RESEARCH PROJECTS

BELARUS

Research Project

Title: Breeding programme for early bearing, high yielding, winter resistant, diseases tolerant walnut varieties (Juglans regia L.) previously for home garden producing quality nuts.

Project objectives: The Belarus climate is very difficult for walnut-growing: a short frost-free season (from 140-147 days in the South-west to 160-170 days in the North-east); cool summers (sum of active temperatures >10°C are 1900-2000°C in the South-west and 2400-2500°C in the North-east); extremely variable and cold winter temperatures (minimum temperatures in critical winters reach -34°C to -36°C in South-west region, and -39°C to -44°C in the North-east).

The aim of research was the introduction of walnut in Belarus at individual horticulture level.

Project summary and results: More than 2.000 local genotypes of Juglans regia L., over than 600 of their own seedlings and more than 150 precocious walnut forms (J. regia f. fertilis Petz. et Kirch) from Ukraine, Tadzhikistan, Lithuania and their own progenies have been studied. The main biological particularities of these walnut trees, quality and chemical content of their nuts have been valued. More than 200 very hardy to low temperatures individuals were selected among these trees.

Some of these walnuts set nuts without pollination. 75 % of seedlings of such plants set fruits without pollination too. Apomixis has the great importance for walnut in regions with cold winters.

The nut kernel of walnut has a rich nutrient content in Belarus too. Differences between the chemical content of species and seedlings were observed. This chemical content of the nut kernel is taken into consideration in our selecting programme.

Precocious forms of walnut (J. regia f. fertilis Petz et Kirch) are very interesting for home gardens. They differ in winter resistance duration of life, time of fruit bearing, time of coming and passing the phenophases, types of flowering, morphological characteristics of trees, of nuts, etc.

Related publications:

LOIKO R.E., 1989. Zymaustoilivast arecha greckaga u Belarusy. Vesci AN BSSR. Ser. s.-g. navuk 4:79.85 (in bellarussian)

LOIKO R.E., 1991. Grecky orech v Belorussyi i drugych raionach severnogo plodovodstva. Minsk. -59s (in russian)

LOIKO R.E., SZYRCO T.S., 1994. Mineralnyi sostav iadra plodov nekotorych vydov roda Orech v Belarusy i na Ukraine. Fiziologia i biochimia kulturnych rastenyi, 26(2):165-169 (in russian)

LOIKO R.E., SZYRCO T.S., KOROTKEVITCZ A.I., KUCHTA P.N., 1996. Chimitczeskyi sostav plodov Juglans regia L. v Belorussyi. Rastitelnye resursy. 26(2): 160-169 (in russian)

LOIKO R.E., 1996. Apomixis of walnut in Belarus. Collection of Scientific Articles. Jelgava. 1996:187-191

LOIKO R.E., 1997. Osobennosty cvetenia i plodonoszenia scoroplodnych form orecha greckogo v Belarusi. Plodovodstvo. Naucznye trudy BelNIIP. Minsk.1997, 11(1):62-76 (in russian)

LOIKO R.E., 1997. Osobennosty rosta i razvitia orecha greckogo v Belarusi. Plodovodstvo. Naucznye trudy BelNIIP. Minsk 1997, 9(1):83-95 (in russian).

LOIKO R.E., 1998. Perspectives on Walnut culture in Belarus. 89th Ann. Rept. of the Norhern nut growers Ass: 147-148

BELGIUM

Research Project

Title: Physiological study of rooting of walnut shoots in vitro and biochemical relationship between root system and wood quality

Project objectives: The lab of hormonology is from its origin involved in studies on the hormonal control of growth and development process. The physiological roles of peroxidases have been extensively investigated in several physiological processes in the interplay between different hormone types, which has allowed the lab to consider the conditions under which these enzymes can be considered as markers.

The first objective was a physiological study of the rooting process in walnut shoot in vitro and in a second time it was a characterisation of walnut quality in relation to culture conditions and a research of relationship between development of the root system and shoot development and wood formation.

Project summary and results: Rooting of micropropagated walnut shoots was achieved through the use of two successive rooting media: a rooting inductive medium with auxin (under darkness) followed by a gelrite-vermiculite medium without auxin (in the light). The rooting process can be divided in a series of interdependent physiological phases and the measurement of endogenous content of growth regulators and specific enzyme activities have been done during the time-course. An early and temporary increase in the endogenous level of IAA and putrescine was the major event of the inductive phase of adventitious rooting since it appears necessary for the reactivating of cell divisions. The auxin treatment induced the rooting process, which is also related with ethylene emission, PAL and peroxidase activities. This treatment favoured also lignin formation. There is a positive relationship between root emergence and lignification.

Related publications:

BISBIS B., KEVERS C., 1998. Changes in phenylammonia lyase and peroxidase activities associated with lignin formation and rooting in walnut shoots in vitro. Bull. Soc. Royale des Sciences de Liège. 67:214

BISBIS B., KEVERS C., DOMMES J., GASPAR Th. Biochemical events related to lignin accumulation by micropropagated shoots during the rooting process. Submitted.

GATINEAU F., FOUCHE J.G., KEVERS C., HAUSMAN J.F., GASPAR Th., 1997. Quantitative variations of indolyl compounds including IAA, IAA-aspartate and serotonin in walnut microcuttings during root induction. Biol. Plant. 39:131-137

GATINEAU F., KEVERS C., GASPAR Th., 1994. Early changes in the serotonin content of walnut shoots during their rooting inductive phase in vitro. Arch. intern. Physiol. Biochim. Biophys. 102:B76

HELOIR M.C., KEVERS C., HAUSMAN J.F., DELTOUR R., GASPAR Th., 1994. Histological origin of roots of micropropagated walnut (Juglans regia) shoots. Arch. intern. Physiol. Biochim. Biophys. 102: pp 5

HELOIR M.C., KEVERS C., HAUSMAN J.F., GASPAR Th., 1996. Changes in the concentrations of auxins and polyamines during rooting of in vitro-propagated walnut shoots. Tree Physiol. 16: 515-519.

KEVERS C., BRINGAUD C., HAUSMAN J.F., GASPAR Th., 1997. Putrescine involvement in the inductive phase of walnut shoots rooting in vitro. Saussurea 28: 47.57

RIPETTI V., KEVERS C., GASPAR Th., 1994. Two successive media for the rooting of walnut shoots in vitro. Changes in peroxidase activity and in ethylene production. Advances Hortic. Science. 8:29-32

BULGARIA

Research Project

Title: Breeding and studies of walnut cultivars

Project objectives: The local walnut cultivars, commonly found in Bulgaria, are characterized by economically valuable fruit traits, but their potential of productivity is limited for the apical type of their bearing. The new Bulgarian cultivar Izvor 10 exhibits a lateral type of bearing, but it is relatively early leafing and its fruits are not large enough. Most of the cultivars, introduced from the U.S.A., are early leafing and very sensitive to spring frosts, when they grow under the Bulgarian conditions. Therefore it is necessary to carry out a breeding programme for developing new cultivars with lateral bearing, late bud-break, disease resistance and valuable economic traits of fruits. The other line in the programme was to introduce cultivars, possessing the above-mentioned characters and suitable to be grown under the Bulgarian conditions.

For the differences in the rootstocks recommended as most suitable for Bulgaria and abroad, it became necessary to conduct a trial on rootstocks specific for the conditions of Bulgaria. In this connection, the local Bulgarian dwarfing form IP-R1 of the Pendula type, is a good compromise for being used both as a rootstock and a donor at sexual hybridization.

To speed up the propagation of valuable hybrids, new cultivars and virus-free plants, it was necessary to use new biotechnological approaches, one of which is the in vitro method. The difficulties and the low efficiency of sexual hybridisation necessitated the in vitro cultivation of embryo cultures to guarantee the production of hybrid plants, necessary for the work of breeding improvement.

The studies, related to the arguments noted above, are still going on, in addition to the starting of new investigations aimed at achieving the following:

1. use of sexual hybridisation and open-pollinisation for producing hybrids with lateral fruit-bearing, late leafing out, disease resistance and economically valuable fruit traits;

2. trials for the rootstocks J. regia, J. nigra, J. hindsii and J. rupestris concerning their effect on cv. Izvor 10 during the period of bearing;

3. anatomical and physiological investigations of the unique form IP-R1 in connection with its use in sexual hybridisation;

4. development of a technological scheme for micropropagation and embryo culture in vitro

Project summary and results: During the first stage of the research work in Bulgaria (1961-1977), a study was conducted on the local walnut plant resources, in result of which 8 Bulgarian cultivars were examined and approved for distribution. They were characterized by economically valuable fruit traits, but they had limited potential productivity for the apical type of bearing. In this connection, during the last years, the Bulgarian cultivar Izvor 10, characterized by a lateral fruit formation of up to 80 %, was developed and commonly distributed.

In result of the breeding improvement work during the period 1976-1986, 423 walnut hybrids were produced and grown. Among them, the hybrids Nos 315, 224 and 1694 were selected in order to be studied and used as source parents in the present work.

