concentrations on shoot proliferation and hyperhydricitya CS
Concentration (mg l 1)
Number of shoots formed per explant
Fresh mass increase (%)
Sucrose
20 30 40
3.3 abb 1.8 c 1.5 c
564.3 bc 504.6 bc 477.4 bc
0 0 6.7
Sorbitol
20 30 40
4.4 a 3.9 a 2.2 bc
777.3 ab 906.5 a 676.6 abc
0 6.7 0
Glucose
20 30 40
2.3 bc 1.6 c 1.1 c
737.5 abc 578.0 abc 430.6 c
6.7 0 13.3
** ** NS
** NS NS
Significantc CS Significant concentration Significant CS concentration
Hyperhydric individuals (%)
a
Fresh mass of shoot tips at the start of the experiment were 19:9 1:3 mg. Mean separation by Duncan's multiple range test at P 0:05. c NS and ** indicate not signi®cant, signi®cant at P 0:01, respectively. b
fructose (Table 3). The difference in concentration affected the shoot number, which decreased as the concentration of each CS increased. A few hyperhydric explants were observed with every CS. The shoot number increased at 0.4% agar and at 0.3% gellan gum and followed 0.6% agar (Table 4). The percentage of fresh mass increase increased at 0.4% Table 4 Effect of gelling agents and their concentrations on shoot proliferation and hyperhydricitya Gelling agent
Concentration (%)
Number of shoots formed per explant (%)
Agar
0.4 0.6 0.8 1.0 1.2
4.4 3.9 3.1 2.4 2.3
Gellan gum
0.1 0.15 0.2 0.25 0.3
2.4 1.7 2.1 3.1 4.5
a b
Fresh mass increase (%)
Hyperhydric individuals (%)
ab ab bc c c
3999.8 1269.3 906.4 802.5 402.1
a cd cd d e
100 33.3 6.7 6.7 0
c c c bc a
1769.2 1306.6 2594.5 1828.1 1666.6
c c b c c
100 93.3 93.3 86.7 33.3
Fresh mass of shoot tips at the start of the experiment were 19:6 0:6 mg. Mean separation by Duncan's multiple range test at P 0:05.
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Table 5 Effect of a double-phase culture system on shoot proliferation and hyperhydricitya Culture system
Number of shoots formed per explant
Fresh mass increase (%)
Hyperhydric individuals (%)
Double-phase culture Improved culture Conventional culture
4.7 ab 4.7 a 2.2 b
1559.4 a 1005.6 b 499.1 b
6.7 0 6.7
a b
Fresh mass of shoot tips at the start of the experiment were 22:7 1:3 mg. Mean separation by Duncan's multiple range test at P 0:05.
agar. The percentage of hyperhydric explants was 33% or more, i.e. over onethird of the total, at 0.4 and 0.6% agar, and at every concentration of gellan gum. The double-phase culture and the improved culture produced a high number of axillary shoots compared with the conventional culture (Table 5). Also, the former culture had a higher fresh mass than the other cultures. The percentage of hyperhydric explants in the three culture systems was few or none. 4. Discussion Moretti et al. (1991) investigated the effect of BA concentration from 0 to 1.0 mg l 1 on shoot proliferation of European pear and reported that shoot proliferation increased as the concentration increased. In wild pear, also, 1.5 and 2.0 mg l 1 BA stimulates axillary shoot production and dry weight compared with from 0 to 1.0 mg l 1 BA (Shibli et al., 1997). In this study, the fresh mass increase did not differ between 1.0 and 2.5 mg l 1 BA, which gave the best results, but the latter value produced a greater shoot number than the former value (Table 1). Van Nieuwkerk et al. (1986) showed that 10.0 (2.2 mg l 1) and 1.0 mM TDZ were equivalent to, or greater than, the number of shoots produced when using 4.4 mM BA, but this increase was not observed in this study. Therefore, we think that 2.5 mg l 1 BA is the best cytokinin and concentration for shoot proliferation. We found no signi®cant effect by the three macroelements against shoot proliferation (Table 2). Although not signi®cant, the highest shoot number was obtained with WP, so we used it for the next experiment. As the nitrogen concentration of WP is about one-fourth of full strength MS, a low concentration of nitrogen or low total salts may produce better results in Japanese pear. AC supplemented to the medium prevented markedly axillary shoot and fresh mass production and produced shoot hyperhydricity, which is a physical disorder and causes the shoots to have an aberrant morphology characterized by translucent and brittle stems and leaves. AC have the property of a strong absorption of
