Investigation of modified WPM medium for the best meristem proliferation of Corylus avellana L

Cultivation of Corylus avellana L. in Turkey is performed generally in the northern regions where it is an important source of livelihood for the local farmers. More than 70% of world hazelnut production is supplied by Turkey, but compared with other countries, Turkey’s hazelnut production area is quite narrow. In this study was aimed to develop an effective in vitro production for seven local cultivars of C. avellana. Therefore, WPM medium supplemented with 6­Benzylaminopurine (BAP) was modified by using single or in combinati­ on of Fe­EDDHA, AgNO3, H3BO3, charcoal and gibberellic acid. In all varieties, the best regeneration rates varying between 68% and 94% were obtained from WPM medium supplemented with 4.4 μM BAP, 27.8 μM Fe­EDDHA and 10g/L Charcoal. Genetic stability of shoots derived from meristem culture using the best medium was analysed using ISSR primers, when the gel images of the PCR products were examined, no polymorphic band was observed in samples col­ lected from seven provinces, and the genetic stability was determined as 100%.


Introduction
Corylus avellana L. plants are grown at altitudes in the range of 20 1500 m in the northern parts of the Turkey, they can also be grown in the southern and eastern creeks, and rarely in the western regions (Aydinoglu, 2010). Annual rainfall is balanced according to the seasons and temperatures, above 700 mm rainfall and temperatures ranging from 8°C to 10°C with the highest between 33°C and 36°C are ideal conditions for the production of C. avellana L. (Koksal et al., 2006). More than 70% of world hazelnut production is produced by Turkey (Zhao et al., 2015). However, Turkey's hazelnut production efficiency is very low compared to other countries. The average yield is 179.2 kg/ha in the US, 168.6 kg/ha in Italy whereas in Turkey it is 91.7 kg/ha (Mennan et al., 2020;Sen and Kahveci, 2020).
The in vitro propagation procedures for clonal multiplication provide the useful alternative tools to traditional propagation of hazelnut culti vars. Development of an efficient procedure for hazelnut micropropagati on can also be beneficial for increase of clones, culti vars and varieties resistant to different pathogens such as hazelnut worm (Curculio nucum), eastern fil bert blight (Anisogramma anomola) and powdery mildew (Erysiphe corylacearum) (Bassil et al., 1992;Lucas et al., 2018).
The transfer of parts of the plant such as tissue, cell, organ, embryo to in vitro environment under aseptic conditions in an artificial nutrient medium for clonal propagation is important to produce a patho genfree plant with protected germplasms and/or to maintain its secondary metabolites production which otherwise is limited for various reasons is called plant tissue culture (Hayta et al., 2017;EspinosaLeal et al., 2018). In addition to the continuous production thro ughout the year by obtaining thousands of plants with the same form and characteristics as the mother plant in a short time by micropropagation method from plant tissue cultures; superior species resistant to factors such as drought, salinity, acidity, and/or cold can also be produced. Micropropagation invol ves plant selection, sterilization, in vitro transfer, clo nal propagation, rooting, conditioning, and transfer to soil. Due to these advantages, micropropagation is one of the most effective methods when compared with other production techniques (Ozudogru et al., 2011;Ozudogru and Kaya, 2012).
Different techniques in plant tissue culture can be used such as shoot tip culture by transferring the shoot tips with growth cone including meristematic doom to in vitro environment, bud node culture by transferring the axillary or apical buds of the shoots to aseptic conditions, meristem culture by transfer ring meristem from the meristematic region to the nutrient medium with the help of a stereomicrosco pe, embryo culture by taking the embryo from the seed template or from the seed and transferring it to the germination medium (Ahmad and Anis, 2007;Usha et al., 2007).
