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Characterization of Prunus domestica L. in vitro regeneration via hypocotyls
Scientia Horticulturae 112 (2007) 462–466 www.elsevier.com/locate/scihorti
Short communication
Characterization of Prunus domestica L. in vitro regeneration via hypocotyls Lining Tian a,*, Susan Sibbald a, Jayasankar Subramanian b, Antonet Svircev c a
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, Canada N5V 4T3 Department of Plant Agriculture-Vineland Station, University of Guelph, 4890 Victoria Avenue, North, Vineland Station, Ontario, Canada L0R 2E0 c Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 4902 Victoria Avenue, North, Vineland Station, Ontario, Canada L0R 2E0
b
Received 28 September 2006; received in revised form 14 December 2006; accepted 12 January 2007
Abstract Several important aspects of regeneration of European plum from hypocotyl explants were studied. Multiple shoots were induced and full plants were recovered for a large number of plum varieties. This indicates that European plum species is, in general, very responsive to in vitro regeneration from hypocotyls. Shoot organogenesis could be induced from both mature and immature seed explants but regeneration efficiency was higher when immature seeds were used. Rooting efficiency for varieties with low rooting tendency could be greatly increased by addition of naphthaleneacetic acid in the medium. Primary shoots, when sub-cultured on fresh induction medium, produced multiple shoots at a high frequency and such multiplication could continue for many cycles. The secondary new shoots could be induced for various plum varieties. Plant recovery from the secondary shoots was as efficient as that from the primary shoots. This new system may be an alternative for plum transformation with a potential for increasing transformation efficiency. The system can be used for propagation of transgenic lines and other genetic clones of various varieties. # 2007 Elsevier B.V. All rights reserved. Keywords: Embryonic axes; Immature seeds; Prunus; Shoot organogenesis
1. Introduction Genetic engineering has been reported in an increased number of fruit plants for various trait improvements (Ko et al., 2000; Ravelonandro et al., 2000; Costa et al., 2002; Belfanti et al., 2004; Broothaerts et al., 2004; Manshardt, 2005). Successful application of this technology has greatly increased the potential for genetic improvement for many fruit plants. European plum (Prunus domestica) is an important fruit species worldwide (Okie and Ramming, 1999). Genetic transformation of European plum via Agrobacterium was described before (Mante et al., 1991); however, transformation was reported with only one or two varieties and the transformation efficiency was very low. Studies on various aspects, such as selection schemes, different Agrobacterium strains, different transformation vectors, and alternative selectable markers did not result in a transformation improvement over the years (Mante et al., 1991;
Abbreviations: IBA, indolebutyric acid; NAA, naphthaleneacetic acid; TDZ, thidiazuron * Corresponding author. Tel.: +1 519 457 1470x230; fax: +1 519 457 3997. E-mail address: [email protected] (L. Tian). 0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2007.01.026
Scorza et al., 1994; Gonzalen-Padilla et al., 2003; Sibbald et al., 2006). Currently, the transformation efficiency in this species still remains very low, with a range of 0–4.2% and an average of only 1.2% (Gonzalen-Padilla et al., 2003), and transformation for many important plum varieties has not been developed yet. In previous reports, important aspects of plum in vitro regeneration were not studied nor optimized. The lack of knowledge and indepth understanding of in vitro regeneration can be a limitation and obstacle to develop efficient transformation technologies and for efficient plant regeneration for other applications. In this report, we studied several important aspects of regeneration of European plum using hypocotyls. The objective of this study was to gain a better knowledge and understanding of plum in vitro regeneration. The information from the study can be used for plant regeneration of different plum varieties and provide a useful base to develop transformation technologies for plum varieties. 2. Materials and methods European plum (P. domestica L.) fruits were collected from Vineland, Ontario, Canada. Thirteen plum varieties, namely,
L. Tian et al. / Scientia Horticulturae 112 (2007) 462–466
