Plant Cell Reports
Plant Cell Reports (1989) 8:403-406
© Springer-Verlag 1989
Shoot regeneration from petioles and leaves of Vitis X labruscana 'Catawba' Z.-M. Cheng and B. I. Reisch Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA Received February 13, 1989/Revised version received July 27, 1989- Communicated by E. D. Earle
Summary. Shoot regeneration and normal plants were obtained from leaf and petiole explants derived from in vitro grown shoots of Vitis X labruscana 'Catawba'. Regeneration was induced in the presence of both 6-benzylaminopurine and indole-3-butyric acid; combinations of 2,4-dichlorophenoxyacetic acid or 2-naphthoxyacetic acid with 6-benzylaminopurine did not permit regeneration from leaf explants. Up to 15% of leaf and 70% of petiole explants regenerated shoots on media with 5.010.0 I.tM BA and 0.1-0.5 ~tM IBA. Incubation in the dark was required to obtain regeneration. About 50% of shoots developed normally following transfer to light. An average of one shoot regenerated from leaf explants and 3.3 shoots regenerated per petiole explant. Regeneration from petioles and leaves was always from the basipetal end. The interaction of 6-benzylaminopurine with indole-3-butyric acid was also examined. Key Words: Morphogenesis
apices (Barlass and Skene 1978, 1980a, 1980b), leaf callus (Favre 1977; Hirabayashi 1985; Stamp and Meredith 1988), internode callus (Krul and Worley 1977; Rajasekaran and Mullins 1981); or from sporophytic reproductive tissues, such as anthers (Rajasekaran and Mullins 1979; Mauro et al. 1986; Stamp and Meredith 1988) and unfertilized ovules (Mullins and Srinivasan 1976; Srinivasan and Mullins 1980). However, most of the available techniques are limited by low frequency of regeneration; genotype and explant specificity; and a process involving two or three steps. The objective of our research was to develop an efficient and reliable regeneration system for Vitis X labruscana 'Catawba'. Preliminary results were reported earlier (Cheng and Reisch 1988). Shoot regeneration from in vitro leaves and petioles was examined. The interaction of cytokinin and auxin, the effect of explant source, and the effect of culture in darkness were also examined.
Auxins - Cytokinins - Grapes Materials and methods
Abbreviations: BA, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, indole-3-butyric acid; MS, Murashige and Skoog (1962) medium; NN69, Nitsch and Nitsch (1969) medium; NOA, 2-naphthoxyacetic acid Introduction
Improved ability to produce plants from tissue cultures of Vitis would expedite efforts in genetic engineering, in vitro mutant isolation, virus removal, rapid multiplication of elite stocks, and the production of haploids and then homozygous diploids. In grapes, varying degrees of organogenesis and somatic embryogenesis have been obtained. Regeneration has been achieved from vegetative tissues, such as shoot
Offprint requests to: Z.-M. Cheng
The grape cultivar 'Catawba' was established in vitro from vines grown at the New York State Agricultural Experiment Station, Geneva, NY. All explants for regeneration experiments were derived from in vitro 'Catawba' shoot cultures. Axillary buds from the basal part of growing primary shoots were isolated in August, 1986 and were washed for 30 minutes in tap water with Micro detergent (International Products Corp., Trenton, NJ, USA). After woody parts and outer layers of bud scales were removed, buds were submerged in 70% ethanol for 2 to 3 min and then were transferred to 1.85% sodium hypochlorite for 15 min, followed by 3 to 5 washes in sterile distilled water. Apex tissues, 2 to 4 mm long, were isolated from buds and inoculated for shoot multiplication on MS basal medium (Murashige and Skoog 1962) supplemented with 4.0 laM BA. Conditions for further growth of shoot tip cultures were described earlier (Reisch 1986). The microoropa~ated shoots were later
404
cultured in MS medium with 2.0 or 4.0 I~M BA prior to excision of explants. All media were solidified with agar (Difco bacto) at 7.0 g/l. The basic medium for regeneration experiments was NN69 (Nitsch and Nitseh 1969). This medium was supplemented with various plant growth regulators or other components. Myo-inositol (100 rag/l), sucrose (20 g/l), BA, 2,4-D, IBA, and NOA were added to the media prior to autoclaving. Leaf blades (1 x 1 cm or larger) were obtained from the in vitro shoot cultures. These were cut into about 5 mm x 5 nun pieces and inoculated abaxial side up. Petioles from shoot cultures were isolated with great care to exclude nearby axillary buds. The orientation of in vitro petioles in a later experiment was noted to determine which end was capable of shoot production. Each treatment included 4 Petri dishes (25 x 100 rnm) with 10 explants each. The cultures for regeneration were grown in the dark (in cardboard boxes) in a culture room at 24-26°C. After 8 weeks of culture, regenerated shoots were transferred to NN69 medium with 0.5 p.M IBA under a 16:8 light:dark photoperiod to induce rooting. To examine the effect of darkness, petiole explants were cultured continuously in a 16:8 light:dark photoperiod or cultured in the dark for two, four, or eight weeks and then moved to a 16:8 light:dark photoperiod. Percentage data were arcsin transformed and then subjected to analyses of variance. F tests were used to determine the significance of treatments and interactions, if applicable.
