1. Introduction
Genetic transformation provides the means for adding single horticultural traits in existing cultivars without modify their commercial characteristics. This capability is particularly valuable for perennial plants and fruit tree species, in which conventional breeding is hampered by their long generation time and juvenile periods, complex reproductive biology, high levels of heterozygosity, limited genetic sources and linkage drag of undesirable traits from wild relatives. In addition, gene transfer technologies for fruit tree species take the inherent advantage of vegetative propagation used for their reproduction, which allowed for the application of a high scale production of the desired transgenic line starting from one successful transformed line. Despite this opportunity, final setting of transformation protocols in this type of species, endures major limiting factors preventing the development of new varieties: a) explants recalcitrance to regenerate adventitious transformed shoots and b) a limited regeneration capability, usually extended to just few genotypes (i.e. cultivar dependence).
This chapter illustrates the road between the establishment of transformation methodologies on particular species of
1.1. Genetic transformation of fruits in the current research era
Genetic improvement of fruit trees is essential for increasing fruit production. For most of these species, the desired new varieties contemplate the presence of agronomic and horticultural traits related to propagation, yield, appearance, quality, disease and pest control, abiotic stress and shelf-life. Incorporation of these traits into the genetic backgrounds of species by conventional breeding needs overcome some major disadvantages, including long juvenile periods and reduced possibility of introgression of the suitable traits (when available) into commercially relevant cultivars. Although currently the use of new technologies based on high throughput platforms for sequencing and genotyping has deeply contributed to accelerate the association of molecular markers and major genes to these relevant traits, there exists a bottle neck in this strategy when phenotyping must be carried out. In addition, breeding by controlled crosses is hampered due to factors specifically related to complex characteristics belonging to these species, such as delayed flowering, unsuccessful fruit setting due to abortive embryos, massive fruit drop, and self-incompatibility barriers found in many of them.
Genetic transformation represents inherent advantages for fruit tree improvement, although in fruit trees this area of research is not a routine procedure. The transversal negative perception about the “transgenic technology” is added to an additional degree of difficulty for setting up adequate technical systems in fruit tree species. Eventually, if a proper regenerative system has been established, any DNA construct designed for either a major gene over-expression or gene silencing (i.e. interfering RNA´s
2. Use of grapevine systems as a model in fruit species
2.1. Grapevine somatic embryogenesis and genetic transformation of somatic embryos
Since its first report in 1976 (Mullins and Srinivasan, 1976), somatic embryogenesis (SE) in
SE process based on the use of Petri dishes is illustrated for both leaves and inflorescences as source explants. This process leads to somatic embryo generation (embryogenic masses) prone for
2.2. Current requirements in grapevine genetic transformation
The convergence of genome sequencing studies on
In general terms, the whole process requires a total time of 24 to 25 weeks and leads to the generation of candidate genetically modified plantlets ready for a primary, PCR-based, screening.
Regardless the strategy (solid media Petri dishes or solid media plus the inclusion of a flasks’ step), both routes share a critical and consistent problem referred to the massive generation of adequate amounts of E or PE masses supporting routine transformation experiments.
2.3. Optimizing somatic embryogenesis platforms. Different strategies
It is accepted that the developmental stage of source explants is of great importance in grapevine SE setting (Martinelli and Gribaudo, 2009). Commonly, SE systems in grapes are initiated from stamens and pistils and responses are variety-dependent. The best developmental stages to initiate embryogenic cultures have been deduced for some genotypes using the basis of phenological stages of inflorescences (Dhekney et al., 2009); whereas stamens and pistils from some cultivars such as ´Pinot Gris´ must be collected at early developmental stages; other genotypes such as ´Merlot´, ´Sauvignon Blanc´ and ´Freedom´ must be induced using explants at more advanced maturity stages.