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dissolved solids (George and Sherington, 1984). Also, a decrease in gelling agent concentration in the medium produces a hyperhydricity of apple shoot (Pasqualetto et al., 1986). The adsorption of BA in the medium by AC may be caused by poor shoot proliferation and the adsorption of agar in the medium may produce the hyperhydricity. Although AC increases plant recovery ef®ciency in many species (George and Sherington, 1984), it is not suitable for multiplication of Japanese pear. The type of carbohydrates affects the growth and the frequency of shoots developed, and shoot proliferation of woody Rosaceous species, such as apple and apricot, is often increased using sorbitol (Pua and Chong, 1984; Welander et al., 1989; Marino et al., 1991, 1993; Karhu, 1997). However, examination of the effect of different CS on Japanese pear has not been made. In this study, sorbitol produced a good result for shoot multiplication compared with sucrose and fructose (Table 3). The concentrations of sorbitol at 20 and 30 g l 1 were more effective than 40 g l 1 in producing new shoots. Therefore, the most ef®cient CS was sorbitol at a concentration 20±30 g l 1. Low concentration of agar medium, such as 0.4 and 0.6%, and gellan gum medium clearly increased hyperhydric explants (Table 4). Using gellan gum or a decrease in gelling agent concentration hastens the hyperhydricity of cultured explants of apple and European pear (Pasqualetto et al., 1986, 1988; Turner and Singha, 1990; Marga et al., 1997), similar to that observed in this study of Japanese pear. These results imply that using gellan gum and low concentrations of agar must be avoided when proliferating Japanese pear shoots. At three higher concentrations of agar, 0.8% agar had a greater effect than 1.0 and 1.2% agar for shoot proliferation. So, the best gelling agent was agar at a concentration 0.8%. The double-phase culture increased the number of shoots about twice, and the rate of fresh mass increased about three times compared with the conventional culture (Table 5). The rate of fresh mass increase in the double-phase culture was greater than that of the improved culture. Although double-phase culture increases shoot proliferation of some fruit trees (Viseur, 1987; Moretti et al., 1991; RodrõÂguez et al., 1991; Murai and Harada, 1995), it is greatly effective with Japanese pear. In this study, we improved markedly the speed of proliferating shoots of Japanese pear cultivars using 2.5 mg l 1 BA, 20±30 g l 1 sorbitol and the doublephase culture system. In the future, we will use this culture system to produce haploids by anther culture and transgenic plants. References Banno, K., Yoshida, K., Hayashi, S., Tanabe, K., 1989. In vitro propagation of Japanese pear cultivars. J. Jpn. Soc. Hortic. Sci. 58, 37±42.
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Bertazza, G., Baraldi, R., Predieri, S., 1995. Light effects on in vitro rooting of pear cultivars of different rhizogenic ability. Plant Cell, Tiss. Organ Cult. 41, 139±143. Chevreau, E., Skirvin, R.M., Abu-Qaoud, H.A., Korban, S.S., Sullivan, J.G., 1989. Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro. Plant Cell Rep. 7, 688±691. George, E.F., Sherington, P.D., 1984. Plant Propagation by Tissue Culture. Eastern Press, Berks, UK. Karhu, S.T., 1997. Sugar use in relation to shoot induction by sorbitol and cytokinin in apple. J. Am. Soc. Hortic. Sci. 122, 476±480. Lane, W.D., Iketani, H., Hayashi, T., 1998. Shoot regeneration from cultured leaves of Japanese pear (Pyrus pyrifolia). Plant Cell, Tiss. Organ Cult. 54, 9±14. Lloyd, G., McCown, B., 1981. Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Proc. Int. Plant Prop. Soc. 30, 421±427. Marga, F., Vebret, L., Morvan, H., 1997. Agar fractions could protect apple shoots cultured in liquid media against hyperhydricity. Plant Cell, Tiss. Organ Cult. 49, 1±5. Marino, G., Magnanini, E., Battistini, S., Righetti, B., 1991. Effect of hormones and main carbon energy sources on in vitro propagation of apricot (Prunus armeniaca L.) cvs. `San Castrese' and `Portici'. Acta Hortic. 293, 355±362. Marino, G., Bertazza, G., Magnanini, E., Altan, A.D., 1993. Comparative effects of sorbitol and sucrose as main carbon energy sources in micropropagation of apricot. Plant Cell, Tiss. Organ Cult. 34, 235±244. Moretti, C., Scozzoli, A., Pasini, D., Paganelli, F., 1991. In vitro propagation of pear cultivars. Acta Hortic. 300, 115±118. Murai, Y., Harada, H., 1995. Improvement of shoot proliferation of Japanese plum, mume and apricot cultivars by double phase culture. Plant Tiss. Cult. Lett. 12, 325±327 (in Japanese). Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 478±497. Ochatt, S.J., Power, J.B., 1988. Plant regeneration from mesophyll protoplasts of Willams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.). Plant Cell Rep. 7, 587±589. Ochatt, S.J., Chevreau, E., Gallet, M., 1992. Organogenesis from `Passe Crassane' and `Old Home' pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants. Theor. Appl. Genet. 83, 1013±1018. Pasqualetto, P.-L., Zimmerman, R.H., Fordham, I., 1986. Gelling agent and growth regulator effects on vitri®cation of `Gala' apple in vitro. J. Am. Soc. Hortic. Sci. 111, 976±980. Pasqualetto, P.-L., Zimmerman, R.H., Fordham, I., 1988. The in¯uence of cation and gelling agent concentrations on vitri®cation of apple cultivars in vitro. Plant Cell, Tiss. Organ Cult. 14, 31±40. Predieri, S., Fasolo Fabbri Malavasi, F., Passey, A.J., Ridout, M.S., James, D.J., 1989. Regeneration from in vitro leaves of `conference' and other pear cultivars (Pyrus communis L.). J. Hortic. Sci. 64, 553±559. Pua, E.C., Chong, C., 1984. Requirement for sorbitol (D-glucitol) as carbon source for in vitro propagation of Malus robusta No. 5. Can. J. Bot. 62, 1545±1549. Reed, B.M., 1995. Screening Pyrus germplasm for in vitro rooting response. Hortscience 30, 1292±1294. RodrõÂguez, R., DõÂaz-Sala, C., Cuozzo, L., Ancora, G., 1991. Pear in vitro propagation using a double-phase culture system. Hortscience 26, 62±64. Shen, X.S., Mulling, M.G., 1984. Pyrus communis L., cultivars `William's Bon ChreÂtien', `Packham's Triumph' and `BeurreÁ Bosc'. Sci. Hortic. 23, 51±57. Shibli, R.A., Ajlouni, M.M., Jaradat, A., Aljanabi, S., Shatnawi, M., 1997. Micropropagation in wild pear (Pyrus syrica). Sci. Hortic. 68, 237±242.
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Singha, S., 1982. In¯uence of agar concentration on in vitro shoot proliferation of Malus sp. `Almey' and Pyrus communis `Seckel'. J. Am. Soc. Hortic. Sci. 107, 657±660. Turner, S.R., Singha, S., 1990. Vitri®cation of crabapple, pear, and geum on gellan gum-solidi®ed culture medium. Hortscience 25, 1648±1650. Van Nieuwkerk, J.P., Zimmerman, R.H., Fordham, I., 1986. Thidiazuron stimulation of apple shoot proliferation in vitro. Hortscience 21, 516±518. Viseur, J., 1987. Micropropagation of pear Pyrus communis L. in a double-phase culture medium. Acta Hortic. 212, 117±124. Wang, Q., 1991. Factors affecting rooting of microcuttings of the pear rootstock BP 10030. Sci. Hortic. 45, 209±213. Wang, Q., 1992. The effect of light, darkness and temperature on micropropagation of the pear rootstock BP 10030. J. Hortic. Sci. 67, 869±876. Welander, M., Wrender, N.T., Brackman, A.S., 1989. Regulation of in vitro shoot multiplication in Syringa, Alnus, and Malus by different carbon sources. J. Hortic. Sci. 64, 361 and ±366. Yeo, D.Y., Reed, B.M., 1995. Micropropagation of three Pyrus rootstocks. Hortscience 30, 620 and ±623.
