WALNUT PROPAGATION TRAINING SHORT COURSE

Murcia, Spain, March, 10th -12th, 2009

Organized by:

COST Action 873: ‘Bacterial Diseases of Stone Fruits and Nuts’

www.cost873.ch

Local organizer

• Diego FRUTOS Executive organizers

• Jose COS • Antonio CARRILLO • Gregorio LOPEZ • Maria A. SANCHEZ

This Short Training Course was welcomed by the Consejería de Agricultura y Agua of Murcia’s Region and by the city of Murcia

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Index Venue and Programme

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Abstracts

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Germination of walnut (Juglans regia L.) seeds for rootstock production. Diego Frutos

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Walnut propagation in Bulgaria Stefan Gandev

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Training on Hot Callusing technique applied to walnut grafting. Damiano Avanzato

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Grafting walnuts in Spain: new techniques. Diego Frutos

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In vitro propagation of walnut Maria A. Sanchez and José Cos

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In vitro walnut micropropagation Juglans regia L. application. Laurence Bourrain

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Discussion

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Conclusions

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List of participants

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Official press note about this meeting

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COST Action 873

WALNUT PROPAGATION TRAINING SHORT COURSE Venue: Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA).

Address: Calle Mayor, s/n. 30.150- La Alberca, Murcia, Spain. Date: March, 10th to 12th, 2009

PROGRAMME Day Hour Act Tuesday, 10th

08:30

Registration

09:15 to 09:30

Welcome to participants Adrian Martinez, Director of IMIDA

09: 30 to 10:15

Germination of walnut (Juglans regia L.) seeds for rootstocks production D. Frutos, IMIDA, Spain

10:15 to 10:45

Coffe break

10:45 to 11:30

Walnut propagation in Bulgaria S. Gandey , Fruit Growing Institute, Plovdiv, Bulgaria.

11:30 to 12:15

Training on Hot Callusing technique applied to walnut grafting. D. Avanzato, Centro di Richerca per la Frutticoltura, Roma, Italia

12:15 to 12:45

Grafting walnuts in Spain: new techniques. D. Frutos, IMIDA, Spain

12:45 to 13:30

In vitro propagation of walnut. J.Cos and M.A. Sanchez, IMIDA, Spain

13:30 to 14:15

In vitro walnut micropropagation. L. Bourrain. CTIFL, France

14:15 Lunch in IMIDA 15:30 to

16:30 Round of Discussion: How to profit walnut grafting techniques for selecting walnuts

16:30 Conclusions Wednesday, 11th 09:00 to Walnut small grafting and packing practices for forcing in

hot room. A. Carrillo and G. López, IMIDA, Spain

10:15 to 10:45

Coffe break

10:45 to 13:30

Practice of whip grafting and packing practices for forcing in hot room A. Carrillo and G. López, IMIDA, Spain

13:30 to14:30

Lunch

Thursday,12th 9:00 to

12:00 Visit to a Walnuts Nursery with Hot Callusing Technique

12:00 Lunch 13:00 End of workshop

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Abstracts

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Germination of walnut (Juglans regia) seeds for rootstock production. Diego Frutos, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), 30.150, La Alberca, Murcia, Spain. diego.frutos@carm.es It has been reported the inhibitors germination occurrence in Persian walnut (J. regia) seeds (Martin et al., 1960). On the other hand, soaking walnut seeds into running water had improved the germination until 83% (Memmedov, 1976). Perhaps such washing treatment could have removed inhibitors out of walnut seed. When walnut seeds were stratified in a sandy bed in the open, where rain water fell, germination percentage increases until 86% (Memmedov, 1976). It has also been reported the effect of gibberellic acid (GA3) in the range of 50 – 500 mg/l for breaking dormancy in filbert seeds (Robert and Lagerstedt, 1968). It looks like that effect of an inhibitor as abscisic acid (ABA) can be counteracted by GA3 (Dorfinger, 1970). On the basis of this references, a set of experiments were carried out in greenhouse and in the open field with the goal of clarifying the germination process in J. regia seeds. Greenhouse experiments Comparing the store procedure of cv. Adams in cold and in environmental temperature, and in dry or wet condition it was found that cold and wet stratification led to the best results (60%), but dry storing in both temperatures also resulted in a germination rounding 40%. However, as expected, wet and ambient temperature resulted in the losing of all seeds because fungi rot. But when Adams seeds kept in dry and ambient temperature were soaked into GA3 in the range of 50 to 200 mg/l, germination rounded 70%. Even the control 0 reaches a germination value rounding 60 %. The effect of soaking Adams seeds for 24 hours before GA3 treatments led to a similar evolution of all treatment, that didn’t show any significant difference in germination. Nevertheless, water soaking pre-treatment seeds gave seedlings lower than no water pre-treatment. These results led to discuss whether water pre-treatment produced a lesser entry of GA3 into seeds. In fact, gradient of humidity between GA3 treatment solutions and tissues of seeds ought to be bigger in no water pre-treatment seeds, which would receive an extra amount of GA3. Therefore, the effect of this extra GA3 absorbed would increase the height of seedlings. To verify this hypothesis a new experiment was established with seeds of an old J. regia seedling located in Garganta de la Olla, Cáceres, Spain. Seeds of it were nominated as Garganteña. They were dried for 24, 48 and 72 hours at 29 ºC, a temperature not to much high to avoid the embryo death. The idea was to increase the gradient of humidity between seeds and GA3 treatment solutions, and then to observe the effect of these gradient differences on the germination percentage. In fact, the more the time of drying, the bigger the gradient ought to be. Results recorded didn’t show any significant difference between GA3 treatments, including the control, at the end of the trial. However, significant differences were found between drying time, but the bigger the drying time, the lower the germination percent. Just opposite to initial hypothesis. What it could occurs this time was an excessive dehydration that could injure the embryos. This result should warn how important seems to be humidity in germination of J. regia seeds.

