Plant Cell Reports
Plant Cell Reports (1991) 10:240-242
:~ Springer-Verlag1991
Plantlet regeneration from cultured leaves of
Cydonia oblongaL. (quince)
Ramon Dolcet-Sanjuan, David W.S. Mok, and Machteld C. Mok Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA Received January 15, 1991/Revised version received May 29, 1991 - Communicated by A. R. Gould
Summary. Adventitious shoots o f Cydonia oblonga Quince A were obtained from leaves cultured on MS-N6 m e d i u m containing thidiazuron ( T D Z ) and et-naphthaleneacetie acid (NAA). The frequency o f regeneration was high (78 % of the cultured leaves with 3.2 shoots per regenerating leaf) at 32 # M T D Z plus 0.3 # M N A A on young leaves obtained from micropropagated shoots. Shoots were rooted by culturing them first on medium containing 5 # M N A A for one week and then o n auxinfree m e d i u m for four weeks. The regeneration protocol may be useful for selection o f somaclonal variants with increased tolerance to low F e and for transformation mediated by Agrobacterium.
Introduction
Cydonia oblonga (quince) is used extensively in Europe as a dwarfing rootstock for pear. Quince is sensitive to low F e availability, resulting i n leaf chlorosis, a prevalent p r o b l e m in pear-growing regions with calcareous soils. Selection of somaclonal variants o f quince with increased tolerance to low F e is one approach to this problem. A pre-requisite for selection o f such variants is adventitious shoot regeneration from callus or from'plant organs. I n this report we describe experiments leading to efficient shoot and plantlet regeneration from quince leaves.
Materials and methods Leaves were obtained from three-week-old micropropagated shoots of (7. oblonga L. East Mailing Quince A (Dolcet-Sanjuanet al., 1990). Transverse incisions, 2 mm apart, were made on the leaves, which were then placed with the abaxial side down in petri dishes containing 25 ml medium. The medium consisted of a combination of MS (Murashigr and Skoog, 1962) and N6 (Chu, t978) minerals (with N, P, K, and S from N6), sucrose (30 g/l), myo-inositol (100 mg/l), thiamine.HCl (1 mg/I), nicotinic acid (I rag/I), pyridoxine.HCI (1
Offprint requests to." M. C. Mok
rag/l) and Difco Bacto-agar (6 g/l). The pH was adjustedto 5.7. The growth regulators, bP-benzylaminopurine(BAP), thidiazuron (TDZ), and or acid (NAA), were dissolved in dimethylsulfoxide and added to the medium after autoclaving (12.5 gl per 25 rni medium) (Schmitzand Skoog, 1970). In the first experimentto determineoptimal growth regulatorconcentrations, BAP (1, 3, 10, 32, and 1130IA4) and TDZ (0.001, 0.0l, 0.1, 1, and 10 gM) concentrationswere tested in combinationwith NAA (0.1, 0.3, 1 and 3 p2d), with 24 leaves per treatment. In the second experimentTDZ concentrationsof 1, 3, 10, 32, and I00/.aM and NAA concentrationsof 0.3, 1 and 3/zM were chosen. Four replicateexperiments were performed, each with 12 leaves per treatment. In subsequent experiments, casein hydrolysate (100 mg/I), yeast extract (100 mg/l), malt extract (100 rag/I), coconut water (100 ml/1), glycine (2 rag/l), folio acid (1 rag/l), biotin (1 mg/l), pantothenlc acid (1 mg/1), putrescine (161 mg/l), and higher sucrose concentration(60 g/l) were tested. Finally, the optimal medium was used to determine if higher regenerationfrequenciescould be attained by careful selectionof leaves (as explants). Two replicate tests were conducted, each with 50 uniform young leaves. All cultures were kept at 25~ in the dark for the first three weeks, and at a 16 h photoperiod (40/.tErn%"~) for the following three weeks. Leaf sectionswith adventitiousshoots were transferred to multiplication mediumcontaining5 p.M BAP (Lane, 1979; Doicet-Sanjuanet al., 1990). To induce rooting, shoots were grown on medium containing 5 ~vl NAA for one week and then on auxin-free medium for four weeks (Maard et al., 1986; Dolcet-Sanjuanet al., 1990). To determinethe anatomy of organogenictissue, leaf sampleswere taken after the three-weekdark period and fixed in FAA (2% formalin, 5% acetic acid, and 60% alcohol). Tissue sections (10 #In) were stained with toluidine blue.
