Bio-agroFood Sciences

Grey mould or bunch rot caused by the fungus Botrytis cinerea is the major cause of fruit and wine losses for the fast-growing New Zealand grapevine industry. The cost of Botrytis rot to the industry worldwide is also enormous. In a collaborative project we have developed protocols for somatic embryogenesis in grapevine and this system is now in use for inducing mutations and selecting for fungal pathogen resistance using toxins from Botrytis (Pathirana
et al. 2008). The availability of pathogenic cultures of Botrytis throughout the year facilitates rapid screening and evaluation of genotypes. For long-term maintenance, we suspended fresh
Botrytis cultures from potato dextrose agar (PDA) plates in Cryobank® beads in glycerol and maintained them at -20oC. The stored cultures were retrieved after more than one month and
their pathogenicity was tested on leaves from in vitro-grown susceptible cultivars. Results indicate that the strains maintain the same pathogenicity as the original cultures. This method
of cold storage is therefore more effective for maintaining B. cinerea cultures than on PDA plates at low temperatures. The latter method requires frequent subculture and often results in
loss of pathogenicity of the original isolates.

Recent developments in molecular biology and other biotechnologies have advanced our knowledge of the history of grapevine domestication and the evolution of this crop. The aim of this chapter is to review recent applications of biotechnology to characterize and improve grapevine germplasm in order to guide the more efficient development of new grapevine cultivars. The chapter focuses on specific strategic areas, including germplasm management and improvement as well as pathogen elimination.

Cryotherapy of grapevine (Vitis spp.) to remove leafroll viruses from infected plants
Ranjith Pathirana1, Angela Carra2, Andrew McLachlan1, Duncan Hedderley1, Bart Panis3, Francesco Carimi2

1 The New Zealand Institute for Plant & Food Research Ltd, Private Bag 11600, Palmerston North 4442, New Zealand
2 Consiglio Nazionale delle Ricerche, Istituto di Genetica Vegetale, U.O.S. di Palermo, Corso Calatafimi 414, I-90129 Palermo, Italia
3Laboratory of Tropical Crop Improvement, Department of Biosciences, Katholieke Universiteit Leuven (K. U. Leuven), 3001 Leuven, Belgium.

Infections by viruses and other pathogens are a threat to the grapevine industry. A robust method for removing all microorganisms from infected tissue is important for cultivar imports, germplasm maintenance and to produce grafted material for the industry. The demonstration of virus and phytoplasma eradication by methods used in cryopreservation of plants has led to the establishment of cryotherapy: a new method for cleaning infected plant material of vegetatively propagated species. Vitrification-based cryopreservation techniques have been shown to be the most adaptable across species. In droplet vitrification, plant tissue pre-treated with vitrification solution is placed on aluminium foil in a droplet of vitrification solution and directly immersed in liquid nitrogen.  Only highly cytoplasmic, non-vacuolar meristematic cells survive the freezing process because only these cells can tolerate the dehydration caused by the vitrification solution; therefore, cryotherapy can be considered a precise method of meristem culture. Using in vitro-sourced apical and axillary buds as explants, we developed a droplet vitrification protocol and plants were regenerated from cryopreserved tissues of the 13 Vitis genotypes used, although regeneration ability was influenced by genotype. Growing plantlets in salicylic acid-supplemented media followed by pre-treatment of explants sourced from those in high sucrose solutions increased regeneration rates after cryopreservation, including that of the previously recalcitrant 41B rootstock. Regeneration from virus-infected plant material was generally poorer than for “clean” plants. The regeneration rates achieved are such that for most varieties cryopreservation of 6 – 15 explants will be sufficient to regenerate at least one plant at 95% probability, providing a cost-effective way of maintaining clonal plant material that avoids the threats associated with field-based collections. To test the suitability of cryotherapy for virus eradication, we used Lakemont Seedless and Chardonnay infected with Grapevine leafroll associated-virus-3 (GLRaV-3, an Ampelovirus), Sauvignon blanc and Pinot gris infected with Grapevine leafroll associated virus-2 (a Closterovirus), and another clone of Sauvignon blanc infected with both Grapevine leafroll associated virus -1 (an Ampelovirus) and GLRaV-3. Plants regenerated after cryotherapy tested negative (DAS-ELISA) for all three viruses, whereas untreated control plants tested positive. Droplet vitrification has the potential to be a novel and precise tool for virus eradication and establishment of high-health grapevine germplasm collections.
Acknowledgement: This work was funded by New Zealand Winegrowers (NZW 10-107 – “Cryopreserved grapevine: a new way to maintain high-health germplasm and cultivar imports with less rigorous quarantine”) and was part of COST Action 871. Work in Italy was part of PO-FESR Linea di intervento 4.1.1.1: “Recupero e valorizzazione dei vitigni tradizionali Siciliani”.

