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The Role of Explant Source and Culture Conditions on Callus Induction and Shoot Regeneration in Sugarbeet (Beta vulgaris L.)
}.PlantPhysiol. Vol. 134.pp. 651-655 (1989)
The Role of Explant Source and Culture Conditions on Callus Induction and Shoot Regeneration in Sugarbeet (Beta vulgaris L.) F. A.
KRENS
and D. JAMAR
Foundation for Agricultural Plant Breeding, SVP, P.O. Box 117, 6700 AC Wageningen, The Netherlands Received November 2,1988 . Accepted January 22,1989
Summary
De novo shoot formation from callus induced on explants of Beta vulgaris (sugarbeet) seedlings was demonstrated to be particularly dependent on the origin of the explants. The organ giving the highest regeneration response was the petiole. Neither the two basal media nor the different hormonal regimes that were tested had a clear effect on shoot formation. Root formation could be stimulated by increasing 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations. Callus formation appeared at high frequencies from leaves and petioles, but was shown to be hormone-dependent when the explants were derived from hypocotyls or cotyledons.
Key words: Beta vulgaris, sugarbeet, callus induction, shoot formation, explant source.
Introduction No reports have yet been published on the routine production of large numbers of transgenic plants in the sugarbeet (Beta vulgaris L.). The susceptibility of sugarbeet to Agrobacterium tumefaciens (Wang et aI., 1985; Paul et aI., 1987; Krens et aI., 1988) is not so much an obstacle as is the lack of a good regeneration system (Ford-Lloyd and Bhat, 1986). Some research on this subject has been reported, however, yielding only a limited number of regenerated shoots or regenerating calli (Hooker and Nabors, 1977; De Greef and Jacobs, 1979; Saunders and Doley, 1986; Saunders and Shin, 1986). Habituated cell lines giving occasional regeneration have also been described (Van Geyt and Jacobs, 1985) Tetu et al. (1987) found higher regeneration frequencies, but they required rather elaborate multiple hormonal sequences. Recently it was found that a special medium enriched with several amino acids and vitamins gave rise to numerous adventitious shoots (Freytag et aI., 1988). However, in many cases the shoots originated either from pre-existing meristems, predetermined cells or from callus formed on parts of the petiole that were not wounded by the cutting. Wounding is essential for A. tumefaciens transformation (Krens et aI., 1985). The present paper reports on the callus-forming response of different explant types of a diploid B. vulgaris genotype on 1989 by Gustav Fischer Verlag. Stuttgart
several different media as well as the subsequent de novo formation of shoots from the wounded areas.
Materials and methods Plant material
The diploid, fertile SVP accession no 31-188 (B. vulgaris) was used in all studies. In vitro culture conditions were essentially as described by Krens et al. (1988). Prior to sterilization seeds were permeabilized by a 15 min submersion in hot water (55°C). Subsequently, the seeds were transferred to 95% (v/v) ethanol (few seconds), 1% (w/v) Na-hypochlorite (15 min) and 3 washes in sterile H 20 (5 min, 10 min and 15 min), respectively. After sterilization the seeds were placed on water! agar [1.5 % (w/v) Daichin agar, Brunschwig Chemie, in distilled H 20] at 22°C in the light (see later) for germination. The germinated seeds were put on MS medium (Murashige and Skoog, 1962) at half strength supplemented with 30 g 1- I sucrose and 8gl- 1 agar (1/2 MS). Culture of the seedlings took place at 22°C with a photoperiod of 16 hours (1000 lux from cool white FTD58W133 fluorescent and Philinea lamps). Callus induction and organogenesis
Unless stated otherwise 6-week-old seedlings were taken from the culture tubes and divided into different organs (leaves, petioles, hy-
652
F. A. KRENS and D. JAMAR '00
Table 1: The numbers of explants from 6-week-old seedlings. 112 MS' Leaves Petioles Hypocotyls Cotyledons
160 174 80 146
(205) (247) (83) (76)
PGo' 153 197 68 151
(126) (150) (57) (52)
112 MS b 132 196 80 120
(233) (202) (69) (41)
eo
PGo b 229 227 70 106
eo
(158) (191) (65) (5)
• Sixteen hormone combinations, b 2mgl~1 BAP. Data in brackets are from 12-week-old seedlings.
