Stable transformation of European plum using and
1. Introduction
With the advancement of genomics research, many genes have been identified and cloned from various plants. Transfer of these genes into plants for gene function studies and for plant improvement is important in the post-genomics era. At this time, the lack of efficient, effective, and high throughput genetic transformation systems in many crops and varieties is a major barrier and a challenge in functional genomics research and for plant trait improvement via biotechnology (Petri and Burgos 2005). Studies and understanding of different aspects and factors in plant transformation are important and are a prerequisite in the development of effective and efficient transformation technologies for various crops and varieties (Gill et al. 2004; Petri and Burgos 2005).
European plum (
Plant transformation is a complicated process which involves various factors, such as plant genotype and variety, regeneration efficiency, culture medium and condition, selectable marker, infection condition, gene construct and
The objective of this research was to study important aspects of
2. Materials and methods
2.1. Plant materials
Plum (
Plum fruits, two weeks prior to maturation, were collected from plum trees in Vineland, Ontario, Canada. Endocarps were cracked open and the seeds were sterilized in 10% commercial bleach solution. The seeds were then rinsed three times with sterile distilled water in the laminar flow hood and were imbibed in final rinsing water overnight. After a removal of the seed coat, the embryonic axis was excised from the cotyledons. Embryonic axis was cut into five sections with one radicle, three hypocotyls and one epicotyl. Hypocotyl and epicotyl segments were employed in transient gene expression studies while the radicle was discarded. For stable transformation studies, only hypocotyl slices were used in the experiments. Transformation was conducted using hypocotyls as explants as described by Mante et al. (1991) and Padilla et al. (2003) with modifications by Tian et al. (2009) and in this study.
2.2. Agrobacterium and vectors
Three
2.3. Agrobacterium infection and plant transformation
Regenerated shoots at about 0.5 -1 cm in length from antibiotic selections were excised from the explants and transferred to fresh shoot induction medium containing the same antibiotics as well as timetin. After another 2-3 subcultures, well established and developed shoots in antibiotic-containing medium were placed in Magenta boxes containing rooting medium. The rooting medium consisted of 1/2-strength MS salts (Murashige and Skoog 1962), 5 µM naphthalene acetic acid (NAA), 0.01 µM kinetin, vitamins (555 µM myo-inositol, 1.2 µM thiamine HCl, 1.4 µM nicotinic acid, 2.4 µM pyridoxine HCl), 10 g L-1 sucrose, and 7 g L-1 Bactoagar. Plants developed in magenta containers were transferred to soil and plants were established in a greenhouse. Plants were analyzed for transformation using different approaches as described in previous studies (Tian et al. 2009).
2.4. Histochemical and Fluorogenic GUS expression assay
Five days after
For fluorometric GUS expression, plant tissues five days after
3. Results and discussion
Efficient infection of
The plum explants were infected with different
Stable transformation were conducted with either kanamycin selection or hygromycin selection depending on the vectors used. It appeared that transient reporter gene expression was well related to the effectiveness of stable transformation in plum (Table 1, Fig. 2& 3). Specifically, higher levels of transient GUS expression after EHA105 and LBA4404 infection led to the effectiveness of stable transformation and consistently generated transgenic lines (Table 1). On the other hand, lower transient GUS expression using strain GV3101 resulted in ineffectiveness of stable transformation (Table 1, Fig. 2& 3). Such relation is consistent using different constructs and with either the kanamycin selection or the hygromycin selection (Table 1).
Vector | Selection scheme | No of Explants | No. of Transformants | Transformation Efficiency | |
pC2301 | EHA105 | Kanamycin | 272 | 6 | 2.2% |
LBA4404 | Kanamycin | 270 | 2 | 0.7% | |
GV3101 | Kanamycin | 272 | 0 | 0% | |
pC1301 | EHA105 | Hygromycin | 272 | 3 | 1.1% |
LBA4404 | Hygromycin | 271 | 2 | 0.7% | |
GV3101 | Hygromycin | 272 | 0 | 0% |
L-cysteine (mg/L) | Number of explants | % of explants transiently expressing GUS gene | Number of transgenic line | Transformation efficiency |
0 | 78 | 80.0 | 2 | 2.6% |
900 | 78 | 22.7 | 0 | 0 |
Our previous studies have indicated that use of culture medium including L-cysteine, which was used in media for transformation improvement (Olhoft et al. 2001), could affect transient GUS gene expression in plum (non-published results). We conducted research to study how transient gene expression was related to stable transformation using medium containing L-cysteine. The results showed that explants cultured in medium with L-cysteine resulted in significantly low levels of transient GUS gene expression (Table 2) as found in previous studies. No transformation was obtained from the explants cultured in the presence of L-cysteine. On the other hand, high level of transient GUS expression was observed in explants without L-cysteine treatment and stable transformation was routinely recovered (Table 2). This study further indicated that transient reporter gene expression was related to stable transformation in European plum. The positive relationship between transient reporter gene expression and stable transformation in plum could be important for studying and evaluating various factors and conditions for transformation, which can be useful in the development and improvement of stable transformation technologies in different plum varieties.