Double-phase in vitro culture using sorbitol increases shoot proliferation and reduces hyperhydricity in Japanese pear M. Kadota*, K. Imizu, T. Hirano Toyama City Agricultural Center, 3-101 Tsukioka-cho, Toyama 939-8132, Japan Accepted 11 October 2000
Abstract This study aimed to improve in vitro shoot proliferation ef®ciency without inducing hyperhydricity in Japanese pear. The shoot number increased at 2.5±10.0 mg l 1 benzyladenine (BA), while shoot fresh mass increased at 1.0 and 2.5 mg l 1 BA. Different macroelement formulation did not affect shoot proliferation, but adding activated charcoal (AC) to the medium inhibited markedly the production of axillary shoots and biomass and many shoots were hyperhydric. Different carbon sources (CS) signi®cantly increased the shoot number and fresh mass, with the best results for shoot proliferation at 20±30 g l 1 sorbitol. With gelling agents, the shoot number increased at 0.4 and 0.6% agar and 0.3% gellan gum, while fresh mass increased at 0.4% agar. The hyperhydric explants were more than 30% at 0.4±0.6% agar and at any concentration of gellan gum. The improved culture (woody plant (WP) supplemented with 20 g l 1 sorbitol, 0.1 mg l 1 3-indolyl-butyric acid (IBA), 2.5 mg l 1 BA and 0.8% agar) and double-phase culture (the same medium using a double-phase liquid-gelling agent solidi®ed culture system) produced a higher number of axillary shoots than the conventional culture (1/2MS supplemented with 0.1 mg l 1 IBA, 1.0 mg l 1 BA, 30 g l 1 sucrose and 0.8% agar), moreover, double-phase culture had a higher fresh mass than the other cultures. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Carbon source; Double-phase culture; Gelling agent; Hyperhydricity; Japanese pear; Pyrus pyrifolia N.; Shoot proliferation; Sorbitol
* Corresponding author. Tel.: 81-76-429-4504; fax: 81-76-429-2449. E-mail address: [email protected] (M. Kadota).
0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 3 4 - X
208
M. Kadota et al. / Scientia Horticulturae 89 (2001) 207±215
1. Introduction Japanese pear (Pyrus pyrifolia N.) is one of the most important deciduous fruit trees in Japan, China, South Korea and New Zealand. Several studies on the micropropagation of European pear (Pyrus communis L.) have been carried out and factors in¯uencing shoot proliferation and rooting (Singha, 1982; Shen and Mulling, 1984; Viseur, 1987; Moretti et al., 1991; RodrõÂguez et al., 1991; Wang, 1991, 1992; Bertazza et al., 1995; Reed, 1995; Yeo and Reed, 1995) have been identi®ed. Relatively, the in vitro behavior of the Japanese pear has been less investigated. In the genus Pyrus, adventitious shoots obtained from leaf segments (Chevreau et al., 1989; Predieri et al., 1989; Lane et al., 1998), protoplasts derived calli (Ochatt and Power, 1988; Ochatt et al., 1992) or anthers (unpublished data) are often stunted and do not elongate satisfactorily until after several subcultures in proliferation phase. Therefore, establishing a reliable and rapid micropropagation of the adventitious shoots derived from various organs or tissues is important when breeding by applying biotechnological techniques and propagating virusfree plants. In Japanese pear, micropropagation by apical meristem culture has been developed (Banno et al., 1989), but the ef®ciency of this propagation system is very low. Therefore, this study aimed to improve in vitro shoot proliferation ef®ciency without inducing hyperhydricity in Japanese pear. 2. Materials and methods 2.1. Plant materials and culture conditions Shoots from Japanese pear cultivar Hosui that had been maintained in vitro by monthly subcultures for 12 months were used for this study. Hosui is one of the most important cultivars in Japan. Shoot cultures were kept in a proliferation medium consisting of half strength MS (Murashige and Skoog, 1962) (1/2MS) with 0.1 mg l 1 3-indolyl-butyric acid (IBA), 1.0 mg l 1 benzyladenine (BA), 30 g l 1 sucrose and 0.8% powder agar (Wako Co. Ltd., Japan). All media were adjusted with 0.1 N NaOH to pH 5.7 and were autoclaved at 1.1 kg cm 2 (1218C) for 20 min. All cultures were incubated under a 16 h photoperiod illuminated by a cool-white ¯uorescent light (50 mmol m 2 s 1) at 25 1 C in a plastic pot (5 15 cm) containing 30 ml of gelled medium (unless otherwise speci®ed). 