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pocotyls and cotyledons). The sizes into which the explants were cut, were as follows: leaves and cotyledons 0.25 - 0.5 cm 2, and petioles and hypocotyls 5 -7 mm. Care was taken to avoid meristematic tissues. The explants were placed on two basal media: MS at half strength (112 MS) and PGo (De Greef and Jacobs, 1979), both supplemented with 30 g I ~ 1 sucrose and 8 g I ~ 1 agar. Four concentrations of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D; 0.01, 0.05, 0.1, 0.2 mgl ~ I) were combined with four concentrations of the cytokinin 6-benzylaminopurine (BAP; 0.05, 0.1, 0.2, 0.25 mgl ~ I), giving sixteen different phytohormone combinations per basal medium. Next to these, BAP was tested at the higher concentration of 2 mgl ~ 1 as the sole phytohormone. CaIlus formation and organogenesis were scored after 37 days of culture under the same physical conditions as the seedlings (Krens et aI., 1988). Shoots appearing within one week were not considered as newly formed regenerants, but most likely as arising from meristems that were still present. They were not included in calculating regeneration frequencies. The number of explants per medium is presented in Table 1. In later experiments performed to determine the variation in shoot formation from petioles and to investigate the role of high BAP levels, the numbers of explants were 315 on PGo (2 mg I ~ 1 BAP) and 296,195 & 870 on PGo (0.05mgl~1 2,4-D + 0.1 mgl~1 BAP; one of the sixteen combinations).
Results
Callus fonnation Within every organ type no preference for a certain hormone regime could be found when the explants were placed on the different (16) combinations (data not shown). Therefore, the percentages of callus formation for the media with variable hormone concentrations were pooled and are shown in Fig. 1 A, in relation to explant source and basal medium composition. In Fig. 1 B the percentage callus formation on the two basal media carrying 2 mg I ~ 1 BAP are presented, also in relation to explant source. On the media with relatively low hormone concentrations the callusing response was high for each organ type, with petioles and hypocotyls giving the best results. When a much higher level of BAP was used, callus formation on leaf and petiole explants was not affected, but on cotyledons and especially on hypocotyls the response was severely attenuated. No clear difference in reaction could be determined between the two basal media.
Shoot fonnation Shoot formation occurred at low frequencies. Again no particular hormone combination proved to be favorable. Fig. 2 demonstrates that mostly no shoots were formed in
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Fig. 1: The percentages of explants forming callus in the culture of four different explant types on two basal media (lI2MS and PGo) supplemented with A) sixteen low 2,4-D and BAP combinations (data are pooled, see text) and B) 2 mgl ~ 1BAP.
~
0.05
0.1
0.2
0.25
0.01
R
R
R
R
0.05
R
R
2,4-0
0.1
0.2
R
R R
Fig. 2: Diagram shows on which combinations of 2,4-D and BAP the regeneration (R) of shoots was observed.
combinations with the higher 2,4-D concentrations. For further calculations the data of the sixteen hormone combinations were pooled. Table 2 shows the conditions and the frequencies with which regeneration occurred. No clear difference between the two basal medium compositions (1/2
Regeneration of sugarbeet Table 2: Percentages of shoot formation on two types of basal media (112 MS and PGo). Explant
Medium supplemented with variable, but low hormone concentrations 1/2 MS
1/2 MS
PGo
(O)a
Medium supplemented with 2mgl- 1 BAP
0 (0.80) 1.02 (0) 2.94 (0) 1.32
a
from 12-week-old seedlings.