Today, a new collection of 386 hybrids, produced after starting of the new breeding programme in 1990, is being maintained. Some of these plants have already started bearing. In result of the studies, 18 seedlings with late flowering, 22 with early bearing initiation, 7 with early spring frost resistance, and 5 with less vigorous growth, were selected. The valuable hybrids Nos 315, 224 and 1694 are going to be comparatively studied in cultivars testing stations, which is a necessary condition for their recognition as walnut cultivars in Bulgaria.

Today, on the basis of the rich germplasm of local and foreign cultivars, a breeding work for new cultivars development is going on. Valuable Bulgarian and foreign cultivars, including cv. Izvor 10, the American cv. Pedro, the promising hybrids No. 315 and No. 1694 and the unique dwarfing form IP-R1 are being used as source parents.

In terms of the rootstock-combinations, the results from the 15-year investigations showed an equal effect of the rootstocks J. hindsii and J. regia on the vegetative and reproductive behaviour of cv. Izvor 10. At this stage, no incompatibility between the tested rootstocks and the studied cultivars was established.

The comparative characteristics between cv. Izvor 10 and the IP-R1 form showed differences in the transpiration and photosynthetic intensities. The higher aminoacid composition of the IP-R1 fruit is interesting. Isoenzyme analyses are currently conducted.

Some parameters for in vitro cultivation of cv. Izvor 10 and the form IP-R1, such as the nutrient media and the conditions for microplant growing at the stage of multiplication, were developed. The main problems arose at the stages of sterile culture initiation and the rooting of propagated plants. In this connection, studies are currently conducted on antioxidants and techniques of setting up explants under in vitro conditions, as well as on plant growth substances and methods of microplant growing at the stages of rooting and adaptation to in vivo conditions.

The work on in vitro cultivation of walnut embryos is at an advanced stage of development. The optimum time for sterile culture initiation of immature embryos, as well as the suitable nutrient media and growing conditions during multiplication, were established. Studies on optimising the parameters during the stages of rooting and adaptation are forthcoming.

Related publications:

KAVARDJIKOV L., KRINKOV H., 1994. Fruit growing - walnuts. Agromagazin, n° 7-8

KORNOVA K., STEPHANOVA A., 1992. Possibilities for developing embryo cultures of Juglans regia L., Second National Scientific Session "Application of in vitro Techniques in Agricultural Crops", Plovdiv, November 1992:175-179

KORNOVA K., STEPHANOVA A., TERZIJSKY D., 1993. In vitro culture of immature embryos and cotyledons of Juglans regia L. Morphological and anatomical analyses of some regenerants. Acta Horticulturae, 311:125-133

KORNOVA K., VISHANSKA J., PENEV P. STEPHANOVA A., 1998. Walnut (Juglans regia L.) micropropagation. IPPS in Bulgaria. Third Scientific Conference, "Propagation of Ornamental Plants", Sofia, Forest University, October 1998: 219-224.

STEPHANOVA A., KORNOVA K., TERZIJSKY D., 1987. Preliminary investigations into the possibilities of somatic embryogenesis and regeneration of plants from cotyledons with local walnut tree varieties. Scientific Works of the Higher Institute of Agriculture, Plovdiv, 32 (2): 141-145.

STEPHANOVA A., TERZIJSKY D., KORNOVA K., 1989. Deviation in regenerants obtained in vitro from embryos and cotyledonous walnut (Juglans regia L.) tissue. Scientific Works of the Higher Institute of Agriculture, Plovdiv, 34 (1): 165-170

STEPHANOVA A., TERZIJSKY D., KORNOVA K., 1998. Electron microscope studies on degeneration processes in primary callus during somatic embryogenesis in walnut (J. regia L.). Collection of papers, presented at the Jubilee Scientific Session "50th Anniversary of the Union of Scientists in Bulgaria- Plovdiv on "Science at the Threshold of the New Millenium", vol 1: 87-90.

FRANCE

Research Project

Title: Intraspecific hybridization breeding programme for late flowering, early bearing, high yielding blight tolerant walnut varieties (Juglans regia L.) producing quality nuts.

Project objectives: During the last 30 years, a study carried out on more than 100 foreign varieties representing almost all the main cultivars in the world, has proved disappointing in France. Their environmental adaptation is often poor due to early bud break and high sensitivity to walnut blight. As for Franquette, the best French variety, they bear nuts relatively late and have a medium production potential. Consequently the aim of the breeding programme carried out in Bordeaux is to associate in a same cultivar, late leafing, lateral fruitfulness, blight tolerance and high nut quality.

Project summary and results: At first (1977-1988) French varieties with late leafing and producing quality nuts were used as female parents. They were crossed with Californian cultivars which have a relatively early bud-break, are lateral bearers and therefore they are very productive with early fruiting. During a second step (1987-1992), crosses were made between the best hybrids obtained and Californian varieties. For each cross, parents were chosen in such way as to minimize inbreeding which has strong depressive effect in walnut. Since 1992, new crosses were made between Franquette and the best hybrids obtained as female parents and lateral bearing genitors from Central Asia and Mediterranean Basin as male parents.

42 crosses have been made so far and 5200 seedlings obtained. A selection for late leafing was made in nursery. 47% of these seedlings ranged for medium late to very late leafing and were planted in selection plots on their own roots. To date, 2000 hybrids have been observed, each cross represented by about 100 individuals. Among these hybrids 10 to 75% showed a lateral fruitfulness according to the fruiting type of genitors. They bore nuts early, usually as early as in their third year after planting, and most of them produced catkins in their 6th year. Therefore a preselection based on low blight sensitivity and quality nut production can be made as early as the 7th or 8th year after planting.

Among the 1800 hybrids already studied 97 individuals have been preselected for the desired characters. They have been planted in the main nut growing areas in France for further observations. The first varieties issued from this programme have been released in 1995, i.e. Fernette and Fernor, and in 1998, i.e. Ferjean. Half a dozen other preselections will be released before 2010.

Related publications:

GERMAIN E., 1979. Création par hybridation de variétés de Noyer (Juglans regia L.) associant une floraison tardive à une mise à fruit très rapide et une productivité élevée. Exposé d'une méthode. Ann. Amélior. Plantes 29 (2): 187-199.

GERMAIN E., LEGLISE P., BAYOL M., 1985. Création, par hybridation de variétés de noyer (Juglans regia L.) associant floraison tardive, mise à fruit rapide et productivité élevée. Premiers résultats. 5ème Colloque Recherches Fruitières, Bordeaux (France), Nov. 1985: 157-174.

GERMAIN E., 1988. Use of the late leafing character in a walnut variety breeding program. Intern. Conf. on Walnut, Yalova (Turkey), Sept. 1988: 95-98.

GERMAIN E., 1990. Inheritance of late leafing and lateral bud fruitfulness in walnut (Juglans regia L.). Phenotypic correlations among some traits of the trees. 1st Intern. Symp. on Walnut Production. Budapest (Hungary), Sept. 1989. Acta Hort. 284: 125-134.

GERMAIN E., 1992. Ereditarieta dei caratteri fogliazione tardiva e fruttificazione laterale nel noce (Juglans regia L.). Frutticoltura, 1: 55-60

GERMAIN E., 1992. Le noyer. In "amelioration des espèces cultivées: objectifs et critères de sélection". Gallais A., Bannerot H. ed.: 620-632, 650-651

GERMAIN E., CHARLOT G., PRUNET JP, 1996. Fernor et Fernette: deux nouvelles variétés, un atout pour la nuciculture française de l'an 2000. Infos Ctifl, 124: 27-29

GERMAIN E., CHARLOT G., PRUNET JP, 1997. Fernor e Fernette: due nove varieta di noce licenziate in Francia. Rivista di Frutticoltura, 4: 86-87

GERMAIN E., GARCIN A., PRUNET JP, 1997. Noix: 2 nouvelles variété: Fernette et Fernor. Fruits et Légumes, 149: 27-28

GERMAIN E., 1997. Effets dépressifs de la consanguinité chez le noyer Juglans regia L. In "Amelioration d'espèces de fruits à coque: noyer, amandier, pistachier". Options Méditerranéennes, Série B, 16: 49-52

GERMAIN E., 1997. Genetic improvement of the Persian Walnut (Juglans regia L.). 3RD International Walnut Congress, Alcobaca (Portugal), 13-16 June 1995. Acta Hort., 442: 21-31

GERMAIN E., 1997. Fiche variétale Fernor. Arbo. Fruit. 502

GERMAIN E., 1998. Fiche variétale Fernette. Arbo Fruit. 518

GERMAIN E., 1999. Fiche variétale Ferjean. Arbo Fruit. 530

Research Project

Title: Selection of walnut fruit varieties resistant (hypersensitive) to cherry leaf roll virus

Project objectives: Transfer hypersensitivity to cherry leaf roll virus from black walnuts, J. nigra and J. major, into Persian walnut J. regia.

