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Approaches applied to induce regeneration, via somatic embryogenesis, in the peach rootstock ‘GF677’ (P. persica × P. amygdalus) and in different peach cultivars using leaves and anthers as starting explants
No full textP. persica L. is considered one of the most recalcitrant fruit trees species in terms of in vitro regeneration and genetic transformation, especially when somatic tissues are used as starting material. In this study, different attempts to induce somatic embryogenesis (SE) in the peach hybrid rootstock ‘GF677’ (P. persica × P. amygdalus) and in several peach cultivars are described, using expanding leaves and anthers as starting explants. The final objective is to eventually apply biotechnological approaches aimed at genetically improve different peach rootstocks and cultivars. The current investigation aimed to determine the proper balance of in vitro plant growth regulators (PGRs) capable of inducing SE in a peach rootstock and different cultivars from expanding leaves of elongated in vitro shoot cultures, or from anthers belonging to unopened flowers of one-year old dormant cuttings. The explants have been cultured in different induction media, which consisted of McCown Woody Plant Medium (WPM) as basal salt supplemented with N6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D). Although none of the media tested induced the development of somatic embryos from the somatic explants treated, cream-colored calli similar to pre-embryogenic structure were obtained when anthers of ‘Big Top’, ‘Tardibelle’ and ‘Big Bang’ were cultured on SE induction media supplemented with 4.4 μΜ BAP in combination with 4.5 or 9 μΜ 2,4-D. Considering the optimization of efficient regeneration protocol through SE in Prunus persica L. as useful tool for peach genetic improvement, it would be worth continuing to test the induction of SE from these kinds of explants by investigating other culture media supplemented with different concentrations and combinations of BAP as cytokinin and 2,4-D as auxin
Peach (Prunus persica L.)
No full textUntil now, the application of genetic transformation techniques in peach has been limited by the difficulties in developing efficient regeneration and transformation protocols. Here we describe an efficient regeneration protocol for the commercial micropropagation of GF677 rootstock (Prunus persica × Prunus amygdalus). The method is based on the production, via organogenesis, of meristematic bulk tissues characterized by a high competence for shoot regeneration. This protocol has also been used to obtain GF677 plants genetically engineered with an empty hairpin cassette (hereafter indicated as hp-pBin19), through Agrobacterium tumefaciens-mediated transformation. After 7-8 months of selection on media containing kanamycin, we obtained two genetically modified GF677 lines. PCR and Southern blot analyses were performed to confirm the genetic status
Micropropagation of walnut: A real opportunity
No full textSince 2005, Vitroplant Italia s.r.l. has been producing micropropagated walnut plants, which have been planted in demonstrative orchards from 2007 with excellent results. Following this first experience, from 2013 there has been an increasing demand for micropropagated walnut plants. The micropropagated 'Chandler' potted plants, 10 cm height (in 0.9-L pot) and 30 cm height (1.7-L pot), were compared with bare root 1-year-old 'Chandler' grafted plants, on rootstock J. regia seedlings, 1.80 m height. The plantation was made for all the plants at the end of April in a demonstrative orchard in Emilia Romagna region (45°N, Italy). Micropropagated plants in 0.9-L pots got an equivalent or higher growth than grafted plants at the third year, while the micropropagated plants in1.7-L pots overtook growing performance of grafted plants already during the first year. The cumulative production per plant since now has been equivalent or higher in micropropagated 'Chandler' orchards than in grafted ones. This result was obtained with less pruning and greater vegetative growth of micropropagated plants, which made easier the training of structured central axis. In the last few years, the most planted type of micropropagated plants was the dormant bare root type. These plants can be obtained in one growing season in the nursery, after early spring acclimation ex vitro. This type of plant was between 0.20 and 0.60 m height and easier to be managed during the first year of growth in the orchard compared to the pot plants. Therefore, in a second demonstrative field trial, 'Chandler' dormant bare root plants from micropropagation were compared with 1-year-old grafted plants on J. regia seedlings 1.80 m height. Micropropagated plants showed better growth rate and more homogeneity with minimal pruning to train the structured central axis. While, a delay in production of female flowers was found in micropropagated 'Chandler' trees during the first 3-4 years, the cumulative production per plant was higher than in grafted plants 8 years after planting
Genetic transformation of <it>Vitis vinifera</it> via organogenesis
Full text linkAbstract Background Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered. Results Transgenic grape plants (V. vinifera, table-grape cultivars Silcora and Thompson Seedless) were produced using a method based on regeneration via organogenesis. In vitro proliferating shoots were cultured in the presence of increasing concentrations of N6-benzyl adenine. The apical dome of the shoot was removed at each transplantation which, after three months, produced meristematic bulk tissue characterized by a strong capacity to differentiate adventitious shoots. Slices prepared from the meristematic bulk were used for Agrobacterium-mediated transformation of grape plants with the gene DefH9-iaaM. After rooting on kanamycin containing media and greenhouse acclimatization, transgenic plants were transferred to the field. At the end of the first year of field cultivation, DefH9-iaaM grape plants were phenotypically homogeneous and did not show any morphological alterations in vegetative growth. The expression of DefH9-iaaM gene was detected in transgenic flower buds of both cultivars. Conclusions The phenotypic homogeneity of the regenerated plants highlights the validity of this method for both propagation and genetic transformation of table grape cultivars. Expression of the DefH9-iaaM gene takes place in young flower buds of transgenic plants from both grape cultivars.</p
