Giorgio Ancora

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

Genetic transformation of potato (Solanum tuberosum): An efficient method to obtain transgenic plants

A quick procedure for efficient transformation of potato (cv. Desiree) has been devised. Leaf disc have been inoculated with an Agrobacterium tumefaciens harbouring a Ti plasmid derived binary vector. Transformed shoots carrying the neomycin phosphotransferase gene were regenerated using a feeder layer technique after 4 weeks on selective medium containing kanamycin. Transgenic plants obtained in large numbers appeared phenotypically normal and expressed the NPT II gene. The present work points out that culture conditions are fundamental to obtain the highest transformation efficiency.

In vitro propagation of caper (Capparis spinosa L.)

SummaryA twenty fold multiplication per twenty days of caper was achieved by culturing nodal shoot segments in the presence of BAP (4 μM) plus IAA (0.3 μM) and GA3 (0.3 μM). The use of a modified MS medium facilitated this response. Plantlet regeneration was induced on single shoots taken from proliferating clusters subcultured for 20 days on a reduced BAP (2 μM) without auxin and gibberellin Higher rooting responses (70%) were obtained after a 20-day incubation period in darkness on solid half-strength MS1 medium plus IAA (30 μM), followed by a subsequent 20 day culture period on half-strength MSI basal medium. Proliferation was mainly due to axillary shoot-bud development as revealed by histological studies. The extensive meristematic activities observed indicated the enormous morphological potential of this species.

Genetic Transformation of Nicotiana clevelandii Using a Ti Plasmid Derived Vector

Summary A procedure for the rapid production of transgenic shoots and F 1 progeny from Nicotiana clevelandii is described. Leaf discs were co-cultivated with a disarmed strain of Agrobacterium tumefaciens harbouring a binary vector system utilizing the neomycin phosphotransferase II as reporter gene. Inoculated leaf discs produced transgenic shoots at high frequency (50 %) after culturing for about three weeks on a regeneration medium which also contained 150 mg/l Kanamycin. The transgenic plants directly initiated flowering in vitro , producing a F 1 transgenic progeny. The life cycle was completed in approx. 4 months (from inoculation to F 1 progeny). Molecular analysis of the neomycin phosphotransferase marker confirmed the insertion, the expression and the transmission of the transgene to the F 1 progeny. Due to the wide use of N. clevelandii as a propagation host for most viruses the technique is of potential use in studies aimed at conferring engineered protection against viruses.

A Study on the Possible Role of Auxin in Potato «Hairy Root» Tissues

Summary The physiological role of auxin in the development of «hairy root» tumors was examined with the use of auxin antagonists in transformed and normal roots of potato cultured in vitro. In particular the use of the auxin polar transport inhibitor, TffiA, progressively led to «dedifferentiation » only in «hairy root» tissues. In addition the competitive inhibitors 2,4,6-T, and PCIB counteract the rooting efficiency of the tumors. The present study suggests that auxin, playing a major role in adventitious tumoral root formation, is synthetized at a high rate, but a precise mechanism of differentiation avoids intracellular accumulation.

cDNA cloning of artichoke mottled crinkle virus RNA and localization and sequencing of the coat protein gene

We report the cDNA cloning of the genomic RNA of artichoke mottled crinkle virus (AMCV), which is a member of Tombusvirus group. AMCV has a monopartite positive sense RNA genome, which is not polyadenylated at the 3′ end. The genome size is 4.8 kb.We have localized and sequenced the open reading frame (ORF) encoding the coat protein. Unlike most monopartite positive-strand RNA plant viruses, the ORF is not located near the 3′ end, but like other members of the Tombusvirus group, CyRSV (cymbidium ringspot virus), TBSV-cherry (tomato bushy stunt virus cherry strain) and CNV (cucumber necrosis virus) it starts ca. 2.7 kb downstream of the 5′ end and stops ca. 1 kb upstream of the 3′ end. This ORF predicts a polypeptide chain of 387 amino acids.Comparison of the coat proteins of AMCV, TBSV-BS3, TBSV-cherry and CNV confirms that, within the Tombusvirus group, there exists a high degree of similarity among coat proteins but that this similarity is not uniformly distributed among domains. In particular, the N-terminal region, thought to make contact with the phosphate groups of the viral RNA, and the C-terminal region, considered the most immunogenic portion of the capsid, are found to be the least homologous.

In vitro morphogenesis in the globe artichoke (Cynara scolymus L.).

Abstract The successful in vitro regeneration of shoots from bracts of globe artichoke (Cynara scolymus L.) is reported. Shoot regeneration was induced in bracts collected from very young (1.5–2 cm in lenght) capitula of cv. Romanesco. Bract cultures were maintained on MS medium containing very different hormonal balances. From all combinations of growth regulators utilized, the most rapid and the most prolific callusing response was obtained using a combination of 5 mg/l NAA and 2 mg/l BAP. Shoot differentiation occured after about 5 weeks, starting from the lower region of the bract explant. Shoot elongation was obtained following transfer to MS medium containing 0.5 mg/l IBA and 0.5 mg/l BAP. Considering the previous negative results using several artichoke explants and the positive response of the bracts under very different culture conditions, the importance of explant origin in the in vitro response is emphasized.

Callus formation from isolated globe artichoke (Cynara scolymus L.) suspension protoplasts

Abstract Viable protoplasts were isolated from suspension culture cells of globe artichoke (Cynara scolymus L.). Protoplast yield, cell wall regeneration and cell division were influenced by several factors, e.g. age of the cell culture, enzyme composition, culture density. First cell divisions were observed after 4–6 days of culture. Upon transfer to solid medium, the cell colonies gave rise to proliferating green calli. Globular structures formed on these calli but failed to develop further.

Morphogenesis and Isoperoxidase Characterization in Tobacco «Hairy Root» Régénérants*

Summary The morphogenetic pattern of tobacco «hairy root» regenerants has been investigated under different culture conditions. Under conditions which promote shoot differentiation in expiants from normal plants hairy root regenerants are shown to differentiate roots in vitro . Both regenerants and their in vitro derived roots contain agropine and show a distinctive isoelectrofocusing pattern of the peroxidase enzyme complex. This pattern, unlike that of the normal plant, is characterized by the permanent activation of the slow migrating (acidic) peroxidase G II group.

Procedure for the regeneration of plants from cell suspension protoplasts of tetraploid potato (Solanum tuberosum L.) cv. Desirée

Abstract A routine procedure for the isolation, culture and plant regeneration of a large number of protoplasts from suspension cultures of Solanum tuberosum cv. Desiree has been developed. An optimal protoplast yield of up to 80% was obtained by gently shaking at 30°C overnight the cell suspension with an enzyme mixture (3% cellulase R10, 1% macerozyme R10 and 1% pectinase in 0.3 M Mannitol). The protoplasts cultured at a density of 7 · 104 prot./ml in a modified MS liquid medium supplemented with napthaleneacetic acid (NAA) (2 mg/l), 6-benzylaminopurine (BAP) (0.5 mg/l), 2,4-dichlorophenoxyacetic acid (2,4-D) (2 mg/l) and 0.4 M Mannitol regenerated cell wall within 2 days and they underwent the first division within 3 days. The first regenerated shoot was obtained approximately 3–4 months after protoplast isolation. The critical factors seem to be associated with the quality of the culture and the enzyme composition used for protoplast isolation rather than with specific culture conditions for the protoplasts.