The use of induction media based on modified MS or Nitsch (described by Li et al., 2008), established the basis for additional improvements in grapevine SE. This time, authors were focused on the yield of the system. Solid cultures are heterogeneous and diffusion-limited; on the other hand, agitated liquid cultures, involve mainly faster, more uniform, efficient, and controllable mass transfer processes. It is accepted that use of liquid cultures offers numerous technical advantages over solid cultures (Archambault et al., 1994). However, the actual evaluation of embryogenic protocols must be carried out on the basis of volumetric productions, true plant organ (i.e. torpedo shape, Jayasankar et al., 2003), system homogeneity, and finally, conversion of embryo cells into whole plants (Archambault et al., 1994). In ´Thompson Seedless´, application of an induction period by six weeks using Li´s modifications generated PE masses that are transferred into maintenance liquid media based on B5 major salts (Gamborg et al., 1968) and vitamins from MS. These flask assays have been the basis for the generation of an air-lift bioreactor as recently described Tapia et al. (2009). The system was designed to improve biomass production of ´Thompson Seedless´ somatic embryos and, at the same time, enabled a preliminary characterization of cell´s behavior during the grapevine SE time-course. The very first improvement derived from the use of liquid systems was that biomass multiplication rate decreased from 60 to about 40 days due to the use of agitated flasks (Figure 2a). Even better, the use of an air-lift bioreactor improved this rate to seven days (Tapia et al., 2009). In addition, a lower than expected sugar consumption was observed during the SE process, suggesting side roles for this substrate during culturing. Li´s procedures described that flasks culturing leaded to the generation of up to 400 mg of biomass, obtaining PE and E cells; in those experiments, bigger inocula leaded to explants’ oxidation. On the contrary, the 2 L vessel’s reactor (Figure 2b) regularly admitted 2 g inocula without affecting the process, including explants viability and duplication of this biomass at the seventh day of batching. Genetic transformation procedures of somatic embryos obtained from this system did not show any difference compared to explants produced by the regular solid media-based system (depicted in Figure 1), generating fully regenerated transgenic plants (Tapia et al., 2009).
2.4. Inducing somatic embryogenesis in recalcitrant germplasms. New strategies
Embryogenic competence can be considered as an exception more than a rule. Several genotypes have shown low or null responses to protocols that have been successfully evaluated in certain varieties. An optimized SE procedure for ´Thompson Seedless´ (Figure 3a) is not as efficient as applied on ´Red Globe´ (Figure 3b). Analyses of factors affecting competence have been recently reported (Dhekney et al., 2009) by exploring changes in the induction phase. The use of MS and Nitsch macro- and micro-elements supplemented with 6-bencyl aminopurine (BAP) at 4.5 and 8.9 µM plus 2,4-dichlorophenoxy acetic acid (2,4-D) at 8.9 and 4.5 µM, respectively, leaded to most of the 18 evaluated cultivars and eight
The new strategies include the above referred use of
3. Genetic transformation of plums in the waiting for a peach transformation system
It has already been almost 20 years since the generation of the most highlighted event in the field of stone fruits genetic transformation: the
Multiple tissues have been used for plant regeneration in the
From these works, new improvement was made in the diploid species
3.1. A new example: RNAi-based PPV resistance in Japanese plums
Holding the research focus on PPV, new strategies against the virus can be evaluated with improved expectations, including new technology development obtained for gene silencing. As mentioned, an additive effect of multicopy T-DNA fragments arranged in C5´s genome and the occurrence of an
Recently, Dolgov et al. (2010) developed and used a coat protein gene-based hairpin inducing plasmid to generate RNAi transgenic
Considering that genome annotation for peach is expected in the very near future, the advantage of transforming a diploid species in the
These assays are carried out in a biosafety greenhouse at La Platina Station of the National Institute of Agriculture, Santiago, Chile.
