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Isozymes in Differentiating Shoot Bud Cultures of Betula pendula Roth.
lsozymes in Differentiating Shoot Bud Cultures of Betula pendula Roth. P.
s. SRIVASTAVA*) and A. STEINHAUER
Lehrstuhl fur Forstgenetik und Forstpflanzenziichtung dec Universitat Gottingen. Biisgenweg 2, D-3400 Gottingen, F.R.G. Received February 19,1981 . Accepted April 24. 1981
Summary Shoot buds of birch cultured in vitro produced callus and differentiated plandets. On defined White's medium supplemented with 2 mg .} - 1 each of GA j • IAA, and BAP, the explants started callusing in 2 weeks and within 4 weeks differentiated roots. After 6 weeks, the callus produced shoot buds. The growth of callus and differentiation of shoots improved on defined Murashige and Skoog's medium fortified with IAA + zeatin + adenine. Excised shoots transferred on this medium grew vigorously and formed normal plantiets. Isozyme investigations reyealed varying patterns in growing and differentiating callus. The growth responses of the callus and a correlation between isozyme expression and differentiation process is discussed.
Key words: &tula pendula, shoot bud culture, plantlets, isozymes. Introduction
Callus cultures of a number of trees have been established. but plantlet regeneration has been obtained in only a few (Srivastava, 1973; for reviews see Johri and Srivastava, 1973; Johri et aI., 1980 a, b; Vasil and Vasil, 1980). This has led to the possibility of vegetative propagation and genetic manipulation of tree species. Very little is known, however, about the inherent changes underlying the process of differentiation. A number of investigators studied the isozyme expression of different plant tissues and found changes in isozymes occurring during cell or callus cultures (Amison and Boll, 1974; Simola and Sopanen, 1970). Isozyme analysis at different stages of culture might throw light on the physiological and biochemical changes underlying the processes of callusing and differentiation. This is important in respect to tissue-specific biochemical characteristics which are expressed and maintained during the culture period (see Scandalios, 1974). In the present studies, an attempt was made to culture and grow the shoot bud explants of &tula pendula progenies obtained from various crosses between red and *) Permanent Address: Department of Botany, S.G.T.B. Khalsa College, University of Delhi. Delhi 110007, India.
Z. Pjlanzenplrysiol. Bd. 103. S. 341-346. 1981.
342
P. S. SRIVASTAVA and A. STEINHAUER
early flowering trees, and to obtain early regeneration which would provide a quick method for vegetative multiplication of genetically defined clones. Also, isozyme analysis was carried out to establish a correlation between morphogenic responses and isozyme expression, which could be used as a genetic marker.
Material and Methods Buds of Betula pendula Roth. were collected from Gottingen. They were surface*sterilized as described by Johri and Srivastava (1972). Before implanting, the explants were washed thoroughly with sterile distilled water. Modified White's medium according to Srivastava et al. (1980) with 3% sucrose, and 5.8 pH was used (WM). The medium was gelled with 0.65% agar and dispensed both in Erlenmeyer flasks and 40 mm petridishes. Growth hormones, indoleacetic acid (lAA), indolebutyric acid (rnA), naphthaleneacetic acid (NAA), gibberellic acid (GA,), kinetin, zeatin, W-benzylaminopurine (BAP), and adenine were also added to the medium, as required. Cultures were maintained on a shelf adjusted to 16/8 h light/dark period with 28±2 °C and 60±5% RH. To induce early regeneration, the callus pieces were transferred to the medium of Murashige and Skoog (1962) (MMS) containing, in mg' 1- ',9250 KNO" 7000 NH,NO" 1260 CaCI,' 2H,O, 1650 MgSO,· 7H, O, 640 KH,PO" 15 H,BO" 12 MnSO, . 4H,O, 8.5 ZnSO,' 7H,O, 0.25 Na,MoO,' 2H,O, 0.25 CuSO,' 5H,O, 50 Na2FeEDTA, 5 niacin, 2 glycine, 2 calcium pantothenate, 0.5 pyridoxine' HCl, 0.5 thiamin' HCI, 20 glutamine, 30 asparagin, 20 arginine, 125 myo-inositol, and 30,000-40,000 sucrose. For isozyme (leucine aminopeptidase, LAP and acid phosphatase, AP) determination, tissues were removed from the culture vials at regular intervals. The callus and differentiated organs were homogenized in tris*HCI (PH 7.5) buffer containing 30 mg soluble polyvinylpyrolidone (PVP). Starch (11.5%) gel zone-electrophoresis was performed using a modified discontinuous system with HJBOJ*NaOH buffer (PH 8.2) at a voltage distribution of 15 V /cm (for details see Poulik, 1957).