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Open field experiments Seeds of cultivar Adams, kept dry at ambient temperature, were treated with 10, 50, 100 and 200 mg/l GA3, for 24 hours. A control 0 and a 24 hours water control were also included. Seeds were sown in the open in Lewis Brown Experimental Farm of the Oregon State University in summer of 1979. In this case, 200 mg/l gave poorer result than water control. The best germination (57 %) was reached with 10 mg/l of GA3, that didn’t show any significant difference with 50 and 100 GA3 mg/l treatments. Control 0 only reaches 8% germination. A new trial comparing 10 and 200 GA3 mg/l treatments was established in Torreblanca (IMIDA) in 1980. This time it was sown five varieties: Lozerone (INRA selection RA 1006), selected for good homogeneity and emerging behaviour in nursery, Franquette, a French reference cultivar, Manregian, used as rootstock in Oregon (USA), Serr, a Californian reference variety, and Garganteña, used in a prior experiment in greenhouse. It was also included a Lozerone Control 0. Results at 10 mg/l GA3 show the best germination in Lozerone (80%). At 200 mg/l GA3 Lozerone reached 60%, Serr was very sensitive to high GA3 concentration, as long as Manregian didn’t show any difference connected to GA3 treatments. Franquette and Garganteña ranged between 40 and 50% of germination at both GA3 concentrations. All cultivars, excepting Serr, gave taller seedlings at 10 mg/l Ga3. It was also evaluated seedlings coming out 40 cultivars of an ex situ collection of IMIDA. In this case, root volume and stem section was correlated with total weight of seedlings. Conclusions

1. 10 mg/l GA3 is a good treatment for J. regia seed germination. This concentration didn’t produce unfavourable effects on seedlings

2. Seed humidity is crucial for J. regia seed germination. 3. Total weight of seedlings are correlated with root volume and with stem section 4. Seedling size seems to be bound to cultivar genotype

References Dorfing, K.L., 1970.- Planta, 93:243. In The germination of seeds. Cited by Mayer and

Poljakoff, p.65. Whealand &Co.Ltd. Ed. Frutos, D.; Martinez, J., 1993.- Evaluación de plantas de semilla de cultivares de nogal

(Juglans regia L.) como portainjertos de la especie. Martin, C.G.; Mason, I.R., Forde, H.I., 1960.- Changes in endogenous growth

substances in the embryos of Juglans regia during stratification. Journ. Amer. Soc. Hor. Sci., 94 (1)

Memmedov, B.A., 1976.- Preparation of walnut seeds for sowing. Temal. Sb. Tr. Azerb. N.II Sadovodtsva, Vinigradarska i Subtropo. Kul’tur, (9): 23-28.

Robbert, W.W., Lagerstedt, H.B., 1969.- Plastic tents for early walnut grafting. Proc. Soc. Nut. Grow. Or. Wa, 55: 98-102.

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Walnut propagation in Bulgaria Stefan Gandev. Fruit Growing Institute, Plovdiv, Bulgaria. e-mail: instov@infotel.bg

In Bulgaria the production of walnut grafted planting material started in the 70-ies of the last century. Patch budding was used and a technology was developed adapted to the climatic conditions in the country. Rootstocks are produced from fruits of common walnut (Juglans regia L.) directly planted in a nursery at the end of October or put for stratification and after that planted in spring. Planting distance is 100 cm between the rows and 20 cm space between the plants within the row. Patch budding is used leaving 1-1,5 mm looseness. The most favourable period for walnut grafting under the climatic conditions of the country is from 20 August till 5 September. At the end of November the grafted rootstocks are earthed up, the buds being covered with a soil layer of 20-25 cm thickness. In spring after the risk of frosts has passed, the grafted plants are uncovered. The wild part is pruned and the binding strip is removed. At the end of the vegetation the grafted trees reach a height of about 150 -180 cm and a graftage thickness of 2-2,5 cm. Immediately after grafting, 70-80 % of callus-formed plants are obtained, the percentage decreasing significantly under the effect of the low winter temperatures. During the last four years the percentage of successfully propagated plants at the end of the second year (before taking out the trees from the nursery) was within 33 % and 47 % (Gandev, S., 2007).