Results and Discussion A combination o f MS and N6 m e d i u m similar to that of Welander (1988) prevented b r o w n i n g o f cultured leaf discs better than MS medium. As shown by Welander (1988) for apple, exposing leaves to a sequence of dark and light treatment improved regeneration as compared to a continuous u ~ o f a 16 hour light period. Therefore, we used a combination o f MS and N6 media, with three weeks o f darkness followed b y three weeks under a 16h photoperiod.
241 The first experiment involving broad ranges of growth regulator concentrations indicated clearly that TDZ could prevent leaf senescence and induce adventitious shoot formation much more effectively than BAP (Table 1). TDZ was active from 0.1/~M to 10/~M, with the highest regeneration frequency at 10/xM TDZ. In contrast, shoot formation was sporadic on medium with BAP. TDZ also stimulated regeneration from leaf discs of apple and pear effectively (Cbevreau et al., 1989; Fasolo et al., 1989; Swartz et al., 1990), although BAP has also been used successfully with these species (Browning et al., 1987; Fasolo et al., 1989; James et al., 1988; Welander, 1989; Predieri et al., 1989). TDZ is generally more active and stable than adenine-type cytokinins (Mok et al., 1982; Mok and Mok, 1985), and the only metabolites formed after long-term incubation were glucosyl derivatives (Mok and Mok, 1985). Thus the efficacy of TDZ in regeneration of quince may be due to the particularly high cytokinin requirement of this species.
vigorous leaves of micropropagated shoots, the frequency of regenerating leaves reached 78 %, with 3.2 shoots per leaf disc. Rapid multiplication of these shoots was achieved by transfer to standard micropopagation medium. The rooting regime consisting of a sequence of NAA-containing and auxin-free medium, developed for micropropagated shoots (Maarri et al., 1986; Dolcet-Sanjuan, 1990), also induced rooting on shoots regenerated from leaves. Anatomical studies revealed that small callus tissues initiated at the cut edges of the leaves, and buds subsequently formed in the calli (Fig. 1). Usually several shoots could be observed in close proximity (Fig. 2), representing possible proliferation of axillary buds from the first shoot or the formation of multiple adventitious buds. Structural analyses of a number of tissues indicated the latter course of development. The occurrence of multiple adventitious shoots may indicate the presence of
Table 1. Adventitious shoot regeneration from leaf discs of Oydonia oblonga Quince A as influenced by TDZ, BAP, and NAb, NAA concentration (p,M) Cytokinin
Concentration (VM)
0
0.1
0.3
1
3
0 TDZ 0.~1 0.01
-
0.1
-
1 10
++ +++
-
++ ++ ++
+
+ ++
BAP 1
3 10 32 100
+
~24 leaves per treatment no shoots + 1-10 shoots + + 11-25 shoots + + + > 25 shoots
Based on the results of the previous experiment, we used 1 to 100 btM TDZ in combination with 0.3 to 3/~M NAA (Table 2). Most treatments resulted in adventitious shoot formation, but the highest proportion of regenerating leaf discs was observed using 32 #M TDZ and 0.3 /xM NAA. The addition of vitamins (folic acid, biotin, and pantothenic acid) or complex organic substances (casein hydrolysate, yeast extract, malt extract, and coconut water), glycine or putreseine did not enhance regeneration. Increased sucrose concentration (60 g/l) slightly repressed the regeneration rate. Regeneration was improved further by careful selection of leaves. Using only the youngest and most
F'gg, 1. Sections of adventitious shoots of C. oblongaformed on leaves cultured for three weeks on medium containing 32/.tM TDZ and 0.3 /~M NAA. A - Shoot formed in callus at edge of leaf disc; B - Apical meristem.
242 Table 2. Effects of TDZ and NAA on regeneration of Cydonia oblonga Quince A, expresse d as percentage of leaves with adventitious shoots and average number of shoots per regenerating leaf disc.
Percentage Thidiazuron
No. shoots/leaf
NAA cone. (/tM)
NAA cone. (#M)
c:one. (b/M)
1 3 10 32 100
0.3
1
6+3 195-3 33_____3 485-6 05-0
17+4 38+4 21+4 255-4 2+ 1
3 6+4 275-5 335-4 31+5 0+0
0,3
1
1,0+0.6 1.2-1-0.4 2.45:2.1 3.65-2.4 -
1,6+1.4 1.95-1.3 2.35:2.5 3.1-t-2.0 1.05-0.0
2.05-1,0 2.2-1-2.4 2.45-1.8 2.55-2.1 --
leaves formed on most shoots were typically chlorotic, although a few green shoots were isolated. We are in the process of generating sufficient numbers of shoots from these selections to determine whether they represent true and stable variants with increased tolerance to low Fe.