Clonal production of plants in a short time is one of the main purposes of plant tissue culture, howe ver, changes may occur in micropropagated plants, all the changes that may occur in the hereditary material are called somaclonal variation, somaclonal variations can be detected by various molecular tests (Samir, 2004;Lattoo et al., 2006;Ozudogru et al., 2011). Nowadays, genetic differences in the germ plasms of many plant species can be detected using molecular markers, nonspecific primers are often preferred in determining the differences between germplasms in methods based on the polymerase chain reaction, these primers are particularly advan tageous for plant species that have not been previ ously studied or have a limited number of researches performed on them (Devarumath et al., 2002;Carvalho et al., 2004;Kaya and Souza, 2017). Although there are a wide variety of molecular mar ker techniques used nowadays, AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplification of Polymorphic DNA), SSRs (Simple Sequence Repeats), ISSRs (Inter Simple Sequence Repeats) techniques are preferred. The method to be used is selected based on the technical facilities accessible and the purpose of the study (Belaj et al., 2003;Kaya, 2015).
The ISSR technique used in many plants is a sim ple, fast, and lowcost technique, it also stands out for its ability to work with a small amount of templa te DNA and provides a high level of information. With all these advantages, the ISSR technique is often pre ferred in determining the difference in germplasm collections (Gilbert et al., 1999;Kaya and Souza, 2017).
When there are some limitations (shoot tip necro sis, big contaminations, sensitivity for surface sterili zation treatments) causing from plant material such as shoot tips, nodal segments, buds in in vitro propa gation studies, seeds can be used as starting material and there are many similar studies in the literature such as micropropagation of Thymus vulgaris (Ozudogru et al., 2011), Citrus spp. (Kaya et al., 2016, Musa spp. (Kaya et al., 2020 b). Similarly, embryos isolated from hazelnut seeds were used as starting material in the current study, as there was a major contamination problem. Thus, the optimal growth medium developed for hazelnut these local varieties in the current study will be a model growth medium for future in vitro studies such as cryopre servation.
In this study, WPM nutrient medium developed by Lloyd and McCown which commonly used in mic ropropagation of woody plants in order to develop an effective environment for in vitro meristem repro duction of hazelnut plant, which is relatively difficult to micropropagate due to some limitations was used (Lloyd and McCown, 1980). The main aim of this study was to determine optimal medium combinati on by using WPM. Seven different cultivars of hazel nuts grown in seven different provinces of Turkey were examined with different combinations of Benzyl Amino Purine and Gibberellic Acid growth regulators, ethylene diamine di2hydroxyphenyl acetate ferric (FeEDDHA), Silver nitrate (AgNO 3 ), boric acid (H 3 BO 3 ) and activated charcoal. In addition, the another aim was to confirmation genetic stability of in vitro grown plants. For this reason, the genetic stability of the plants growing in in vitro conditions that gave the best results in the study was analysed by the ISSR technique.

Determination of genetic stability
In order to determine the effects of the modified WPM nutrient medium that gives the best regenera tion with the meristem culture method on the gene tic stability of the plant, DNA isolation was performed manually following the 2× CTAB protocol developed by Ferdous et al. (2012). Following the DNA isolation, the protocol developed by Williams et al. (1990)  (GA) 8T UBC810] to perform an ISSR PCR reaction. The products obtained from the ISSR PCR were run on an agarose gel electrophoresis containing 1.5% agarose and the bands profiles were visualized under UVtransilluminator. The "Similarity Ratio" formula developed by Sokal and Sneath (1963)  The DNA of the samples were obtained from in vitro embryogrown leaves (in WPM medium supple mented with 4.44 µM BA), leaves of shoots that developed after subculture (in WPM medium contai ning 4.44 µM BA and 27.8 µM FeEDDHA), and post meristem culture grown shoot leaves (in WPM medi um containing 4.44 µM BA, 27.8 µM FeEDDHA and 10 g L 1 charcoal) were used.