4. Towards peach genetic transformation
In parallel to the systems developed for plum, the race for a
Most of the studies describing genetic transformation in peach have described the use of
Transversal stem segments isolated from mature plants were infected with the
4.1. Combining trials to obtain genetically modified peaches
As depicted, explants from
In our hands, the use of leaves as starting explants in the transformation pipeline for ´O´Henry´ did not produce a consistent number of buds, on the contrary to the reported by Gentile et al. (2002). Despite this and after several years of evaluations, the use of immature cotyledons leaded us to propose a combined procedure based on these procedures for regeneration and transformation of this and other commercial cultivars. ´O´Henry´ and ´Rich Lady´ immature cotyledons have been cultivated in LP modified medium (Gentile et al., 2002) generating viable explants that can produce either direct budding in MP modified medium (Gentile et al., 2002) supplemented with BAP (5 µM) and NAA (concentrations between 3 and 5 µM) or lead to formation of white structures (as shown in Figure 7) that, in presence of LP medium and 2,4-D (1 µM), will become into green buds after 60-90 days of culturing.
These results are quite similar to those obtained using mature cotyledons from
By this methodology, trees expressing GFP have been generated (Figures 8 and 9); interestingly, the behavior of a 35S RNA Cauliflower Mosaic Virus promoter driving the
5. Conclusions
A compact view about different strategies to reach successful genetic transformation experiments in grapes and stone fruits (plums and peach) has been presented using results from our close experimentation. This time, the focus has been on mixing different ideas from procedures used in both genera and their corresponding results.
In case of
For
Acknowledgments
The author gratefully acknowledges to all colleagues and students joined since 2001 to the genetic transformation and molecular biology group at La Platina Station in Santiago de Chile. Special thanks to the current group members: Grapevine genetic transformation team: Catalina Álvarez, Hayron Canchignia, Álvaro Castro, María de los Ángeles Miccono, Christian Montes, Marisol Muñoz, Blanca Olmedo, Luis Ortega, Eduardo Tapia, and Evelyn Sánchez. Stone fruits transformation team: Manuel Acuña, Paola Barba, Daniel Espinoza, Julia Rubio, Catalina Toro, and Wendy Wong. Thanks to all of you!All the Figures´ composition was made by team´s members using original results. Special thanks to compositions made by Manuel Acuña (plum and peach work), Álvaro Castro (SE work) and Eduardo Tapia (bioreactor work).Local advances presented in this chapter are funded by joint-venture programs between the governmental agencies FONDEF-Chile, PBCT-Chile, CONICYT-Chile, and INNOVA-Chile and the Fruit Technology Consortium “Biofrutales” S.A. This publication is funded by the INIA-CSIC Grant 501646-70.
References
- 1.
Ammirato P. 1983 Embryogenesis. In: D.A. Evans, W.R. Sharp, P.V. Ammirato, & Y. Yamada (Eds.), McMillan,0-02949-230-0 York, USA. - 2.
Araya S. Prieto H. Hinrichsen P. 2008 An efficient buds culture method for the regeneration via somatic embryogenesis of table grapes cvs. Red Globe and Flame Seedless. ,47 4 0042-7500 - 3.
Archambault J. Williams R. Lavoie L. Pepin M. Chavarie C. 1994 Production of somatic embryos in a helical ribbon impeller bioreactor. ,44 8 930 EOF 43 EOF 0006-3592 - 4.
Bhansali R. Drive J. Durzan D. 1990 Rapid multiplication of adventitious somatic embryos in peach and nectarine by secondary embryogenesis. ,9 5 0721-7714 - 5.
Canli F. Tian L. 2008 shoot regeneration from stored mature cotyledons of sweet cherry (Prunus avium L.) cultivars. Scientia Horticulturae,116 1 34 EOF 40 EOF 0304-4238 - 6.
Da Câmara. Machado M. Da Câmara. Machado A. Hanzer V. Mattanovich D. Himier G. Katinger H. 1989 Regeneration of shoots from leaf discs and stem micro-cuttings of fruit trees as a tool for transformation. - Acta Horticulturae235 85 92 978-9-06605-333-5 Thessaloniki, Greece, June 12-18, 1988. - 7.
Da Câmara. Machado M. Da Câmara. Machado A. Hanzer V. Weiss H. Regner F. Steinkeliner H. Mattanovich D. Plail R. Knapp E. Kalthoff B. Katinger H. 1992 Regeneration of transgenic plants of containing the coat protein gene of Plum Pox Virus. Plant Cell Reports,11 1 0721-7714 - 8.