Acknowledgment. The financial support from INIA (Spain) to R.D.-S. is gratefully acknowledged. Research support was provided by the Oregon Agricultural Experiment Station (paper no, 9481)
References
Fig. 2. Multiple shoots on C. oblonga leaf cultured for six weeks on medium containing 32/zM TDZ and 0.3 p,M NAA.
a number of competent cells close to each other and possibly of common origin. Alternatively, the first bud may stimulate formation of additional buds. The regeneration protocol described here may be used to recover somaclonal variants with tolerance to low Fe or for Agrobacterium-mediatedtransformation. We have tested adventitious shoots obtained from over 500 independent regeneration sites on Fe-deficient medium. New
Browning G, Ognjanov V, Passey AJ, James DJ (1987) J Herr Sci 62:305-311 Chevreau E, Skirvin RM, Abu-Qaoud HA, Korban SS, Sullivan JG (1989) Plant Cell Rep 7:688-691 Chu CC (1978) In: Proceedings of Symposium on Plant Tissue Culture. Science Press, Peking, pp 43-50 Dolcet-Sanjuan R, Mok DWS, Mok MC (1990) Plant Cell Tiss Org Cult 16:191-199 Fasolo F, Zimmerman RI-I, Fordham I (1989) Plant Cell Tiss Org Cult 16:75-87 James DJ, Passey AJ, Barbara DJ, Bevan M (1989) Plant Cell Rep 7:658-661 James DJ, Passey AJ, Rugini E (1988) J Plant Physiol 132:148-154 Lane WD (1979) Plant Sci Lett 16:33%342 Maarri, K AI, Amaud Y, Miginiae E (1986) Sci Hortic 28:315-321 Mok MC, Mok DWS (1985) Physiol Plant 65:427-432 Mok MC, Mok DWS, Armstrong DJ, Shudo K, lsogai Y, Okamoto T (1982) Phytochemistry 21:1509-1511 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Predieri S, Fasolo F (1989) Plant Cell Tiss Org Cult 17:133-142 Scbmitz RY, Skoog F (1970) Plant Physiol 45:537-538 Swartz HJ, Bors R, Mohamed F, Naess K (1990) Plant Cell Tiss Org Cult 21:179-184 Welander M (1988) I Plant Physiol 132:738-744
Plant Cell Reports (1991) 10:240-242
:~ Springer-Verlag1991
Plantlet regeneration from cultured leaves of
Cydonia oblongaL. (quince)
Ramon Dolcet-Sanjuan, David W.S. Mok, and Machteld C. Mok Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA Received January 15, 1991/Revised version received May 29, 1991 - Communicated by A. R. Gould
Summary. Adventitious shoots o f Cydonia oblonga Quince A were obtained from leaves cultured on MS-N6 m e d i u m containing thidiazuron ( T D Z ) and et-naphthaleneacetie acid (NAA). The frequency o f regeneration was high (78 % of the cultured leaves with 3.2 shoots per regenerating leaf) at 32 # M T D Z plus 0.3 # M N A A on young leaves obtained from micropropagated shoots. Shoots were rooted by culturing them first on medium containing 5 # M N A A for one week and then o n auxinfree m e d i u m for four weeks. The regeneration protocol may be useful for selection o f somaclonal variants with increased tolerance to low F e and for transformation mediated by Agrobacterium.
Introduction
Cydonia oblonga (quince) is used extensively in Europe as a dwarfing rootstock for pear. Quince is sensitive to low F e availability, resulting i n leaf chlorosis, a prevalent p r o b l e m in pear-growing regions with calcareous soils. Selection of somaclonal variants o f quince with increased tolerance to low F e is one approach to this problem. A pre-requisite for selection o f such variants is adventitious shoot regeneration from callus or from'plant organs. I n this report we describe experiments leading to efficient shoot and plantlet regeneration from quince leaves.