In vitro shoot regeneration from leaves of Pyrus communis L. rootstock and cultivars
Full text linkThe influence of TDZ on adventitious shoot regeneration from leaves of the pear cultivars Conference and Abate Fetel, and the rootstock Farold((R))87 was investigated. Our main aim was to set up efficient in vitro regeneration protocols for all these pear genotypes by using expanding leaves from elongated shoot cultures as starting plant material. Our best results in terms of percentage of regeneration were achieved by using half-strength Murashige and Skoog basal medium supplemented with 1 mu M NAA, combined with 13.5 mu M TDZ for Conference (87.3%) and Farold((R))87 (53.3%), and 9 mu M TDZ for Abate Fetel (68%). The impact on leaf organogenesis of the antibiotics timentin, cefotaxime, and carbenicillin, alone or in combination, usually used for the control of Agrobacterium overgrowth, and of kanamycin, commonly used for the selection of putatively transformed plants, were also evaluated to be exploited in future transformation trials. In general, the use of carbenicillin (475 mM), cefotaxime/carbenicillin (210/238 mM) and cefotaxime (630 mM) did not negatively affect the regeneration efficiency of Conference, Abate Fetel and Farold((R))87, respectively. The use of 4 mu M kanamycin should be suitable to select transformed shoots from Abate Fetel and Farold((R))87 leaves, while a lower concentration or a different selection strategy should be applied for Conference. We report new regeneration and selection protocols usable for the application of new biotechnologies in the genetic improvement of pear cultivars and rootstocks
In vitro regeneration, via organogenesis, from leaves of the peach rootstock GF677 (P. persica × P. amygdalus)
No full textThe optimization of an efficient in vitro adventitious shoot regeneration protocol represents a key step for the application of biotechnological approaches aimed to genetically improve P. persica L. rootstocks and cultivars. However, peach is well-known as one of the most recalcitrant fruit trees species in terms of in vitro regeneration, especially when adult tissues are used as starting explants. The present study represents the first successful attempt to induce adventitious regeneration from leaves of the peach rootstock GF677 (Prunus persica L. × P. amygdalus L.). Basically, expanded leaves from elongated in vitro shoot cultures were placed in different regeneration media consisting of McCown Woody Plant Medium (WPM) as basal salt, supplemented with different combinations and concentrations of plant growth regulators (PGRs). In this study, low regeneration rates (from 2% up to 12%) were recorded when leaf explants of GF677 were cultured on regeneration media enriched with thidiazuron (TDZ) used alone or in combination with α-naphtalene acetic acid (NAA), or 2,4-dichlorophenoxyacetic acid (2,4-D). Although at low percentage, adventitious shoot regeneration was observed in this study, and the preliminary results obtained can be considered as promising starting point for further studies aimed to increase the percentage of in vitro organogenetic explants of this important commercial genotype. In conclusion the establishment of efficient regeneration method through organogenesis for the peach hybrid rootstock GF677 might represent a step forward for genetic improvement of P. persica L., as the production of plants through somatic embryogenesis of peach rootstocks and cultivars has been arduous to date
Qualità vivaistica di piante di fragola (Fragaria x ananassa Duch.) provenienti da propagazione in vitro e in vivo
No full textAttualmente la tecnica della micropropagazione in fragola, secondo la regolamentazione UE, può essere utilizzata solo per la produzione di materiale esente virus e non è prevista in altre fasi della produzione vivaistica.
Con lo scopo di valutare la possibilità di adottare la tecnica in vitro per la produzione vivaistica di piante madri di fragola, è stata avviata una prova sperimentale per confrontare piante madri di Alba, varietà commerciale diffusa in UE, propagate con protocolli standard in vitro e in vivo (piante frigo)
Peach (Prunus Persica L.)
No full textUntil now, the application of genetic transformation techniques in peach has been limited by the difficultiesin developing efficient regeneration and transformation protocols. Here we describe an efficient regenerationprotocol for the commercial micropropagation of GF677 rootstock (Prunus persica × Prunusamygdalus).The method is based on the production, via organogenesis, of meristematic bulk tissues characterizedby a high competence for shoot regeneration.This protocol has also been used to obtain GF677 plants genetically engineered with an empty hairpincassette (hereafter indicated as hp-pBin19), through Agrobacterium tumefaciens-mediated transformation.After 7–8 months of selection on media containing kanamycin, we obtained two genetically modifiedGF677 lines. PCR and Southern blot analyses were performed to confirm the genetic status
Biotecnologie alternative: silenziamento genico post-trascrizionale per indurre resistenza a Sharka nelle drupacee
No full textLe drupacee, in particolare il pesco (Prunus persica), rappresentano le
specie da frutto coltivate più importanti del bacino del Mediterraneo
e anche in Italia, ma sono soggette a molte perdite agronomiche ed economiche causate da infezioni virali. Una delle malattie più virulente è rappresentata dalla Sharka, causata dal “Plum Pox Virus” (PPV). Al momento non ci sono mezzi di lotta diretta contro tali infezioni, ma solo mezzi di prevenzione che causano grossi problemi di sostenibilità
ambientale ed enormi perdite di reddito per gli agricoltori. Le tecniche di trasformazione genetica potrebbero essere impiegate anche per introdurre geni di resistenza individuati in Prunus spp. o geni per il silenziamento genico post-trascrizionale (PTGS) capaci di indurre resistenza
a virus. Quest’ultima tecnologia può essere utilizzata per introdurre resistenza alla Sharka in varietà e portinnesti di pesco e delle altre principali drupacee
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