0 0 2.14 0 Data in brackets are
(0) (0) (0) (0)
0 5.29 1.43 0
Leaves Petioles Hypocotyls Cotyledons
PGo (0) (0) (1.50) (0)
0 4.08 1.25 0
(0) (1.00) (0) (0)
MS and PGo) could be demonstrated. The results from the first experiment suggest that 2 mg 1- I BAP induced more shoots than low hormone combinations. In a later experi-
Fig. 3 A and B: Shoots developing on callus formed at the site of wounding of petiole explants. Note the clear separation between the petiole and site of occurrence of the shoot in Fig. 3 B.
653
ment the difference proved to be not reproducible, since PGo (2mgl- 1 BAP) gave 1.6% shoot formation (315 explants) and PGo (0.05mgl- 1 2,4-D + 0.1 mgl - I BAP) 1.7% (296 explants). Two subsequent experiments using only PGo (0.05/0.1) yielded shoot formation with frequencies of 3.0 % (870 explants) and 4.6% (195 explants), demonstrating a rather great variation among as well as within experiments. The hypocotyl and particularly the petiole were the best explant types (see also Fig. 3). From leaves, not a single shoot could be regenerated under any of the conditions tested. In order to study the role of age of the seedlings on shooting ability, the original experiment was repeated with 12-weekold material (see Table 1). It was found that with this older starting material shoot formation was greatly reduced (see Table 2).
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Fig. 4: The effect of 2,4-D and BAP on root formation of all explant types.
Root formation In the same experiments the occurrence of roots was also monitored. At the lowest BAP concentration (0.05 mg 1-1 BAP) a clear auxin effect was observed. Increasing 2,4-D concentrations led to increasing numbers of explants showing root formation and an increased elongation of the roots carrying many well-developed root hairs. This effect, however, could not be observed at the higher BAP levels, where root formation was already strongly inhibited at 0.1 mg 1-1 BAP (Fig. 4).
as long as the concentration of 2,4-D was not too high. The concentration of BAP could be varied from 0.05 to 2.0 mg I-I without a significant positive effect. In another study even higher concentrations of BAP (up to 4mgl-l) have been tried on leaf explants without any effect (Krens et al., 1988). All explant types responded similarly with root formation: at very low BAP concentrations increasing 2,4-D stimulated the formation of roots according to the model of Skoog and Miller (1957), and at a slightly higher concentration of BAP this response towards auxin was abolished. The most important factor for shoot formation appeared to be the origin of the explant. Here, for the first time a comparison is presented between different organs as a source of explants. Petioles provided the best results, whereas in our hands leaves failed to produce a single shoot. Great variation in the frequencies was observed among different experiments. An explanation could be that tissue explants carried meristematic parts varying in frequency from one experiment to the other. However, it must be emphasized that care was taken during cutting not to include meristems or meristem parts. In addition to this, the shoots appearing within one week after cutting were not used in further calculations. With an average of 2.66 %, frequencies were still rather low. In a recent report, Freytag et al. (1988) obtained higher frequencies of shoot formation with some of the genotypes that they tested. They used a modified MS medium with extra amino acids and vitamins but without high cytokinin levels. However, many of their regenerants originated from silent meristems or pre-determined cells within the petiole. Here, as shown in Fig. 3, shoots emerged from callus formed on the cut egdes of the petiole and not directly on unwounded areas of the petiole. This is probably a prerequisite for transformation by Agrobacterium tumefaciens.