Results and Discussion The buds did not show any change for 7 days on WM + 2 mg . 1- ' each of GA" IAA, and BAP. After 10 days, in 60.% ohhe cultures, the buds opened and formed normal leaf-like structures. In the remaining 40% the buds showed callusing. After 20 days almost 85% of th~ cultures started callusing (Fig. 1 A). The calli turned green and in 15% of the cultures, anthocyanin synthesis also took place. The smaller buds (3 mm) invariably produced callus whereas the larger buds (5 mm) developed leaves and the cut ends callus. The callus produced on the above medium started rooting after another 4 weeks. The roots were ca. 10 mm with a well-developed, pink-coloured root cap, and root hairs. After 6 weeks, shoot buds also differentiated. Numerous shoot buds differentiated when the callus was transferred to MMS+IAA (2 mg' I-')+BAP (5 mg . 1-') + adenine (20 mg' 1-') (Fig. 1 B). Some of these shoot buds developed further after 2 weeks and formed characteristic normal leaves. Further growth of the shoots was arrested, however. Various concentrations and combinations of IAA and BAP were Z Pjlanzenphysiol. Ed. 103. S. 341-346.1981.
Isozymes in cultured shoot buds
343
Fig. 1 A - D: In vitro growth of cultured shoot buds: (A) Profuse callusing of explants on WM+2 mg . 1-1 each of GAl, IAA, and SAP; (B) Differentiation of shoot buds on MMS + IAA
(2 mg· I- I)+BAP (5 mg· I- I)+adenine (20 mg· I-I); (C) excised shoot grown on MMS+lAA (2 mg· l- I)+zeatin (2 mg· 1- 1) + adenine (30 mg . 1- 1); note normal growth of shoot and well-differentiated leaves; (0) formation of complete plantlet.
tried in order to obtain the best possible combination for maximum growth of the tissue and differentiation of plandets. No combination of IAA and BAP supported further growth (Fig. 2). However. when the differentiating callus was transferred to MMS medium supplemented with IAA (2 mg . I- I)+zeatin (2 mg· I-I)+adenine (30 mg . I-I). the growth of shoots was remarkably fast and within 2 weeks they
Fig. 2: Effect of interaction of IAA and BAP on tissue growth.
Z. Pjlanzenphysiol. Bd. 103. S. 341-346.1981.
344
P.
S. SRIVASTAVA
and A.
STEINHAUER
Fig. 3: A complete plant (before transplanting to soil); note well-differentiated root system.
became 20 mm long with 4 pairs of well-developed leaves (Fig. 1 C). These shoots when excised and grown on the same medium grew vigorously and developed into
normal plandets (Fig. 1 D). If the buds were initially cultured on MMS+IAA (2 mg' l-')+GA, (2 mg . l-')+BAP (5 mg' l-')+casein hydrolysate (1000 mg . 1-') callusing was much better. Subsequent transfer of pieces of these calli on MMS + IAA (2 mg' l-')+zeatin (2 mg' l- ')+adenine (30 mg' 1- ') resulted in plandet formation. As many as 25 - 30 plandets could be recovered from each piece of callus. The plandets (Fig. 3) thus obtained could be transferred to soil. In all we tried bud culture of progenies from 15 different crosses. Of these, only five proved responsive for quick regeneration.
