WP9 Analysis for cold tolerance in oat P8- Luigi Cattivelli and Fulvia Rizza, Experimental Institute for Cereal Research,via S.Protaso 302, Fiorenzuola d’Arda (PC), Italy. P9 – Daniela Murariu, Suceava Genebank, Romania AVEQ - Clermont-Ferrand, September2007 Valeria Terzi

Analysis for Cold Tolerance Field testing to explore the genetic variability for cold tolerance in winter (and spring) oats and to identify genotypes with superior frost tolerance will be employed. The experiment will be carried out using the project working collection developed within the project. A field evaluation of frost tolerance will be carried out in a nursery of small plots (1 m2) for 2 years in different locations (Fiorenzuola d’Arda –North Italy; Suceava Genebank – Romania) to control the environmental effect over the genetic expression of the single genotype.

Among winter cereals, oat is the most frost sensitive and its insufficient level of winterhardiness is the main factor limiting sowing and yield of winter oat in Northern Italy and Central Europe.

WINTERHARDINESS IN OAT: IS THERE GENETIC VARIABILITY FOR THIS TRAIT?

Sixty-two genotypes with different origin (Europe, North America) were grown in Northern Italy (Fiorenzuola) in 1996-’97 Frost damage was estimated by a visual score (0: no damage 9: all plants killed)

A BROAD GENETIC VARIABILITY FOR WINTERHARDINESS EXISTS IN OAT Range between 2.5 and 8.59 (mean 5.8)

Frost reduces yield potential of winter genotypes LSD 0.05 Highest yield potential is expressed in winter sowing, however only highly resistant cvs yielded more in winter than in spring sowing. The extent of yield decrease in winter sowing was related to the degree of frost injury detected in field

. Frost resistance, the most important component of winterhardiness, is an inducible process, promoted by cold acclimation (HARDENING) at low non-freezing temperature and associated with a number of biochemical and molecular changes

How to measure frost resistance?

LOW TEMPERATURE and LIGHT INTENSITY affect the PSII excitation pressure which plants are exposed to and, consequently, their tolerance to stress

Explore the genetic variability for PSII stability and frost resistance in a set of oat cvs

Chlorophyll fluorescence responds to changes in PSII photochemistry and therefore represents a convenient and rapid tool to evaluate the capacity of the photosynthetic machinery at low temperature. F v F m -F = ---- F m F m The ratio of variable to maximal fluorescence (F v /F m ) in dark-adapted state measures the maximum quantum yield for PSII photochemistry and represents a diagnostic probe for measuring low temperature stress-induced injury of photosynthesis = 0.8 in dark-adapted health plants

Experimental unit (PAM 2000) Rapid Reliable Sensitive, Not destructive

The ratio Fv/Fm was determined to evaluate the frost resistance of 16 cultivars of different origin, growth habit and year of release 1 AINTREE GBR W 2 ARGENTINA ITA W 3 BULBAN AUS S 4 COKER USA W 5 DONATA ITA W 6 EMPEROR GBR W 7 FLAVIA ITA W 8 FULVIA ITA W 9 GENZIANA ITA S 10 ORIGINE FRA W 11 PREVISION ESP W 12 PRIMULA ITA S 13 ROGAR 8 ITA W 14 TROPICALE FRA S 15 WEIBULL SWE S 16 YEATS USA W The standard treatment: 1 week-old plants were hardened at 3-1°C (day/night) 200  E m -2 s -1 for 4 weeks and subjected to a freezing treatment (–12°C, –13°C)

The maximum quantum yield of the Photosystem II (PSII) photochemistry was evaluated during hardening and after freezing, through the chlorophyll fluorescence parameter F v /F m, Rizza et al. (Plant Breed.120: , 2001) days Fv/Fm EFFECT OF A HARDENING TREATMENT (4 weeks at 3-1°C) ON Fv/Fm (mean ± stand.dev for 16 oat genotypes) EFFECT OF A FREEZING STRESS ( –12°C) ON Fv/Fm (mean ± stand.dev for 16 oat genotypes) HARDENED NON HARDENED

a) Plants at first-leaf stage hardened at 3-1°C, 200  E m -2 s -1 4 weeks 6 cvs showed the highest F v /F m : ‘Coker’ ‘Donata’ ‘Emperor’ ‘Flavia’ ‘Fulvia’ ‘Yeats’ Only 4 maintained a value close to 0.8 Lsd 0.05 A (genotype) Lsd 0.05 A (genotype) cvs showed the highest F v /F m (for 3 it was close to 0.8): ‘Coker’ ‘Donata’ ‘Emperor’ ‘Yeats’ winter spring Lsd 0.05 A (genotype) 0.12 Lsd 0.05 A (genotype) 0.17