In order to eliminate temperature as a factor influencing the grafting success, a new method of walnut propagation started to be introduced into practice, called epicotyl grafting (Suk-In, H., L. Moon-Ho, J. Yong-Seok, 2006). A technology was developed at the Fruit-Growing Institute, which can be applied in industrial-scale epicotyl grafting of walnut in nurseries. Walnut seeds of common walnut (Juglans regia L.), selected for obtaining rootstocks, are immersed in water at the middle of December. After 4-5 days they are taken out of water and put for stratification in tied plastic bags at a temperature of 3-6° С. After the seeds crack open, they are temporary planted in pots of 14 l on two floors. The substrate for covering the seeds consists of peat and perlite at 2:1 ratio. Each pot contains about 30-40 seeds. Thus processed, the seeds are left for germinating in a non-heated greenhouse. When the seeds form a well-developed root and a juvenile epicotyl, split grafting is performed with 5-8 cm long one-year old scions. The scions are collected from mother trees during dormancy and they are stored in a refrigerator until the time of grafting. During the grafting procedure the vascular cambium of the scion and the rootstock are adhered to each other. The graft is fixed with plastic folio, which is not tight in order to allow the exit of surplus moisture at the place of grafting. The root tip of each rootstock is cut in order to stimulate the formation of new lateral rootlets. The grafted plants are dipped into fungicide solution to prevent from walnut bacteriosis (Xanthomonas campestris p.v. juglandis Pierce) and walnut anthracnose (Gnomonia leptostyla Fries) and then placed horizontally in plastic basins. The roots are covered with moist substrate of pine saw-dust and perlite at 1:1 ratio. The place of grafting is not covered with substrate, just the opposite – it is isolated from the moist substrate with a polyethylene band. Each plastic basin with grafted plants is put in a nylon bag which is tied to provide air humidity to the plants. Thus handled, the basins are placed on shelves in a room with controlled temperature of 25-27° С. After 14-20 days the plants form callus and start growing..

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When the new growth increment reaches 4-6 сm, the plants are temporarily planted in plastic bags of 15/20 сm size and left for adaptation. After adaptation the successfully propagated plants are about 60 %. They are either planted directly in the nursery or potted in 14 l containers

References Gandev, S. (2007). Budding and grafting of the walnut (Juglans regia L.) and their effectiveness in Bulgaria. Bulgarian Journal of Agricultural Science, 13, 683-689.

Suk-In, H., L. Moon-Ho, J. Yong-Seok (2006). Study on new vegetative propagation method ‘Epicotyl grafting’ in walnut trees (Juglans Spp.). Acta Hort. 705: 371-374.

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Training on Hot Callusing technique applied to walnut grafting Damiano Avanzato. CRA-Centro di Ricerca per la Frutticoltura. Roma, Italia. E-mail: damiano.avanzato@entecra.it

The training will face the aspects linked to the walnut graft, that is a propagation

step not so easy to be performed, in fact, the grafting results varies country to country. However the success on graft is the result of several factors, such as the selection of suitable budsticks, the use of vigorous rootstocks, and especially the right combination between humidity and temperature on the graft-point during 4 weeks of callusing. The training will concern the specific background of the Hot Callusing, a technique developed many year ago in Oregon on hazelnut grafting, and later on, extended to walnut too. The influence of seed selection, seed treatment, seedling nursery growing (in open field as well in the pot), and the pre-treatment of the mother tree donor of the budsticks, will be analyzed with respect the its influence to the graft-take.

The production of good vigorous rootstock starts since the sown of the seeds. The best results in terms of germination and production of uniform seedlings it depend from the use of selected seeds from selected mother plant. Sometime, the nurseries uses a seeds mélange represented of nuts recovered from some stock out of the commercial selling standard. Grafting seedling quality is affected by sown density also. Another factor that affects the result is the selection of suitable shoots as donor of budsticks. The easy and cheapest way to prepare the budstick is the use of shoots collected from walnut orchards. This is the worse choice because the competition between fruit and shoot growing don’t favorites the production of good budsticks. The best grafting material come from devoted tree submitted to specific pruning technique. Such shoot, having a size above of 7-8 mm, always must be well wooded, free of diseases attacks, and free of flower buds. To select the best shoot, is recommended to submit it to a forced banding: the suitable shoots are those that don’t break easily. The shoot not immediately used, can be stored in plastic bug at 4° C.