Project summary and results: Hypersensitivity to cherry leaf roll virus appears to be absent from the Persian species (J. regia) but present in all black walnut species. Hypersensitivity is inherited as a single, dominant gene. In order to introduce the hypersensitivity into J. regia varieties without including undesirable traits from the black walnut species, crosses followed by several backcrosses have been made.

In each back-cross generation, half of the seedlings are hypersensitive and therefore are potential parents for the next generation. Screening methods for hypersensitivity among seedling populations including inoculation of the virus through grafting and immunodetection of the virus have been used since 1998. These methods are labour-intensive and time-consuming. Attempts to use the molecular markers linked to hypersensitivity and described by Woeste et al (1996) are under way. Among the hypersensitive individuals, phenotype selection includes both vegetative (late budburst) and reproductive traits (production, quality of the nut).

The backcross-breeding programme began in 1987 with a population of 630 first backcross (BC1) walnut hybrids. Until now, both interspecific crosses and backcrosses have been made under open-pollination conditions. Today, seven populations of second backcross (BC2) including 241 individuals are currently observed: [(J. nigra ´ J. regia) ´ J. regia) ´ J. regia: 4 progenies and 96 individuals, (J. major ´ J. regia) ´ J. regia) ´ J. regia: 3 progenies and 145 individuals].

The third backcross is planed in 2002-2003. Pollination will be made under controlled conditions and will involve a lateral fruitfulness variety as the male parent.

BC2 plant material under study:

Related publications:

DOSBA F., LANSAC M., GERMAIN E., MAZY K., ROVIRA M.1990. Le virus du cherry leaf roll (CLRV): relation avec le dépérissement du noyer et comportement de différentes espèces ou hybrides interspécifiques de Juglans. Fruits 45(2):171-175.

DOSBA F., GERMAIN E. 1993. Behaviour of the progeny of Juglans interspecific hybrids towards cherry leaf roll virus (CLRV). II Intern. Walnut Meeting, Tarragona (Spain), 21-25 Oct. 1991. Acta Horticulturae 311:68-72.

Mc GRANAHAN G., LESLIE C., WOESTE K., 1997. Back-cross breeding walnuts for resistance to the cherry leaf roll virus. 3rd Intern. Walnut Congress, Alcobaca (Portugal), 13-16 June 1995. Acta Horticulturae, 442:121-127

WOESTE K., McGRANAHAN G., BERNATZKY R. 1996. The identification and characterization of a genetic marker linked to hypersensitivity to the cherry leafroll virus in walnut. Molecular Breeding 2:261-266.

Research Project

Title: Selection of walnut rootstocks vigorous and tolerant to cherry leafroll virus

Project objectives: To breed a rootstock which benefits from the enhanced capacity of growth of the interspecific hybrid without risking the walnut black line disease.

Project summary and results: New varieties, i.e. 'Fernette', 'Fernor' and 'Ferjean', adapted to the French growing conditions have been released from the French breeding program since 1995. They show a high percentage of lateral bud fruitfulness and come into commercial production about twice as fast as traditional French cultivars. They are characterized by a very high productivity. A vigorous rootstock providing a good balance between production and vegetative growth during all the life of the tree is needed for these lateral fruitfulness varieties. The interspecific hybrids between Black walnuts and Persian walnut have this good level of vigor to be used as rootstock but, associated with lateral fruitfulness varieties, they show a major drawback. With these new varieties, flowering is early, and therefore infection with cherry leafroll virus, may occur at a very young stage. After infection, cherry leafroll virus can invade the tree very rapidly due to its small development and cause fatal necrosis of the graft union very early in the life of the orchard. Consequently, the ideal rootstock for these lateral fruitfulness varieties would be one with the response to black line of J. regia, i.e. tolerance to cherry leafroll virus, combined with the vigour of interspecific hybrids.

Tolerant BC1s are subjected to a phenotypic selection in nursery on the following traits: vigour, late budburst, apical dominance and few branching. Due to the dominance of the hypersensitive gene, all the J. nigra ´ J. regia interspecific hybrids are hypersensitive. In the first backcross (BC1) with J. regia, only half of the progeny inherited the hypersensitivity of J. nigra. The current procedures for identifying hypersensitivity versus tolerance backcross progeny include grafting onto J. regia rootstocks, inoculation of the rootstock with the virus through grafting and immunodetection of the virus within the rootstock. The progenies are identified as hypersensitive or tolerant depending on the presence or absence of virus-induced black line and/or immunodetection of the virus within the scion. These methods are labour-intensive and time-consuming. Attempts to use the molecular markers linked to hypersensitivity and described by Woeste et al (1996) are under way.

Since 1987, 632 BC1s (306 from J. nigra and 326 from J. major) originated from 22 combinations have been observed. 44 BC1s have been selected in nursery. Among 23 BC1s tested against hypersensitivity, 15 were identified as tolerant and 13 have been propagated in vitro. 7 BC1s have been favourably evaluated against Agrobacterium and Phytophthora. The first rootstock trial has been planted in 1999 near Bordeaux.

Related publications:

DOSBA F., LANSAC M., GERMAIN E., MAZY K., ROVIRA M.1990. Le virus du cherry leaf roll (CLRV): relation avec le dépérissement du noyer et comportement de différentes espèces ou hybrides interspécifiques de Juglans. Fruits 45 (2): 171-175.

DOSBA F., GERMAIN E. 1993. Behaviour of the progeny of Juglans interspecific hybrids towards cherry leaf roll virus (CLRV). II Intern. Walnut Meeting, Tarragona (Spain), 21-25 Oct. 1991. Acta Horticulturae 311: 68-72.

GERMAIN E., DOSBA F., FOS E. 1994. Création de nouveaux porte-greffe de noyer vigorisants et tolérants au virus du cherry leaf roll. 10ème colloque sur les Recherches Fruitières, Angers 15-16 mars 1994: 277-284

WOESTE K., McGRANAHAN G., BERNATZKY R. 1996. The identification and characterization of a genetic marker linked to hypersensitivity to the cherry leafroll virus in walnut. Molecular Breeding 2: 261-266.

Research Project

Title: Ecophysiology of the walnut tree (Juglans regia L.)

Project objectives: The scientific purpose of the project is to better understand the biology of the walnut tree, especially the dynamics of growth and development as influenced by microclimate and mechanical constraints. Special emphasis is given to carbon, water and nitrogen relations and bud biology. The ultimate goal is to build a simulation structure-function model based on the interactions between the tree, the environment and practices as pruning. This model should integrate processes studied in the lab.

Related publications:

AMEGLIO T., ARCHER P. - 1996. Représentativité du potentiel de base sur sols à humidité hétérogène. Agronomie, 16: 493-503.

AMEGLIO T., ARCHER P., COHEN M., VALANCOGNE C., DAUDET F.A., DAYAU S., CRUIZIAT P. - 1998. Significance and limits in the use of predawn leaf water potential for tree irrigation. Plant and Soil, 207, (2): 155-167.

AMEGLIO T., CRUIZIAT P., BERAUD S. - 1995. Alternance tension/pression de la sève dans le xylème chez le noyer pendant l'hiver. Conséquences sur la conductance hydraulique des rameaux. C. R. Acad. Sci. Paris, Sér. III: Sci. Vie, 318: 351-357.

AMEGLIO T., EWERS F.W., COCHARD H., MARTIGNAC M., VANDAME M., BODET C., CRUIZIAT P., 2001. Winter stem xylem pressure in walnut trees: effects of carbohydrates, cooling and freezing. Tree Physiology, 21(6): 387-394.

BALANDIER P., LACOINTE A., LE ROUX X., SINOQUET H., CRUIZIAT P., LE DIZES S., 2000. SIMWAL: a structural-functional model simulating single walnut tree growth in response to climate and pruning. Annals of Forest Science, 57 (5/6): 571-585

CHENEVARD D., FROSSARD J.S., JAY ALLEMAND C. - 1997. Carbohydrate reserves and CO2 balance of hybrid walnut (Juglans nigra no 23 x Juglans regia) plantlets during acclimatisation. Scientia Hortic., 68 (1/4): 207-217.

CHENEVARD D., JAY ALLEMAND C., GENDRAUD M., FROSSARD J.S. - 1995. The effect of sucrose on the development of hybrid walnut microcuttings (Juglans nigra x Juglans regia). Consequences on their survival during acclimatization. Ann. Sci. for., 52(2):147-156.

COCHARD H., GRANIER A., 1999. Fonctionnement hydraulique des arbres forestiers. Revue Forestière Française, 51:121-134.

COMBES D., SINOQUET H., VARLET-GRANCHER C., 2000. Preliminary measurement and simulation of the spatial distribution of the Morphogenetically Active Radiation (MAR) within an isolated tree canopy. Annals of Forest Science, 57 (5/6): 497-511

DAUDET FA, LE ROUX X., SINOQUET H., ADAM B., 1999. Wind speed and leaf boundary layer conductance variation within tree crown: consequences on leaf-to-atmosphere coupling and tree functions. Agricultural and Forest Meteorology, 97: 171-185.