A CRUCIAL ASPECT OF STRESS RESISTANCE IS THE CAPACITY OF A PLANT TO RESPOND EARLY TO AN ENVIRONMENTAL STIMULUS

b) Plants pre-hardened at 12-7°C, 200  E m -2 s -1, 4 weeks (second-third leaf) 6 cvs showed the highest Fv/Fm (close to 0.8) ‘Aintree’ ‘Coker’ ‘Donata’ ‘Emperor’ ‘Flavia’ ‘Yeats’ 3 cvs showed the highest Fv/Fm (close to 0.8): ‘Coker’ ‘Donata’ ‘Emperor’ ‘Origine’ ‘Yeats’ Lsd 0.05 A (genotype) 0.14 Lsd 0.05 A (genotype) 0.17

16 oat genotypes Data were highly correlated in oat, suggesting that the capacity to respond precociously to an environmental stimulus (like a moderate drop in temperature) has an important role in the acquisition of the tolerance to a more severe stress

C) Plants pre-hardened at 12-7°C, 700  E m -2 s -1, 4 weeks (second-third leaf) The improvement of the PSII stability after freezing was especially evident in the cultivars with intermediate level of resistance Lsd 0.05 A (genotype) 0.13 Lsd 0.05 A (genotype) 0.15

Pre-hardened plants 12-7°C, 200  E Pre-hardened plants 12-7°C, 700  E

Growth at 12/7°C, associated to higher light intensity (700  E) implied a morphological adaptation in frost resistant and frost sensitive cultivars Frost resistantFrost sensitive

Molecular analysis: COR14 accumulation in pre-hardened plants 1 week at 12-7°C4 weeks at 12-7°C 200  E m -2 s  E m -2 s -1 P R W C D Y SUSCEPTIBLE RESISTANT P: ‘Primula’ C: ‘Coker’ R: ‘Rogar 8’ D: ‘Donata’ W:‘Weibull’ Y: ‘Yeats’

HOW DOES A RESISTANT OAT BEHAVE WITH RESPECT TO RESISTANT CVS OF OTHER CEREALS SPECIES? 1 PICASSO rye 2 CHEYENNE wheat 3 OFANTO durum wheat 4 AUBRAC triticale 5 NURE barley 6 PAMINA barley 7 COKER oat

Rye wheat durum triticale barley barley oat wheat mean of three experiments (-14, -15°C) Survival rate % a) Plants at first-leaf stage hardened at 3- 1°C, 200  E m -2 s -1 4 weeks b) Plants pre- hardened at 12-7°C, 200  E m -2 s -1, 4 weeks C) Plants pre- hardened at 12-7°C, 700  E m -2 s -1, 4 weeks mean of three experiments (-5, -6°C) mean of three experiments (-5, -6°C)

Rye Triticale Wheat Barley Oat Durum Wheat Rye Triticale Wheat Barley Oat Durum Wheat 12/7°C 200  E12/7°C 700  E

CONCLUSION AND PERSPECTIVES Significant differences in frost resistance were detected within winter cultivars The most resistant showed a behaviour comparable to that of a winter resistant barley, suggesting that it should be possible to improve the resistance of winter oat to a level similar to that of winter barley. Basis for a possible winter sowing in oat and for breeding. The freezing evaluation test represents a useful tool for the evaluation of frost tolerance AVEQ project is a unique opportunity to evaluate biodiversity for this trait.

PEOPLE INVOLVED IN FROST TOLERANCE STUDIES Fulvia Rizza Luigi Cattivelli Donata Pagani Fabio Reggiani Cristina Crosatti