Discussion In this report the factors which were studied for their role in callus formation and organogenesis were the age of the starting material (the seedlings), the explant source, i.e. the organ from which the explants were taken, the basal medium composition and phytohormones. Normally, six-week-old seedlings were used to provide explants. When twelve-week-old seedlings were taken, the number of explants showing callus formation (data not shown) did not deviate from the number obtained with sixweek-old material and was very high (up to 100 %). However, shoot formation was greatly reduced. The basal medium composition had no effect on callus formation nor on organogenesis. The two media were prepared according to their original formulations (Murashige and Skoog, 1962; De Greef and Jacobs, 1979) without organic additives. Two synthetic growth regulators, 2,4-D and BAP, were added as phytohormones in different combinations giving auxin/ cytokinin ratios ranging from a to 4. In general these different ratios had no effect on callus formation except that at a high BAP concentration of 2 mg I-I hypocotyls and cotyledons reacted quite differently than leaves or petioles did. Especially with hypocotyls, callus formation was severely inhibited. This might suggest differences in endogenous hormone levels or in sensitivity to them between the organs. For shoot regeneration the ratio did not appear to be crucial
References DE GREEF, W. and M. JACOBS: In vitro culture of the sugarbeet: description of a cell line with high regeneration capacity. Plant Sci. Lett. 17, 55-61 (1979). FORD-LLOYD, B. V. and S. BHAT: Problems and prospects for the use of protoplasts in beet breeding. In: HORN, W., C. J. JENSEN, W. ODENBACH, and O. SCHIEDER (eds.): Genetic Manipulation in Plant Breeding, 437-440, Walter de Gruyter & Co., Berlin (1986). FREYTAG, A. H., S. C. ANAND, A. P. RAo-ARDELLI, and L. D. OWENS: An improved medium for adventitious shoot formation and callus induction in Beta vulgaris L. in vitro. Plant Cell Reports 7, 30 - 34 (1988). HOOKER, M. P. and M. W. NABORS: Callus initiation, growth, and organogenesis in sugarbeet (Beta vulgaris L.). Z. Pflanzenphysiol. 84,237-246 (1977). KRENS, F. A., L. MOLENDIjK, G. J. WULLEMS, and R. A. SCHILPEROORT: The role of bacterial attachment in the transformation of cell-wall regenerating tobacco protoplasts by Agrobacterium tume/aciens. Planta 166, 300-308 (1985). KRENS, F. A., C. ZIjLSTRA, W. v. D. MOLEN, D. JAMAR, and H. J. HUIZING: Transformation and regeneration in sugar beet (Beta vulgaris L.) induced by «shooter» mutants of Agrobacterium tume/aciens. Euphytica S, 185 -194 (1988). MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 (1962).
Regeneration of sugarbeet PAUL, H ., C. ZIJLSTRA, J. E. LEEUWANGH, F. A. KRENS, and H. J. HUIZING: Reproduction of the beet cyst nematode Heterodera schachtii Schm. on transformed root cultures of Beta vulgaris L. Plant Cell Reports 6, 379-381 (1987). SAUNDERS, J. W. and W. P. DOLEY: One step shoot regeneration from callus of whole plant leaf explants of sugarbeet lines and a somaclonal variant for in vitro behavior. J. Plant Physiol. 124, 473 -479 (1986). SAUNDERS, J. W. and K. SHIN: Germplasm and physiologic effects on induction of high-frequency hormone autonomous callus and subsequent shoot regeneration in sugarbeet. Crop. Sci. 26, 1240-1245 (1986).
655
SKOOG, F. and C. O . MILLER: Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Bot. 11, 118-131 (1957). TETu, T., R. S. SANGWAN, and B. S. SANGWAN-NoRREEL: Hormonal control of organogenesis and somatic embryogenesis in Beta vul· garis callus. J. Exp. Bot. 38,506-517 (1987). VAN GEYT, J. P. C. and M. JACOBS: Suspension culture of sugarbeet (Beta vulgaris L.). Induction and habituation of dedifferentiated and self-regenerating cell lines. Plant Cell Reports 4, 66-69 (1985). WANG, Y. Q., C. E. Lo, Y. Y. LIAN, Q. Q. SHAO, X. C. JIANG, Z. Q. ZHOU, A. S. LEE, and Y. Q. CHEN: Teratome induction and gene transfer in Beta. Acta Agron. Sinica 11, 159-162 (1985).
The Role of Explant Source and Culture Conditions on Callus Induction and Shoot Regeneration in Sugarbeet (Beta vulgaris L.) F. A.