2.2. Shoot proliferation experiments First, the effect of cytokinins and their concentrations on shoot proliferation and hyperhydricity were studied. The shoots were transferred to 1/2MS medium containing 30 g l 1 sucrose, 0.1 mg l 1 IBA, 0.8% powder agar and 0.1, 1.0, 2.5,
M. Kadota et al. / Scientia Horticulturae 89 (2001) 207±215
209
5.0 and 10 mg l 1 BA or 0.1 and 1.0 mg l 1 thidiazuron (TDZ). To determine the effect of three different basal salts mixtures (macro and micro) and activated charcoal (AC), the shoots were transferred to MS, 1/2MS and woody plant (WP) (Lloyd and McCown, 1981) medium supplemented with 30 g l 1 sucrose, 0.1 mg l 1 IBA, 2.5 mg l 1 BA and 0.8% powder agar with or without 0.3% AC. To investigate the effect of carbon sources (CS) at different concentrations, the shoots were transferred to WP supplemented with 0.1 mg l 1 IBA, 2.5 mg l 1 BA, 0.8% powder agar and one of the following CS, sucrose, sorbitol, or glucose. Each CS was provided at three levels: 20, 30 and 40 g l 1. To compare the effect of gelling agents and their concentrations, the shoots were transferred to WP supplemented with 20 g l 1 sorbitol, 0.1 mg l 1 IBA, 2.5 mg l 1 BA solidi®ed by 0.1, 0.15, 0.2, 0.25 and 0.3% gellan gum (Wako Co. Ltd.) or 0.4, 0.6, 0.8, 1.0 and 1.2% powder agar. Finally, the effect of a double-phase culture system was evaluated. The shoots were transferred to WP supplemented with 20 g l 1 sorbitol, 0.1 mg l 1 IBA, 2.5 mg l 1 BA and 0.8% powder agar (the improved culture), or the same medium using a double-phase liquid-gelling agent solidi®ed culture system. For double-phase culture, 10 ml of liquid medium composed of the same as solid phase minus agar was added after horizontally positioning the primary explants. At the same time, the conventional culture of Banno et al. (1989), which is consisting of 1/2MS with 0.1 mg l 1 IBA, 1.0 mg l 1 BA, 30 g l 1 sucrose and 0.8% powder agar, was compared. 2.3. Data analysis All experiments were carried out completely randomly and were repeated three times with three shoots within ®ve pots per treatment. After 35 days from the start of the experiment, the number of shoots formed per explant, percentage of fresh mass increase and percentage of hyperhydric shoot were recorded and the former two were analyzed using Duncan's multiple range test at P 0:05. Also, for the experiment on the effect of macroelements and AC, the number of shoots formed per explant and rate of fresh mass increase were subjected to a two-way ANOVA with macroelements and AC. For the experiment on the effect of CS at different concentrations, the two indicators of shoot proliferation were subjected to a twoway ANOVA with CS and CS concentrations. 3. Results Hyperhydric shoots were observed in the experiment on the effect of macroelements and the experiment on the effect of gelling agents, but they were subcultured in the same fresh medium, they stopped growing within 2±3 months and then died. The number of shoots increased at 2.5±10.0 mg l 1 BA, and the
210
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Table 1 Effect of cytokinins on shoot proliferation and hyperhydricitya Cytokinin
Concentration (mg l 1)
Number of shoots formed per explant
±b
Fresh mass increase (%)
1.1 cc
BA