On graft-take, an important role plays the temperature and the moist around the graft-point. The importance of these factors was well known in the Mediterranean region from many years ago. An ancient grafting technique (applied in Campania-Italy as well as in Majorca-Spain) consisted on filling the graft-point with clay (to reduce the night/day temperature’s excursion) and wrapping the area around the graft-point with soil (to keep moist it). It is more than 25 year that an industrial tool called “Hot callusing” has been developed to heat continuously the graft-point: in fact, heating the graft-point for 4 weeks at 27°, help the graft-take. Using the Hot callusing tool, time grafting can start at beginning of winter (in cold greenhouse) up to the beginning of spring (on open field). The Hot callusing tools can consist of a pipe heated by hot water or electric wire and can be performed in several ways. For example, to avoid the transplanting stress, the tools can be applied on potted plants. The Hot Callusing is able to perform good results on walnut graft-takes, however for nursery production it has some bottle necks. For example, after callusing plants must be kept in greenhouse for some weeks: if transferred early to outside condition, the callus could be damaged from the late frost. In terms of quality, normally, the plants submitted to Hot callusing reach the standard size on the second year, however, this tool is a reliable technique in case of propagation of changing genetic material, such will occur between the COST’ partners.

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Grafting walnuts in Spain: new techniques. Diego Frutos. IMIDA. 30150, La Alberca, Murcia. E-mail: diego.frutos@carm.es First walnut grafts were made by rapprochement, reaching ten until 75% of takes (Mortillet, 1863). Wood quality has been reported as essential for succeeding walnut grafts. So, for getting good quality scions it was recommended to remove them out of mother plant specially cultured for this purpose (Germain and Jalinat, 1974). These scions were usually harvested in summer for getting out of each one 3-5 buds used for patch budding (Bergougnoux and Grospierre, 1975). This technique requires vigorous material, that usually it is not available in old walnut trees. For this reason it has been recommended pruning 3-4 years-old branches in walnuts of median age for increasing in 5-6% the availability of scions good enough as grafting material (Novak, 1974). The use of weak scions, available in big amount onto adult walnut trees, is crucial for simplifying walnut selection. This was the main reason for developing walnut grafting techniques, as Hot Callusing (HC) and Small Grafting Technology (SGT), at the IMIDA.

Temperature and humidity conditions It has been reported since a long time ago the benefit of good temperature conditions for taking grafts in walnuts (Corsa, 1896, Olivier, 1901). 27 ºC looks like to be the optimal temperature for walnut grafting success (Sitton, 1931). Grafted walnuts have been forced into humid peorlite at 27ºC for 3 weeks (Hartmann, 1974) or into humid sawdust for 10-15 days (Tsurkan and Chebotar, 1972; Ristevski and Georgiev, 1977) or in polyethilen bags for 12-15 days (Sharma and Dhuria, 1981). These grafts can latter be stocked at ambient temperature (Avdeev, 1987). Takes of grafting walnuts was increased by a white plastic tent covering grafts (Lagerstedt, 1972; Robert and Lagerstedt, 1972). Taking awareness of how important was temperature for succeeding with grafts, Lagerstetd developed a pipe for hot callusing that produces good results in filbert. This technology was transferred to nursery industry in Oregon (Lagerstedt, 1981). As soon as the news of this device arrived to IMIDA it was built and tried with walnut grafting, but unfortunately it didn’t success. Grafts didn’t produce callus at all in the outer side of union, and most of time formed a poor callus in the inner side. Even wetting scion cuts in IBA at 1200 mg/l, as recommended by Bugarcic and Mitrovic (1985), before forcing, the results were no goods at all. Ten it was thought that perhaps humidity closed to the forced graft union should benefit grafting walnut takes. In fact, 70-80% of air relative humidity round graft union was reported as beneficial for grafting walnut takes (Komanic, 1967). Besides, good air humidity conditions around the graft union could avoid detrimental effects of wound flooding (Lagerstedt and Robert, 1972; Prataviera et al., 1983). Then, a new device was built having account of temperature and humidity benefits on walnut grafting takes (Frutos, 1988). This first prototype was patented by Instituto Nacional de Investigaciones Agrarias (INIA) in 1982 (nº 517589). In this case the source of hot was an electric resistance, and humidity was added whit humid peorlite. Soon it was seen their weak points: a) no electricity and humidity must be work together, and b) it was necessary to improve the air humidity conditions around graft union instead surround it with humid peorlite. Both inconvenient were solved by using a hot running water pipe and a plastic drip irrigation pipe placed inside an thermally isolated channel, whit a cover thermally isolated too. This technology was transfer to nursery industry.