KRENS
and D. JAMAR
Foundation for Agricultural Plant Breeding, SVP, P.O. Box 117, 6700 AC Wageningen, The Netherlands Received November 2,1988 . Accepted January 22,1989
Summary
De novo shoot formation from callus induced on explants of Beta vulgaris (sugarbeet) seedlings was demonstrated to be particularly dependent on the origin of the explants. The organ giving the highest regeneration response was the petiole. Neither the two basal media nor the different hormonal regimes that were tested had a clear effect on shoot formation. Root formation could be stimulated by increasing 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations. Callus formation appeared at high frequencies from leaves and petioles, but was shown to be hormone-dependent when the explants were derived from hypocotyls or cotyledons.
Key words: Beta vulgaris, sugarbeet, callus induction, shoot formation, explant source.
Introduction No reports have yet been published on the routine production of large numbers of transgenic plants in the sugarbeet (Beta vulgaris L.). The susceptibility of sugarbeet to Agrobacterium tumefaciens (Wang et aI., 1985; Paul et aI., 1987; Krens et aI., 1988) is not so much an obstacle as is the lack of a good regeneration system (Ford-Lloyd and Bhat, 1986). Some research on this subject has been reported, however, yielding only a limited number of regenerated shoots or regenerating calli (Hooker and Nabors, 1977; De Greef and Jacobs, 1979; Saunders and Doley, 1986; Saunders and Shin, 1986). Habituated cell lines giving occasional regeneration have also been described (Van Geyt and Jacobs, 1985) Tetu et al. (1987) found higher regeneration frequencies, but they required rather elaborate multiple hormonal sequences. Recently it was found that a special medium enriched with several amino acids and vitamins gave rise to numerous adventitious shoots (Freytag et aI., 1988). However, in many cases the shoots originated either from pre-existing meristems, predetermined cells or from callus formed on parts of the petiole that were not wounded by the cutting. Wounding is essential for A. tumefaciens transformation (Krens et aI., 1985). The present paper reports on the callus-forming response of different explant types of a diploid B. vulgaris genotype on 1989 by Gustav Fischer Verlag. Stuttgart
several different media as well as the subsequent de novo formation of shoots from the wounded areas.
Materials and methods Plant material
The diploid, fertile SVP accession no 31-188 (B. vulgaris) was used in all studies. In vitro culture conditions were essentially as described by Krens et al. (1988). Prior to sterilization seeds were permeabilized by a 15 min submersion in hot water (55°C). Subsequently, the seeds were transferred to 95% (v/v) ethanol (few seconds), 1% (w/v) Na-hypochlorite (15 min) and 3 washes in sterile H 20 (5 min, 10 min and 15 min), respectively. After sterilization the seeds were placed on water! agar [1.5 % (w/v) Daichin agar, Brunschwig Chemie, in distilled H 20] at 22°C in the light (see later) for germination. The germinated seeds were put on MS medium (Murashige and Skoog, 1962) at half strength supplemented with 30 g 1- I sucrose and 8gl- 1 agar (1/2 MS). Culture of the seedlings took place at 22°C with a photoperiod of 16 hours (1000 lux from cool white FTD58W133 fluorescent and Philinea lamps). Callus induction and organogenesis
Unless stated otherwise 6-week-old seedlings were taken from the culture tubes and divided into different organs (leaves, petioles, hy-
652
F. A. KRENS and D. JAMAR '00
Table 1: The numbers of explants from 6-week-old seedlings. 112 MS' Leaves Petioles Hypocotyls Cotyledons
160 174 80 146
(205) (247) (83) (76)
PGo' 153 197 68 151
(126) (150) (57) (52)
112 MS b 132 196 80 120
(233) (202) (69) (41)
eo
PGo b 229 227 70 106
eo
(158) (191) (65) (5)
• Sixteen hormone combinations, b 2mgl~1 BAP. Data in brackets are from 12-week-old seedlings.
