Genetic improvement of olive (Olea europaea L.) by conventional and in vitro biotechnology methods.

In olive (Olea europaea L.) traditional methods of genetic improvement have up to now produced limited results. Intensification of olive growing requires appropriate new cultivars for fully mechanized groves, but among the large number of the traditional varieties very few are suitable. High-density and super high-density hedge row orchards require genotypes with reduced size, reduced apical dominance, a semi-erect growth habit, easy to propagate, resistant to abiotic and biotic stresses, with reliably high productivity and quality of both fruits and oil. Innovative strategies supported by molecular and biotechnological techniques are required to speed up novel hybridisation methods. Among traditional approaches the Gene Pool Method seems a reasonable option, but it requires availability of widely diverse germplasm from both cultivated and wild genotypes, supported by a detailed knowledge of their genetic relationships. The practice of "gene therapy" for the most important existing cultivars, combined with conventional methods, could accelerate achievement of the main goals, but efforts to overcome some technical and ideological obstacles are needed. The present review describes the benefits that olive and its products may obtain from genetic improvement using state of the art of conventional and unconventional methods, and includes progress made in the field of in vitro techniques. The uses of both traditional and modern technologies are discussed with recommendations.

Somatic embryogenesis and shoot regeneration from transgenic roots of the cherry rootstock Colt (Prunus avium×P. pseudocerasus) mediated by pRi 1855 T-DNA of Agrobacterium rhizogenes.

Hairy roots were obtained after inoculation with Agrobacterium rhizogenes strain NCPPB 1855 of the in-vitro-grown shoots of the cherry rootstocks Colt (Prunus avium×P. pseudocerasus) and Mazzard F12/1 (P. avium L.). Not all putatively transgenic roots were able to grow in hormone-free medium. Mazzard F12/1 roots, induced with A. rhizogenes, did not differentiate any shoot or embryo, while both somatic embryos and shoots differentiated from the transgenic roots of Colt in medium containing 1 mg/l 6-benzylaminopurine and 1 mg/l 1-naphthaleneacetic acid. Somatic embryos were capable of secondary embryogenesis, but few developed into whole plants. DNA hybridization showed both a different number of bands and signal intensity in each of the five transgenic shoot clones and embryos examined. In a morphogenetic in vitro test, leaf explants of the transgenic shoot clones showed an increased capacity to differentiate roots, although clones differed in their sensitivity to the hormone ratio. Clones from the transgenic shoots had not only an increased rooting ability when grown in vitro but also exhibited various hairy root phenotypes when cultured in vitro and when transferred into the greenhouse.

A novel strategy for the induction and maintenance of shoot regeneration from callus derived from established shoots of apple [Malus×Domestica Borkh.] cv. Golden Delicious.

A novel strategy for the production and maintenance of morphogenic callus for 1 year from mature leaf explants of apple has been developed using micropropagated primary leaves of cv. Golden Delicious. The technique required second generation adventitious buds produced from cultured primary leaves also produced from established shoot cultures. The age at which buds were capable of producing morphogenic callus was critical and found to be when leaflets were 2-3 mm in length. Medium composition affected the maintenance but not the induction of shoot regeneration from callus and the best combination was found to be high calcium, low ammonium and low hormone levels. Adventitious shoots were rooted in vitro and established glasshouse-grown plants showed no phenotypic differences from the plants derived from shoot proliferation. The great advantage of this technique for an increased efficiency of recovery of transgenic plants from transformed cells is discussed and the acquisition and maintenance of cell competence with respect to the formation of shoots in culture is explained.

Somatic embryogenesis and plant recovery from mature tissues of olive cultivars (Olea europaea L.) "canino" and "moraiolo".

A cyclic system of somatic embryogenesis from mature tissues of olive (Olea europaea L.) and subsequent plant recovery were developed. The primary embryos originated from morphogenetic masses derived from petioles of shoots regenerated from tissues of two micropropagated cultivars: Canino and Moraiolo. The rejuvenation acquired by the shoots by regeneration, directly from petiole tissues or indirectly from petiole callus, seems to be essential for the subsequent somatic embryogenesis induction. Cyclic embryogenesis, both from normal embryos or teratoma, was obtained on modified olive medium (OMe) plus 0.5 µM; 6dimethylaminopurine, 0.44 µM 6-benzylaminopurine, 0.25 µM 3-indolebutyric acid and 0.42 mM cefotaxime. The production of normal embryos was higher, faster and often more clustered on a filter paper liquid medium or on a media solidified with phytagel than with agar. The capacity to produce continuous cycles of successive embryos has been maintained for over two years only in the dark, since the light inhibited embryo induction. The embryogenetic capacity was qualitatively and quantitatively enhanced by adding 0.42 mM cefotaxime. Mature embryos germinated easily by increasing the amount of liquid medium with shake culture. Although the majority of embryos appeared vitrified when transplanted to Jiffy-7 pots, they subsequently grew normally and were similar to those derived from nonvitrified embryos. The plantlets obtained from somatic embryos appeared to be morphologically similar to those produced from axillary buds.

Increase of rooting ability in the woody species kiwi (Actinidia deliciosa A. Chev.) by transformation with Agrobacterium rhizogenes rol genes.

The woody species kiwi (Actinidia deliciosa A. Chev.), a male and late flowering clone of the cv Hayward, has been transformed by a T-DNA fragment encompassing rol A, B, C genes of A. rhizogenes. Transgenic plants, regenerated from leaf disc callus, showed the typical "hairy root" phenotype as described in herbaceous species. Explants from these plants (both leaf discs or 3 to 4 node leafy microcuttings) showed an increased ability to produce roots. Since root formation is one of the limiting factors in the vegetative propagation of woody species, the results have been discussed in relation to the use of A. rhizogenes rol genes in improving root morphogenesis in trees.