V70033, California Blue, Vanette, Bluefre, Bluebell, V72481, Italian, Victoria, Stanley, Shropshire Damson, Veeblue, President, and V72511 were evaluated for in vitro regeneration. The varieties chosen represent fruits with different maturity times from plum grown in Canada and some regions of USA. Maturity dates range from early maturing (approximately 13 weeks after flowering) to late maturing (approximately 21 weeks after flowering). The selection includes an industry standard variety (such as Stanley), old traditional blue plums (such as Italian and Bluefre), and small fruit (such as Shropshire Damson). Thus, plum varieties selected in the study represent all major varieties in plum growth- and market-season and have the major characteristics of plum fruits. However, as plum is open-pollinated, plum varieties may not be the true varieties in a strict genetics sense. For the effect of development stage on regeneration study, fruit was sampled at six times throughout the season ranging from 8 weeks before maturity until 1 week post-maturity. Fruit flesh was removed and the stones were washed with tap water, cleaned with a sodium hypochlorite solution (0.05%), and rinsed under running water for 5 min. Stones were dried on a lab bench at room temperature (20–25 8C) for 3–4 days and then stored at 4 8C. The stones were cracked open carefully and the seeds were collected, disinfected with a 0.5% sodium hypochlorite solution containing 0.005% Tween 20 for 15 min and rinsed three times with sterile water. Seeds were then soaked in sterile water at room temperature overnight to soften tissue texture for easier dissection. The embryonic axis was excised from cotyledons and the hypocotyl was carefully removed. The hypocotyl was cut into three slices across the axis (each was about 0.5–1 mm thickness; Fig. 1A) under a dissection microscope and slices were cultured on induction medium. Induction medium consisted of MS salts (Murashige and Skoog, 1962), 555 mM inositol, 1.2 mM thiamine HCl, 4.1 mM nicotinic acid, 2.4 mM pyridoxine HCl, 2.5 mM indolebutyric acid (IBA), 25 g L 1 sucrose, 7 g L 1 Bactoagar (Difco), and was adjusted to pH 5.9 before autoclaving for 30 min (in 1 L containers). The medium was allowed to cool and 7.5 mM thidiazuron (TDZ) added before pouring approximately 35 mL into 100 mm 25 mm Petri dishes. About 20 hypocotyl segments were placed in each Petri dish. Explants on this induction medium were incubated at 25 8C under a 16-h photoperiod with a Photosynthetic Photon Flux of 50 mmol m 2 s 1. Shoot induction was evaluated after 1 month in culture. Shoots induced from explants were excised and transferred to a medium consisting of half strength MS medium supplemented with 2.5 mM IBA, 10 g L 1 sucrose and 7 g L 1 agar, in a 100 mm 25 mm Petri dish for rooting (basal rooting medium). Rooted plants were transferred to Magenta vessels (Magenta Corp., Chicago, IL) containing the same rooting medium for further development. Naphthaleneacetic acid (NAA) effect on plum rooting was studied in four genotypes that were difficult to root in basal medium, namely, Vanette, Bluebell, President, and V72511. Shoots of similar size for all of the varieties were placed on the
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Fig. 1. Regeneration of plum from hypocotyls and new shoot development from primary shoots. (A) Hypocotyl explant preparation (E: epicotyl; 1–3: hypocotyl slices; and R: radicle). (B) Shoot development 1 month after the explants were introduced into the culture. (C) Closer view of shoot induction and development on a hypocotyl slice. (D) Efficient root development on modified rooting medium. (E) Plantlet development in Magenta containers. (F) Plant recovery of different plum varieties in the greenhouse. From left: Vanette, Italian, California Blue, V70033, Bluebell, Victoria, V72511, Stanley, V72481, Veeblue, President, and Bluefre. (G) Secondary shoot development from a primary shoot 10 days after the primary shoot was placed on fresh induction medium. New shoot development was indicated by arrows. (H) New shoot growth from primary shoots after 1 month. (I) Dissection study of secondary shoot initiation. New shoots were developing from axillary meristem tissue. (J) High frequency of new shoot induction and development from primary shoots.
basal rooting medium and a modified medium containing 5 mM NAA and 0.01 mM kinetin instead of 2.5 mM IBA. Rooting efficiency was evaluated 5 weeks after shoots were placed on rooting media.