ESCOBAR GUTIERREZ A., DAUDET F.A., GAUDILLERE J.P., MAILLARD P., FROSSARD J.S. - 1998. Modelling of allocation and balance of carbon in walnut (Juglans regia L.) seedlings during heterotrophy-autotrophy transition. J. Theor. Biol., 194 (1): 29-47.

FROSSARD J.S., CRUIZIAT P. - 1995. Physiologie intégrée de l'arbre fruitier. Analyse et modélisation du fonctionnement de l'arbre en relation avec le climat. Fruits, 50 (3): 221-224.

GODIN C., COSTES E., SINOQUET H., 1999. A method for describing plant architecture which integrates topology and geometry. Annals of Botany, 84: 343-357.

LACOINTE A., KAJJI A., DAUDET F.A., ARCHER P., FROSSARD J.S. - 1995. Seasonal variation of photosynthetic carbon flow rate into young walnut and its partitioning among the plant organs and functions. J. Plant Physiol., 146 (3): 222-230.

LACOINTE A., in press. Carbon allocation among tree organs: a review of basic processes and representation in functional-structural tree models. Annals of Forest Science.

LE DIZES S., CRUIZIAT P., LACOINTE A., SINOQUET H., LE ROUX X., BALANDIER P., JACQUET P. - 1997. A model for simulating structure-function relationships in walnut tree growth processes. Silva Fennica, 31 (3): 313-328.

LE ROUX X., GRAND S., DREYER E., DAUDET F.A., 1999. Parameterisation and testing of a biochemically-based photosynthesis model in walnut (Juglans regia L.) trees and seedlings. Tree Physiology, 19: 481-492.

LE ROUX X., SINOQUET H., VANDAMME M., 1999. Spatial distribution of leaf dry weight per area and leaf nitrogen content in relation to local radiation regime within an isolated tree crown. Tree Physiology, 19: 181-188.

MAILLARD P., DAUDET F.A., CASTELL F., DELEENS E., 1999. Effects of two elevated CO2 on sink-source relationships for carbon and nitrogen during germination and early growth of Juglans regia L. seedlings. Annals of Forest Science, 56: 59-69.

PANTERNE P., BURGER J., CRUIZIAT P. - 1995. Modélisation de l'évolution du potentiel hydrique dans une section d'axe ligneux. C. R. Acad. Sci., Série 3: Sci. Vie, 318 (11): 1119-1124.

PANTERNE P., BURGER J., CRUIZIAT P. - 1998. A model of the variation of water potential and diameter within a woody axis cross-section under transpiration conditions. Trees, 12: 293-301.

SINOQUET H., ADAM B., RIVET P., GODIN C. - 1997. Interactions between light and plant architecture in an agroforestry walnut tree. Agrofor. Forum, 8 (2): 37-40.

SINOQUET H., LE ROUX X., in press. Short-term interactions between tree foliage and the aerial environment: an overview of modelling approaches available for tree structure-function models. Annals of Forest Science.

SINOQUET H., RIVET P. - 1997. Measurement and visualization of the architecture of an adult tree based on a three-dimensional digitising device. Trees, 11 (5): 265-270.

SINOQUET H., RIVET P., GODIN C. - 1997. Assessment of the three-dimensional architecture of walnut trees using digitising. Silva Fennica, 31 (3): 265-273.

SPERRY J.S., SALIENDRA N.Z., POCKMAN W.T., COCHARD H., CRUIZIAT P., DAVIS S.D., EWERS F.W., TYREE M.T. - 1996. New evidence for large negative xylem pressures and their measurement by the pressure chamber method. Plant Cell Environ., 19: 427-436.

Research Project

Title: Cambial activity and wood formation in walnut trees

Project objectives: In order to study the physiological determinism of different wood properties, different approaches were developed with the following objectives: 1) To enhance the cambium activity and wood production through the modulation of auxin content in cambial cells. 2) To identify the chemical structures responsible of wood colouring. 3) To better understand the molecular and biochemical processes involved in heartwood formation. 4) To develop a new concept of heartwood extension in stem through the effects of endogenous chemical and mechanical factors.

Project summary and results: The experiments were conducted on wood samples collected either on stump sprouts or on adult trees aged 20 at different seasonal periods. The study of cambium activity consisted in cloning and sequencing of the cdc2 gene involved in the cell cycle. Its expression was studied by northern blot in the cambial zone during the growth season indicating a high accumulation of these transcripts during July when radial growth is optimal. Promoter of the gene will be identified in order to built vectors (cambium specific promoter: gene coding enzyme(s) involved in auxin synthesis) and tested by genetic transformation. Moreover, phenolics were analysed to identify chromophores responsible of wood colour that appears during heartwood formation. Four families of compounds are directly involved in such process: naphthoquinones, ellagitannins, gallic tannins and proanthocyanidines. Recently, it was shown that flavonoids are specifically accumulated in the transition zone where genes coding enzymes involved in flavonoid synthesis are highly expressed. Thus, active processes that occurred in living cells bordering heartwood result in specific activation of genes involved in phenolic synthesis from available carbohydrates. Chemical and mechanical factors that may be involved in such activation are under study.

Related publications:

BURTIN P., JAY-ALLEMAND C., JANIN G., 1996. Relation entre les composés phénoliques solubles du bois de noyer (Juglans sp) et sa couleur. 4è Colloque des Sciences et Industrie du bois, Nancy: 107-114.

BURTIN P., CHARPENTIER JP, JAY-ALLEMAND C., 1998. Hydrojuglone b-D-glucopyranoside: a major precursor of walnut wood colour. XIXth International Conference on Polyphenols, Lille, 1-4 September 1998, 1:163-164.

BURTIN P., JAY-ALLEMAND C., CHARPENTIER JP, JANIN JP, 1998. Natural wood colouring process in Juglans sp (J. nigra, J. regia and hybrid J. nigra 23 x J. regia) depends on native phenolic compounds accumulated in the transition zone between sap and heartwood. Trees, 12: 258-264.

BURTIN P., JAY-ALLEMAND C., CHARPENTIER JP, JANIN G., 1999. Modifications of hybrid walnut (Juglans nigra 23 x Juglans regia) wood colour under various steaming conditions. Relationship with phenolic composition. Holzforschung (in press).

CLAUDOT A.C., DROUET A., JAY-ALLEMAND C., 1992. Tissue distribution of phenolic compounds in annual shoots from adult and rejuvenated hybrid trees. Plant Physiol. Biochem., 30: 565-572.

COUEE I., LABEL P., JAY-ALLEMAND C., 1997. A critical review of the book: "biochemistry and molecular biology of wood by Takayoshi Higushi". Ann. Sci. For., 54: 502-503.

JANIN G., BERTI S., JAY-ALLEMAND C., BARGELLI S., CHARRIER B., MOSEDALE J., GUILLOUZO A., 1995. The Aesthetic Properties of Oak and Walnut Wood Appraised by an Opinion Poll of Professional and Non-professional Woodworkers and End Users. Xème congrès IUFRO, Août 1995, Tampere, Finlande. 1page

JAY-ALLEMAND C., BURTIN P., CHARPENTIER JP, DUROUX L., LABEL P., COUEE I., BRETON C., JANIN G., BONNET-MASIMBERT M., 1996. Mécanismes physiologiques et moléculaires impliqués dans la formation du bois de cœur. 9ème journées scientifiques, Biotechnocentre, Seillac, 24-25 octobre 1996. Bio. Cell. (Elsevier), 87: 3.

LABEL P., BERITOGNOLO I., BURTIN P., DEHON L., COUEE I., BRETON C., CHARPENTIER JP, JAY-ALLEMAND C., 1999. Cambial activity and xylem differentiation in walnut (Juglans sp) In "Cell and Molecular Biologie of Wood Formation", Savidge R., Barnett J. et Napier R., ed., BIOS Scientific Publishers, London, p 24 (in press)

LABEL P., LEVERT I., BRETON C., BERITOGNOLO I., CHARPENTIER JP, JAY-ALLEMAND C., 2001. Gene expression in cambial zone of hybrid walnut stem related to radial growth. 4th Intern. Walnut Symp., Bordeaux (France), 13-16 Sept 1999. Acta Hort., 544: 323-326

MAGEL E., JAY-ALLEMAND C., ZIEGLER H., 1992. Formation of heartwood substances in the stem of Robinia pseudoacacia. Distribution of reserve carbohydrates and heartwood extractives across the trunk. IUFRO, Edited by Association pour la Recherche sur le Bois en Lorraine, Nancy, France, August 23-28, 1992. All division 5 conference "Forest product", 1: 232.

MAGEL E., JAY-ALLEMAND C., ZIEGLER H., 1994. Formation of heartwood substances in the stemwood of Robinia pseudoacacia. II-Distribution of nonstructural carbohydrats and wood extractives across the trunk. Trees, 8: 165-171.

