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This report discusses the successful cryopreservation of almond and apple apices using encapsulation and desiccation techniques. The study demonstrates the survival and regrowth of the apices after cryopreservation, providing potential for preservation of clonal genotypes and genetic resources of these economically important species.
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CRYMCEPT REPORT PARTNER 5
Partner 5 Team and Working Package WP2;WP5; WP6; WP8 FRUIT TREES INSTITUTE (Dr. CARMINE DAMIANO) SUBCONTRACTOR WP2 UNIVERSITY OF ROME “TOR VERGATA” (Prof.CINZIA FORNI) WP5;WP6UNIVERSITY OF ROME “TOR VERGATA” (Prof. SIMONE BENINATI)
Partner 5 WP2 WP5 WP6 WP8 Provision of plant material and cryopreservation of shoot tip Dr. C. Damiano
INTRODUCTION… Almond and Apple are species of great economic importance in temperate regions. Usually, these species can be both propagated vegetatively to maintain clonal genotypes and genetic resources and traditionally maintained as whole plants in field genebanks
INTRODUCTION In vitro cultures of almond (rootstock M51, cv Supernova) and apple (cv Annurca, cv Gala) used in this study were obtained from apices, sampled from one-year old plants belonging to the germplasm collection of the Fruit Trees Institute and introduced in vitro.
MATERIAL & METHODS… ALMOND: In vitro plantlets of rootstock M51 were cultured on a modified (by adding thiamine) MS medium containing 30 g/l sucrose, 0.3 mg/l benzyl adenine (BA) and 0.05 mg/l indole acetic acid (IAA), solidified with 3.8 g/l agar and 8.0 g/l pectin. Plantlets of the cv. Supernova were cultured on the same medium but with 0.2 mg/l BA. APPLE : In vitro plantlets (cv Annurca, cv Gala) were grown on MS a medium containing BAP 0.5 mg/l, IBA 0.1 mg/l, and 6 g/l agar. Plants were grown at 24±1oC, 16h light/8h dark, light intensity of 37 µmol.m-2.s-1. Subcultures were made every 15 d.
MATERIAL & METHODS… After dissection, the apices (2-3 mm long) were encapsulated in 3% alginate beads according to the method of Dereuddre et al. (1990). Encapsulated apices were pregrown for different periods (from1 to 7 days) in liquid medium added with sucrose concentrations ranging between 0.5 M and 1.25 M.
MATERIAL & METHODS… Desiccation was performed by placing the beads in air-tight containers (3.5 x 4.5 cm; 5 beads per container) with 8 g silica gel for 0 to 30 h. After desiccation, beads were transferred to standard medium (desiccation controls) or placed in 2-ml sterile polypropylene cryotubes (10 beads/tube) and immersed rapidly in liquid nitrogen (LN), where they were kept for at least 72 h.
MATERIAL & METHODS Normally developed apices (i.e. production of new leaves and/or expansion of a small shootlet) 21 d after freezing were considered regrown. Thirty out of 36 apices were used for each experiment.
RESULTS… Almond: the highest survival rate was obtained for apices pretreated for 3 days with 0.75M sucrose and desiccated down to a bead MC of 20.3% (9 hours). Apices, which were alive after cryopreservation remained green and started producing new shoots within one week. However, they could grow and develop into whole plantlets only if they were extracted from the bead and inoculated directly on culture medium.
RESULTS… Apple: The best combination, after the freezing, was that with 0.75M of sucrose for 1 day of preculture. Instead in the combinations with 5 and 7 days of preculture, the explants died and they didn’t grow after treatment with liquid nitrogen. As regards the dehydratation, the best period was that of 14 hours, equivalent to a water content of 19%.