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L. Tian et al. / Scientia Horticulturae 112 (2007) 462–466
Plantlets that were 4–5 cm in height were transferred from Magenta vessels to a commercial potting mix (PromixTM). Transferred plantlets were partially covered with empty Magenta containers for acclimatization during the first week in the greenhouse and were then exposed to standard greenhouse conditions where the night/day temperatures were 18–25/20–30 8C, respectively, depending on the weather and season. Automated shading was employed when energy levels reached above 950 W m 2. Plants were fertilized weekly with N–P–K fertilizer (20/20/20). One month after the explants were placed on induction medium, primary shoots that developed from explanted hypocotyls were cut out and individually transferred to fresh induction medium as described previously. Primary shoots were evaluated 1 month later for the presence and number of secondary shoots. This cycle was repeated 11 times for cv Stanley. Secondary shoot induction was also studied with five other varieties namely: Bluebell, Italian, President, V72511, and Veeblue. All experiments were conducted as a randomized complete block design. Data was analyzed using the MINITAB program (Release 11, MINITAB Inc.). 3. Results and discussion Shoots started becoming visible after 2–3 weeks and mostly occurred along the edges of the explanted hypocotyl slices. After 1 month, shoots were well elongated and became distinct (Fig. 1B and C). When these shoots were long enough (approximately 0.5–1.0 cm) they were excised and cultured with the basal end inserted in the rooting medium. These shoots rooted readily in most cultivars tested (Fig. 1D) and full plantlets developed within 4 weeks after transfer to Magenta vessels (Fig. 1E). Plantlets acclimated to greenhouse conditions readily (Fig. 1F). Thirteen plum cultivars were evaluated for in vitro response. Due to different maturation times for the various cultivars, they were evaluated at the same physiological age. In general, most of the cultivars tested in this study were responsive to in vitro regeneration; however, regeneration differed among cultivars. For example, ‘Italian’ and ‘Stanley’ showed higher regeneration (58% and 57%, respectively), while ‘Shropshire Damson’ showed poor regeneration (10%). Most varieties showed regeneration at frequencies of 20–30% (data not shown). Complete plants were regenerated and established in a greenhouse for 12 of 13 cultivars evaluated (Fig. 1F). Plantlets developed from ‘Shropshire Damson’ did not survive under ex vitro conditions, indicating that European plum regeneration, like many other species is also genotype dependent (Litz and Gray, 1992). Plants developed from this procedure (as of now with ‘Italian’, ‘Stanley’ and ‘Vanette’) also survived well in the field thus demonstrating the potential use of this system for in vitro regeneration. Earlier reports on regeneration had been successful only in one or two varieties (Mante et al., 1991; Scorza et al., 1994). Here, we demonstrate regeneration of a large number of plum cultivars. This provides useful information regarding genetic improvement of a large number of plum varieties (Okie and Ramming, 1999; Bellini et al.,
2001) through biotechnology. Transformation with only selected genotypes for a species is often the major obstacle for genetic improvement of perennial fruits (Petri and Burgos, 2005). Typically over 10 cultivars are needed to cover a fruiting season of 2–3 months in these crops. Therefore, demonstrating regeneration in a number of cultivars, as shown in this study, is a very important advancement. Previously, plum shoot induction was studied only with mature seeds (Mante et al., 1991). To study if immature seeds can also respond to in vitro regeneration, seeds of ‘Stanley’ were collected at different dates at 2-week intervals during development and evaluated for regeneration. Collection ranged from late July to late September in 2003. At the earliest stage of collection, seeds contained fully developed embryos with thick cotyledons; therefore, those collected at later stages exhibited only physiological maturity. Younger seeds approximately 72 days after bloom exhibited poor regeneration (Fig. 2). Seeds started to show response 85 days after bloom. Our studies suggest that seeds must pass a certain developmental stage to survive and be physiologically fit to respond in vitro. The regeneration capability gradually increased along with seed development and maturation, and reached the highest response level about 103 days after bloom, which marks the onset of ripening (as indicated by the skin colour turning from green to blue). Regeneration capacity decreased when seeds reached full maturation. Such an observation has also been reported in some other fruit species (Lane and Cossio, 1986; Durzan, 1990; Cervera et al., 1998). Results from this study showed that shoots could be induced not only from mature seeds, but also from immature seeds. This is particularly important in a perennial species as it increases the window of culture. Extended capability for regeneration can be useful as sample collection does not then need to be restricted to a particular time point of seed development. Thus, explant collection can be more flexible. It is clear from our studies that immature seeds can also be stored for up to 6 months without losing regeneration capability. In vitro induced shoots of European plums usually exhibited low rooting efficiency (Tian et al., unpublished results), which is the major obstacle for full plant recovery. Given the low transformation efficiency of European plum (Gonzalen-Padilla et al., 2003), increase of rooting efficiency and thus increase of shoot survival rate is important for recovery of transgenic lines.
Fig. 2. Regeneration response of plum seeds at different stages of development. Stanley plum seeds were collected at six different times at about 2-week intervals during seed development. A minimum of 175 explants were used for evaluation of each stage.
L. Tian et al. / Scientia Horticulturae 112 (2007) 462–466
Fig. 3. Effect of NAA on rooting of different plum varieties. A minimum of 32 shoots were used for a treatment for a variety. Bar represents the standard error of the mean. Light bar: original rooting medium and darker bar: modified rooting medium.