The initial results with buds and bud callus indicate that the expression of isozymes, LAP and AP, is altered under in vitro conditions. In the control buds, LAP expressZ Pjlanzenpbysiol. Ed. 103. S. 341 -346.1981.
Isozymes in cultured shoot buds
345
ed only one band and after 4 weeks, when the buds have initiated callusing, 2 bands were discernible. Three AP bands could be seen initially but after 4 weeks one band disappeared. Later, after 6 weeks, the shoot and.plantiet-differentiating callus exhib-
ited no change in AP bands but one additional DAP band appeared (Fig. 4). front
- - -- - - -- ----
._._,_._-_._._._._,- - -- -_._._._._,_._._.a
b
c
d
acid phosphatase
a
b
c
d
leucif'/f! aminopeptidase
start L-~~~~~~~--L-~~~~~~~~
Fig. 4: Isozyme pattern at different stages of callus growth. Expression of an additional band of LAP indicates the shift in morphogenic expression and initiation of bud formation. (a) explant at the time of culture, (b) callus initiation, (c) initiation of organ differentiation, (d) later stage.
Isozymes provide a means of marking the sequential development processes in plant tissue cultures and therefore represent the differential gene activity of the differentiating cells. Changes in isozyme patterns are correlated with physiological and
developmental states of intact plants (Scandalios, 1974). This has also been found in cultured plant cells. In the case of peroxidases, for example, changes due to exogenous
supply of growth regulators were shown by Lee (1972 a, b). Similar effects were observed by Lavee and Galston (1968) in Pelargonium cultures. In Phaseolus vulgaris, however, Bassiri and Carlson {1978} found a great uniformity of isozyme patterns within and between the calli of the same or different origin.
In our studies, the isozymes (LAP and AP) showed varying patterns depending upon the morphogenic response of the callus. Striking changes in isozyme expression during the process of germination are well established. Since this process involves growth and differentiation, this too may be the case in differentiating cultured tissues. In shoot buds, callus initiation and differentiation represent an alteration in the normal morphogenic response and, therefore, a change in the physiological and genetical expression leading to changes in isozymes. The expression of isozyme bands at a particular stage of callus development and prior to differentiation may prove to be a suitable marker to predict the morphogenic
responses of tissues. The rapid regeneration of plandets from shoot bud explants of &tula pendula may prove to be useful for obtaining genetically defined clones. Acknowledgement This work is suppo~ed by a Deutsche Forschungsgemeinschaft grant. The technical assistance of Miss C. Becker and Mr. S. Krakuhn is highly appreciated.