MAGEL E.A., MONTIES B., DROUET A., JAY-ALLEMAND C., ZIEGLER H., 1995. Heartwood formation: biosynthesis of heartwood extractives and secondary lignification. EUROSILVA, "Contribution to Forest tree Physiology", Dourdan (France), 7-10 Novembre, 1994. H. Sandermann and M. Bonnet-Masimbert (Eds). Les Colloques, INRA Paris. 76: 35-56.

Research Project

Title: Micropropagation and rhizogenesis in walnut trees

Project objectives: This research project has two main objectives: 1) To study some molecular and biochemical mechanisms involved in adventitious thizogenesis processes at cellular level. 2) To develop an efficient method to propagate juvenile and adult clones adapted to industrial production. Basic research on rooting is focused on: i) the role of auxins for carbohydrate mobilisation in microshoot, ii) the effect of phenolics regulated by b-glucosidase or chalcone synthase activities, iii) transcription factors able to modulate root formation and development. Expression of such genes cloned is under study through the generation of walnut genetically transformed. Applied research is devoted to study factors that promote root development and microcutting acclimatisation for plant production at commercial scale.

Project summary and results: This work was conducted on interspecific hybrids (Juglans nigra x Juglans regia) showing interesting genetic characters in terms of growth and adaptation to environment. A novel in vitro plant model was developed on Juglans regia using peculiar cotyledon fragments producing roots systematically and somatic embryos of "Early Mature Trees" forming flowers in vitro on microcuttings. The study of rhizogenesis was conducted either on microshoots to analyse auxins, phenolics and carbohydrates during adventitious root initiation or on cotyledon fragments to make cDNA libraries at different times of root formation and to identify genes related to this morphogenetic process. Main results are:

1) Exogenous auxin application results in an internal auxinic gradient along the microshoot that promotes carbohydrate translocation from the shoot tip to the basal part where roots emerge.

2) Naphthoquinones as hydrojuglone glucoside (HJG) and juglone (JUG) could modulate adventitious root initiation and development. Just after section of the microshoot, HJG decrease dramatically whereas JUG increase gradually in the zone where roots can be formed. A significant positive correlation was established between rooting and juglone content.

3) Chalcone synthase (CHS) activity in microshoot tissues was strongly inhibited by a walnut RNA antisense introduced by genetic transformation via Agrobacterium tumefaciens. Transformed lines with very low CHS activity and flavonoid content showed higher rooting capacities (80-90%).

4) A b-glucosidase (WBG8.9) was purified to homogeneity and was found to have a high affinity with HJG. This enzyme is induced in tissues during which root primordium is formed. However, its role that could result in glucose, H+ and 02° release in cells is not clear understood yet.

5) Cloning and sequencing of genes related to rhizogenesis were undertaken from cDNA libraries. Three genes coding transcription factors were identified. Their expression in walnut tissues and their function studied by means of genetic transformation (RNA sense and antisense) are being explored.

Related publications:

BARBAS E., CHAILLOU S., CORNU D., DOUMAS P., JAY-ALLEMAND C., LAMAZE T., 1993. Orthophosphate nutrition of in vitro propagated hybrid walnut (Juglans nigra x Juglans regia). trees: Pi (32Pi) uptake and transport in relation to callus and shoot development. Plant Physiol. Biochem. 31: 41-49

BARBAS E., JAY-ALLEMAND C., DOUMAS P., CHAILLOU S., CORNU D., 1993. Effects of gelling agents on growth, mineral composition and naphtouinone content of in vitro explants of hybrid walnut tree (Juglans nigra x Juglans regia). Ann. Sci. For., 50: 177-186.

CHENEVARD D., JAY-ALLEMAND C., FROSSARD J.S., GENDRAUD M., 1995. Morphological and biochemical factors affecting the survival rate of microcuttings of two hybrid walnut (Juglans nigra x Juglans regia) clones during their acclimatization. Ann. Sci. For. 52: 147-156.

CHENEVARD D., FROSSARD J.S., JAY-ALLEMAND C., 1997. Carbohydrates reserves and CO2 balance of hybrid walnut plantlets during acclimatisation. Scientia Horticulturae. 68: 207-217.

CLAUDOT A.C., DROUET A., JAY-ALLEMAND C., 1992. Tissue distribution of phenolic compounds in annual shoots from adult and rejuvenated hybrid trees. Plant Physiol. Biochem., 30: 565-572.

CLAUDOT A.C., JAY-ALLEMAND C., MAGEL E.A., DROUET A., 1993. Phenylalanine ammonialyase, chalcone synthase and polyphenolic compounds in adult and rejuvenated hybrid walnut tree. Trees, 7: 92-97

DUROUX L., FONTAINE E., BRETON C., CHARPENTIER JP, DOUMAS P., JAY-ALLEMAND C., 1997. Histological and biochemical characterization of adventitious root formation in walnut cotyledon fragments. In "Biology of root Formation and Development", Altman & Waisel Eds.. Plenum Press, New-York. 9: 75-84

DUROUX L., DELMOTTE F., LANCELIN JM, KERAVIS G., JAY-ALLEMAND C., 1998. Insight into naphtoquinone metabolism: b-glucosidase-catalysed hydrolysis of hydrojuglone-b-D-glucopyranoside. Biochem. J., 333: 275-283.

DUROUX L., ERMEL-FONTAINE F., BRETON C., CHARPENTIER JP, CAPELLI P., BRUANT B., LABEL P., COUEE I., JAY-ALLEMAND C., 1998. How could we progress in the knowledge of the mechanisms that govern the adventitious root formation from aerial plant tissues Bulletin de la Société Royale des Sciences de Liège. 67: 105-115.

EL EUCH C., JAY-ALLEMAND C., PASTUGLIA M., DOUMAS P., CHARPENTIER JP, CAPELLI P., JOUANIN L., 1996. Modification de l'expression du métabolisme phénolique chez le noyer et réactivité in vitro. Réunion de la Société botanique de France, Montpellier (France), 22 sept. 1995. Acta Botanica Gallica, 143 (6): 547-553.

EL EUCH C., JAY-ALLEMAND C., PASTUGLIA M., DOUMAS P., CHARPENTIER JP, CAPELLI P., JOUANIN L., 1998. Expression of Antisens Gene Chalcone Synthase in Transformed Hybrid Walnut Microcuttings: Effect on Flavonoid Content and Rooting Percentage. Plant Mol. Biol., 38: 467-479.

ERMEL FF, VISOZO Z., CHARPENTIER JP, CATESSON AM, JAY-ALLEMAND C., COUEE I., 1999. Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants. Planta (accepted for publication).

GUTMANN M., CHARPENTIER J.P, DOUMAS P., JAY-ALLEMAND C., 1996. Histological investigation of walnut cotyledon fragments for a better understanding of in vitro adventitious root initiation. Plant Cell. Rep. 15: 345-349

JAY-ALLEMAND C., PONS V. de, DOUMAS P., CAPELLI P., SOSSOUNTZOV S., CORNU D., 1991. In vitro root development from walnut cotyledons: a new model to study the rhizogenesis processes in woody plants. C.R. Acad. Sci. Paris, 312: 369-375

JAY-ALLEMAND C., CAPELLI C., CORNU D., 1992. Root development of in vitro hybrid walnut microcuttings in a vermiculite containing gelrite medium. Scientia Horticulturae, 51: 335-342

JAY-ALLEMAND C., DOUMAS P., SOTTA B., MIGINIAC E., 1995. Juvenility and physiology of rhizogenesis in two woody species. EUROSILVA, Contribution to Forest Tree Physiology, Dourdan (France), 7-10 novembre 1994. H. Sandermann and M. Bonnet-Masimbert, Eds. Les Colloques, INRA Paris. 76: 79-99.

JOUANIN L., El EUCH C., PASTUGLIA M., CAPELLI P., DOUMAS P., JAY-ALLEMAND C., 1996. Characterization of antisense chalcone synthase transgenic microcuttings. In Ahuja MR, Boerjan W, Neale DB, eds "Somatic Cell Genetics and Molecular Genetics of Trees", Forestry Sciences, Vol. 49, Kluwer Academic Publishers, Dordrecht, The Netherlands: 147-152.

Research Project

Title: Genetics and breeding of Juglans varieties for wood production

Project objectives: To achieve production of walnut varieties improved for wood production, several steps must be followed:

1) Sampling of material from natural areas or seed orchards to increase the currently available number of genotypes in France. The main species are:

Juglans regia, Euro-Asian species. Available in France from regional French clonal collection of more trees and collections of genotypes from different countries. Available in the natural area through collaborations.

Juglans nigra, American species (Eastern USA). Available from improved seed orchards in the USA and from scattered trees from unknown genetic background in France.

Juglans major, American species (South-western USA and Northern Mexico) and Juglans hindsii, American species (Northern California). Both are available from germplasm banks in the USA and from their natural growing areas.