Addition of higher concentrations of NAA in the medium has been reported to increase rooting (Gonzalen-Padilla et al., 2003) in ‘Stanley’. Our studies using different plum varieties with different genetic backgrounds support the previous observation and significantly extended the previous research. In this study, rooting efficiency was increased from 8% to 60% for V72511 and from 16% to 78% for ‘President’ (Fig. 3). Shoots induced were usually transferred to medium without TDZ for rooting. However, if induced shoots were transferred to fresh induction medium, secondary shoots developed around the base of the primary shoots (Fig. 1G) and this trend continued for at least 11 cycles without any significant loss in
Fig. 4. New shoot development from primary shoots of Stanley over 11 cycles. Primary shoots developed were transferred and placed on fresh induction medium and new shoot development from the primary shoots was recorded after 1 month. (A) Percentage of primary shoots producing new shoots and (B) average number of new shoots per primary shoot. Bar represents the standard error of the mean.
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efficiency (Fig. 4). Over the cycles, a high percentage (about 90% or above) of primary shoots produced new shoots (Fig. 4A) and as many as 10 new shoots could develop from a primary shoot (Fig. 4B). The secondary shoot development from primary shoots was also observed in different plum varieties (data not shown), indicating this capability is not unique to a specific plum variety. Closer observation and shoot dissection study showed that the new shoots developed from axillary meristem tissue of the primary shoots (Fig. 1I). Secondary shoots behaved just like the primary shoots as far as rooting and plant recovery were concerned. Secondary shoot development provides a continuous supply of explants for further research that can be season independent. The system can also be used for large-scale propagation of various genetic clones. In this report, we studied and characterized several different aspects of plum regeneration via hypocotyls. Continuous indepth studies on in vitro regeneration from different aspects will contribute to knowledge advancement on plum regeneration and will be useful for transformation technology improvement. Information obtained may be useful for plant regeneration and development of transformation technologies for other Prunus species. Acknowledgements We would like to thank Dr. R. Scorza for providing research materials and technical advice. We thank B. Lay for helping with plum materials and T. Vallarino, K. Leach, and G. Seward for their technical support for the research. References Belfanti, E., Silfverberg-Dilworth, E., Tartarini, S., Patocchi, A., Barbieri, M., Zhu, J., Vinatzer, B.A., Gianfranceschi, L., Gessler, C., Sansavini, S., 2004. The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. P. Natl. Acad. Sci. U.S.A. 101, 886–890. Bellini, E., Nencetti, V., Nin, S., 2001. Genetic improvement of plum in Florence. Acta Hort. (ISHS) 577, 19–24. Broothaerts, W., Keulemans, J., Van Nerum, I., 2004. Self-fertile apple resulting from S-RNase gene silencing. Plant Cell Rep. 22, 497–501. Cervera, M., Juarez, J., Navarro, A., Pina, J.A., Ruran-Vila, N., Navarro, L., Rena, L., 1998. Genetic transformation and regeneration of mature tissues of woody plants bypassing the juvenile stage. Trans. Res. 7, 51–59. Costa, M.G.C., Otoni, W.C., Moore, G.A., 2002. An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and production of transgenic plants containing carotenoid biosynthetic genes. Plant Cell Rep. 21, 365–373. Durzan, D.J., 1990. Adult vs juvenile explants: directed totipotency. In: Rodriguez, R., Sanchez Tames, R., Durzan, D.J. (Eds.), Plant Aging: Basic and Applied Approaches. NATO-ASI on Molecular Basis of Plant Aging, vol. 186. Plenum Press, New York, pp. 19–25. Gonzalen-Padilla, L.M., Webb, K., Scorza, R., 2003. Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plant (Prunus domestica L.). Plant Cell Rep. 22, 38–45. Ko, K., Norelli, J.L., Reynoird, J.P., Boresjza-Wysocka, E., Brown, S.K., Aldwinckle, H.S., 2000. Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple. Biotechnol. Lett. 22, 373–381. Lane, W.D., Cossio, F., 1986. Adventitous shoots from cotyledons of immature cherry and apricot embryos. Can. J. Plant Sci. 66, 953–959.
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Litz, R.E., Gray, D.J., 1992. Organogenesis and somatic embryogenesis. In: Litz, R.E., Hammerschlag, F.A. (Eds.), Biotechnology of Perennial Fruit Crops. CAB International, Wallingford, UK, pp. 3–34. Manshardt, R., 2005. Genetic modification in fruit breeding: the papaya example. In: Proceedings of the International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species, Daytona Beach, FL, USA, October 10–14, 2005, p. 93. Mante, S., Morgens, P.H., Scorza, R., Cordts, J.M., Callahan, A.M., 1991. Agrobacterium-mediated transformation of plum (Prunus domestica L.) hypocotyl slices and regeneration of transgenic plants. Bio/Tech. 9, 853–857. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant 15, 473–497. Okie, W.R., Ramming, D.W., 1999. Plum breeding worldwide. HortTechnology 9, 162–176.
