V. Hanzer

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus

SummaryA system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.

Improved detection methods for fruit tree phytoplasmas

Phytoplasmas infecting fruit trees are considered quarantine organisms in Europe and North America. Detection often is hampered by their extremely irregular distribution in host plants. A sensitive, specific and quick diagnostic test would be highly desirable for routine detection, mainly to avoid using infected planting material. PCR methods require tedious preparation of DNA; also, the available primers are highly specific and exhibit some homology to chloroplast and plastid DNA. To address these problems, we compared several DNA preparation protocols for purity of DNA, cost and time required. We also developed new primers using rDNA sequence information from an Austrian isolate of European Stone Fruit Yellows (ESFY). These primers operate at high annealing temperatures and, thus, increase the specificity and decrease the risk of false positives. The primers could reliably detect the European phytoplasmas (AP, ESFY and PD) within a collection of isolates maintained in micropropagated periwinkle. Thus, they are suitable as general primers for phytoplasma detection. The primers also can be used for strain identification by direct PCR followed by RFLP analysis as demonstrated with micropropagated fruit tree material. Finally, an IC-PCR method that uses the primers for AP detection was found very sensitive and suitable for large-scale testing of apple materialin vivo andin vitro.

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.

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].

Expression of the plum pox virus coat protein region in Escherichia coli

A cDNA complementary to the 3′ end of plum pox virus (PPV) RNA was sequenced. The sequence was investigated for the presumable coat protein cistron by computer-aided translation. A fragment containing the stop codon of the polyprotein gene and a putative virus-specific protease cleavage site was subcloned into an E. coli expression vector. It is shown by immunological analysis that the coat protein cistron is located within the subcloned region.

Characteristics of Large-Scale Production of Phyto-Sanitary Improved In-Vitro Fruit Tree Cultivars Using In-Vitro Elimination Treatments and Serodiagnostics

We present results of five years of testing in-vitro cultures of over 30 apple and 20 stone fruit cultivars in a virus elimination programme. Our aim was to develop efficient large-scale screening designed for the special features of in-vitro plants to improve reliability in selection of truly negative in-vitro elite material following elimination treatment by thermotherapy and meristem preparation.