Z. Pjlanzenplrysiol. Ed. 103. S. 341 - 346.1981.
346
P. S. SRIVASTAVA and A. STEINHAUER
References AJ.NlSON, P. G. and W. G . BOLL: Isozymes in cell cultures of bush bean (Phaseolus vulgaris cv. Contender): isoenzymatic changes during the callus culture cycle and differences between stock cultures. Can. J. Bot. 52,2621- 2629 (1974). BASSIlU, A. and P. S. CAJU.SON: Isozyme patterns and differences in plane parts and their callus cultures in common bean. Crop Sci. 18, 955-958 (1978). jOHRI, B. M. and P. S. SRIVASTAVA: In vitro growth responses of mature endosperm of Ricinus communis L. In: Adv. Plant Morph., V. Puri Comm. Vol., 339-358. Sarita Prakashan, India, 1972. - - Morphogenesis in endosperm cultures. Z. Pflanzenphysiol. 10,285-304 (1973). jOHRI, B. M., P. S. SRIVASTAVA, and A. P. RASTE: Plane tissue culture and crop improvemene. Indian J. agric. Sci. 50, 103 ~ 127 (1980 a). - - - Endosperm culture. In: VASIL, I. K. (Ed.): Perspectives in Plane Cell and Tissue Culture, Int. Rev. Cyto!., Supp!. liB, 157 -182. Academic Press, New York, 1980 b. LAVE£, S. and A. W. GALSTON: Hormonal control of peroxidase activity in cultured Pelargonium pith. Am. J. Bot. 55, 890-893 (1968). Lu, T. T.: Changes in IAA oxidase isoenzymes in tobacco tissues aher treatment with 2,4-dichlorophenoxyacetic acid. Plant Physio!. 49, 957 -960 (1972 a). - Interaction of cytokinin, auxin, and gibberellin on peroxidase isozymes in tobacco tissues cultured in vitro. Can. J. Bot. 50,2471-2477 (1972 b). MUIlASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physio!. Plant. 15,473-497 (1962). POUUK, M. D.: Starch-gel electrophoresis in a discontinuous system of buffers. Nature (London) 80, 1477 (1957). SCANDAUoS,j. G.: Isozymes in development and differentiation. Ann. Rev. Plant Physiol. 25, 225-258 (1974). SIMOLA, L. K. and T. SOPANEN: Changes in the activity of certain enzymes of Acer pseudopla· tanus L. cells at four stages of the growth cycle in suspension culture. Physio!. Plant. 23, 1212-1222 (1970). SRIVASTAVA, P. 5.: Formation of triploid plantlets in mature endosperm cultures of Putranjiva roxburghii. Z. Pflanzenphysio!. 69,270-273 (1973). SRIVASTAVA, P. 5., A. VllGA, and]. BRUINSMA: Growth in vitro of fertilized ovules of pea, Pisum sativum L. with and without pods. Z. Pflanzenphysio!. 98, 347-354 (1980). VASIL, I. K. and VIMLA VASIL: Clonal propagation. In: VASIL, I. K. (EeL): Perspectives in Plant Cell and Tissue Culture, Int. Rev. Cytol., Suppl. 11A, 145-173. Academic Press, New York. 1980.
Z PjlanzenpJry.iol. Ed. 103. S. 341-346.1981.
s. SRIVASTAVA*) and A. STEINHAUER
Lehrstuhl fur Forstgenetik und Forstpflanzenziichtung dec Universitat Gottingen. Biisgenweg 2, D-3400 Gottingen, F.R.G. Received February 19,1981 . Accepted April 24. 1981
Summary Shoot buds of birch cultured in vitro produced callus and differentiated plandets. On defined White's medium supplemented with 2 mg .} - 1 each of GA j • IAA, and BAP, the explants started callusing in 2 weeks and within 4 weeks differentiated roots. After 6 weeks, the callus produced shoot buds. The growth of callus and differentiation of shoots improved on defined Murashige and Skoog's medium fortified with IAA + zeatin + adenine. Excised shoots transferred on this medium grew vigorously and formed normal plantiets. Isozyme investigations reyealed varying patterns in growing and differentiating callus. The growth responses of the callus and a correlation between isozyme expression and differentiation process is discussed.
Key words: &tula pendula, shoot bud culture, plantlets, isozymes. Introduction
Callus cultures of a number of trees have been established. but plantlet regeneration has been obtained in only a few (Srivastava, 1973; for reviews see Johri and Srivastava, 1973; Johri et aI., 1980 a, b; Vasil and Vasil, 1980). This has led to the possibility of vegetative propagation and genetic manipulation of tree species. Very little is known, however, about the inherent changes underlying the process of differentiation. A number of investigators studied the isozyme expression of different plant tissues and found changes in isozymes occurring during cell or callus cultures (Amison and Boll, 1974; Simola and Sopanen, 1970). Isozyme analysis at different stages of culture might throw light on the physiological and biochemical changes underlying the processes of callusing and differentiation. This is important in respect to tissue-specific biochemical characteristics which are expressed and maintained during the culture period (see Scandalios, 1974). In the present studies, an attempt was made to culture and grow the shoot bud explants of &tula pendula progenies obtained from various crosses between red and *) Permanent Address: Department of Botany, S.G.T.B. Khalsa College, University of Delhi. Delhi 110007, India.