Petri, C., Burgos, L., 2005. Transformation of fruit trees: useful breeding tool or continued future prospect? Transgenic Res. 14, 15–26. Ravelonandro, M., Scorza, R., Callahan, A., Levy, L., Jacquet, C., Monsion, M., Damsteegt, V., 2000. The use of transgenic fruit trees as a resistance strategy for virus epidemics: the plum pox (sharka) model. Virus Res. 71, 63–69. Scorza, R., Ravelonandro, M., Callahan, A.M., Cordts, J., Fuchs, M., Dunez, J., Gonsalves, D., 1994. Transgenic plums (Prunus domestica L.) express the plum virus coat protein gene. Plant Cell Rep. 14, 18–22. Sibbald, S., Wang, X., Wen, Y., Tian, L., 2006. Genetic transformation of Prunus domestica using hygromycin resistance as selection for plum pox virus research. Joint Meeting of the American Phytopathological Society, Canadian Phytopathological Society, Mycological Society of America, Quebec City, Quebec, Canada, July 29–August 2, 2006.
Short communication
Characterization of Prunus domestica L. in vitro regeneration via hypocotyls Lining Tian a,*, Susan Sibbald a, Jayasankar Subramanian b, Antonet Svircev c a
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, Canada N5V 4T3 Department of Plant Agriculture-Vineland Station, University of Guelph, 4890 Victoria Avenue, North, Vineland Station, Ontario, Canada L0R 2E0 c Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 4902 Victoria Avenue, North, Vineland Station, Ontario, Canada L0R 2E0
b
Received 28 September 2006; received in revised form 14 December 2006; accepted 12 January 2007
Abstract Several important aspects of regeneration of European plum from hypocotyl explants were studied. Multiple shoots were induced and full plants were recovered for a large number of plum varieties. This indicates that European plum species is, in general, very responsive to in vitro regeneration from hypocotyls. Shoot organogenesis could be induced from both mature and immature seed explants but regeneration efficiency was higher when immature seeds were used. Rooting efficiency for varieties with low rooting tendency could be greatly increased by addition of naphthaleneacetic acid in the medium. Primary shoots, when sub-cultured on fresh induction medium, produced multiple shoots at a high frequency and such multiplication could continue for many cycles. The secondary new shoots could be induced for various plum varieties. Plant recovery from the secondary shoots was as efficient as that from the primary shoots. This new system may be an alternative for plum transformation with a potential for increasing transformation efficiency. The system can be used for propagation of transgenic lines and other genetic clones of various varieties. # 2007 Elsevier B.V. All rights reserved. Keywords: Embryonic axes; Immature seeds; Prunus; Shoot organogenesis
1. Introduction Genetic engineering has been reported in an increased number of fruit plants for various trait improvements (Ko et al., 2000; Ravelonandro et al., 2000; Costa et al., 2002; Belfanti et al., 2004; Broothaerts et al., 2004; Manshardt, 2005). Successful application of this technology has greatly increased the potential for genetic improvement for many fruit plants. European plum (Prunus domestica) is an important fruit species worldwide (Okie and Ramming, 1999). Genetic transformation of European plum via Agrobacterium was described before (Mante et al., 1991); however, transformation was reported with only one or two varieties and the transformation efficiency was very low. Studies on various aspects, such as selection schemes, different Agrobacterium strains, different transformation vectors, and alternative selectable markers did not result in a transformation improvement over the years (Mante et al., 1991;
Abbreviations: IBA, indolebutyric acid; NAA, naphthaleneacetic acid; TDZ, thidiazuron * Corresponding author. Tel.: +1 519 457 1470x230; fax: +1 519 457 3997. E-mail address: [email protected] (L. Tian). 0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2007.01.026
Scorza et al., 1994; Gonzalen-Padilla et al., 2003; Sibbald et al., 2006). Currently, the transformation efficiency in this species still remains very low, with a range of 0–4.2% and an average of only 1.2% (Gonzalen-Padilla et al., 2003), and transformation for many important plum varieties has not been developed yet. In previous reports, important aspects of plum in vitro regeneration were not studied nor optimized. The lack of knowledge and indepth understanding of in vitro regeneration can be a limitation and obstacle to develop efficient transformation technologies and for efficient plant regeneration for other applications. In this report, we studied several important aspects of regeneration of European plum using hypocotyls. The objective of this study was to gain a better knowledge and understanding of plum in vitro regeneration. The information from the study can be used for plant regeneration of different plum varieties and provide a useful base to develop transformation technologies for plum varieties. 2. Materials and methods European plum (P. domestica L.) fruits were collected from Vineland, Ontario, Canada. Thirteen plum varieties, namely,