Construct | Agro strain | No. of total explants | Lines recovered | Efficiency |
pPV-1 | GV3101 | 444 | 0 | 0.0% |
LBA4404 | 1019 | 17 | 1.7% | |
pPV-2 | GV3101 | 330 | 1 | 0.3% |
LBA4404 | 601 | 11 | 1.8% | |
pPV-3 | GV3101 | 283 | 0 | 0.0% |
LBA4404 | 769 | 20 | 2.6% | |
Summary | GV3101 | 1057 | 1 | 0.09% |
LBA4404 | 2389 | 48 | 2.0% |
Medium | Number of explants | Number of transgenic lines | Transformation efficiency (%) |
MS | 300 | 6 | 2.0 |
B5 | 310 | 19 | 6.1 |
Plant genetic transformation is usually conducted
4. Conclusion
Genetic transformation efficiency in
Acknowledgments
The authors would like to thank Dr. Jay Subramanian for providing plum research materials and for research discussion and Dr. A. Wang for providing some transformation constructs. We also thank Dr. Ralph Scorza for providing some Agrobacterium strains and for transformation research discussion. We thank Susan Sibbald, Janny Lac and Kaalak Reddy for technical assistance.
References
- 1.
Bond J. E. Roose M. L. 1998 mediated transformation of the commercially important citrus cultivar Washington navel orange. Plant Cell Rep.18 229 234 - 2.
Capote N. Pérez-Panadés J. Monzó C. Carbonell E. Urbaneja A. Scorza R. Ravelonandro M. Cambra M. 2008 Assessment of the diversity and dynamics of Plum pox virus and aphid populations in transgenic European plums under Mediterranean conditions. Transgenic Res.17 367 377 - 3.
Cervera M. Lopez M. M. Navarro L. Pena L. 1998 Virulence and supervirulence of in woody fruit plants. Physiological and Molecular Plant Pathology52 67 78 - 4.
Chen L. Zhang S. Beachy R. N. Fauquet C. M. 1998 A protocol for consistent, large-scale production of fertile transgenic rice plants. Plant Cell Rep.18 25 31 - 5.
and Laimer da Câmara Machado, M.da Câmara. Machado A. Katinger 1994 Coat protein-mediated protection against plum pox virus in herbaceous model plants and transformation of apricot and plum. Euphytica77 129 134 - 6.
De Bondt A. Eggermont K. Druart P. De Vil M. Goderis I. Vanderleyden J. Broekaert W. F. 1994 -mediated transformation of apple (Malus x domestica Borkh.): An assessment of factors affecting gene transfer efficiency during early transformation steps. Plant Cell Rep.13 587 593 - 7.
Gamborg O. L. Miller R. A. Ojima K. 1968 Nutrient requirements of suspension cultures of soybean root cell. Exp. Cell Research50 151 158 - 8.
Gill M. I. S. Zora S. Agrez V. 2004 Factors affecting -mediated genetic transformation in fruit and nut crops- an overview. J. Food, Agriculture & Environment2 327 347 - 9.
Hartmann W. 1994 Plum production in Germany. Acta Hort359 17 25 - 10.
Hily J. M. Scorza R. Malinowski T. Zawadzka B. Ravelonandro M. 2004 Stability of gene silencing-based resistance to Plum pox virus in transgenic plum ( L.) under field conditions. Transgenic Res.13 427 436 - 11.
Hoekema A. Hirsch P. R. Hooykaas P. J. J. Schilperoort R. A. 1983 A binary plant vector strategy based on separation of vir- and T-region of the Ti-plasmid. Nature303 179 180 - 12.