Materials and methods Leaves were obtained from three-week-old micropropagated shoots of (7. oblonga L. East Mailing Quince A (Dolcet-Sanjuanet al., 1990). Transverse incisions, 2 mm apart, were made on the leaves, which were then placed with the abaxial side down in petri dishes containing 25 ml medium. The medium consisted of a combination of MS (Murashigr and Skoog, 1962) and N6 (Chu, t978) minerals (with N, P, K, and S from N6), sucrose (30 g/l), myo-inositol (100 mg/l), thiamine.HCl (1 mg/I), nicotinic acid (I rag/I), pyridoxine.HCI (1
Offprint requests to." M. C. Mok
rag/l) and Difco Bacto-agar (6 g/l). The pH was adjustedto 5.7. The growth regulators, bP-benzylaminopurine(BAP), thidiazuron (TDZ), and or acid (NAA), were dissolved in dimethylsulfoxide and added to the medium after autoclaving (12.5 gl per 25 rni medium) (Schmitzand Skoog, 1970). In the first experimentto determineoptimal growth regulatorconcentrations, BAP (1, 3, 10, 32, and 1130IA4) and TDZ (0.001, 0.0l, 0.1, 1, and 10 gM) concentrationswere tested in combinationwith NAA (0.1, 0.3, 1 and 3 p2d), with 24 leaves per treatment. In the second experimentTDZ concentrationsof 1, 3, 10, 32, and I00/.aM and NAA concentrationsof 0.3, 1 and 3/zM were chosen. Four replicateexperiments were performed, each with 12 leaves per treatment. In subsequent experiments, casein hydrolysate (100 mg/I), yeast extract (100 mg/l), malt extract (100 rag/I), coconut water (100 ml/1), glycine (2 rag/l), folio acid (1 rag/l), biotin (1 mg/l), pantothenlc acid (1 mg/1), putrescine (161 mg/l), and higher sucrose concentration(60 g/l) were tested. Finally, the optimal medium was used to determine if higher regenerationfrequenciescould be attained by careful selectionof leaves (as explants). Two replicate tests were conducted, each with 50 uniform young leaves. All cultures were kept at 25~ in the dark for the first three weeks, and at a 16 h photoperiod (40/.tErn%"~) for the following three weeks. Leaf sectionswith adventitiousshoots were transferred to multiplication mediumcontaining5 p.M BAP (Lane, 1979; Doicet-Sanjuanet al., 1990). To induce rooting, shoots were grown on medium containing 5 ~vl NAA for one week and then on auxin-free medium for four weeks (Maard et al., 1986; Dolcet-Sanjuanet al., 1990). To determinethe anatomy of organogenictissue, leaf sampleswere taken after the three-weekdark period and fixed in FAA (2% formalin, 5% acetic acid, and 60% alcohol). Tissue sections (10 #In) were stained with toluidine blue.
Results and Discussion A combination o f MS and N6 m e d i u m similar to that of Welander (1988) prevented b r o w n i n g o f cultured leaf discs better than MS medium. As shown by Welander (1988) for apple, exposing leaves to a sequence of dark and light treatment improved regeneration as compared to a continuous u ~ o f a 16 hour light period. Therefore, we used a combination o f MS and N6 media, with three weeks o f darkness followed b y three weeks under a 16h photoperiod.
241 The first experiment involving broad ranges of growth regulator concentrations indicated clearly that TDZ could prevent leaf senescence and induce adventitious shoot formation much more effectively than BAP (Table 1). TDZ was active from 0.1/~M to 10/~M, with the highest regeneration frequency at 10/xM TDZ. In contrast, shoot formation was sporadic on medium with BAP. TDZ also stimulated regeneration from leaf discs of apple and pear effectively (Cbevreau et al., 1989; Fasolo et al., 1989; Swartz et al., 1990), although BAP has also been used successfully with these species (Browning et al., 1987; Fasolo et al., 1989; James et al., 1988; Welander, 1989; Predieri et al., 1989). TDZ is generally more active and stable than adenine-type cytokinins (Mok et al., 1982; Mok and Mok, 1985), and the only metabolites formed after long-term incubation were glucosyl derivatives (Mok and Mok, 1985). Thus the efficacy of TDZ in regeneration of quince may be due to the particularly high cytokinin requirement of this species.