L. Tian et al. / Scientia Horticulturae 112 (2007) 462–466
V70033, California Blue, Vanette, Bluefre, Bluebell, V72481, Italian, Victoria, Stanley, Shropshire Damson, Veeblue, President, and V72511 were evaluated for in vitro regeneration. The varieties chosen represent fruits with different maturity times from plum grown in Canada and some regions of USA. Maturity dates range from early maturing (approximately 13 weeks after flowering) to late maturing (approximately 21 weeks after flowering). The selection includes an industry standard variety (such as Stanley), old traditional blue plums (such as Italian and Bluefre), and small fruit (such as Shropshire Damson). Thus, plum varieties selected in the study represent all major varieties in plum growth- and market-season and have the major characteristics of plum fruits. However, as plum is open-pollinated, plum varieties may not be the true varieties in a strict genetics sense. For the effect of development stage on regeneration study, fruit was sampled at six times throughout the season ranging from 8 weeks before maturity until 1 week post-maturity. Fruit flesh was removed and the stones were washed with tap water, cleaned with a sodium hypochlorite solution (0.05%), and rinsed under running water for 5 min. Stones were dried on a lab bench at room temperature (20–25 8C) for 3–4 days and then stored at 4 8C. The stones were cracked open carefully and the seeds were collected, disinfected with a 0.5% sodium hypochlorite solution containing 0.005% Tween 20 for 15 min and rinsed three times with sterile water. Seeds were then soaked in sterile water at room temperature overnight to soften tissue texture for easier dissection. The embryonic axis was excised from cotyledons and the hypocotyl was carefully removed. The hypocotyl was cut into three slices across the axis (each was about 0.5–1 mm thickness; Fig. 1A) under a dissection microscope and slices were cultured on induction medium. Induction medium consisted of MS salts (Murashige and Skoog, 1962), 555 mM inositol, 1.2 mM thiamine HCl, 4.1 mM nicotinic acid, 2.4 mM pyridoxine HCl, 2.5 mM indolebutyric acid (IBA), 25 g L 1 sucrose, 7 g L 1 Bactoagar (Difco), and was adjusted to pH 5.9 before autoclaving for 30 min (in 1 L containers). The medium was allowed to cool and 7.5 mM thidiazuron (TDZ) added before pouring approximately 35 mL into 100 mm 25 mm Petri dishes. About 20 hypocotyl segments were placed in each Petri dish. Explants on this induction medium were incubated at 25 8C under a 16-h photoperiod with a Photosynthetic Photon Flux of 50 mmol m 2 s 1. Shoot induction was evaluated after 1 month in culture. Shoots induced from explants were excised and transferred to a medium consisting of half strength MS medium supplemented with 2.5 mM IBA, 10 g L 1 sucrose and 7 g L 1 agar, in a 100 mm 25 mm Petri dish for rooting (basal rooting medium). Rooted plants were transferred to Magenta vessels (Magenta Corp., Chicago, IL) containing the same rooting medium for further development. Naphthaleneacetic acid (NAA) effect on plum rooting was studied in four genotypes that were difficult to root in basal medium, namely, Vanette, Bluebell, President, and V72511. Shoots of similar size for all of the varieties were placed on the
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Fig. 1. Regeneration of plum from hypocotyls and new shoot development from primary shoots. (A) Hypocotyl explant preparation (E: epicotyl; 1–3: hypocotyl slices; and R: radicle). (B) Shoot development 1 month after the explants were introduced into the culture. (C) Closer view of shoot induction and development on a hypocotyl slice. (D) Efficient root development on modified rooting medium. (E) Plantlet development in Magenta containers. (F) Plant recovery of different plum varieties in the greenhouse. From left: Vanette, Italian, California Blue, V70033, Bluebell, Victoria, V72511, Stanley, V72481, Veeblue, President, and Bluefre. (G) Secondary shoot development from a primary shoot 10 days after the primary shoot was placed on fresh induction medium. New shoot development was indicated by arrows. (H) New shoot growth from primary shoots after 1 month. (I) Dissection study of secondary shoot initiation. New shoots were developing from axillary meristem tissue. (J) High frequency of new shoot induction and development from primary shoots.
basal rooting medium and a modified medium containing 5 mM NAA and 0.01 mM kinetin instead of 2.5 mM IBA. Rooting efficiency was evaluated 5 weeks after shoots were placed on rooting media.