Dhekney S. Li Z. Compton M. Gray D. 2009 Optimizing initiation and maintenance of Vitis embryogenic cultures 44 5 1400 1406 0018-5345 - 9.
Dolgov S. Mikhailov R. Shulga O. 2005 Genetic engineering approach in stone fruits breeding. ,0567-7572 Daytona Beach, Florida, USA, October 10-14, 2005. - 10.
Dolgov S. Mikhaylov R. Serova T. Shulga O. Firsov A. 2010 Pathogen-derived methods for improving resistance of transgenic plums (Prunus domestica L.) for Plum pox virus infection - Julius-Kühn-Archiv,133 140 978-3-93003-767-4 Neustadt, Germany, July 5-10, 2009. - 11.
Driver J. Kuniyuki A. 1984 propagation of Paradox walnut rootstock. HortScience,19 4 0018-5345 - 12.
Gamborg O. Miller R. Ojima K. 1968 Nutrient requirements of suspension cultures of soybean root cells. ,50 1 151 EOF 8 EOF - 13.
Gentile A. Monticelli S. Damiano C. 2002 Adventitious shoot regeneration in peach ( (L.) Batsch). Plant Cell Reports,20 11 1011 EOF 1016 EOF 0721-7714 - 14.
González-Padilla I. Webb K. Scorza R. 2003 Early antibiotic selection and efficient rooting and acclimatization improved the production of transgenic plum plants ( L.). Plant Cell Reports,22 1 38 EOF 45 EOF 0721-7714 - 15.
Gray D. 1995 Somatic embryogenesis in grape. In: Somatic embryogenesis in woody plants-2 P. Gupta, S. Jain, & R. Newton (Eds.), 191-217, Kluwer Academic,0-79233-070-6 The Netherlands. - 16.
Hammerschlag F. 1982 Factors affecting establishment and growth of peach shoots . HortScience,17 1 0018-5345 - 17.
Hammerschlag F. Bauchan G. Scorza R. 1985 Regeneration of peach plants from callus derived from immature embryos. ,70 3 248 EOF 251 EOF 0040-5752 - 18.
Hammerschlag F. Bauchan G. Scorza R. 1987 Factors influencing multiplication and rooting of peach cultivars. Plant Cell Tissue Organ Culture,8 3 235 EOF 242 EOF 0167-6857 - 19.
Hammerschlag F. Oowns L. Smigocki A. 1989 mediated transformation of Peach cells derived from mature plants that were propagated in vitro. Journal of the American Society for Horticultural Science,114 3 0003-1062 - 20.
Hily J. Scorza R. Malinowski T. Zawadzka B. Ravelonandro M. 2004 Stability of gene silencing-based resistance to in transgenic plum (Prunus domestica L.) under field conditions. Transgenic Research,13 5 427 EOF 436 EOF 0962-8819 - 21.
Hily J. Scorza R. Webb K. Ravelonandro M. 2005 Accumulation of the long class of siRNA is associated with resistance to in a transgenic woody perennial plum tree. Molecular Plant Microbe Interaction,18 8 794 EOF 9 EOF 0894-0282 - 22.
Hinrichsen P. Reyes M. Castro A. Blanchard E. Araya S. Garnier M. Reyes F. Dell’Orto P. Moynihan M. Prieto H. Muñoz C. 2005 Genetic transformation of grapevines with and antimicrobial peptide genes for improvement of fungal tolerance. Proceddings of the VII International Symposium on Grapevine Physiology and Biotechnology- Acta Horticulturae,689 469 EOF 978-9-06605-718-0 Davis, California, USA, June 21-25, 2004. - 23.
Iocco O. Franks T. Thomas M. 2001 Genetic transformation of major wine grape cultivars of L. Transgenic Research,10 2 105 EOF 112 EOF 0962-8819 - 24.