Experimental design, data collection, and statistical analysis
For each cultivar, more than 100 embryos of seeds were used for in vitro culture establishment. Three Petri dishes (replicates), each containing 10 shoot tips, were used for each in vitro proliferation treatment, and each experiment was repeated at least three times. Data of in vitro proliferation were recorded 4 weeks after culture initiation and consis ted of the percentage of shoot tips that generated at least one elongated shoot. Regeneration percentage was compared by multiple X2 test by the SPSS prog ram (IBM SPSS Statistics 21.0) and statistical analysis was also performed with ANOVA, followed by the LSD test at P≤0.05 (Marascuilo and McSweeney, 1977).

Results
After surface sterilization, the embryos germina ted in WPM medium containing 4.44 µM BAP were transferred to the regeneration medium, from the samples that were used as starting material, 71% to 85% sterile material was obtained as a result of surfa ce sterilization. The obtained clean materials showed successful regeneration between 81.1% and 100%. Shoot tips grown in vitro were subcultured, and rep roduced in 30day periods, and used as the material in meristem culture study. The desired amount of in vitro material was obtained from regenerated plants for use in meristem culture applications.
The best regeneration rate from eight different WPM media in different combinations tested for meristem regeneration was obtained from WPM media containing 4.44 µM BAP, 27.8 µM FeEDDHA, and 10 gL 1 charcoal. The regeneration rate obtained on this medium ranged from 68% to 94% for the seven hazelnut local cultivars tested (Fig. 2).
Polymerase chain reactions were carried out using six different primers to determine the genetic stabi lity of regenerated hazelnut varieties. In order to determine genetic stability, clones of each sample developed from a single embryo were used. PCR pro ducts obtained from the PCR using six different ISSR primers were run on an agarose gel and visualized. A total of 324 bands with a length of 120 bp and 2800 bp were scored from the band profiles obtained. In the light of the band profiles obtained, no polymorp hic bands were observed in samples collected from the seven provinces, and the genetic stability was determined as 100% (Fig. 3).

Discussion and Conclusions
In Turkey, for hazelnut production, the certified seedlings have not still been studied and conventio nal production does not meet all requirements of Hazelnut plants. Because these plant cultivars can be affected very quickly by climatic conditions and fruit yield may decrease accordingly, physical factors such as the reduction in the mineral and organic matter content of the soil, and biological factors such as hazelnut pests and diseases can further decrease its yield. In this case, it is extremely important to exami ne the factors that cause low productivity and to determine the measures that could eliminate these factors or reduce their effect (Amaral et al., 2006;Aydinoglu, 2010;Ciarmiello et al., 2014).
Increasing trends in areas such as the develop ment of systems for densely planting tree seedlings, selection of new parents for nutrition programs, development of new cultures in order to better eva luate the existing limited areas, increased the need for modern techniques in fruit tree cultivation. Traditional methods, which were carried out to meet the demand for more highquality plant material are insufficient for they are both time consuming and insufficient for standard and healthy production, the refore, the application of in vitro techniques in fruit growing may be a suitable alternative way to overco me these problems. Developments in plant biotech nology, particularly in the fields of in vitro cultures and molecular biology, have been a powerful tool in the control and conservation of plant biodiversity (Withers, 1995). Nowadays, biotechnological met hods comprise the most suitable methods for patho genfree short, medium, and longterm protection of ornamental, medicinal, and aromatic plants as well as woody plant species, which are in danger of extincti on and poses rare economical value. In vitro conser vation methods are especially important for the pro tection of vegetatively propagated plant species with seeds that are intolerant to desiccation (Engelmann, 2011). In addition to the advantages mentioned above, in vitro techniques provide a reliable platform for international plant material exchange, enable large collections using minimal space, provide valu able materials for species recovery, and facilitate molecular research and ecological studies (Tandon et al., 2005).