RESULTS Cryopreservation techniques Regrowth
CONCLUSIONS Next steps: Apices sampled after encapsulation/dehydration for analyses Cryopreservation with vitrification techniques Innovative cryopreservation systems
Provision of Plant Material: Main Focus To set up and eventually modified protocol of analyses on plant material Apple cv Annurca The apices collected from the in vitro plantlets of were provided to the subcontractor WP2 (proteins determination), WP5 and WP6 (transglutaminases and polyamines) for further analyses
Partner 5 WP2 Proteins Prof. C. Forni
Plant material Main focus on Apple cv. Annurca. Efforts have been made to use the same sample for SDS gel electrophoresis (WP2) as well as for polyamine(WP5) and transglutaminase (WP6) analyses. The protein work started with the optimization of protein extraction method .
Protein analyses Standard extraction protocol has been modified to avoid the presence of substances which may interfere with the analyses, such as phenolic compounds and polysaccharides. The extraction protocol allow us to get results from 200 mg of fresh materials. A reliable separation of protein in SDS-PAGE has been obtained.
Protein analyses: Methods Extraction 200 mg of plant material Problems to be solved Solution adopted Phenols interference Homogenization step in PVPP (Sigma) Dialysis overnight against 50 mM Tris-HCl Buffer, pH 7 Polysaccharides interference
Same sample for protein, polyamine and TGase Extraction 200 mg of plant material Changes made: extraction buffer solution suitable for protein extraction, polyamine and TGase 0.5 M Tris-HCl pH 7 Sampling for the other analyses made After homogenization in PVPP, centrifugation and sonication
Main results 1 D SDS- PAGE Pattern obtained from 200 mg of plant material avoiding protein precipitation with TCA, which interfere with polyamine analysis
Partner 5 WP5 Polyamine analysis Prof. S. Beninati
POLYAMINE ANALYSIS… The first target of the project has been reached developing a new assay for free and protein-bound polyamine in the extracts of apple shoot apex. The technique includes the amine derivatizaton with o-phtalaldehyde and the HPLC separation of the fluorescent derivatives (method in publication). Calibration curves have been elaborated in order to obtain reliable results even for minimal amount of polyamines. The actual sensitivity is in the range of 5-10 pmoles per mg of protein. The method will be further applied to frozen material, and the data compared with the control samples.
POLYAMINE ANALYSIS… The chromatographic analysis performed in plantlet extracts revealed the presence of putrescine (1,15 nmoles/mg protein), spermidine (64,17 pmoles/mg protein) and spermine (12,02 nmoles/mg protein). Only spermidine levels were detected in shoot apex extracts (23,74 pmoles/mg protein), and the amount of both putrescine and spermine was undetectable.
HPLC determination of polyamines in apple plantlet and shoot apex extracts
Partner 5 WP6 Transglutaminase analysis Prof. S. Beninati
TRANSGLUTAMINASE ANALYSIS… The first step was to determine the presence of transglutaminase activity in the apple plantlet and shoot apex extracts, using an in vitro enzyme assay. The enzyme activity found in plantlet was 1,16 pmoles of [3H]-putrescine incorporated/mg protein, whereas in shoot apex was 0,9 pmoles of [3H]-putrescine incorporated/mg protein.
TRANSGLUTAMINASE ANALYSIS… The second experimental step has been the application of the in vivo transglutaminase assay in the shoot apex. The samples were incubated with [3H]-putrescine for about 20 hours at 25 °C and the substrates modified by the endogenous transglutaminase have been analyzed.
TRANSGLUTAMINASE ANALYSIS… The enzyme activity found is 0,067 pmoles of [3H]-putrescine incorporated/mg protein. This activity is sufficient to hypothesize the post-translational modification of the substrate protein by transglutaminase. This type of assay will be suitable to determine also the content of g-glutamyl-polyamines, the activity of the endogenous PAO (polyamine oxidase) and the metabolism of the free polyamines.
Transglutaminase activity in apple extracts pmol 3H-put /mg protein Plantlet Apex In vitro assay In vivo assay
THANKS FOR YOUR ATTENTION Partner 5 CRYMCEPT ROME, NOVEMBER 2003 WP2 WP8 WP5 WP6