Jaillon O. Aury J. Noel B. Policriti A. Clepet C. Casagrande A. Choisne N. Aubourg S. Vitulo N. Jubin C. Vezzi A. Legeai F. Hugueney P. Dasilva C. Horner D. Mica E. Jublot D. Poulain J. Bruyère C. Billault A. Segurens B. Gouyvenoux M. Ugarte E. Cattonaro F. Anthouard V. Vico V. Del Fabbro C. Alaux M. Di Gaspero G. Dumas V. Felice N. Paillard S. Juman I. Moroldo M. Scalabrin S. Canaguier A. Le Clainche I. Malacrida G. Durand E. Pesole G. Laucou V. Chatelet P. Merdinoglu D. Delledonne M. Pezzotti M. Lecharny A. Scarpelli C. Artiguenave F. Pè M. E. Valle G. Morgante M. Caboche M. Adam-Blondon A. F. Weissenbach J. Quétier F. Wincker P. 2007 French-Italian Public Consortium for Grapevine Genome Characterization. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. ,449 7161 - 25.
Jayasankar S. Bondada B. Li Z. Gray D. 2003 Comparative anatomy and morphology of (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses. American Journal of Botany,90 90 973 EOF 9 EOF 0002-9122 - 26.
Kundu J. Briard P. Hily J. Ravelonandro M. Scorza R. 2008 Role of the 25-26 nt siRNA in the resistance of transgenic graft inoculated with Plum pox virus. Virus Genes¸36 1 215 EOF 220 EOF 0920-8569 - 27.
Li A. Jayasankar S. Gray D. 2001 Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (). Plant Science,160 5 877 EOF 887 EOF 0168-9452 - 28.
Li Z. Dhekney S. Dutt M. Gray D. 2008 An improved protocol for -mediated transformation of grapevine (Vitis vinifera L.). Plant Cell Tissue and Organ Culture,93 3 311 EOF 321 EOF 0167-6857 - 29.
Malinowski T. Cambra M. Capote N. Zawadzka B. Gorris M. Scorza R. Ravelonandro M. 2006 Field trials of plum clones transformed with the coat protein (PPV-CP) gene. Plant Disease,90 8 1012 EOF 1018 EOF 0191-2917 - 30.
Mante S. Scorza R. Cordts J. 1 19 1 EOF 11 EOF 0167-6857 - 31.
Mante S. Morgens P. Scorza R. Cordts J. Callahan A. 1991 -mediated transformation of plum (Prunus domestica) hypocotyls slices and regeneration of transgenic plants. Nature Biotechnology,9 9 1087-0156 - 32.
Martinelli L. Bragagna P. Poletti V. Scienza A. 1993 Somatic embryogenesis from leaf- and petiole-derived callus of . Plant Cell Reports,12 4 207 EOF 0721-7714 - 33.
Martinelli L. Gribaudo I. 2001 Somatic embryogenesis in grapevine. In:. K.A. Roubelakis-Angelakis (Ed.),393 410 Kluwer Academic Publishers,0-79236-949-1 The Netherlands. - 34.
Martinelli L. Candioli E. Costa D. Poletti V. 2001 Morphogenic competence of secondary somatic embryos with a long culture history. Plant Cell Reports,20 4 279 EOF 284 EOF 0721-7714 - 35.
Martinelli L. Gribaudo I. Bertoldi D. Candioli E. Poletti V. 2001a High efficiency somatic embryogenesis and plant germination in grapevine cultivars Chardonnay and Brachetto a grappolo lungo. ,40 3 0042-7500 - 36.
Martinelli L. Gribaudo I. 2009 Strategies for effective somatic embryogenesis in grapevine: an appraisal. In: , K. A. Roubelakis-Angelakis (Ed.),461 494 Springer,978-9-04812-304-9 Dordrecht, The Netherlands. - 37.
Meng X. Zhou W. 1981 Induction of embroid and production of plantlets from endosperm of peach. Acta Agriculturae Universitatis Pekinensis,7 0479-8007 - 38.
Mikhaylov R. Dolgov S. V. 2007 Transgenic plum ( L.) plants obtained by Agrobacterium-mediated transformation of leaf explants with various selective agents. Proceedings of the International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species- Acta Horticulturae,738 978-9-06605-219-2 Daytona Beach, Florida, USA, October 10-14, 2005. - 39.