Z. Pjlanzenplrysiol. Bd. 103. S. 341-346. 1981.
342
P. S. SRIVASTAVA and A. STEINHAUER
early flowering trees, and to obtain early regeneration which would provide a quick method for vegetative multiplication of genetically defined clones. Also, isozyme analysis was carried out to establish a correlation between morphogenic responses and isozyme expression, which could be used as a genetic marker.
Material and Methods Buds of Betula pendula Roth. were collected from Gottingen. They were surface*sterilized as described by Johri and Srivastava (1972). Before implanting, the explants were washed thoroughly with sterile distilled water. Modified White's medium according to Srivastava et al. (1980) with 3% sucrose, and 5.8 pH was used (WM). The medium was gelled with 0.65% agar and dispensed both in Erlenmeyer flasks and 40 mm petridishes. Growth hormones, indoleacetic acid (lAA), indolebutyric acid (rnA), naphthaleneacetic acid (NAA), gibberellic acid (GA,), kinetin, zeatin, W-benzylaminopurine (BAP), and adenine were also added to the medium, as required. Cultures were maintained on a shelf adjusted to 16/8 h light/dark period with 28±2 °C and 60±5% RH. To induce early regeneration, the callus pieces were transferred to the medium of Murashige and Skoog (1962) (MMS) containing, in mg' 1- ',9250 KNO" 7000 NH,NO" 1260 CaCI,' 2H,O, 1650 MgSO,· 7H, O, 640 KH,PO" 15 H,BO" 12 MnSO, . 4H,O, 8.5 ZnSO,' 7H,O, 0.25 Na,MoO,' 2H,O, 0.25 CuSO,' 5H,O, 50 Na2FeEDTA, 5 niacin, 2 glycine, 2 calcium pantothenate, 0.5 pyridoxine' HCl, 0.5 thiamin' HCI, 20 glutamine, 30 asparagin, 20 arginine, 125 myo-inositol, and 30,000-40,000 sucrose. For isozyme (leucine aminopeptidase, LAP and acid phosphatase, AP) determination, tissues were removed from the culture vials at regular intervals. The callus and differentiated organs were homogenized in tris*HCI (PH 7.5) buffer containing 30 mg soluble polyvinylpyrolidone (PVP). Starch (11.5%) gel zone-electrophoresis was performed using a modified discontinuous system with HJBOJ*NaOH buffer (PH 8.2) at a voltage distribution of 15 V /cm (for details see Poulik, 1957).
Results and Discussion The buds did not show any change for 7 days on WM + 2 mg . 1- ' each of GA" IAA, and BAP. After 10 days, in 60.% ohhe cultures, the buds opened and formed normal leaf-like structures. In the remaining 40% the buds showed callusing. After 20 days almost 85% of th~ cultures started callusing (Fig. 1 A). The calli turned green and in 15% of the cultures, anthocyanin synthesis also took place. The smaller buds (3 mm) invariably produced callus whereas the larger buds (5 mm) developed leaves and the cut ends callus. The callus produced on the above medium started rooting after another 4 weeks. The roots were ca. 10 mm with a well-developed, pink-coloured root cap, and root hairs. After 6 weeks, shoot buds also differentiated. Numerous shoot buds differentiated when the callus was transferred to MMS+IAA (2 mg' I-')+BAP (5 mg . 1-') + adenine (20 mg' 1-') (Fig. 1 B). Some of these shoot buds developed further after 2 weeks and formed characteristic normal leaves. Further growth of the shoots was arrested, however. Various concentrations and combinations of IAA and BAP were Z Pjlanzenphysiol. Ed. 103. S. 341-346.1981.