Results In preliminary experiments on NN69 medium, petioles regenerated shoots from 2 of 6 explants on either 5.0 lxM BA or 5.0 ~VI BA with 0.1 IxM IBA. Leaf explants regenerated shoots from 4 of 7 explants on medium with 5.0 IxM BA and 0.1 lxM IBA and from 3 of 8 on 10.0 paM BA and 0.1 lxM IBA. No leaves regenerated shoots in the presence of 5.0 or 7.5 tiM BA alone. To further examine regeneration from leaf explants, an experiment was conducted in media containing three levels of BA (0.0-10.0 ttM) combined with either 2,4-D (0.0-0.2 lxM), NOA (0.0-0.2 ttM) or IBA (0.0-1.0 ~tM). Shoots regenerated only on media containing both BA and IBA (Fig.l). The percentage of regeneration ranged from 2.5% to 15% based upon the percentage of explants forming at least one shoot. The effect of BA was significant at the 5% level while IBA and the BA/IBA interaction were significant at p
Plant Cell Reports (1989) 8:403-406
© Springer-Verlag 1989
Shoot regeneration from petioles and leaves of Vitis X labruscana 'Catawba' Z.-M. Cheng and B. I. Reisch Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA Received February 13, 1989/Revised version received July 27, 1989- Communicated by E. D. Earle
Summary. Shoot regeneration and normal plants were obtained from leaf and petiole explants derived from in vitro grown shoots of Vitis X labruscana 'Catawba'. Regeneration was induced in the presence of both 6-benzylaminopurine and indole-3-butyric acid; combinations of 2,4-dichlorophenoxyacetic acid or 2-naphthoxyacetic acid with 6-benzylaminopurine did not permit regeneration from leaf explants. Up to 15% of leaf and 70% of petiole explants regenerated shoots on media with 5.010.0 I.tM BA and 0.1-0.5 ~tM IBA. Incubation in the dark was required to obtain regeneration. About 50% of shoots developed normally following transfer to light. An average of one shoot regenerated from leaf explants and 3.3 shoots regenerated per petiole explant. Regeneration from petioles and leaves was always from the basipetal end. The interaction of 6-benzylaminopurine with indole-3-butyric acid was also examined. Key Words: Morphogenesis
apices (Barlass and Skene 1978, 1980a, 1980b), leaf callus (Favre 1977; Hirabayashi 1985; Stamp and Meredith 1988), internode callus (Krul and Worley 1977; Rajasekaran and Mullins 1981); or from sporophytic reproductive tissues, such as anthers (Rajasekaran and Mullins 1979; Mauro et al. 1986; Stamp and Meredith 1988) and unfertilized ovules (Mullins and Srinivasan 1976; Srinivasan and Mullins 1980). However, most of the available techniques are limited by low frequency of regeneration; genotype and explant specificity; and a process involving two or three steps. The objective of our research was to develop an efficient and reliable regeneration system for Vitis X labruscana 'Catawba'. Preliminary results were reported earlier (Cheng and Reisch 1988). Shoot regeneration from in vitro leaves and petioles was examined. The interaction of cytokinin and auxin, the effect of explant source, and the effect of culture in darkness were also examined.