Mullins M. Srinivasan C. 1976 Somatic embryos and plantlets from an ancient clone of grapevine (cv. Cabernet-Sauvignon) by apomixes . Journal of Experimental Botany,27 100 1022 EOF 1030 EOF 0022-0957 - 40.
Murashige T. Skoog F. 1962 A revised medium for rapid growth and bioassay with tobacco tissue culture. ,15 43 0031-9317 - 41.
Padilla I. Golis A. Gentile A. Damiano C. Scorza R. 2006 Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes. ,84 3 309 EOF 314 EOF 0167-6857 - 42.
Pennisi E. 2010 Sowing the seeds for the ideal crop. ,327 5967 802 EOF 803 EOF 0036-8075 - 43.
Pérez-Clemente R. Pérez-Sanjuán A. García-Férriz L. Beltrán J. Cañas L. 2004 Transgenic peach plants ( L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an in vivo marker. Molecular Breeding,14 4 419 EOF 427 EOF 1380-3743 - 44.
Polák J. Pívalová J. Kundu M. Jokes M. Scorza R. Ravelonandro M. 2008 Behaviour of transgenic -resistant Prunus domestica L. Clone C5 grown in the open field under a high and permanent infection pressure of the PPV-rec strain. Journal of Plant Patholology,90 1-Supplement S1.33 EOF S1 EOF S1.36,1125-4653 - 45.
Pooler M. Scorza R. 1995 Regeneration of peach ( (L.) Batsch) rootstock cultivars from cotyledons of mature stored seed. HortScience,30 2 355 EOF 0018-5345 - 46.
Qu J. Ye J. Fang R. 2007 Artificial microRNA-mediated virus resistance in plants. ,81 12 6690 EOF 9 EOF 0002-2538 X. - 47.
Rajasekaran K. Mullins M. 1983 Influence of genotype and sex-expression on formation of plantlets by cultured anthers of grapevines. ,3 3 233 EOF 238 EOF 0249-5627 - 48.
Salunkhe C. Rao P. Mhatre M. 1997 Induction of somatic embryogenesis and plantlets in tendrils of L. Plant Cell Reports,17 1 65 EOF 67 EOF 0721-7714 - 49.
Scorza R. Cordts J. Mante S. 1990 Long-Term somatic embryo production and regeneration from embryo-derived peach callus. ,280 978-9-06605-214-7 Bologna, Cesena, Italy, May 30-June 3, 1989. - 50.
Scorza R. Cordts J. Gray D. Emershad R. Ramming D. 1996 Producing transgenic ´Thompson Seedless´ grape ( L.) plants. Journal of American Society for Horticultural Science,121 4 616 EOF 0003-1062 - 51.
Scorza R. Callahan A. Levy L. Damsteegt V. Webb K. Ravelonandro M. 2001 Post-transcriptional gene silencing in resistant transgenic European plum containing the Plum pox potyvirus coat protein gene. Transgenic Research,10 3 201 EOF 209 EOF 0962-8819 - 52.
Scorza R. Georgi L. Callahan A. Petri C. Hily J. Dardick C. Damsteegt V. Ravelonandro M. 2010 Hairpin coat protein (hpPPV-CP) structure in ‘HoneySweet’ C5 plum provides PPV resistance when genetically engineered into plum (Prunus domestica) seedlings. Proceeding of the 21st International Conference on Virus and Other Graft Transmissible Diseases of Fruit Crops- Julius-Kühn-Archiv427 141 EOF 146 EOF 1868-9892 Neustadt, Germany July 5- 10, 2009. - 53.
Scorza R. Sherman W. 1996 Peaches. In: . J. Wiley & J. Moore (Eds.), Wiley, 047131014X, New York, USA. - 54.
Smigocki A. Hammerschlag F. 1991 Regeneration of plants from peach embryo cells infected with a shooty mutant strain of . Journal of the American Society for Horticultural Science,116 6 0003-1062 - 55.
