Modification of fruit tree architecture by biotechnology approaches

Italy produces high quantity of fruit whose major part is exported in other countries. Other countries invest a large amount of their economical resources into the area of genetic improvement and plant development study. In many cases plant growers and farmers of our country must pay royalties to other countries for plant breeding technologies, for using improved cultivars. Nonetheless, these cultivars are not always adapt to our needs and environmental conditions. Cherries and apricots are of great economic interest both for areas of high intensity investments and for marginal ones, these have problems linked with strong apical dominance (cherry, and in some cases apricot), impeding the cultivation of modern fruit production technologies, problems linked with flowering period, with fruit ripening, with problems of self-incompatibility and cold tolerance. The objective of this research, which involves seven Research Units from several Italian public labs, is to improve the knowledge about growth and development of fruit crops, through the studies of two important factors such as light and plant growth regulators and their interactions. In this context biotechnologies are used for two main reasons: the first one is to evaluate their possible use as a method of genetic improvement and the second is to understand how light and hormones act and integrate in order to determine the development and architecture of the plant. Plant photoreceptors are involved in light dependent responses. Overexpression of phytocrome a and b (belonging to the multigene family encoding phytocrome) has been demonstrated to induce phenotipic consequences such as short hypocotyl in transgenic Arabidopsis seedlings (Wagner, 1991) and reduction of apical dominance in transgenic fruit trees (Muleo e Iacona,1998). The genes that we are going to use for plant transformation are phytochrome A (phyA) of rice, phytochrome B (phyB) of Arabidopsis thaliana and the rol genes of A. rhizogenes. Both, phyA and phyB, control the biochemical signal regulating the expression of the strategy of plants development (avoidance shade syndrome), the proteins of the rol genes interact with the signal chain of the hormones which have a preponderant role in the plant development. We expect that the over expression of phytochrome genes will induce phenotypic modifications affecting apical dominance, reduced canopy development, functional and dimensional development of root system, flowering time, reduction of juvenile period. Nevertheless the use of genetic transformation requires a suitable regeneration systems. Plant regeneration from fruit tree is in most cases difficult to achieve. In spite of several reports on Prunus spp only a few literature referring to regeneration from mature tissues of Prunus avium cultivars is available (Yang et al. 1991, Hammat and Grant 1998, Negri et al. 1998). In our lab we are testing regeneration ability from mature explants of several Prunus avium cultivars and a regeneration protocol for cultivar Hedelfinger has been set up. Nevertheless Hedelfinger is demonstrated to be a recalcitrant genotype as indicated by the very poor regeneration frequencies obtained and by the fact shoot regeneration has quite a discontinuous trend. At the present time other commercially relevant cultivars, such as Lapins and Burlat C1, are now under testing to determine the conditions to induce regeneration. Regenerated plantlets from cultured tissues can undergo somatic stable variation and meiotically heritable variation caused by the in vitro process. So, random and frequent genetic variability occurring in cell cultures is preserved among the population of regenerants. Despite this, very little is understood about how somaclonal variation can occur and be regulated, the event is particularly intriguing because it is presumably under the control of many factors. Transgenic plants are expected to integrate and express one or more foreign gene in an otherwise unmodified genomic environment. Indeed, less attention has been given for the occurrence of somaclonal variation in transgenic plants also if in some case it has been documented at the morphological and agronomic level or in other cases molecular evidence has also been given (Sala et al, 2000). We have performed an early screening method for selection of putative somaclones based on morphological and physiological traits regulated by the light quality (Piagnani et al 2001, a) Regenerated and micropropagated shoots, were incubated for four weeks under continuous light supplied by different types of lamps, filtered through photoselective layers in order to obtain several spectral outputs. When grown under different light spectral quality one somaclone, named HS, shows a different pattern of growth and development compared to the wild type with the main modifications related to apical dominance and chlorophyll production: somaclone HS shows reduced apical dominance compared to the wild type and a different pattern of chlorophyll a and b production. Light quality has played a different role on chlorophylls content in somaclone HS in respect of cultivar H. In particular blue light significantly promoted Chl a + Chl b content only in somaclone HS both in vitro and in ex vitro (glasshouse) conditions (Piagnani et al 2001, b). Darkness affects Chl a + Chl b at a greater extent in HS than in H which has shown to be able to accumulate higher amounts of chlorophylls than somaclone, also in this case both in vitro and in ex vitro. DNA analyses of HS, conducted using Inter Simple Sequence Repeat (ISSR), has revealed polymorphism between the somaclone HS and wild type propagated by microcuttings. As regarding DNA analysis of non-coding region of chloroplast DNA (trn) and mitochondrial DNA, NADreductase gene region, no differences between H and HS have been detected. A total of 15 ISSR primers have been screened for the amplification of genomic DNA. ISSR5 primer did not amplify any DNA fragment, while amplifications profiles obtained through ISSR6 and ISSR8 primers were able to reveal polymorphism between H and HS, in which, respectively, 2 fragments of about 830 and 760 bp are present in HS, and 2 fragments of about 1430 and 1080 bp are present in H. Out of a total number of 80 analysed fragments 4 were polymorphic. References Hammatt, N., Blake, P.S., Hand, P., 1998. Characterisation and use of apparent rejuvenation achieved during micropropagation of mature Prunus avium L. In : Davey MR, Alderon PG, Lowe KC and Power JB (eds) Tree Biotechnology: towards the millennium. Pp 45-63. Nottingham University Press, Nottingham. Muleo e Iacona,1998. [regolazione dell’habitus vegetativo da parte del citocromo, evidenze fenotipiche in piante di ciliegio sovraesponenti il citocromo A di riso]. Atti AGRO-BIO-FRUT, incontro tecnico su “La trasformazione genetica delle piante da frutto: stato d’avanzamento delle ricerche in atto”. Cesena, 8 maggio, pp 14-15. [In Ital.] Negri, P., Magnanini, E., Cantoni, L., Berardi, G., Sansavini, S. [Piante arboree transgeniche: prime esperienze sul trasferimento di geni per il controllo dell’habitus vegetativo]. Rivista di Frutticoltura 5: 91-97, 1998.[In Ital.] Piagnani C., 1998. [Organogenesi e aspetti della la coltura in vitro di cultivar di ciliegio dolce. Atti AGRO-BIO-FRUT, incontro tecnico su “La trasformazione genetica delle piante da frutto: stato d’avanzamento delle ricerche in atto”]. Cesena, 8 maggio, 1998, 12-13. [In Ital.] Piagnani C., Iacona C., Intrieri M. C., Muleo R., 2001, a . A new somaclone of Prunus avium shows diverse growth pattern under different spectral quality of radiation. Biologia Plantarum, accepted May 10 2001 Piagnani C.,. Iacona C,. Intrieri M. C, and Muleo R. 2001. b A Somaclonal variant in 'Hedelfinger' sweet cherry. Proc. ISHS IV Int. 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