Auxins - Cytokinins - Grapes Materials and methods
Abbreviations: BA, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, indole-3-butyric acid; MS, Murashige and Skoog (1962) medium; NN69, Nitsch and Nitsch (1969) medium; NOA, 2-naphthoxyacetic acid Introduction
Improved ability to produce plants from tissue cultures of Vitis would expedite efforts in genetic engineering, in vitro mutant isolation, virus removal, rapid multiplication of elite stocks, and the production of haploids and then homozygous diploids. In grapes, varying degrees of organogenesis and somatic embryogenesis have been obtained. Regeneration has been achieved from vegetative tissues, such as shoot
Offprint requests to: Z.-M. Cheng
The grape cultivar 'Catawba' was established in vitro from vines grown at the New York State Agricultural Experiment Station, Geneva, NY. All explants for regeneration experiments were derived from in vitro 'Catawba' shoot cultures. Axillary buds from the basal part of growing primary shoots were isolated in August, 1986 and were washed for 30 minutes in tap water with Micro detergent (International Products Corp., Trenton, NJ, USA). After woody parts and outer layers of bud scales were removed, buds were submerged in 70% ethanol for 2 to 3 min and then were transferred to 1.85% sodium hypochlorite for 15 min, followed by 3 to 5 washes in sterile distilled water. Apex tissues, 2 to 4 mm long, were isolated from buds and inoculated for shoot multiplication on MS basal medium (Murashige and Skoog 1962) supplemented with 4.0 laM BA. Conditions for further growth of shoot tip cultures were described earlier (Reisch 1986). The microoropa~ated shoots were later
404
cultured in MS medium with 2.0 or 4.0 I~M BA prior to excision of explants. All media were solidified with agar (Difco bacto) at 7.0 g/l. The basic medium for regeneration experiments was NN69 (Nitsch and Nitseh 1969). This medium was supplemented with various plant growth regulators or other components. Myo-inositol (100 rag/l), sucrose (20 g/l), BA, 2,4-D, IBA, and NOA were added to the media prior to autoclaving. Leaf blades (1 x 1 cm or larger) were obtained from the in vitro shoot cultures. These were cut into about 5 mm x 5 nun pieces and inoculated abaxial side up. Petioles from shoot cultures were isolated with great care to exclude nearby axillary buds. The orientation of in vitro petioles in a later experiment was noted to determine which end was capable of shoot production. Each treatment included 4 Petri dishes (25 x 100 rnm) with 10 explants each. The cultures for regeneration were grown in the dark (in cardboard boxes) in a culture room at 24-26°C. After 8 weeks of culture, regenerated shoots were transferred to NN69 medium with 0.5 p.M IBA under a 16:8 light:dark photoperiod to induce rooting. To examine the effect of darkness, petiole explants were cultured continuously in a 16:8 light:dark photoperiod or cultured in the dark for two, four, or eight weeks and then moved to a 16:8 light:dark photoperiod. Percentage data were arcsin transformed and then subjected to analyses of variance. F tests were used to determine the significance of treatments and interactions, if applicable.
Results In preliminary experiments on NN69 medium, petioles regenerated shoots from 2 of 6 explants on either 5.0 lxM BA or 5.0 ~VI BA with 0.1 IxM IBA. Leaf explants regenerated shoots from 4 of 7 explants on medium with 5.0 IxM BA and 0.1 lxM IBA and from 3 of 8 on 10.0 paM BA and 0.1 lxM IBA. No leaves regenerated shoots in the presence of 5.0 or 7.5 tiM BA alone. To further examine regeneration from leaf explants, an experiment was conducted in media containing three levels of BA (0.0-10.0 ttM) combined with either 2,4-D (0.0-0.2 lxM), NOA (0.0-0.2 ttM) or IBA (0.0-1.0 ~tM). Shoots regenerated only on media containing both BA and IBA (Fig.l). The percentage of regeneration ranged from 2.5% to 15% based upon the percentage of explants forming at least one shoot. The effect of BA was significant at the 5% level while IBA and the BA/IBA interaction were significant at p