Stamp J. Meredith C. 1988 Somatic embryogenesis from leaves and anthers of grapevine. ,35 Nos. 3-4,235 EOF 250 EOF 0304-4238 - 56.
Tapia E. Sequeida A. Castro A. Montes C. Zamora P. Prieto P. 2009 Development of grapevine somatic embryogenesis using an air-lift bioreactor as an efficient tool in the generation of transgenic plants. ,139 1 95 EOF 101 EOF 0168-1656 - 57.
Teycheney P. Y. Tavert G. Delbos R. Ravelonandro M. Dunez J. 1989 The complete nucleotide sequence of RNA (strain D). Nucleic Acids Research,17 23 10115 EOF 6 EOF 0305-1048 - 58.
Tian L. Wen Y. Jayasankar S. Sibbald S. 2007 Regeneration of Lindl (Japanese plum) from hypocotyls of mature seeds. In Vitro Cellular and Developmental Biology- Plant,43 4 343 EOF 347 EOF 1054-5476 - 59.
Torregrosa L. Iocco P. Thomas M. 2002 Influence of Agrobacterium strain, culture medium, and cultivar on the transformation efficiency of L. American Journal of Enology and Viticulture,53 3 183 EOF 0002-9254 - 60.
Urtubia C. Devia J. Castro A. Zamora P. Aguirre C. Tapia E. Barba P. Dell` Orto. P. Moynihan M. Petri C. Scorza R. Prieto H. 2008 -mediated genetic transformation of Prunus salicina. Plant Cell Reports,27 9 1333 EOF 1340 EOF 0721-7714 - 61.
Velasco R. Zharkikh A. Troggio M. Cartwright D. Cestaro A. Pruss D. Pindo M. Fitzgerald L. Vezzulli S. Reid J. Malacarne G. Iliev D. Coppola G. Wardell B. Micheletti D. Macalma T. Facci M. Mitchell J. Perazzolli M. Eldredge G. Gatto P. Oyzerski R. Moretto M. Gutin N. Stefanini M. Chen Y. Segala C. Davenport C. Demattè L. Mraz A. Battilana J. Stormo K. Costa F. Tao Q. Si-Ammour A. Harkins T. Lackey A. Perbost C. Taillon B. Stella A. Solovyev V. Fawcett J. A. Sterck L. Vandepoele K. Grando S. M. Toppo S. Moser C. Lanchbury J. Bogden R. Skolnick M. Sgaramella V. Bhatnagar S. K. Fontana P. Gutin A. Van de Peer Y. Salamini F. Viola R. 2007 A high quality draft consensus sequence of the genome of a heterozygous grapevine variety ,2 12 e1326,1932-6203 - 62.
Watson J. Fusaro A. Wang M. Waterhouse P. 2005 RNA silencing platforms in plants. ,579 26 5982 EOF 7 EOF 0014-5793 - 63.
Wesley S. Helliwell C. Smith N. Wang M. Rouse D. Liu Q. Gooding P. Singh S. Abbott D. Stoutjesdijk P. Robinson S. Gleave A. Green A. Waterhouse P. 2001 Construct design for efficient, effective and high-throughput gene silencing in plants. ,27 6 581 EOF 590 EOF 0960-7412 - 64.
Wong W. Barba P. Álvarez C. Castro A. Acuña M. Zamora P. Rosales M. Dell’Orto P. Moynihan M. Scorza R. Prieto H. 2010 Evaluation of the resistance of transgenic C5 plum ( l.) Against four Chilean Plum pox virus isolates through micro-grafting. Chilean Journal of Agricultural Research,70 3 372 EOF 380 EOF 0365-2807 - 65.
Yancheva S. Druart Ph. Watillon B. 2002 -mediated transformation of plum (Prunus domestica L.). Proceeding of the VII International Symposium on Plum and Prune Genetics, Breeding and Pomology- Acta Horticulturae,577 215 EOF 220 EOF 978-9-06605-895-8 Plovdiv, Bulgaria, August 20-24, 2001. - 66.
Ye X. Brown S. Scorza R. 1994 Genetic transformation of peach tissue by particle bombardment. ,119 2 0003-1062