2) Establishment of collections in experimental field sites and containers: their goal is to provide easily accessible material for experiments and to act as ex situ gene conservation banks.

3) Structure of genetic variation for selected traits: results will be available from the provenance and progeny tests installed (see step 2). Selected traits are as follows:

* adaptation (cold, summer drought, late frost); * pest and disease resistance (Armillaria, Phytophtora, crown gall); * height growth (juvenile growth and total adult height); * wood quality, i.e. architecture bole (straightness and length without lateral branches), color, wood density, etc.

4) Study of flower biology: detection of genotypes suitable for inter-specific crosses: results will be available from experiments performed on container collections (see step 2) in the nursery. The creation of interspecific hybrids is currently considered as the most promising way to yield high performant varieties for wood production. However, within species improvement will not be excluded from the program.

5) Selection of parents for intra- and inter-specific crosses: from steps 3 and 4, parents with desired traits and the ability to combine when inter (or possibly intra) specific crosses are performed will be chosen.

6) Progeny tests with material from intra- and inter-specific crosses. Progeny tests can also be performed within species using material collected in natural areas from individual mother trees.

7) Creation of pure and hybrid species seed orchards using parents with the best General and Specific Combining Ability as determined from step 6.

Project summary and results: So far, little has been done. Step 1 has been started. Regional conservation collections are underway or achieved in several regions of France. INRA has collections of several Juglans species in a few sites. Step 5 has also been started with a test comparing 39 Caucasian Juglans regia progenies which are good candidates for intraspecific improvement.

Related publications:

See reports of the EEC AIR1-CT92-0142 contract "Development of walnut trees for wood and fruit production as an alternative and extensive system to agricultural crops", JAY-ALLEMAND C. coordinator.

Research Project

Title: Integration of morphological criteria in the description of young tree architecture and fruiting potentials in walnut progenies

Project objectives: Since the end of 70's, a breeding programme has been developed at the French Institut National de la Recherche Agronomique (INRA) in Bordeaux (France), aimed at breeding new varieties for late flowering to avoid spring frost damage, high productivity, lateral fruiting, and good nut quality sustainable over the years (Germain, 1980).

Our objectives are twofold:

- To determine, on existing cultivars, if morphological (e.g. spur density, axis length) traits are related to fruiting ones (e.g. nut weight variability) in Juglans regia.

- To apply these results when analysing seedling populations or when describing the behaviour of recently introduced foreign cultivars.

Project summary and results: Studies were carried out on five genotypes, the cultivar "Lara", a selection from a French nurseryman, together with "Fernor" and three genotypes, "H93-63", "H94-101" and "H96-7" released from the INRA breeding programme, were chosen. These genotypes all belong to the lateral fruiting type but with various lateral branching densities and fruiting behaviour. H94-101 is characterised by high branching density and weak axes, whereas H96-7 has a low branching density with longer and stronger axes. First results (Lauri et al., 1999) show that at least two factors are involved in nut weight variability: length of preformed subtending axis and number of nuts developing from the inflorescence. Nut weight homogeneity was greater for genotypes with longer preformed subtending axes than for genotypes with short preformed subtending axes. For these last genotypes, short preformed subtending axes (of under 6 cm) or the presence of more than one nut per inflorescence was shown to significantly reduce nut weight and increase nut weight heterogeneity. Studies on walnut progenies will be developed within the next few years.

Related publications:

GERMAIN E., 1990. Inheritance of late leafing and lateral bud fruitfulness in walnut (Juglans regia L.), phenotypic correlations among some traits of the trees. Ist Intern. Symp. on Walnut Production, Budapest (Hungary) 25-29 Sept, 1989. Acta Horticulturae 284: 125-134

LAURI PE, DELORT F., GERMAIN E., REYNET P., 1999. Factors affecting nut weight in walnut (Juglans regia L.) - An analysis of genotypes with contrasting branching patterns. IV Intern. Walnut Symp., Bordeaux (France) 13-16 Sept, 1999. Acta Horticulturae 544: 265-273.

Research Project

Title: Architectural and morphological variability in Juglans regia L., Juglans nigra L., and interspecific hybrids (MJ209xRA and NG23xRA)

Project objectives: Walnut trees are traditionally used for wood and fruit production in France. The first objective was to characterise the architectural development of these species in order to determinate the time of fork formation on the main stem. The fork formation has directly an influence on the wood quality and the final size of the butt log used as timber. Another objective was to determinate the architectural variability according to genotype (species, varieties, hybrids), local environmental conditions and cultural practices.

Project summary and results: The architecture of a plant depends on the nature and relative arrangement of each of its parts. At any given time, the architecture is the expression of an equilibrium between endogenous growth processes and exogenous constraints exerted by the environment. The architectural analysis method allows to identify these endogenous processes of growth and branching and to understand the influence of environmental factors by means of observation. Considering the plant as a whole, from its germination to its death, architectural analysis is essentially a dynamic approach of plant development. Architectural description consists in morphological observations and drawings of varying number of individuals at different ages and growing in various environmental conditions. The results are summarised in a set of diagrams that represent the sequence of development of the studied species. The quantitative analysis of annual shoots allows to represent the growth and branching processes by probabilistic models.

In Walnut trees, several types of annual shoots are distinguished by the growth pattern and, in consequence, by branching pattern. Each morphological type of annual shoot may be related to well defined development stages of tree development and/or with a precise architectural position. The interspecific architectural variations mainly concern (1) the growth direction and the curvature of the young branches, (2) the time of the fork formation, (3) the branching pattern of annual shoots and (4) the axes structure (alternation between floriferous and vegetative shoots). Both last morphological criteria vary also between varieties (Lozeronne, Franquette, Parisienne, Solèze, Serr, Hartley, Pedro, Lara). The environmental factors (light, soils) modulate only in quantitative way the sequence of architectural development of species.

Related publications:

BARTHÉLÉMY D., SABATIER S., PASCAL O., 1995. Le développement architectural du noyer commun Juglans regia L. (Juglandaceae). Forêt Entreprise, 103: 61-68.

BARTHÉLÉMY D., SABATIER S., PASCAL O., 1997. Le développement architectural du noyer noir Juglans nigra L. (Juglandaceae). Forêt Entreprise, 115: 40-47.

BARTHÉLÉMY D., MERCURIO R., SABATIER S., 1998. L'architettura della chioma del noce comune. In: "Il noce comune par la produzione legnosa". R. Giannini et R. Mercurio (Eds). Avenue Media, Bologne, Italie, pp. 57-65.

DUCOUSSO I., SABATIERr S., BARTHÉLÉMY D., GERMAIN E., 1995. Comparaison de quelques caratéristiques morphologiques des pousses annuelles et branches de cime de 7 variétés de noyer commun, Juglans regia L. (Juglandaceae). In: Architecture des Arbres Fruitiers et Forestiers, J. Bouchon (Ed.), Montpellier, France 23-25/11/1993, INRA Editions. Les Colloques, Vol. 74: 91-108.

JAY-ALLEMAND C., BARTHÉLÉMY D., SABATIER S., BRUANT B., 1995. Gradients morphologiques et biochimiques chez un Noyer hybride (Juglans regia x Juglans nigra). In: Architecture des Arbres Fruitiers et Forestiers, J. Bouchon (Ed.), Montpellier, France 23-25/11/1993, INRA Editions. Les Colloques, Vol. 74, pp. 125-138.

SABATIER S., 1999. Variability morphologique et architecturale de deux espèces de Noyers: Juglans regia L. et Juglans nigra L. et de deux noyers hybrides interspécifiques. Thèse de Doctorat, Université Montpellier II, 140 p.

SABATIER S., BARTHÉLÉMY D., 2001. Bud structure in relation to shoot morphology and position on the vegetative annual shoots of Juglans regia L., (Juglandaceae). Annals of Botany. 87 (1): 117-123.

SABATIER S., BARTHÉLÉMY D., 2001. Annual shoot morphology and architecture in Persian Walnut, Juglans regia L. (Juglandaceae). In: 5th International walnut symposium, Bordeaux, France 12-16/09/1999, ISHS. Acta Horticulturae, 544: 255-264.

SABATIER S., BARTHÉLÉMY D., BECQUEY J., PERRIER S., 2000. Taille et architecture chez de jeunes Noyers hybrides. Forêt-Entreprise, 132: 54-58.

SABATIER S., BARTHÉLÉMY D., DUCOUSSO I., GERMAIN E., 1995. Nature de la pousse annuelle chez le Noyer commun, Juglans regia L. var. "Lara" (Juglandaceae). In: Architecture des Arbres Fruitiers et Forestiers, J. Bouchon (Ed.), Montpellier, France 23-25/11/1993, INRA Editions. Les Colloques, Vol. 74, pp. 109-123.

SABATIER S., BARTHÉLÉMY D., DUCOUSSO I., GERMAIN E., 1998. Modalités d'allongement et morphologie des pousses annuelles chez le noyer commun, Juglans regia L. cv. "Lara" (Juglandaceae). Canadian Journal of Botany, 76: 1253-1264.