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Small Graft Technology (SGT) This procedure consists on beheading walnut rootstock at the neck level and ten

inserting an herbaceous scion having two leafs between both cotyledons and closing the wound whit mastic made out of gypsum and clay. Keeping graft wounds at 20-28 ºC and 60-70 % HR for 2 or 3 weeks it was possible to observe the growth of scion (Czu-Cty, 1961). This is the older and lonely reference found on this subject.

In IMIDA it has been developed a walnut graft procedure combining germination techniques and grafting woody plant material by inserting a scion between cotyledons. Young seedlings were planted in post and grafted, and then placed into a box cover with a polyethylene film in order to avoid air drying. This box must be ten kept into a climatic room at 27ºC and whit light to avoid yellowing of scion.

Whip grafted walnuts can also success in boxes as commented above. In this case roots must be cover with peorlite and scion must remain in the air. Both techniques reach high percent of takes after keeping grafts 15-18 days in these environmental conditionas.

References

Avdeev, V.A., 1987. Raising walnut and fruit mulberry transplants by bench grafting. Inst Sadov., Vinogr.,Ovoschchadvosdstva, (3): 40-43.

Bergougnoux, F., Grospierre, P.,1975. Multiplication. In Le noyer,Chapter V. Invuflec: pp, 73-82.

Bugarcic, V., Mitrovic, M., 1985. Effect of phytohormones on take of bench-grafted walnuts. Jugoslovenko Vocarstvo, 19(73/74(3/4): 389-395.

Corsa, W.P., 1896. Nut culture of the United States. USDA, Div. of Pomology, 58:13-16. Czu-Cty, 1961. Methods of grafting walnuts. Sadovodstvo, 3:25-26. Frutos, D., 1988. Walnut grafting by hot callusing. 2nd colloque Noyer-Noisetier, Bordeaux. Germain and Jalinat, 1974. Une technique pouvant etre adoptee pou la production de greffons

de noyer:la formation de pieds mere sur un axe central. CTIFL Doc nº 43. Hartmann, W., 1974. Studies on grafting walnuts. Erwerbsobstbau, 16(5):77-80. Komanic, I.G., 1967. Characteristics of walnut grafting. Izv. Akad. Nauk. Mold. SSR. Ser. Biol.

Him. Nauk, 7: 30-35. Lagerstedt, H.B., 1981. A new device for hot-callusing. HortScience, 16(4):529-530. Lagertstedt, H.B., Robert, W.W., 1972.- Walnut grafting in Oregon. In 63 Ann. Rep. North. Nut

Grws. Ass. Mortillet, P.D., 1863. Le noyer, sa culture, ses varietes.Rev. Hort., 186:499. Novak, Yu. V., 1974. Stimulating shoots formation in walnut trees by grown prinning. Lesov. I

Agrolesomelior. Resp. Mezhved. Temat. Nauch. Sbornik, (38): 115-119. Olivier, G.W., 1901. Grafting walnut and hickories. Amer. Gard., 22:307-308. Prataviera, A.G., Kuniyiki, A.M., Ryugo, K., 1983. Growth inhibitors in xilem exudates of

Persian walnuts (Juglans regia L.) and their possible role in graft failure. Journ. Amer. Soc. for Hort. Sci., 108(6): 1043-1045.

Ristevski, B., Georgiev, D., 1977. Bench grafting alnuts. Jugoslovensko Vocarstvo, 10(39/40):573-578.

Robert, W.W., Lagerstedt, H.B., 1972. Plastic tents for early walnut grafting. Proc. Soc. Nut. Grw. Or-Wa, 55: 98-102.

Sharma,S.D., Dhuria, H.S., 1981. Standardization of suitable and interval for walnut propagation under controlled conditions. Progressive Horticulture, 13(3/4): 43.46.

Sitton, B.G., 1931. Vegetative propagation of black walnut. Mich.State College Agric. Exp. Sta. Tech. Bull., 119.

Tsurkan, I.P., Chebotar, E.I., 1972. Whip grafting walnuts. Sadovostvo, (10:3) – 31.

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In vitro propagation of walnut Maria A. Sánchez, (e-mail:mangeles.sanchez3@carm.es) , and José Cos (e-mail: josee.cos@carm.es ), Instituto Murciano de Investigación y Desarrollo Agrário y Alimentario (IMIDA). 30.150, La Alberca, Murcia, Spain.

An in vitro procedure for J.regia rootstocks micropropagation has been carried

out. This procedure allowed getting clonal rootstocks with good agronomic characteristics as vigour, resistance/tolerance to Armillaria-like fungus and root knot nematodes (Meloidogine sp.).

Stage1: Getting explants out of the mother plants

Small Grafting Technology was first applied for providing explants before of their in vitro establishment. This technology has facilitate timing of works and the use of woody, half-herbaceous and/or herbaceous explants in the best sanitary conditions, as well as growth cycle control, according to works organization real needs.