70
eo 00
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40 30
0
(]I
20
C
pocotyls and cotyledons). The sizes into which the explants were cut, were as follows: leaves and cotyledons 0.25 - 0.5 cm 2, and petioles and hypocotyls 5 -7 mm. Care was taken to avoid meristematic tissues. The explants were placed on two basal media: MS at half strength (112 MS) and PGo (De Greef and Jacobs, 1979), both supplemented with 30 g I ~ 1 sucrose and 8 g I ~ 1 agar. Four concentrations of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D; 0.01, 0.05, 0.1, 0.2 mgl ~ I) were combined with four concentrations of the cytokinin 6-benzylaminopurine (BAP; 0.05, 0.1, 0.2, 0.25 mgl ~ I), giving sixteen different phytohormone combinations per basal medium. Next to these, BAP was tested at the higher concentration of 2 mgl ~ 1 as the sole phytohormone. CaIlus formation and organogenesis were scored after 37 days of culture under the same physical conditions as the seedlings (Krens et aI., 1988). Shoots appearing within one week were not considered as newly formed regenerants, but most likely as arising from meristems that were still present. They were not included in calculating regeneration frequencies. The number of explants per medium is presented in Table 1. In later experiments performed to determine the variation in shoot formation from petioles and to investigate the role of high BAP levels, the numbers of explants were 315 on PGo (2 mg I ~ 1 BAP) and 296,195 & 870 on PGo (0.05mgl~1 2,4-D + 0.1 mgl~1 BAP; one of the sixteen combinations).
Results
Callus fonnation Within every organ type no preference for a certain hormone regime could be found when the explants were placed on the different (16) combinations (data not shown). Therefore, the percentages of callus formation for the media with variable hormone concentrations were pooled and are shown in Fig. 1 A, in relation to explant source and basal medium composition. In Fig. 1 B the percentage callus formation on the two basal media carrying 2 mg I ~ 1 BAP are presented, also in relation to explant source. On the media with relatively low hormone concentrations the callusing response was high for each organ type, with petioles and hypocotyls giving the best results. When a much higher level of BAP was used, callus formation on leaf and petiole explants was not affected, but on cotyledons and especially on hypocotyls the response was severely attenuated. No clear difference in reaction could be determined between the two basal media.
Shoot fonnation Shoot formation occurred at low frequencies. Again no particular hormone combination proved to be favorable. Fig. 2 demonstrates that mostly no shoots were formed in
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....
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Fig. 1: The percentages of explants forming callus in the culture of four different explant types on two basal media (lI2MS and PGo) supplemented with A) sixteen low 2,4-D and BAP combinations (data are pooled, see text) and B) 2 mgl ~ 1BAP.
~
0.05
0.1
0.2
0.25
0.01
R
R
R
R
0.05
R
R
2,4-0
0.1
0.2
R
R R
Fig. 2: Diagram shows on which combinations of 2,4-D and BAP the regeneration (R) of shoots was observed.
combinations with the higher 2,4-D concentrations. For further calculations the data of the sixteen hormone combinations were pooled. Table 2 shows the conditions and the frequencies with which regeneration occurred. No clear difference between the two basal medium compositions (1/2
Regeneration of sugarbeet Table 2: Percentages of shoot formation on two types of basal media (112 MS and PGo). Explant
Medium supplemented with variable, but low hormone concentrations 1/2 MS
1/2 MS
PGo
(O)a
Medium supplemented with 2mgl- 1 BAP
0 (0.80) 1.02 (0) 2.94 (0) 1.32
a
from 12-week-old seedlings.
0 0 2.14 0 Data in brackets are
(0) (0) (0) (0)
0 5.29 1.43 0
Leaves Petioles Hypocotyls Cotyledons
PGo (0) (0) (1.50) (0)
0 4.08 1.25 0
(0) (1.00) (0) (0)
MS and PGo) could be demonstrated. The results from the first experiment suggest that 2 mg 1- I BAP induced more shoots than low hormone combinations. In a later experi-
Fig. 3 A and B: Shoots developing on callus formed at the site of wounding of petiole explants. Note the clear separation between the petiole and site of occurrence of the shoot in Fig. 3 B.