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Plantlets that were 4–5 cm in height were transferred from Magenta vessels to a commercial potting mix (PromixTM). Transferred plantlets were partially covered with empty Magenta containers for acclimatization during the first week in the greenhouse and were then exposed to standard greenhouse conditions where the night/day temperatures were 18–25/20–30 8C, respectively, depending on the weather and season. Automated shading was employed when energy levels reached above 950 W m 2. Plants were fertilized weekly with N–P–K fertilizer (20/20/20). One month after the explants were placed on induction medium, primary shoots that developed from explanted hypocotyls were cut out and individually transferred to fresh induction medium as described previously. Primary shoots were evaluated 1 month later for the presence and number of secondary shoots. This cycle was repeated 11 times for cv Stanley. Secondary shoot induction was also studied with five other varieties namely: Bluebell, Italian, President, V72511, and Veeblue. All experiments were conducted as a randomized complete block design. Data was analyzed using the MINITAB program (Release 11, MINITAB Inc.). 3. Results and discussion Shoots started becoming visible after 2–3 weeks and mostly occurred along the edges of the explanted hypocotyl slices. After 1 month, shoots were well elongated and became distinct (Fig. 1B and C). When these shoots were long enough (approximately 0.5–1.0 cm) they were excised and cultured with the basal end inserted in the rooting medium. These shoots rooted readily in most cultivars tested (Fig. 1D) and full plantlets developed within 4 weeks after transfer to Magenta vessels (Fig. 1E). Plantlets acclimated to greenhouse conditions readily (Fig. 1F). Thirteen plum cultivars were evaluated for in vitro response. Due to different maturation times for the various cultivars, they were evaluated at the same physiological age. In general, most of the cultivars tested in this study were responsive to in vitro regeneration; however, regeneration differed among cultivars. For example, ‘Italian’ and ‘Stanley’ showed higher regeneration (58% and 57%, respectively), while ‘Shropshire Damson’ showed poor regeneration (10%). Most varieties showed regeneration at frequencies of 20–30% (data not shown). Complete plants were regenerated and established in a greenhouse for 12 of 13 cultivars evaluated (Fig. 1F). Plantlets developed from ‘Shropshire Damson’ did not survive under ex vitro conditions, indicating that European plum regeneration, like many other species is also genotype dependent (Litz and Gray, 1992). Plants developed from this procedure (as of now with ‘Italian’, ‘Stanley’ and ‘Vanette’) also survived well in the field thus demonstrating the potential use of this system for in vitro regeneration. Earlier reports on regeneration had been successful only in one or two varieties (Mante et al., 1991; Scorza et al., 1994). Here, we demonstrate regeneration of a large number of plum cultivars. This provides useful information regarding genetic improvement of a large number of plum varieties (Okie and Ramming, 1999; Bellini et al.,
2001) through biotechnology. Transformation with only selected genotypes for a species is often the major obstacle for genetic improvement of perennial fruits (Petri and Burgos, 2005). Typically over 10 cultivars are needed to cover a fruiting season of 2–3 months in these crops. Therefore, demonstrating regeneration in a number of cultivars, as shown in this study, is a very important advancement. Previously, plum shoot induction was studied only with mature seeds (Mante et al., 1991). To study if immature seeds can also respond to in vitro regeneration, seeds of ‘Stanley’ were collected at different dates at 2-week intervals during development and evaluated for regeneration. Collection ranged from late July to late September in 2003. At the earliest stage of collection, seeds contained fully developed embryos with thick cotyledons; therefore, those collected at later stages exhibited only physiological maturity. Younger seeds approximately 72 days after bloom exhibited poor regeneration (Fig. 2). Seeds started to show response 85 days after bloom. Our studies suggest that seeds must pass a certain developmental stage to survive and be physiologically fit to respond in vitro. The regeneration capability gradually increased along with seed development and maturation, and reached the highest response level about 103 days after bloom, which marks the onset of ripening (as indicated by the skin colour turning from green to blue). Regeneration capacity decreased when seeds reached full maturation. Such an observation has also been reported in some other fruit species (Lane and Cossio, 1986; Durzan, 1990; Cervera et al., 1998). Results from this study showed that shoots could be induced not only from mature seeds, but also from immature seeds. This is particularly important in a perennial species as it increases the window of culture. Extended capability for regeneration can be useful as sample collection does not then need to be restricted to a particular time point of seed development. Thus, explant collection can be more flexible. It is clear from our studies that immature seeds can also be stored for up to 6 months without losing regeneration capability. In vitro induced shoots of European plums usually exhibited low rooting efficiency (Tian et al., unpublished results), which is the major obstacle for full plant recovery. Given the low transformation efficiency of European plum (Gonzalen-Padilla et al., 2003), increase of rooting efficiency and thus increase of shoot survival rate is important for recovery of transgenic lines.