Isozymes in cultured shoot buds
343
Fig. 1 A - D: In vitro growth of cultured shoot buds: (A) Profuse callusing of explants on WM+2 mg . 1-1 each of GAl, IAA, and SAP; (B) Differentiation of shoot buds on MMS + IAA
(2 mg· I- I)+BAP (5 mg· I- I)+adenine (20 mg· I-I); (C) excised shoot grown on MMS+lAA (2 mg· l- I)+zeatin (2 mg· 1- 1) + adenine (30 mg . 1- 1); note normal growth of shoot and well-differentiated leaves; (0) formation of complete plantlet.
tried in order to obtain the best possible combination for maximum growth of the tissue and differentiation of plandets. No combination of IAA and BAP supported further growth (Fig. 2). However. when the differentiating callus was transferred to MMS medium supplemented with IAA (2 mg . I- I)+zeatin (2 mg· I-I)+adenine (30 mg . I-I). the growth of shoots was remarkably fast and within 2 weeks they
Fig. 2: Effect of interaction of IAA and BAP on tissue growth.
Z. Pjlanzenphysiol. Bd. 103. S. 341-346.1981.
344
P.
S. SRIVASTAVA
and A.
STEINHAUER
Fig. 3: A complete plant (before transplanting to soil); note well-differentiated root system.
became 20 mm long with 4 pairs of well-developed leaves (Fig. 1 C). These shoots when excised and grown on the same medium grew vigorously and developed into
normal plandets (Fig. 1 D). If the buds were initially cultured on MMS+IAA (2 mg' l-')+GA, (2 mg . l-')+BAP (5 mg' l-')+casein hydrolysate (1000 mg . 1-') callusing was much better. Subsequent transfer of pieces of these calli on MMS + IAA (2 mg' l-')+zeatin (2 mg' l- ')+adenine (30 mg' 1- ') resulted in plandet formation. As many as 25 - 30 plandets could be recovered from each piece of callus. The plandets (Fig. 3) thus obtained could be transferred to soil. In all we tried bud culture of progenies from 15 different crosses. Of these, only five proved responsive for quick regeneration.
The initial results with buds and bud callus indicate that the expression of isozymes, LAP and AP, is altered under in vitro conditions. In the control buds, LAP expressZ Pjlanzenpbysiol. Ed. 103. S. 341 -346.1981.
Isozymes in cultured shoot buds
345
ed only one band and after 4 weeks, when the buds have initiated callusing, 2 bands were discernible. Three AP bands could be seen initially but after 4 weeks one band disappeared. Later, after 6 weeks, the shoot and.plantiet-differentiating callus exhib-
ited no change in AP bands but one additional DAP band appeared (Fig. 4). front
- - -- - - -- ----
._._,_._-_._._._._,- - -- -_._._._._,_._._.a
b
c
d
acid phosphatase
a
b
c
d
leucif'/f! aminopeptidase
start L-~~~~~~~--L-~~~~~~~~
Fig. 4: Isozyme pattern at different stages of callus growth. Expression of an additional band of LAP indicates the shift in morphogenic expression and initiation of bud formation. (a) explant at the time of culture, (b) callus initiation, (c) initiation of organ differentiation, (d) later stage.
Isozymes provide a means of marking the sequential development processes in plant tissue cultures and therefore represent the differential gene activity of the differentiating cells. Changes in isozyme patterns are correlated with physiological and
developmental states of intact plants (Scandalios, 1974). This has also been found in cultured plant cells. In the case of peroxidases, for example, changes due to exogenous
supply of growth regulators were shown by Lee (1972 a, b). Similar effects were observed by Lavee and Galston (1968) in Pelargonium cultures. In Phaseolus vulgaris, however, Bassiri and Carlson {1978} found a great uniformity of isozyme patterns within and between the calli of the same or different origin.