Stage2: In vitro explants establishment Herbaceous material getting out of small grafted walnuts must be collected

during Df-Df2 phenological walnut state. Ten nodal segments about 2 cm long with an axillary bud can be prepared. These segments were soaked down in tap water for 30 minutes. After that, three successive washes were made, for 30 minutes each, into distilled water, soap and some few drops of "Tween-20." Disinfection was done inside of a sterile Laminar Air Flux (LAF), by dipping explants into ethanol 70% (v / v) for 30 seconds, and ten in and 5% commercial bleach for 20 minutes. All the media tried in this stage - MS, NGE, WPM and DKW- perform well. It must be unused woody and semi-woody materials, harvested at Af-CF2 phenologinal state, because their high contamination rate. The use of L-cysteine in aqueous solution at 50 mg/l helped prevent browning of culture medium and oxidation and browning of explants in the establishment phase.

Stage3: Proliferation

In this stage the best growing medium was DKW with a micronutrients and iron concentration 50% enriched, supplemented with BAP between 1 and 1.5 mg/l. White light gave good results in proliferation rate and height of explants. No differences between red and white light were found. On the other hand, use of different gelling agents – Phytagel TM, bacteriological Agar Pronadisa ®, and 2-2-1, this latter developed in the Fruit Trees Department - didn’t show any significant difference in explant proliferation. However, 2-2-1 gave the best explant quality. Results of carbohydrate sources type tried were similar. In this aspect, 25 to 45 g/l sucrose and/or glucose showed good results. Stage 4: Rooting IBA concentration and macroelements interact, in some cases, in root differentiation process. For a particular medium, rooting varies with the plant material, what suggests the convenience of adjusting media ‘a la carta’ specific for each material. i.e., the genotype Vigor bajo performed better in DKW diluted four times and supplemented with 3 mg / l IBA, kept for 10 days in darkness. However, the genotype Peralta, DKW

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medium with the concentration of macronutrients reduced to half or to a quarter, supplemented with 6 mg / l IBA, kept for 10-12 days in darkness it was recommended. Finally, the best rooting of genotype Armillaria was reached whit DKW medium with a macronutrients concentration reduced to half and/or a quarter, when was supplemented with 6 mg / l IBA, and was kept 8 days in darkness. There are evidences of root primordia at the base of the callus in many in vitro rooted walnut plants. Such grew later normally during the hardening stage, resulting in nursery viable plants. Stage 5: Weaning

Rooted in vitro walnut selections acclimation coming from stage 4 reached survival rates varying within the range of 45 to 70%. These percentages should be increased in future studies.

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In vitro walnut micropropagation Juglans regia L. application Laurence BOURRAIN. CTIFL, Centre de Balandran 30 127 Bellegarde – France E-mail: bourrain@ctifl.fr Key words: Juglans regia L., micropropagation, mycorrhizae, own-rooted varieties, rootstock, walnut, weaning. For lateral-bearing varieties production, the Ctifl carried out a program on the selection of vigorous CLR (Cherry Leaf Roll) virus free rootstock. In order to multiply homogeneous selected rootstock clones for evaluation, the Ctifl in vitro laboratory developed an in vitro multiplication technique. The now well established procedure for the micropropagation of Juglans regia L. allows for the production of homogeneous plants. This technique also allows to produce own-rooted varieties. The micropropagation is divided in 4 stages:

- stage 1 : explant preparation and in vitro establishment - stage 2 : multiplication of vitroplants - stage 3 : production of rooted plants - stage 4 : weaning

Stage 1: Establishment The initial material came from 1-2 year old seedlings or from 1 year old grafted plants if the selected material comes from mature trees. The development of the plants takes place in greenhouses in order to produce clean material. Phytosanitary treatments are provided if necessary. The in vitro establishment is generally performed between March and May. After classical disinfection with sodium hypochlorite, single bud microcuttings are established on DKW medium supplemented with 0.5-1mg/l BA and 0.01-0.05 mg/l IBA, 30 g/l glucose, 9-10 g/l agar, pH 6. Growth chamber conditions: temperature 26-28 °C, photoperiod 16 hours. Stage 2: Multiplication The vitroplants multiplication is done by axillary budding. DKW supplemented with 0.7-1 mg/ BA, 0.01 mg/l IBA, 30 g/l glucose, 9-10 g/l agar, pH6 The growth conditions are the same as for the establishment stage. The plants are subcultured every 5 weeks. Stage 3: Rooting The rooting is carried out in two stages: Induction: 7-8 days in darkness at 22 °C on MS medium supplemented with IBA 3 mg/l, 30 g/l sucrose, 9-10 g/l agar, pH 6. Rooting expression: after induction the plants are transferred onto a root development medium. Modified MS (macroelements diluted to ¼) without growth regulator, 30 g/l glucose, 9-10 g/l agar, pH 6, 190 ml exfoliated vermiculite (Vermex M) for 150 ml of agar medium, photoperiod 16 h, 23-24 °C.