vigorous leaves of micropropagated shoots, the frequency of regenerating leaves reached 78 %, with 3.2 shoots per leaf disc. Rapid multiplication of these shoots was achieved by transfer to standard micropopagation medium. The rooting regime consisting of a sequence of NAA-containing and auxin-free medium, developed for micropropagated shoots (Maarri et al., 1986; Dolcet-Sanjuan, 1990), also induced rooting on shoots regenerated from leaves. Anatomical studies revealed that small callus tissues initiated at the cut edges of the leaves, and buds subsequently formed in the calli (Fig. 1). Usually several shoots could be observed in close proximity (Fig. 2), representing possible proliferation of axillary buds from the first shoot or the formation of multiple adventitious buds. Structural analyses of a number of tissues indicated the latter course of development. The occurrence of multiple adventitious shoots may indicate the presence of
Table 1. Adventitious shoot regeneration from leaf discs of Oydonia oblonga Quince A as influenced by TDZ, BAP, and NAb, NAA concentration (p,M) Cytokinin
Concentration (VM)
0
0.1
0.3
1
3
0 TDZ 0.~1 0.01
-
0.1
-
1 10
++ +++
-
++ ++ ++
+
+ ++
BAP 1
3 10 32 100
+
~24 leaves per treatment no shoots + 1-10 shoots + + 11-25 shoots + + + > 25 shoots
Based on the results of the previous experiment, we used 1 to 100 btM TDZ in combination with 0.3 to 3/~M NAA (Table 2). Most treatments resulted in adventitious shoot formation, but the highest proportion of regenerating leaf discs was observed using 32 #M TDZ and 0.3 /xM NAA. The addition of vitamins (folic acid, biotin, and pantothenic acid) or complex organic substances (casein hydrolysate, yeast extract, malt extract, and coconut water), glycine or putreseine did not enhance regeneration. Increased sucrose concentration (60 g/l) slightly repressed the regeneration rate. Regeneration was improved further by careful selection of leaves. Using only the youngest and most
F'gg, 1. Sections of adventitious shoots of C. oblongaformed on leaves cultured for three weeks on medium containing 32/.tM TDZ and 0.3 /~M NAA. A - Shoot formed in callus at edge of leaf disc; B - Apical meristem.
242 Table 2. Effects of TDZ and NAA on regeneration of Cydonia oblonga Quince A, expresse d as percentage of leaves with adventitious shoots and average number of shoots per regenerating leaf disc.
Percentage Thidiazuron
No. shoots/leaf
NAA cone. (/tM)
NAA cone. (#M)
c:one. (b/M)
1 3 10 32 100
0.3
1
6+3 195-3 33_____3 485-6 05-0
17+4 38+4 21+4 255-4 2+ 1
3 6+4 275-5 335-4 31+5 0+0
0,3
1
1,0+0.6 1.2-1-0.4 2.45:2.1 3.65-2.4 -
1,6+1.4 1.95-1.3 2.35:2.5 3.1-t-2.0 1.05-0.0
2.05-1,0 2.2-1-2.4 2.45-1.8 2.55-2.1 --
leaves formed on most shoots were typically chlorotic, although a few green shoots were isolated. We are in the process of generating sufficient numbers of shoots from these selections to determine whether they represent true and stable variants with increased tolerance to low Fe.
Acknowledgment. The financial support from INIA (Spain) to R.D.-S. is gratefully acknowledged. Research support was provided by the Oregon Agricultural Experiment Station (paper no, 9481)
References
Fig. 2. Multiple shoots on C. oblonga leaf cultured for six weeks on medium containing 32/zM TDZ and 0.3 p,M NAA.
a number of competent cells close to each other and possibly of common origin. Alternatively, the first bud may stimulate formation of additional buds. The regeneration protocol described here may be used to recover somaclonal variants with tolerance to low Fe or for Agrobacterium-mediatedtransformation. We have tested adventitious shoots obtained from over 500 independent regeneration sites on Fe-deficient medium. New
Browning G, Ognjanov V, Passey AJ, James DJ (1987) J Herr Sci 62:305-311 Chevreau E, Skirvin RM, Abu-Qaoud HA, Korban SS, Sullivan JG (1989) Plant Cell Rep 7:688-691 Chu CC (1978) In: Proceedings of Symposium on Plant Tissue Culture. Science Press, Peking, pp 43-50 Dolcet-Sanjuan R, Mok DWS, Mok MC (1990) Plant Cell Tiss Org Cult 16:191-199 Fasolo F, Zimmerman RI-I, Fordham I (1989) Plant Cell Tiss Org Cult 16:75-87 James DJ, Passey AJ, Barbara DJ, Bevan M (1989) Plant Cell Rep 7:658-661 James DJ, Passey AJ, Rugini E (1988) J Plant Physiol 132:148-154 Lane WD (1979) Plant Sci Lett 16:33%342 Maarri, K AI, Amaud Y, Miginiae E (1986) Sci Hortic 28:315-321 Mok MC, Mok DWS (1985) Physiol Plant 65:427-432 Mok MC, Mok DWS, Armstrong DJ, Shudo K, lsogai Y, Okamoto T (1982) Phytochemistry 21:1509-1511 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Predieri S, Fasolo F (1989) Plant Cell Tiss Org Cult 17:133-142 Scbmitz RY, Skoog F (1970) Plant Physiol 45:537-538 Swartz HJ, Bors R, Mohamed F, Naess K (1990) Plant Cell Tiss Org Cult 21:179-184 Welander M (1988) I Plant Physiol 132:738-744