Fig. 2. Regeneration response of plum seeds at different stages of development. Stanley plum seeds were collected at six different times at about 2-week intervals during seed development. A minimum of 175 explants were used for evaluation of each stage.
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Fig. 3. Effect of NAA on rooting of different plum varieties. A minimum of 32 shoots were used for a treatment for a variety. Bar represents the standard error of the mean. Light bar: original rooting medium and darker bar: modified rooting medium.
Addition of higher concentrations of NAA in the medium has been reported to increase rooting (Gonzalen-Padilla et al., 2003) in ‘Stanley’. Our studies using different plum varieties with different genetic backgrounds support the previous observation and significantly extended the previous research. In this study, rooting efficiency was increased from 8% to 60% for V72511 and from 16% to 78% for ‘President’ (Fig. 3). Shoots induced were usually transferred to medium without TDZ for rooting. However, if induced shoots were transferred to fresh induction medium, secondary shoots developed around the base of the primary shoots (Fig. 1G) and this trend continued for at least 11 cycles without any significant loss in
Fig. 4. New shoot development from primary shoots of Stanley over 11 cycles. Primary shoots developed were transferred and placed on fresh induction medium and new shoot development from the primary shoots was recorded after 1 month. (A) Percentage of primary shoots producing new shoots and (B) average number of new shoots per primary shoot. Bar represents the standard error of the mean.
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efficiency (Fig. 4). Over the cycles, a high percentage (about 90% or above) of primary shoots produced new shoots (Fig. 4A) and as many as 10 new shoots could develop from a primary shoot (Fig. 4B). The secondary shoot development from primary shoots was also observed in different plum varieties (data not shown), indicating this capability is not unique to a specific plum variety. Closer observation and shoot dissection study showed that the new shoots developed from axillary meristem tissue of the primary shoots (Fig. 1I). Secondary shoots behaved just like the primary shoots as far as rooting and plant recovery were concerned. Secondary shoot development provides a continuous supply of explants for further research that can be season independent. The system can also be used for large-scale propagation of various genetic clones. In this report, we studied and characterized several different aspects of plum regeneration via hypocotyls. Continuous indepth studies on in vitro regeneration from different aspects will contribute to knowledge advancement on plum regeneration and will be useful for transformation technology improvement. Information obtained may be useful for plant regeneration and development of transformation technologies for other Prunus species. Acknowledgements We would like to thank Dr. R. Scorza for providing research materials and technical advice. We thank B. Lay for helping with plum materials and T. Vallarino, K. Leach, and G. Seward for their technical support for the research. References Belfanti, E., Silfverberg-Dilworth, E., Tartarini, S., Patocchi, A., Barbieri, M., Zhu, J., Vinatzer, B.A., Gianfranceschi, L., Gessler, C., Sansavini, S., 2004. The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. P. Natl. Acad. Sci. U.S.A. 101, 886–890. Bellini, E., Nencetti, V., Nin, S., 2001. Genetic improvement of plum in Florence. Acta Hort. (ISHS) 577, 19–24. Broothaerts, W., Keulemans, J., Van Nerum, I., 2004. Self-fertile apple resulting from S-RNase gene silencing. Plant Cell Rep. 22, 497–501. Cervera, M., Juarez, J., Navarro, A., Pina, J.A., Ruran-Vila, N., Navarro, L., Rena, L., 1998. Genetic transformation and regeneration of mature tissues of woody plants bypassing the juvenile stage. Trans. Res. 7, 51–59. Costa, M.G.C., Otoni, W.C., Moore, G.A., 2002. An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and production of transgenic plants containing carotenoid biosynthetic genes. Plant Cell Rep. 21, 365–373. Durzan, D.J., 1990. Adult vs juvenile explants: directed totipotency. In: Rodriguez, R., Sanchez Tames, R., Durzan, D.J. (Eds.), Plant Aging: Basic and Applied Approaches. NATO-ASI on Molecular Basis of Plant Aging, vol. 186. Plenum Press, New York, pp. 19–25. Gonzalen-Padilla, L.M., Webb, K., Scorza, R., 2003. Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plant (Prunus domestica L.). Plant Cell Rep. 22, 38–45. Ko, K., Norelli, J.L., Reynoird, J.P., Boresjza-Wysocka, E., Brown, S.K., Aldwinckle, H.S., 2000. Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple. Biotechnol. Lett. 22, 373–381. Lane, W.D., Cossio, F., 1986. Adventitous shoots from cotyledons of immature cherry and apricot embryos. Can. J. Plant Sci. 66, 953–959.
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