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Stage 4: Weaning A well aerated substrat (Steckmedium, Klasman France for example) is recommended as well as a temperature of 22-28°C during the acclimatization phase. The use of endomychorrizes has a positive effect on the acclimatization of difficult to wean clones. The growth of the terminal bud, which has stooped during the acclimatization phase or during initial nursery establishment, can be restarted using a growth regulator (Promalin for example). References: Navaltel JC., Bourrain L., 2001. Plant production of walnut Juglans regia L. by in vitro

multiplication. Acta Horticulturae 544 : 465-471 Bourrain L., Navaltel JC., Blal B., Parat J., 1999. Les mychorizes : des champignons

symbiotiques en aide aux pépiniéristes. Infos Ctifl n°154 : 36-39 Navaltel JC., Bourrain L., Parat J., Blal B., 1996. Effect of application of a pre commercial

AMF inoculant during the weaning stage of micropropagated walnut and pear – COST 822 Physiology and control of plant propagation in vitro – Proceedings of the work shop held at Humboldt University, Berlin 1996

Bourrain L., Navaltel JC., 1995. Micropropagation du noyer Juglans regia L. Deuxième partie : acclimatation en serre et élevage du plant en pépinière. Infos Ctifl n°112 : 28-33

Navaltel JC., Bourrain L., 1994. Influence of the physical structure of the medium on in vitro rooting. Advances in horticultural science, 1 : 57-59

Bourrain L., Navaltel JC., 1994. Micropropagation du noyer Juglans regia L. Première partie : production in vitro. Infos Ctifl n°98 : 40-48

Jay-Allemand C., Capelli P., Cornu D., 1992. Root development on in vitro hybrid walnut microcuttings in a vermiculite containing gelrite medium. Scient. Hort., 51 : 335-342

Driver J.A., Kunyuki A.H., 1984. In vitro propagation of Paradox walnut rootstock. Hortsci, 19 : 507-509

Murashigue T., Skoog F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15 : 473-497

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Discussion

Lacking in walnut Xanthomonas arboricola pv. juglandis (Xaj) resistance is hard of accepting a priori because X. arboricola pv. pruni (Xap) resistance have been reported some cultivars of stone fruit species. Xaj and Xap are two pathovarieties of the same bacterium species. This fact lead to think that J. regia should not behave as an exceptional species, without Xaj resistant genotypes.

In the framework of COST Action 873, the main selection objective of WG3 for walnut (J. regia) consists on finding Xaj resistant genotypes. Already has been tried such a selection out of native walnut population of Asturias, Spain, where it was possible to find genotypes without Xaj symptoms in nature. Unfortunately when these genotypes were tested in lab all of them showed Xaj cankers to a bigger or lesser extent. However diverse degrees of tolerance were pointed out. These results led to think that Asturian walnuts without Xaj symptoms would be placed in Xaj free areas of Asturias.

Anyway, areas with bigger possibilities of finding Xaj resistant walnuts genotypes must be those holding a number of old native seedlings in areas of humid summers with presence of Xaj. Main candidate areas with these characteristics are Carpathians and Balkans mountains, or humid areas of Mediterranean countries.

Old walnut seedlings yield weak scions, very difficult to graft by traditional procedures. In fact, old grafting/budding methods require one-year-old vigorous scions that seldom occur in old walnut trees. Besides, these old methods, thought mainly to be made in the open field during growing season, are strongly dependent of local temperature conditions. Using these techniques, the process of selecting old walnut seedlings is a heavy and low-efficient job. To get that with these techniques, next steps are required: a) selection of trees in field during summer for recording the wished agronomic characteristics, b) come back to visit the selected walnut tree in winter for pruning some thick limbs and promote some vigorous shouts; c) optionally, c1) harvesting in next summer these vigorous shouts, keep them in good conditions and patch budding them, or c2) in spring of the following year, harvesting scions and make whip or cleft grafting in the open field, d) growing budded/grafted walnuts during one more active growth season in the open field, and e) remove then out of nursery in next winter grafted trees, for planting them in ex situ collection, and/or for built a mother plants orchard for good-quality-scion production.

In contrast with the above described procedure, grafting techniques shown in this meeting have been considered as very efficient tools for selecting old walnut seedlings out of native population. Hot Callusing and Small Grafting Technology (SGT) bear high percent of takes with weak one-year-old scions. A protocol for applying both methods will be prepared.

Two main considerations were done by assistant persons in the meeting. On one hand, walnut populations with potential resistance are now identified. On the other hand, technology for making efficiently walnut grafting is now available. Both considerations lead to recommend the organization of a Task Force for prospecting and evaluating Xaj resistance/tolerances out of native walnut populations.