0.1 1.0 2.5 5.0 10.0
TDZ
0.1 1.0
1.1 2.2 4.2 4.0 5.6
66.9 c
c b a a a
294.7 640.6 559.6 374.0 204.1
1.5 bc 1.1 c
bc a a b bc
301.8 bc 96.4 c
a
Fresh mass of shoot tips at the start of the experiment were 80:1 5:7 mg. Proliferation medium with no cytokinin. c Mean separation by Duncan's multiple range test at P 0:05. b
percentage of shoot fresh mass increased at 1.0 and 2.5 mg l 1 BA (Table 1). TDZ had no stimulatory effect on the two indicators of proliferation. Hyperhydric shoots were not observed. The different macroelements did not affect shoot proliferation, but adding AC to the medium markedly inhibited the production of axillary shoots and biomass and many normal shoots were hyperhydric (Table 2). The different CS concentrations increased signi®cantly the shoot number and the percentage of fresh mass increase, with sorbitol producing a greater increase than sucrose and Table 2 Effect of macroelements and AC on shoot proliferation and hyperhydricitya Macroelement MS MS 1/2MS 1/2MS WP WP
d
Significant macroelement Significant AC Significant macroelement AC a
ACb
Number of shoots formed per explant 4.0 1.3 4.7 1.5 5.0 1.1 NS ** NS
ac b a b a b
Fresh mass increase (%)
Hyperhydric individuals (%)
499.0 152.5 628.9 226.1 591.2 130.0
0 73.3 0 66.7 0 60.0
a b a b a b
NS ** NS
Fresh mass of shoot tips at the start of the experiment were 78:5 4:8 mg. Proliferation medium with () or without ( ) 0.3% AC. c Mean separation by Duncan's multiple range test at P 0:05. d NS and ** indicate not signi®cant, signi®cant at P 0:01, respectively. b
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Table 3 Effect of CS and their concentrations on shoot proliferation and hyperhydricitya CS
Concentration (mg l 1)
Number of shoots formed per explant
Fresh mass increase (%)
Sucrose
20 30 40
3.3 abb 1.8 c 1.5 c
564.3 bc 504.6 bc 477.4 bc
0 0 6.7
Sorbitol
20 30 40
4.4 a 3.9 a 2.2 bc
777.3 ab 906.5 a 676.6 abc
0 6.7 0
Glucose
20 30 40
2.3 bc 1.6 c 1.1 c
737.5 abc 578.0 abc 430.6 c
6.7 0 13.3
** ** NS
** NS NS
Significantc CS Significant concentration Significant CS concentration
Hyperhydric individuals (%)
a
Fresh mass of shoot tips at the start of the experiment were 19:9 1:3 mg. Mean separation by Duncan's multiple range test at P 0:05. c NS and ** indicate not signi®cant, signi®cant at P 0:01, respectively. b
fructose (Table 3). The difference in concentration affected the shoot number, which decreased as the concentration of each CS increased. A few hyperhydric explants were observed with every CS. The shoot number increased at 0.4% agar and at 0.3% gellan gum and followed 0.6% agar (Table 4). The percentage of fresh mass increase increased at 0.4% Table 4 Effect of gelling agents and their concentrations on shoot proliferation and hyperhydricitya Gelling agent
Concentration (%)
Number of shoots formed per explant (%)
Agar
0.4 0.6 0.8 1.0 1.2
4.4 3.9 3.1 2.4 2.3
Gellan gum
0.1 0.15 0.2 0.25 0.3
2.4 1.7 2.1 3.1 4.5
a b
Fresh mass increase (%)
Hyperhydric individuals (%)
ab ab bc c c
3999.8 1269.3 906.4 802.5 402.1
a cd cd d e
100 33.3 6.7 6.7 0
c c c bc a
1769.2 1306.6 2594.5 1828.1 1666.6
c c b c c
100 93.3 93.3 86.7 33.3
Fresh mass of shoot tips at the start of the experiment were 19:6 0:6 mg. Mean separation by Duncan's multiple range test at P 0:05.
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Table 5 Effect of a double-phase culture system on shoot proliferation and hyperhydricitya Culture system
Number of shoots formed per explant
Fresh mass increase (%)
Hyperhydric individuals (%)
Double-phase culture Improved culture Conventional culture
4.7 ab 4.7 a 2.2 b
1559.4 a 1005.6 b 499.1 b
6.7 0 6.7
a b
Fresh mass of shoot tips at the start of the experiment were 22:7 1:3 mg. Mean separation by Duncan's multiple range test at P 0:05.