Traditional propagation procedures for hazelnut through shoots is time consuming, limited and ineffi cient for large scale production. These days, many hazelnut cultivars have been identified for plantation in Turkey and it immediately needs to develop effici ent procedures for rapid clonal propagation. The plant tissue culture systems offer an useful tools for large scale production of plantlets (Damiano et al., 2005;Bacchetta et al., 2008). The current study aimed to develop and integrated procedure to clonal propagation of Turkish local hazelnut cultivars with optimal combination of growth media supplemented with plant growth regulators/additives in tissue cul ture techniques.
Meristem culture has been used for the purificati on of pathogens from plants since the 1930s (Dodds and Roberts, 1986;Nehra and Kartha, 1994). In this technique, the difference in the amount of patho gens in different plant cells is utilized such that the apical and root meristem cells of the plant are very small due to their continuous division characteristics hence the probability of viruses in these cells is very low, therefore, these tissues are highly preferred in the production of pathogenfree and/or diseasefree plants (Kaya et al., 2020 a). Although it cannot be sta ted that these parts of the plant are completely free from viruses, the probability can be increased when applying different treatment combinations together (Sherwood, 1993). In this study, meristem culture examinations were carried out using meristematic regions of length between 0.10.8 mm extracted from axenic (virusfree) shoots of plants belonging to hazelnut varieties collected from seven different pro vinces that were grown in vitro using clonal propaga tion. The microshoots regenerated in vitro were sub cultured in 46 week periods at standard conditions and the best regeneration rate among the eight diffe rent WPM media containing different ingredient combinations tested for meristem regeneration was obtained from WPM medium containing 4.44 µM BAP, 27.8 µM FeEDDHA and activated charcoal, and the regeneration rate obtained on this medium was between 68% and 94% for seven different hazelnut local cultivars tested.
The iron component of medium is an essential micronutrient for Hazelnut in vitro cultures and these cultures can also be very sensitive to the iron source and concentration. In in vitro cultures of hazelnut, beneficial effect of FeEDDHA as source of iron, have been reported in previous studies (Nas and Read, 2001;Garrison et al., 2013). In the current study, the significant multiplication rates in hazelnut local culti vars were achieved by using medium supplemented with FeEDDHA as iron source.
In the current study, the media supplemented with activated charcoal showed the beneficial effect on in vitro cultures of all hazelnut local cultivars. In in vitro cultures, because of their absorbtion ability (growth regulators, inhibitors, other compounds), the activated charcoal can promote growth (Pan and Staden, 1998).
Morphological differences that make up biological and pharmaceutical characters are under the influen ce of environmental and/or genetic factors (Hay and Waterman, 1995). In recent years, DNAbased mole cular markers have been used to determine genetic differences in the germplasms of many plant species, the use of various DNA markers in plant research has become increasingly common as they provide a lar ger number of markers than allozymes. In various DNAbased PCR methods, mostly nonspecific pri mers are used to determine the differences between (and within) species. These methods, which are pre ferred because they do not require sequence know ledge in target species, are particularly suitable for plant varieties on which none or very little research has been done before (Walton, 1993).
In this study, the genetic stability of hazelnut sam ples collected from seven different provinces after meristem culture was evaluated by PCR using six dif ferent ISSR primers. Polymorphism was not detected in the cultivars as a result of visualizing the PCR pro ducts in agarose gel. In this context, we can say that the nutrient medium optimized for meristem culture does not have a negative effect on the genetic stabi lity of the hazelnut varieties tested.
It is known that plants propagated in vitro by shoot tip, axillary bud, and meristems, maintain their genetic stability during cell division and differentiati on under in vitro conditions (Shenoy and Vasil, 1992;Ostry et al., 1994). However, it should not be over looked as there is a risk of alteration of genetic stabi lity induced during the in vitro applications (types and concentrations of growth regulators, nutrient conditions). There are many studies in the literature regarding this (Rani et al., 1995;Giménez et al., 2001), these changes in genetic stability are often inherited and can be passed down from generation to generation during micropropagation (Breiman et al., 1987).