653
ment the difference proved to be not reproducible, since PGo (2mgl- 1 BAP) gave 1.6% shoot formation (315 explants) and PGo (0.05mgl- 1 2,4-D + 0.1 mgl - I BAP) 1.7% (296 explants). Two subsequent experiments using only PGo (0.05/0.1) yielded shoot formation with frequencies of 3.0 % (870 explants) and 4.6% (195 explants), demonstrating a rather great variation among as well as within experiments. The hypocotyl and particularly the petiole were the best explant types (see also Fig. 3). From leaves, not a single shoot could be regenerated under any of the conditions tested. In order to study the role of age of the seedlings on shooting ability, the original experiment was repeated with 12-weekold material (see Table 1). It was found that with this older starting material shoot formation was greatly reduced (see Table 2).
654
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2,4-0 ooncenll'Otlan
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Fig. 4: The effect of 2,4-D and BAP on root formation of all explant types.
Root formation In the same experiments the occurrence of roots was also monitored. At the lowest BAP concentration (0.05 mg 1-1 BAP) a clear auxin effect was observed. Increasing 2,4-D concentrations led to increasing numbers of explants showing root formation and an increased elongation of the roots carrying many well-developed root hairs. This effect, however, could not be observed at the higher BAP levels, where root formation was already strongly inhibited at 0.1 mg 1-1 BAP (Fig. 4).
as long as the concentration of 2,4-D was not too high. The concentration of BAP could be varied from 0.05 to 2.0 mg I-I without a significant positive effect. In another study even higher concentrations of BAP (up to 4mgl-l) have been tried on leaf explants without any effect (Krens et al., 1988). All explant types responded similarly with root formation: at very low BAP concentrations increasing 2,4-D stimulated the formation of roots according to the model of Skoog and Miller (1957), and at a slightly higher concentration of BAP this response towards auxin was abolished. The most important factor for shoot formation appeared to be the origin of the explant. Here, for the first time a comparison is presented between different organs as a source of explants. Petioles provided the best results, whereas in our hands leaves failed to produce a single shoot. Great variation in the frequencies was observed among different experiments. An explanation could be that tissue explants carried meristematic parts varying in frequency from one experiment to the other. However, it must be emphasized that care was taken during cutting not to include meristems or meristem parts. In addition to this, the shoots appearing within one week after cutting were not used in further calculations. With an average of 2.66 %, frequencies were still rather low. In a recent report, Freytag et al. (1988) obtained higher frequencies of shoot formation with some of the genotypes that they tested. They used a modified MS medium with extra amino acids and vitamins but without high cytokinin levels. However, many of their regenerants originated from silent meristems or pre-determined cells within the petiole. Here, as shown in Fig. 3, shoots emerged from callus formed on the cut egdes of the petiole and not directly on unwounded areas of the petiole. This is probably a prerequisite for transformation by Agrobacterium tumefaciens.
Discussion In this report the factors which were studied for their role in callus formation and organogenesis were the age of the starting material (the seedlings), the explant source, i.e. the organ from which the explants were taken, the basal medium composition and phytohormones. Normally, six-week-old seedlings were used to provide explants. When twelve-week-old seedlings were taken, the number of explants showing callus formation (data not shown) did not deviate from the number obtained with sixweek-old material and was very high (up to 100 %). However, shoot formation was greatly reduced. The basal medium composition had no effect on callus formation nor on organogenesis. The two media were prepared according to their original formulations (Murashige and Skoog, 1962; De Greef and Jacobs, 1979) without organic additives. Two synthetic growth regulators, 2,4-D and BAP, were added as phytohormones in different combinations giving auxin/ cytokinin ratios ranging from a to 4. In general these different ratios had no effect on callus formation except that at a high BAP concentration of 2 mg I-I hypocotyls and cotyledons reacted quite differently than leaves or petioles did. Especially with hypocotyls, callus formation was severely inhibited. This might suggest differences in endogenous hormone levels or in sensitivity to them between the organs. For shoot regeneration the ratio did not appear to be crucial
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