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
2
Index
Venue and Programme
4
Abstracts
5
Germination of walnut (Juglans regia L.) seeds for rootstock production.
Diego Frutos
6
Walnut propagation in Bulgaria
Stefan Gandev
8
Training on Hot Callusing technique applied to walnut grafting.
Damiano Avanzato
10
Grafting walnuts in Spain: new techniques.
Diego Frutos
11
In vitro propagation of walnut
Maria A. Sanchez and José Cos
13
In vitro walnut micropropagation Juglans regia L. application.
Laurence Bourrain
15
Discussion
17
Conclusions
18
List of participants
19
Official press note about this meeting
20
3
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
09:15 to
09:30
09: 30 to
10:15
Registration
Welcome to participants
Adrian Martinez, Director of IMIDA
Germination of walnut (Juglans regia L.) seeds for
rootstocks production
D. Frutos, IMIDA, Spain
Coffe break
10:15 to
10:45
10:45 to
11:30
11:30 to
12:15
12:15 to
12:45
12:45 to
13:30
13:30 to
14:15
14:15
15:30 to
16:30
16:30
Wednesday, 11th
09:00 to
10:15 to
10:45
10:45 to
13:30
13:30
to14:30
Thursday,12th
9:00 to
12:00
12:00
13:00
Walnut propagation in Bulgaria
S. Gandey , Fruit Growing Institute, Plovdiv, Bulgaria.
Training on Hot Callusing technique applied to walnut grafting.
D. Avanzato, Centro di Richerca per la Frutticoltura, Roma, Italia
Grafting walnuts in Spain: new techniques.
D. Frutos, IMIDA, Spain
In vitro propagation of walnut.
J.Cos and M.A. Sanchez, IMIDA, Spain
In vitro walnut micropropagation.
L. Bourrain. CTIFL, France
Lunch in IMIDA
Round of Discussion:
How to profit walnut grafting techniques for selecting walnuts
Conclusions
Walnut small grafting and packing practices for forcing in
hot room.
A. Carrillo and G. López, IMIDA, Spain
Coffe break
Practice of whip grafting and packing practices for forcing
in hot room
A. Carrillo and G. López, IMIDA, Spain
Lunch
Visit to a Walnuts Nursery with Hot Callusing Technique
Lunch
End of workshop
4
Abstracts
5
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. [email protected]
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.
6
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.
7
Walnut propagation in Bulgaria
Stefan Gandev. Fruit Growing Institute, Plovdiv, Bulgaria.
e-mail: [email protected]
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 FruitGrowing 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..
8
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.
9
Training on Hot Callusing technique applied to walnut grafting
Damiano Avanzato. CRA-Centro di Ricerca per la Frutticoltura. Roma, Italia.
E-mail: [email protected]
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.
10
Grafting walnuts in Spain: new techniques.
Diego Frutos. IMIDA. 30150, La Alberca, Murcia. E-mail: [email protected]
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.
11
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 (CzuCty, 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, 7382.
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.
12
In vitro propagation of walnut
Maria A. Sánchez, (e-mail:[email protected]) , and José Cos (e-mail:
[email protected] ), 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
13
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.
14
In vitro walnut micropropagation Juglans regia L. application
Laurence BOURRAIN. CTIFL, Centre de Balandran 30 127 Bellegarde – France
E-mail: [email protected]
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.
15
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 : 335342
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
16
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.
17
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
[email protected]
France
Laurence Bourrain
[email protected]
Greece
Alexandros Papachatzis
[email protected]
Greece
Peter Roussos
[email protected]
Italy
Damiano Avanzato
[email protected]
Italy
Davide Giovanardi
[email protected]
Romania
Gheorghe Achim
[email protected]
Romania
Mihai Botu
[email protected]
Spain
Antonio Carrillo
[email protected]
Spain
Atanasio Arrieta
[email protected]
Spain
Alfonso Guevara
[email protected]
Spain
Carmen Frutos
[email protected]
Spain
David Correa
[email protected]
Spain
Diego Frutos
[email protected]
Spain
Domingo López
[email protected]
Spain
Francisco Sánchez
Spain
Gregorio López
[email protected]
Spain
Maria Ángeles Sánchez
[email protected]
Spain
Maria Belén López
[email protected]
Spain
Margarita Pérez
[email protected]
Spain
José Cos
[email protected]
19
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”.
20
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.
21