Holsters M. Silva B. Van Vliet F. Genetello C. De Block M. Dhaese P. Depicker A. Inzé D. Engler G. Villarroel R. others 1980 The functional organization of the nopaline plasmid pTiC58. Plasmid3 212 230 - 13.
Hood E. E. Gelvin S. B. Melchers L. S. Hoekema A. 1993 New helper plasmids for gene transfer to plants. Transgenic Res.2 208 218 - 14.
Jefferson R. A. Kavanaugh T. A. Bevan M. W. 1987 GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J.6 3901 3907 - 15.
Joyce P. Kuwahata M. Turner N. Lakshmanan P. 2010 Selection system and co-cultivation medium are important determinants of -mediated transformation of sugarcane. Plant Cell Rep.29 173 183 - 16.
Kaufmane E. Ikase L. Trajkovski V. Lacis G. 2002 Evaluation and characterization of plum genetic resources in Sweden and Latvia. Acta Hort.577 207 213 - 17.
Kikhailov R. V. Shuga O. A. Dolgov S. V. 2008 Genetic transformation os somatic tissues and molecular biology approach for improving resistance to sharka disease of plum (Prunuc domestica L.) varieties. First International Symposium on Biotechnology of Fruit Species. September1 5 Dreston, Germany. - 18.
Le Gall O. Torregrosa L. Danglot Y. Candresse T. Bouquet A. 1994 -mediated genetic transformation of grapevine somatic embryos and regeneration of transgenic plants expressing the coat protein of grapevine chrome mosaic nepovirus (GCMV). Plant Sci.102 161 170 - 19.
Malinowski T. Zawadzka B. Ravelonandro M. Scorza R. 1998 Preliminary report on the apparent breaking of resistance of a transgenic plum by chip bud inoculation of PPV-S. Acta Virol.42 241 243 - 20.
Malinowski T. Cambra M. Capote N. Zawadzka B. Gorris M. T. Scorza R. Ravelonandro M. 2006 Field trials of plum clones transformed with the Plum pox virus coat protein (PPV-CT) gene. Planr Disease90 1012 1018 - 21.
Mante S. Morgens P. H. Scorza R. Cordts J. M. Callahan A. M. 1991 -mediated transformation of plum (Prunus domestica L.) hypocotyl slices and regeneration of transgenic plants. Bio/Technol9 853 857 - 22.
Murashige T. Skoog F. 1962 A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant.15 473 497 - 23.
Okie W. R. Ramming D. W. 1999 Plum breeding worldwide. HortTechnology9 162 176 - 24.
Olhoft P. M. Somers D. A. 2001 L-Cysteine increases -mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep.20 706 711 - 25.
Padilla I. M. G. Webb K. Scorza R. 2003 Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants ( L.). Plant Cell Rep.22 38 45 - 26.
Petri C. Alburquerque N. Garc-Castillo Ãa. S. Egea J. Burgos L. 2004 Factors affecting gene transfer efficiency to apricot leaves during early mediated transformation steps. Journal of Hort. Sci. Biotech.79 704 712 - 27.
Petri C. Burgos L. 2005 Transformation of fruit trees. Useful breeding tool or continued future prospect? Transgenic Res.14 15 26 - 28.
Petri C. Webb K. Hily J. M. Dardick C. Scorza R. 2008 High transformation efficiency in plum ( L.): A new tool for functional genomics studies in Prunus spp. Mole. Breed.22 581 591 - 29.
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 - 30.
Scorza R. Ravelonandro M. Callahan A. M. Cordts J. M. Fuchs M. Dunez J. Gonsalves D. 1994 Transgenic plums ( L.) express the plum pox virus coat protein gene. Plant Cell Rep.14 18 22 - 31.
Scorza R. Callahan A. Levy L. Damsteegt V. Webb K. Ravelonandro M. 2001 Post-transcriptional gene silencing in Plum pox virus resistant transgenic European plum containing the Plum pox potyvirus coat protein gene. Transgenic Res.10 201 209 - 32.
Tian L. Sibbald S. Subramanian J. Svircev S. 2006 Characterization of L. in vitro regeneration via hypocotyls. Sci. Hort..112 462 466 - 33.
Tian L. Canli F. A. Wang X. Sibbald S. 2009 Genetic transformation of L. using the hpt gene coding for hygromycin resistance as the selectable marker. Sci. Hort.119 339 343 - 34.
Yancheva S. D. Druart P. Watillon B. 2002 - nediated transformation of plum (Prunus domestica L.). Acta Hort.577 215 217