M. Laimer da Câmara Machado

Localization of fruit tree viruses by immuno-tissue printing in infected shoots of Malus sp. and Prunus sp.

Immuno-tissue printing protocols for the localization of apple chlorotic leaf spot virus (ACLSV), stem grooving virus (SGV) and plum pox virus (PPV) in shoots of Prunus and Malus in vitro have been established for routine diagnosis in a virus elimination program. Since these viruses belong to different virus genera, the protocols were adapted according to the properties of the virus under investigation. Accumulation of ACLSV was highest in the base of the stem and decreased towards the apex of the shoots. ACLSV was found in the epidermis, the cortex, in the vascular bundles, but seldom in the pith tissue of in vitro apple shoots. ACLSV immuno-tissue printing was as sensitive as ELISA and the intensity of color signals in immuno-tissue prints correlated with absorbance values by two-step ELISA. SGV could be detected by immuno-tissue prints at infectivity levels, where it reacted negative in ELISA. SGV accumulated in the vascular bundles, occurred locally in the parenchymatic tissue, was found in high amounts in young leaves near the meristem, but not within the meristem. PPV was detected in all tissue types of stem sections with an irregular pattern reflecting the in vivo situation causing problems with detection. Discrimination of poorly and heavily infected shoots was possible with the naked eye.

A new, efficient method using 8-hydroxy-quinolinol-sulfate for the initiation and establishment of tissue cultures of apple from adult material

Applying the new method for culture initiation 16 different cultivars of Malus domestica could be established in vitro from shoot tips of adult orchard trees. Actively growing shoot tips were cleaned and surface disinfested, dissected to 2–3 mm and placed on a modified MS-medium with 4.4 μM BA. Explants were covered for 24 h with 200 μl of a 0.1% solution of 8-hydroxy-quinolinol-sulfate (8-HQS) and transferred to a medium containging both auxin and cytokinin after 2 weeks. The application of 8-HQS induced a strong reduction of the infection rate and inhibited the browning of the explants and the media. After 7 days yields of 50–90% sterile explants could be obtained in comparison to 100% losses of untreated shoot tips. After 60 days variable rates of actively growing shoots could be observed, depending on the genotypes. The described method allows a successful establishment of fruit trees from adult orchard material on one hand by strongly reducing the browning, caused by the oxidation of polyphenolic compounds by polyphenoloxidases, and on the other hand 8-HQS can strongly increase the yield of explants without contamination, independently from the vegetation period and the phytosanitary state of the donor material.

Improved Virus Detection in Rosaceous Fruit Trees in vitro

In vitro cultivation techniques are widely used for micropropagation and germplasm storage [1,2]. Moreover, in vitro culture techniques are the only effective tools for eliminating certain pathogens from elite germplasm. Many fruit tree viruses are well identified and characterised [3,4]. Conventional laboratory diagnostics, based on serology, have been developed and are routinely used for orchard surveys [5–8]. Drawbacks in the certification of fruit plants are, however, encountered since many viruses are either latent, irregularly distributed or highly localised. Thus, the risk of false results is increased especially with field plants [9,10]. Little has been published on the virus screening of in vitro cultures; however, optimisation of sampling of in vitro plants may be achieved if carried out in combination with studies on virus localisation using immuno-tissue printing [11–13].

Genetic Transformation in Prunus armeniaca L. (Apricot)

The apricot belongs to the family Rosaceae, subfamily Prunoideae, genus Prunus L., (Loschung and Passecker 1954). Most cultivated apricots belong to one species, Prunus armeniaca L. Its cultivars are unique among fruit trees, their ecological adaptation being so limited, that a given cultivar is usually grown commercially only in one area of one country (Mehlenbacher et al. 1991).