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Conclusions

With the purpose of increasing the success possibilities in the search of walnut genotypes resistant to Xaj, it has been proposed during the meeting the organization of a Task Force whit the next objectives:

1. To propose a meeting in Bulgaria or in Romania for establishing a protocol of finding walnut Xaj resistant genotypes.

2. To identify the critical period of top Xaj symptoms occurrence. 3. To prospect the European walnut populations whit high probability of

finding walnut genotypes resistant to Xaj. 4. Testing in lab whether selected genotypes are really resistant. The participant persons in the meeting had proposed as head of this Task Force

to Dr. Damiano Avanzato.

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LIST OF PARTICIPANTS

Country Name, first name E-mail

Bulgaria Stefan Gandev instov@infotel.bg

France Laurence Bourrain bourrain@ctifl.fr Greece Alexandros Papachatzis papachad@teilar.gr Greece Peter Roussos roussosp@aua.gr Italy Damiano Avanzato damiano.avanzato@entecra.it

Italy Davide Giovanardi d_giova81@yahoo.it Romania Gheorghe Achim stpomvl@onix.ro Romania Mihai Botu stpomvl@onix.ro Spain Antonio Carrillo antonio.carrillo@carm.es

Spain Atanasio Arrieta atanasai@princast.es

Spain Alfonso Guevara joefreestyle@hotmail.com Spain Carmen Frutos cfrutosruiz@hotmail.com Spain David Correa david.correa@carm.es Spain Diego Frutos diego.frutos@carm.es

Spain Domingo López domingo.lopez2@carm.es Spain Francisco Sánchez Spain Gregorio López gregorio.lopez2@carm.es Spain Maria Ángeles Sánchez mangeles.sanchez3@carm.es

Spain Maria Belén López mariab.lopez3@carm.es Spain Margarita Pérez margarita.perez3@carm.es Spain José Cos josee.cos@carm.es

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Official press note. Information available in www.carm.es

Nevera.03.2009 Expertos europeos confirman que en Murcia existe la técnica más avanzada de propagación del nogal como especie frutal

Según el investigador Diego Frutos, “a la luz de los conocimientos actuales aportados por el IMIDA es posible desarrollar una factoría de multiplicación de nogal por injerto, sin necesidad de producir patrones en semillero” Un grupo de investigadores europeos procedentes de Francia, Italia, Grecia, Bulgaria y España, confirmaron que es en Murcia donde actualmente existe la técnica más avanzada de propagación del nogal como especie frutal, resultado de los trabajos realizados por el Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA). Dicho grupo participó entre el 10 y el 12 de marzo en un curso celebrado en Murcia, en la sede del IMIDA, para transferir conocimientos a otros investigadores y estudiantes de postgrado sobre la propagación del nogal. Esta especie se encuentra muy amenazada en Europa por la bacteria Xaj, la cual está ocasionando graves pérdidas en las poblaciones nativas de nogales europeos. El IMIDA presentó en el citado curso sus nuevas tecnologías sobre propagación por injerto con aplicación de calor en la unión y la novedosa técnica de forzado integral de pequeños injertos en cámara climatizada, las cuáles ya han sido transferidas a la industria murciana. Dichas técnicas facilitan y simplifican la costosa selección de variedades que presenten resistencia o tolerancia a la bacteria Xaj en poblaciones de nogales procedentes de semilla. Según el investigador del IMIDA y coordinador del curso, Diego Frutos, “a la luz de los conocimientos actuales aportados por el IMIDA es posible desarrollar una factoría de multiplicación de nogal por injerto, sin necesidad de producir patrones en semillero. Directamente se puede ya injertar sobre nueces germinadas en sus primeros estados de crecimiento. De esta forma puede producirse una planta injertada, desde que se recolecta la nuez hasta que se planta en el campo, en menos de un año.” Los investigadores participantes en el curso visitaron un vivero de nogal que ha incorporado la tecnología transferida por el IMIDA, donde pudieron comprobar la efectividad de la misma. Al término del curso se constituyó un grupo de trabajo para evaluar las poblaciones nativas de nogal en los Cárpatos y en los Balcanes, que, según Frutos, “por sus climas con veranos húmedos son las principales candidatas al hallazgo de formas resistentes a la citada bacteria”.

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Acción COST 873 El mencionado curso está enmarcado en la Acción COST 873, sobre “Enfermedades bacterianas de los frutales de hueso y de los frutos secos”. COST (Cooperation Scientific and Technique) es una organización intergubernamental de investigación científica y técnica que viene trabajando desde 1971, con la participación de 34 países. Las redes de investigación COST se denominan Acciones y la cooperación toma la forma de acciones concertadas entre instituciones de investigación de los países miembros. La Acción COST 873 comprende cuatro grupos de trabajo y al IMIDA le ha correspondido coordinar el grupo sobre Recursos Genéticos, bajo la responsabilidad del investigador Diego Frutos.

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