The Sicilian grape cultivar ‘Nero d’Avola’ is among the oldest and most cultivated in the island, taking part in the production of several red wines exported worldwide, including DOC wines (Etna Rosso and Cerasuolo di Vittoria). Due to the ancient origin and repeated clonally propagation of the cultivar, phenotypic variability has been observed. Clone identification in this important cultivar has so far relied on phenotypic and chemical traits analyses, often affected by environmental conditions. Genetic markers, such as microsatellites, are particularly useful for cultivar identification, parentage testing, pedigree reconstruction and population structure studies. In the present paper, microsatellites were used to analyze the intra-varietal genetic diversity among 118 plants of ‘Nero d’Avola’, collected in 30 vineyards displaced in different areas of Sicily. Out of 22 microsatellites, 11 showed polymorphism among samples and 15 different phylogenetic groups were identified. Results show that ‘Nero d’Avola’ actually comprises different genetic profiles, although most of clones share a common origin.

A robust method for removing all microorganisms from infected tissue is important for cultivar imports, germplasm maintenance and to produce healthy grafted material for the grapevine industry. In droplet vitrification method of cryopreservtion, plant tissue pre-treated with a vitrification solution is placed on aluminium foil in a droplet of vitrification solution and directly immersed in liquid nitrogen. Only highly cytoplasmic, non-vacuolar meristematic cells survive freezing. Therefore, cryopreservation can be considered a precise method of meristem culture and is developing into a new method for virus eradication in horticultural species called cryotherapy. To test the suitability of cryotherapy for virus eradication, we used Chardonnay and Lakemont Seedless infected with Grapevine leafroll associated-virus-3 (GLRaV-3, an Ampelovirus), Pinot gris and Sauvignon blanc 316 infected with Grapevine leafroll associated virus-2 (a Closterovirus), and another clone of Sauvignon blanc i...

Despite many reports on regeneration of Vitis after cryopreservation, there is no cryopreserved collection of its germplasm. Some Vitis genotypes are reported to be recalcitrant to cryopreservation. Droplet vitrification, considered to be an emerging generic method of cryopreservation, has been applied only to a limited extent in Vitis. In the present study, we first tested the toxicity of plant vitrification solution in both axillary and apical buds in six diverse Vitis accessions.  Droplet vitrification was then applied using 50 % predicted survival time of apical and axillary buds in vitrification solution after pre-treatment of donor plantlets with salicylic acid, a substance known to have a protective role in abiotic stress responses. Results showed that axillary buds are more tolerant of vitrification solution than apical buds and required longer treatment time. Pre-treatment of donor plantlets with 0.1 mM salicylic acid resulted in a significantly higher protection to cryopreserved buds, but serial dehydration in sucrose alone had little effect. Pre-treatment with salicylic acid enabled successful cryopreservation of previously recalcitrant rootstock 41B, albeit at a low regeneration rate. For other genotypes, cryopreservation of 6–11 explants will be sufficient to regenerate at least one plant at 95 % probability. This is the first report of successful cryopreservation of a set of diverse Vitis genotypes by droplet vitrification, and we show that pre-treatment of donor plantlets with salicylic acid is critical for the success. This research will contribute to conservation of Vitis germplasm in a cost effective way avoiding the risks associated with field-based collections.