SABATIER S., BARTHÉLÉMY D., DUCOUSSO I., GERMAIN E., 1999. Modalités d'allongement et morphologie de pousses annuelles issues de greffe chez le noyer commun, Juglans regia L. cv. "Lara" (Juglandaceae). Canadian Journal of Botany, 77: 1-9.

SABATIER S., BARTHÉLÉMY D., DUCOUSSO I., GERMAIN E., GUÉDON Y., 1998. Structure de scions d'un an de Noyer commun, Juglans regia L., variété Lara greffés sur trois porte-greffe (Juglans nigra, J. regia, J. nigra x J. regia). In: Architecture et modélisation en arboriculture fruitière, 11ème colloque sur les recherches fruitières, Montpellier, France 5-6/03/1998, INRA-Ctifl, pp. 75-84.

Research Project

Title: Study of the behaviour of new INRA walnut varieties with lateral fruitfulness

Project objectives: To appraise the behaviour of the new varieties created in the varietal breeding programme carried out by INRA at Bordeaux. The new hybrids, crosses between French and American varieties, bear fruit on lateral shoots and therefore make it possible for fruit bearing to start earlier and to be more productive than on traditional varieties. These varieties will improve the competitiveness of French walnut production but must also meet all the other criteria for cultivation (late budbreak, low susceptibility to diseases) and for high quality nuts (size, shell hardness, kernel percentage, colour of kernel, proportion of waste, etc.).

Project summary and results: Selection of INRA hybrids which start producing fruit early, a yield 30% higher than traditional varieties, a high quality of nut and of kernel and a low susceptibility to disease. Observations carried out of the tree (vigour, habit, date of leafing out, date of maturity of the nuts), of the nuts (shape, size, seal shell, thickness) and of the kernel (weight, colour, fullness, ease of shelling, kernel percentage). A study was also made of the agronomic behaviour and the disease resistance of the different varieties.

The studies are based on the developmental orchards at Toulenne (INRA), at Chatte (SENuRA) and at Creysse, in partnership with the Ctifl:

- 1st stage in the trial orchard (3 trees minimum per variety);

- 2nd stage in the behaviour orchard (20 trees minimum per variety).

The observation of each variety will last at least 15 years in order to take full account of the behaviour of the tree and of the variety as it ages.

At present three new INRA varieties are listed in the French C.T.P.S. Register: Fernor (a quality nut equivalent to Franquette), Fernette (pollinator) and Ferjean (nut for the kernel market).

Related publications:

PRUNET JP., 2001. Radiographie des quatre principales variétés de noix française: Franquette, Pieral-Lara®, Fernor, Fernette.

Research Project

Title: Study of the behaviour of new Juglans regia Ctifl rootstocks and of INRA interspecific hybrids

Project objectives: To put on the market, at a competitive price, new more vigorous rootstocks needed to optimise the behaviour and yield of the new French hybrid varieties (since these are 10-25% more vigorous than traditional seedlings, the trees start bearing fruit earlier with an increased quantity and quality of yield). Even if the study programmes and the cultivation of cultivars on their own roots are still relevant, this programme for improving rootstocks is essential since the more vigour is introduced, the better will be the production of varieties fruiting on lateral shoots.

Project summary and results: It is essential to have a good balance between vigour and yield in the new varieties that bear on lateral shoots. A certain number of factors still need to be studied further and mastered, in particular the hardening off by the nurserymen of plants obtained by the in vitro multiplication process established by the Ctifl. Work carried out:

- Planting of the first Ctifl clones of Juglans regia produced in vitro in comparative trials at Creysse and SENuRA to study the agronomic behaviour of the plants hardened off and grafted at the Pondaillan nurseries in Souillac (Lot).

- Testing of susceptibility to certain diseases, in partnership with the INRA of Bordeaux.

- Establishment of a repository of rootstocks.

- Testing the behaviour of cultivars on their own roots.

The first three cultivars of INRA interspecific hybrid rootstocks are in in vitro production and will be compared with the Ctifl Juglans regia rootstocks.

At present three pre-selected Ctifl rootstocks, which had done well in the trials for distinction, homogeneity and stability demanded by the CTPS, have been listed in the Register of fruiting plants. These are RG2 (LIBA) and RG15 (CHEREBA), and more recently RG12 (NOBA). The observations from the trials started in 1994 are encouraging for the RG2 clone, cultivars on their own roots (Lara, Franquette) in comparison with the best grafting rootstocks obtained from seedlings.

Related publications:

PRUNET JP., GARCIN A., GERMAIN E., 1998. De nouveaux porte-greffe adaptés aux variétés de noyer à fructification sur brindilles latérales. Infos-Ctifl, 147: 34-37.

Research Project

Title: Study of systems of management and of pruning for traditional varieties and for new lateral fruit bearing varieties

Project objectives:

* To optimise the production of traditional orchards, an important goal which means bringing them to bear fruit earlier and improving the yield of traditional varieties. The studies carried out by the Creysse Station take account of the aspects of density of plantation, structural pruning and reshaping in intensive plantations.

* To perfect new systems of managing orchards with new lateral fruit bearing varieties formed in the axis. The varieties Lara and Fernor do not have the same habit and need a management system adapted to their specific characteristics.

The task of the Station is to verify the effectiveness and usefulness in technical and economic terms of the suggested improvements:

- shortening the unproductive period of young trees,

- improving yield and quality,

- reducing production costs (by machine pruning).

Project summary and results: The studies concerned:

- the densities of plantation of Franquette, Lara, Fernor, Ferjean;

- the structural pruning of the trees: goblet, structured vertical axis and free vertical axis, taking into account the new techniques of bending the branches in leaf (arching) to encourage fruiting, to create a balanced shape and reduce competition among trees planted very densely, seen especially in hedgerows;

- different methods of maintenance pruning: pruning by hand, machine pruning by vertical trimming at 1 m 20 from the axis, and by oblique trimming at intervals of 2 to 4 years.

Summary of results:

Trials of plantation density with Franquette planted in 1988 (to compare the effects of plantation distance): progressive thinning of the block to 8 x 8m in the winter of 1999/2000 which had a cumulative production 1.600 kg higher than a block planted at 11 x 11m.

Maintenance pruning of Lara trained in a hedgerow at 8 x 4m with free axis and structured axis: the cumulative yields at the 12th leaf stage are 20 t per hectare. Pruning by machine, which saves a considerable amount of time, did not, in the conditions of this study, reduce the yield

Related publications:

PRUNET JP., GARCIN A., GINIBRE T., VERHAEGHE A., 2000. Un choix de conduite pour la variété Fernor. Infos-Ctifl, 164: 36-39.

Research Project

Title: Chemical control of bacterial walnut blight

Project objectives: Walnut blight is the major disease in walnut orchards of France. The causal bacterium Xanthomonas arboricola pv Juglandis, can infect leaves, catkins, buds, nuts and can induce up to 50 % losses of crops.

The studies carried out by Creysse Station concern:

- combining copper with other products; fungicides or products which strengthen the natural defences of the tree to improve its resistance against attacks of bacterial blight;

- adjusting the treatment timetable;

- studying the physiological behaviour of the trees in response to the way they are managed, and the soil and climatic conditions of the orchard.

Project summary and results: During the last years, different copper compounds (sulphate, hydroxide, oxide) were tested alone or supplemented with EBDC material, boron or iron.

Variety Franquette, plantation 1988, rootstock J. regia, density: 156 trees/ha, sand alluvial soil. For all the study modes, from the stage Df2-Ef to Gf, one treatment every 8 days (4 treatments), then one treatment during fruit increasing. Five sprays were applied on the cultivar Franquette during female flowering and the beginning of fruit increasing. From June to the harvest, the disease intensity was measured by counting the percent on nuts dropped because of bacterial blight. Apical necrosis was distinguished from the lateral.

A correlation was noted between disease intensity and duration of moistenning in May. Only Bordeaux mixture showed a limited efficiency. Copper hydroxide, even with restricted dose, was more effective. Copper mixed with iron or boron was not more effective neither. In fact, copper compounds plus EBDC material were the most effective. But EBDC has an effect on auxiliary faun and increases the level of mites. All these treatments had a similar effect on lateral and apical necrosis. It is necessary to check during several years the technical (efficacy, consequences on auxiliary faun, toxicity, washing reaction) and economical interest (rate efficacy, cost) of these treatments.

In the context of studies of other factors playing a role in the physiological behaviour of trees and their susceptibility to bacteria, Creysse Station, in partnership with the SENuRA, has analysed the green organs (leaves, sap, etc.) in more than 20 orchards with different pedo-climatic conditions in the French Southwest area of production. The Ctifl, together with Avignon University, has traced the ways by which the bacteria enter and circulate within these organs.

