Materials and walnut propagation procedures at Industrial Plants Ltd, Kazanlak, Bulgaria

Our team of professionals involved in walnut micropropagation at Industrial Plants Ltd, Kazanlak, Bulgaria (IndPl) are continuing their work on walnut micropropagation despite the major problems regarding the multiplication of walnuts in vitro. As we mentioned earlier, the protocol applied at Bosques Naturales S. A  (BN) proved not to be suitable. We undertook the task of elaborating new protocols for walnut micropropagation solely due to the fact that the protocol provided by the specialists at BN and which is surely viable at BN as we witnessed, did not work in the laboratory conditions at IndPl. Despite our efforts and the spectacular results obtained, our achievements and new protocols are not suitable for production either, due to lack of repeatability, instability and lack of reliability due to the physiological characteristics of the walnut plantlets, which are currently not known and understood. For example, the composition of the media in one multiplication cycle greatly influences the growth habits of the plantlets not only in the present multiplication cycle but in 1-2 subsequent cycles, in most cases in a negative way (large callus at the base of the plantlets, growth inhibition) although the growth habit in the present multiplication cycle was positive (e. g. intense axillary shoot proliferation).


We are going to present, below, a few elements of our practical approach, at IndPl.


Replicating the BN modification of the DKW basal media by using stock solutions. At IndPl we are continuing to use mostly the prepackaged  DKW, BN modification, but we successfully tested replicas of this basal medium prepared from stock solutions and components available at IndPl (Fig. 1)

Fig. 1. Components of the replica for the BN modification of DKW. From left to right: DKW sulphates stock, DKW macronutrients stock, micronutrients, vitamins, myo-inositol, iron source (FeNaEDDHA)

The above replication was used solely for experimental series and not for maintenance and production, but the results were similar to the ones obtained by using the prepackaged DKW, BN modification with the other necessary additives.

At IndPl we elaborated two main media compositions for regular maintenance and multiplication, one of them based on the protocol elaborated at BN (Table 1) and the other one is radically different, based on our experience at IndPl (Table 2).


Table 1. The composition of our maintenance and multiplication medium based on the BN multiplication protocol

We recommend both of them for testing and improvement and we do not recommend them as a multiplication stage in the production process. Both are also suitable for the elongation stage prior to the root induction stage in the two-stage rooting protocol. 

Table 2. The composition of our maintenance and multiplication medium based on experience at IndPl

As we already mentioned, we performed intense axillary shoot proliferation, but the results were not repeatable and thus currently not suitable for publication and/or the elaboration of a production protocol and we are currently doing experiments for the improvement of axillary shoot proliferation.


Currently we are maintaining the walnut plant material by using the media presented in Table 1 and we are selecting and subculturing the available plant material. Currently at IndPl there are 350 jars of walnut cultures available (Fig. 2), maintained and multiplied with the above-mentioned media, with a practical multiplication rate of 1.5 per one-month cycle.

We faced severe challenges due to endogenous and exogenous contamination and we discarded 20 crates 24 jars each this year, that is 480 jars containing 12 explants each.

In Fig. 4 we present several stages of the walnut germplasm growth on the aforementioned culture medium.


We are currently testing different types of explants (one and two-node) as a precursor for a regeneration and elongation stage before a large-scale in vitro rooting test (Fig. 3).

Fig. 2, A, B and C. Walnut in vitro germplasm maintained at IndPl
Fig. 3, A, B, C. Small explants tersted at IndPl for a pre-rooting stage
Fig. 4. Walnut germplasm growth and development on a standard medium: A, B, C – three-day old explants already sprouted; D, E, F – two-week old cultures; G, H, I – 17 and 18 day-old cultures; J – one-month old cultures on deadline, ready for subculture

Woodnat at designers encounter

On June the 28th of 2018, a large Encounter of designers and industrials was hold in Ferrol (Galicia). The Regional Government organized the meeting, which included the presence of the Regional Minister. It counted with the most relevant actors in the field of design and industry. More than 200 people attended to the meeting, which counted with cutting-edge personalities such as Benjamín Castro (the Adidas art director) to Esteban Carballido (the head of Televes product development).


Woodnat coordinator took part on the encounter, which lasted all day long. It included parallel actions to the speeches. There was a design based meal and an exhibition of design pieces. Thanks to the generous invitation of the director of the encounter, Seistag presented a piece of furniture that used walnut wood instead of iron nails.


The activity was developped under the moto of “Design for Innovation”. This is part of a strategic line of development for the region. The forestry resources and wood industry are key stakeholders for that strategy. Proving this, the meeting counted with the speeches of CMD (the regional cluster for wood/forestry industry) and Finsa, the largest Company of that sector. 

Woodnat Project at SEB annual convention and assembly

Woodnat project has been presented to SEB ( Société des Experts Bois) during the General Assembly. Experts of this well known French association come from various sector of activity; trading, consulting, research, engineering ... During the GA, President Thierry Charloux proposed to organize in May 2019 a visit to Galicia to discover the potential of the forest industry in this part of Spain. Of course Woodnat project and especially Bosques Naturales plantation will be at the center of this business trip. President Charloux will enter in contact with Elias Cueto from Seistag to organize the schedule of this visit.

On June 15th & 16th, the Wood Experts Society (Société des Experts Bois, SEB in french) has had its General assembly at the Higher Technical School of Wood Biel (CH). 

Among industrial and construction sites (visits and the presentation of the different research areas of the School, the woodnat project has been mentioned and voted to be the next field visit of the SEB, planned for Mai 2019, in order to have an overview of this integrative project from the seed to the plant and then to the end user.


The Wood nat project has triggered a lot of interest from the SEB Experts and we can expect good dissemination through their respective networks.

Veneer analysis

The European walnut veneer was subject to studies following the original work plan of the WOODNAT Project. One of the issues studied by LOSAN ROMANIA was the colour of the wood. Walnut is known for being dark, but European walnut isn’t. Moreover, the young trees tend to be very clear, so they don’t look like walnut. This is a characteristic that isn’t well received  by the market.


The trials carried out during 2017 demonstrated the importance of log cooking before slicing. The cooking process contributes to two changes on the thin veneer surface. On the one hand, there are fewer marks during slicing when the log is cooked. On the other hand, by cooking the posterior colour of the veneer is darker.


The colour analysis is not a simple question. Measuring colour needs specific tools and knowledge. In order to carry out the study, the technicians of LOSAN ROMANIA used a spectrophotometer with which the CIELab coordinates of the wood surface were established.


The colour difference between cooked and uncooked logs was calculated by comparing the average coordinates of the samples. By means of this we know that: (i) the luminosity “L” decreases when cooking; and (ii) the “a*” parameter and (iii) the “b*” parameter also increase. This means the wood is darker, redder and yellower.