agar. The percentage of hyperhydric explants was 33% or more, i.e. over onethird of the total, at 0.4 and 0.6% agar, and at every concentration of gellan gum. The double-phase culture and the improved culture produced a high number of axillary shoots compared with the conventional culture (Table 5). Also, the former culture had a higher fresh mass than the other cultures. The percentage of hyperhydric explants in the three culture systems was few or none. 4. Discussion Moretti et al. (1991) investigated the effect of BA concentration from 0 to 1.0 mg l 1 on shoot proliferation of European pear and reported that shoot proliferation increased as the concentration increased. In wild pear, also, 1.5 and 2.0 mg l 1 BA stimulates axillary shoot production and dry weight compared with from 0 to 1.0 mg l 1 BA (Shibli et al., 1997). In this study, the fresh mass increase did not differ between 1.0 and 2.5 mg l 1 BA, which gave the best results, but the latter value produced a greater shoot number than the former value (Table 1). Van Nieuwkerk et al. (1986) showed that 10.0 (2.2 mg l 1) and 1.0 mM TDZ were equivalent to, or greater than, the number of shoots produced when using 4.4 mM BA, but this increase was not observed in this study. Therefore, we think that 2.5 mg l 1 BA is the best cytokinin and concentration for shoot proliferation. We found no signi®cant effect by the three macroelements against shoot proliferation (Table 2). Although not signi®cant, the highest shoot number was obtained with WP, so we used it for the next experiment. As the nitrogen concentration of WP is about one-fourth of full strength MS, a low concentration of nitrogen or low total salts may produce better results in Japanese pear. AC supplemented to the medium prevented markedly axillary shoot and fresh mass production and produced shoot hyperhydricity, which is a physical disorder and causes the shoots to have an aberrant morphology characterized by translucent and brittle stems and leaves. AC have the property of a strong absorption of
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dissolved solids (George and Sherington, 1984). Also, a decrease in gelling agent concentration in the medium produces a hyperhydricity of apple shoot (Pasqualetto et al., 1986). The adsorption of BA in the medium by AC may be caused by poor shoot proliferation and the adsorption of agar in the medium may produce the hyperhydricity. Although AC increases plant recovery ef®ciency in many species (George and Sherington, 1984), it is not suitable for multiplication of Japanese pear. The type of carbohydrates affects the growth and the frequency of shoots developed, and shoot proliferation of woody Rosaceous species, such as apple and apricot, is often increased using sorbitol (Pua and Chong, 1984; Welander et al., 1989; Marino et al., 1991, 1993; Karhu, 1997). However, examination of the effect of different CS on Japanese pear has not been made. In this study, sorbitol produced a good result for shoot multiplication compared with sucrose and fructose (Table 3). The concentrations of sorbitol at 20 and 30 g l 1 were more effective than 40 g l 1 in producing new shoots. Therefore, the most ef®cient CS was sorbitol at a concentration 20±30 g l 1. Low concentration of agar medium, such as 0.4 and 0.6%, and gellan gum medium clearly increased hyperhydric explants (Table 4). Using gellan gum or a decrease in gelling agent concentration hastens the hyperhydricity of cultured explants of apple and European pear (Pasqualetto et al., 1986, 1988; Turner and Singha, 1990; Marga et al., 1997), similar to that observed in this study of Japanese pear. These results imply that using gellan gum and low concentrations of agar must be avoided when proliferating Japanese pear shoots. At three higher concentrations of agar, 0.8% agar had a greater effect than 1.0 and 1.2% agar for shoot proliferation. So, the best gelling agent was agar at a concentration 0.8%. The double-phase culture increased the number of shoots about twice, and the rate of fresh mass increased about three times compared with the conventional culture (Table 5). The rate of fresh mass increase in the double-phase culture was greater than that of the improved culture. Although double-phase culture increases shoot proliferation of some fruit trees (Viseur, 1987; Moretti et al., 1991; RodrõÂguez et al., 1991; Murai and Harada, 1995), it is greatly effective with Japanese pear. In this study, we improved markedly the speed of proliferating shoots of Japanese pear cultivars using 2.5 mg l 1 BA, 20±30 g l 1 sorbitol and the doublephase culture system. In the future, we will use this culture system to produce haploids by anther culture and transgenic plants. References Banno, K., Yoshida, K., Hayashi, S., Tanabe, K., 1989. In vitro propagation of Japanese pear cultivars. J. Jpn. Soc. Hortic. Sci. 58, 37±42.
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