In our studies, the isozymes (LAP and AP) showed varying patterns depending upon the morphogenic response of the callus. Striking changes in isozyme expression during the process of germination are well established. Since this process involves growth and differentiation, this too may be the case in differentiating cultured tissues. In shoot buds, callus initiation and differentiation represent an alteration in the normal morphogenic response and, therefore, a change in the physiological and genetical expression leading to changes in isozymes. The expression of isozyme bands at a particular stage of callus development and prior to differentiation may prove to be a suitable marker to predict the morphogenic
responses of tissues. The rapid regeneration of plandets from shoot bud explants of &tula pendula may prove to be useful for obtaining genetically defined clones. Acknowledgement This work is suppo~ed by a Deutsche Forschungsgemeinschaft grant. The technical assistance of Miss C. Becker and Mr. S. Krakuhn is highly appreciated.
Z. Pjlanzenplrysiol. Ed. 103. S. 341 - 346.1981.
346
P. S. SRIVASTAVA and A. STEINHAUER
References AJ.NlSON, P. G. and W. G . BOLL: Isozymes in cell cultures of bush bean (Phaseolus vulgaris cv. Contender): isoenzymatic changes during the callus culture cycle and differences between stock cultures. Can. J. Bot. 52,2621- 2629 (1974). BASSIlU, A. and P. S. CAJU.SON: Isozyme patterns and differences in plane parts and their callus cultures in common bean. Crop Sci. 18, 955-958 (1978). jOHRI, B. M. and P. S. SRIVASTAVA: In vitro growth responses of mature endosperm of Ricinus communis L. In: Adv. Plant Morph., V. Puri Comm. Vol., 339-358. Sarita Prakashan, India, 1972. - - Morphogenesis in endosperm cultures. Z. Pflanzenphysiol. 10,285-304 (1973). jOHRI, B. M., P. S. SRIVASTAVA, and A. P. RASTE: Plane tissue culture and crop improvemene. Indian J. agric. Sci. 50, 103 ~ 127 (1980 a). - - - Endosperm culture. In: VASIL, I. K. (Ed.): Perspectives in Plane Cell and Tissue Culture, Int. Rev. Cyto!., Supp!. liB, 157 -182. Academic Press, New York, 1980 b. LAVE£, S. and A. W. GALSTON: Hormonal control of peroxidase activity in cultured Pelargonium pith. Am. J. Bot. 55, 890-893 (1968). Lu, T. T.: Changes in IAA oxidase isoenzymes in tobacco tissues aher treatment with 2,4-dichlorophenoxyacetic acid. Plant Physio!. 49, 957 -960 (1972 a). - Interaction of cytokinin, auxin, and gibberellin on peroxidase isozymes in tobacco tissues cultured in vitro. Can. J. Bot. 50,2471-2477 (1972 b). MUIlASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physio!. Plant. 15,473-497 (1962). POUUK, M. D.: Starch-gel electrophoresis in a discontinuous system of buffers. Nature (London) 80, 1477 (1957). SCANDAUoS,j. G.: Isozymes in development and differentiation. Ann. Rev. Plant Physiol. 25, 225-258 (1974). SIMOLA, L. K. and T. SOPANEN: Changes in the activity of certain enzymes of Acer pseudopla· tanus L. cells at four stages of the growth cycle in suspension culture. Physio!. Plant. 23, 1212-1222 (1970). SRIVASTAVA, P. 5.: Formation of triploid plantlets in mature endosperm cultures of Putranjiva roxburghii. Z. Pflanzenphysio!. 69,270-273 (1973). SRIVASTAVA, P. 5., A. VllGA, and]. BRUINSMA: Growth in vitro of fertilized ovules of pea, Pisum sativum L. with and without pods. Z. Pflanzenphysio!. 98, 347-354 (1980). VASIL, I. K. and VIMLA VASIL: Clonal propagation. In: VASIL, I. K. (EeL): Perspectives in Plant Cell and Tissue Culture, Int. Rev. Cytol., Suppl. 11A, 145-173. Academic Press, New York. 1980.
Z PjlanzenpJry.iol. Ed. 103. S. 341-346.1981.