Related publications:

GARCIN A., EL MATAOUI M., TICHADOU S., PRUNET JP, GINIBRE T., PENET C., 2001. La bactériose du noyer: nouvelles connaissances pour une vieille maladie. Synthèse des travaux réalisés (1995-2000). Infos-Ctifl, 171: 27-30.

GARDAN L., 1986. La bactériose du noyer. Phytoma, 382: 35-41.

RADIX P., 1994. The influence of soil nature on polyphenols in walnut tissues. A possible explanation of differences in the expression on walnut blight. Agronomie, 18: 927-637.

STATION DE CREYSSE, 1997. Protection phytosanitaire du noyer, note n°8: 20 pp.

Research Project

Title: Study of the quality of nuts and of kernels.

Project objectives: More and more, buyers and consumers expect a nut and a kernel of irreproachable quality. The French kernel should have a good reputation, compared with the rest of the European market, for its shape, its colour, its healthiness and its taste. It is therefore necessary to improve the quality of products put on the market but also to provide proof of this quality in terms of taste, not only of appearance (a kernel that is whole and light in colour). The creation of a quality standard for whole nuts and kernels would meet this need.

Project summary and results: The Experimental Station of Creysse is concerned with three aspects:

- Establishing the HACCP method in the chain of walnut production to identify all the risks and danger points that might be encountered and to guarantee the health and taste standard of the product. The next step is to define the methods required to counter these risks and to ensure that these methods are put in place effectively, in partnership with all concerned.

- Identifying and introducing techniques for treating kernels that will reduce or eliminate the bacterial flora before or after packaging.

- The establishment of a standard of reference relating to taste quality (sensory analysis) and physico-chemical aspects (colour, kernel percentage, volatile components affecting oxidation, etc.).

First results

An enquiry pursued over 5 years has highlighted certain factors which influence the intrinsic quality of the nut and of the kernel: the length of stay on the ground and waiting time before being dried; the drying of nut or kernel which may affect the development of bacterial flora; premature harvesting by shaking, which tends to increase the glassiness of the kernels after drying (too high a level of water or immaturity of the nuts). There is no relationship between this glassiness and the biochemical characteristics of the nuts, but the organoleptic quality is poorer.

Related publications:

LE BARS J., BOUDRA H., PRUNET J.P., 1995 - guide pratique: les moisissures et les mycotoxines: prévention de la contamination de la noix, INRA, Ctifl/Station Expérimentale de Creysse.

ROMERO A., ALETTA N., NINOT A., LOPEZ A. 1993 Indices chimiques qui permettent l'évaluation du potentiel de conservation de noix. Colloque scientifique sur la maîtrise de la qualité des noix. Technologies de séchage et de tri - Bordeaux (France)

LAVIALLE E. 1993 - Le séchage des noix - Guide pratique - Ctifl/Station Expérimentale de Creysse

Research Project

Title: Methods of forecasting walnut yield

Project objectives: Having an accurate estimate on the expected yield is an important factor in managing the trade in walnuts more effectively. With this information it is possible to make decisions at an early stage in the year and to take measures before the actual harvest period which will help to attenuate the effects of annual fluctuations in the volume produced, with consequent benefits to both buyers and producers.

Project summary and results: Two such methods have been developed at the Creysse Experimental Station. The results obtained from these methods have been compared with the actual production of the growers who operate in association with the French Fruit and Vegetable Committee on the great South-West region (Comité Economique Agricole Fruits et légumes du Bassin Grand Sud-Ouest). Their production data are the only reliable ones available and cover about half the tonnage produced in South-West France.

Method 1: forecasting from pollen emission.

This method is based on the fact that the female flowering rate and the effect on weather conditions can be estimated from the natural emission of pollen during the phase of flowering and of pollinisation. Observations over the last 5 years appear to show an inverse correlation between the emission and the subsequent yield. This method does not however take into account conditions that may affect production after the flowering period.

Method 2: forecasting by counting a sample of nuts.

Principle of a ratio between the average number on walnuts on 100 bearing shoots on the current year (the fertility coefficient) and the actual yield. The use of this method in 44 representative orchards in the Perigord each year since 1997 has made it possible to estimate the harvest in this area with reasonable accuracy.

Related publications:

BESSELAT et al., 1994. Prévision de récolte précoce: bilan et perspectives d'emploi d'un nouvel outil basé sur l'analyse pollinique de l'atmosphère. CR. Coll. Yield forecasting, U.E./DG VI, FAO, Villefranche sur mer 24-27 octobre. Méthodes de prévision de rendements. Office des Publications Officielles des communautés Européennes Ed. Luxembourg 1997: 153-161.

COUR et al, 1992. Les méthodes aéropalynologiques: un inventaire des domaines d'application en agriculture, en biogéographie et en bioclimatologies. C.R. Coll. Méthodes aéropalynologiques, phénologie de la floraison et prévision des rendements, Bordeaux (France), 10-11 mars. 2 p.

Research Project

Title: Improving the efficiency of the walnut industry in the South East of France

Project objectives: The objectives of the SENuRA project are defined by the professionals of the walnut industry in the South East of France and fall into a technical and economic framework without neglecting the environment.

To obtain these objectives, work is carried out in the following areas:

- plant material: the behaviour study of vigorous clonal rootstocks, multiplied by micropropagation; the observation of new INRA hybrids and varieties in the local context.

- cultivation itinerary: cost evaluation according to the cultivation system (plantation, irrigation, production). Soil maintenance which favours less polluting methods; hydromineral nutrition.

- phytosanitary protection: fight against pests (codling moth and mites) and walnut tree diseases (walnut blight, anthracnose): testing new products, better knowledge of the factors which give rise to problems, observations and thresholds for starting treatments; the study of causes of walnut tree withering.

- harvest and post-harvest: harvest mechanization (collecting, washing) quality control (drying, humidity of walnut); nut breaking; tracing the origins of a product.

- others: predicting the harvest (by trapping pollen and by counting branches) organic agriculture, comparing production methods, technical and economic studies; alternative management methods.

Project summary and results: The main results obtained in the last few years (1994-1999) are hereby summarized:

- plant material: 67 varieties are currently being observed at the SENuRA and 3 varieties have been selected from the INRA hybridisation program: "Fernor", "Fernette" and "Ferjean".

- Three clones of Juglans regia rootstock from a Ctifl selection program were multiplied in vitro and recorded in the official catalogue.

- cultivation itinerary: optimisation of behaviour and density to increase yield to both traditional and new varieties. New pruning methods shorten the non-productive period. Supplying the walnut producers with tools to optimise the water and mineral supplies. Developing alternative methods to chemical weed-killers: thermic or mechanical for young orchards, a program based on contact herbicides for mature orchards. Technical and economic studies to control production costs; awareness of the different types of charges enables a choice adapted to the orchard.

- phytosanitary protection against walnut blight has been relatively well controlled in orchard planted on soils favorable to walnut tree cultivation but the problem of withering still exists. The SENuRA studies new products or uses smaller doses to fight against this disease or to activate the natural defence of the tree. Some very good results have been obtained in the fight against walnut codling moth while protecting the environment (preservation of auxiliary fauna) by using the granulosis virus or the sexual confusion method.

- A survey on the walnut tree mite was carried out over the whole area which revealed in particular the correlation between phytosanitary behaviour and the outbreak of mites. Identification of phytoseiidae and continuation of research on the free eriothyd mites to find ways to fight against it and optimise phytosanitary behaviour while preserving the balance between pests and auxiliary fauna. Researches were carried out on the causes of walnut tree withering: root diseases, compaction, and testing responses to these (decompaction, nematicide?) were studied.

- harvest and post-harvest: elaboration of a technical itinerary to obtain a product which is wholesome and of good gustative quality. This itinerary begins with a harvest when the walnuts are at the correct maturity, and then the mechanization of the harvest and the washing and drying operations. Each stage is subjected to processing studies or testing new machines. The SENuRA carries out technological monitoring. A study out of nut marking was carried out. The SENuRA is working on tools for quality control, in particular to measure the water content of walnuts; it has set up a reference method using steam. Two measuring tools are being studied, one for commercial use and the other for producers.

- Miscellaneous: for predicting the harvest, several methods are tested: by trapping pollen and by counting branches. Some interesting results were obtained for estimating the grades of the walnuts; a synthesis has to be carried out to measure the reliability of these methods. Some trials in organic farming are carried out, partly on mature orchards to optimise behaviour patterns and reduce the large yield losses compared to conventional farming methods and partly on young orchards where different maintenance methods are tested on the plantation lines.

Related publications:

Annual experimentation reports of the SENuRA

Annual National Experimentation Communication Net sheets and reports of trials carried out at the SENuRA.

INFO NOIX, periodical (3 examples a year)

Agricultural notification "Spécial Noyer" distributed by the SRVP Rhône-Alpes

3 technical leaflets: drying, irrigation, phytosanitary protection

Participation in the compilation of the book "Le Noyer" (The walnut tree), published by the Ctifl in 1999.

Participation in the compilation of the book "Noix et Cerneaux, qualité et consommation" published by the Ctifl in 1996.