Hazelnut micropropagation method

Abstract

The invention relates to a micropropagation method for rapid propagation of hazelnut (Corylus avellana L.) plants by tissue culture, comprising special nutrient media developed for the propagation stage and a technique for rooting.

Description

[0001] HAZELNUT MICROPROPAGATION METHOD

[0002] Technical Field of the Invention

[0003] The invention relates to a micropropagation method for rapid propagation of hazelnut (Corylus avellana L.) plants by tissue culture, comprising special nutrient media developed for the propagation stage and a technique for rooting.

[0004] State of the Art

[0005] Hazelnut is the most widely cultivated nut crop in the world after walnut and almond. The most important nuts produced in Turkiye are hazelnuts, walnuts, pistachios and almonds, respectively. Cultivated varieties of hazelnut are grown in Turkiye, Italy, Spain, USA, Chile, China, Iran, Greece, France, Azerbaijan, Russia and Georgia. Hazelnut is the name of the shrub and tree species that make up the Corylus genus, a member of the birch (Betulaceae) family. Hazelnut species important for hazelnut cultivation and commercial production are Corylus avellana L. (Common hazelnut), Corylus colurna L. (Turkish hazelnut) and Corylus maxima Mill. (Lambert hazelnut). In order to renew hazelnut orchards and to ensure that newly developed high-yielding and high-quality hazelnut varieties can be delivered to the farmers quickly, hazelnut saplings should be massively multiplied. In plant propagation by tissue culture, a basic nutrient medium containing macro- and micronutrients and vitamins is used. By adding growth regulators or different compounds to this, a special nutrient medium is obtained that provides the healthiest proliferation and growth according to the plant species. MS nutrient medium discovered by Murashige and Skoog is effective in many plant species

[0001] . Over time, researchers have developed nutrient mediums suitable for their plants by using this medium in different doses (1 / 2, 1 / 4, 3 / 4) according to the needs of the plants, changing the vitamin mixture, and adding new chemicals (macro / micro elements). DKW nutrient medium is developed for walnuts, and NRM nutrient medium is developed for hybrid hazelnut varieties [2,3]. These 3 basic nutrient mediums (MS, DKW, NRM) are frequently used in tissue culture studies conducted on hazelnuts. In current micropropagation techniques, there is a multiplication rate (3-5 new shoots) in plants that are not problematic in the proliferation stage, and each shoot in each subculture produces new shoots at this rate. In hazelnut, this ratio cannot be maintained at the same level and continuously during the multiplication stage. This prevents the mass production of the plant in a short time. In embodiments in the state of the art, basal sprouts of the plants in hazelnut nurseries are used as propagation material. These sprouts can arise from the main plant or as a result of the germination of seeds that fall from the branches. This situation causes deterioration of variety purity in foreign pollinated hazelnut plants and variety mixture in orchards. For this reason, hazelnut plants need to be propagated vegetatively. On the other hand, a certain number of basal sprouts, which are formed annually, cannot ensure rapid propagation. In the state of the art, Nas (2004) utilized dormant cuttings taken from field-grown hybrid hazelnut (C. avellana L. x C. americana Marsh.) plants and forced outgrowth by immersing the basal end of twigs in forcing solution [3]. Buds taken from fresh shoots formed as a result of the sprouting of dormant cuttings were cultured in MS medium and modified DKW medium containing or not containing 1.5 mg / l, 3 mg / l or 4 mg / l 6- benzyl amino purine (BAP) and polyamines (32.21 mg / l putrescine + 29.05 mg / l spermidine + 10.10 mg / l spermine). Data regarding the evaluated criteria were obtained after 2 subcultures. The effects of culture medium (MS or DKW) and BAP doses on explant reactions were found to be insignificant. When added to the medium, polyamines were observed to be very effective on shoot length and the number of buds per shoot. When added to the medium, polyamines were observed to increase the average shoot length by 83% and the average number of buds per shoot by 41 %. It was observed that the base shoots on the medium containing polyamines could grow up to 4.0 cm, while the shoots on the medium without polyamines could grow up to 2.0 cm. The findings showed that when polyamines were added to the medium, they could facilitate the cultivation of explants and increase their propagation capacity. However, no more than two subcultures were made with the polyamine-containing nutrient media used in the study, and changes that may occur in the propagation and development of the plants were not revealed.

[0006] In another document in the state of the art, Liu et al. (2009) compared NRM, DKW, WPM, NN and MS nutrient media in hybrid hazelnut varieties and reported that the shoot length and number of new shoot buds were higher in NRM nutrient media compared to others [4], They reported that when 5 mg / l 6-benzyl amino purine (BAP), 0.01 mg / l indole-3-butyric acid (IBA), 32.21 mg / l putrescine, 29.05 mg / l spermidine and 10.10 mg / l spermine were added to NRM nutrient media, it increased the development and growth of buds. However, in which subculture the data was taken was not stated. They reported that waiting for 20 seconds in 1 g / l IBA for rooting was 95% effective on the rooting percentage. The high rooting level obtained in hybrid hazelnuts with this method cannot be achieved in common hazelnuts (Corylus avellana L.).

[0007] In order to ensure mass production of hazelnut saplings in a short time, it has become necessary to make a development in this field due to the limited and inadequate solutions in conventional methods and the low proliferation rate and rooting problems encountered in micropropagation.

[0008] Brief Description and Aims of the Invention

[0009] The invention relates to a micropropagation method for rapid propagation of hazelnut (Corylus avellana L.) plants by tissue culture, comprising special nutrient media developed for the propagation stage and a technique for rooting.

[0010] One aim of the invention is to provide mass production of hazelnut plants in a short time. By means of the arrangements made in the nutrient medium and the techniques applied in this invention, propagation continues in each subculture (2-3 shoots), and rooting is provided at a rate of 100% in healthy plants. Thus, mass production of hazelnut plants is provided in a short time.

[0011] Another aim of the invention is to provide a solution to the low rate of propagation and rooting problems encountered in the propagation and rooting stages in seedling production studies with tissue culture in hazelnuts. The special nutrient medium used in the method of the invention is shaped on the NRM basic nutrient medium. The amount of FeEDDHA (iron chelate), which was originally 100 mg / l in the medium, was increased to 250 mg / l as a result of literature information and observations made in subcultures. a-Tocopherol (vitamin E) was not used. Said method comprises the use of polyamines in NRM nutrient medium, their combinations and amounts, and their order of use. By means of this special nutrient medium, a regular increase in the propagation stage in tissue culture studies on hazelnuts and 100% rooting in healthy plants is achieved with the technique applied in rooting.

[0012] Another aim of the invention is to ensure the regular and continuous propagation of hazelnut plants in the tissue culture method. At this stage, the special nutrient medium obtained using the method of the invention is in three stages. The nutrient medium contents for multiplication in each stage differ in terms of growth regulators and polyamines and are applied in a specific order. Only putrescine is added as a polyamine to the nutrient medium in the third stage. The shoots are transferred as clusters to ensure regular and continuous multiplication.

[0013] Another aim of the invention is to develop a method that will provide 100% rooting of Corylus avellana L. in particular. In the studies conducted in the state of the art, the nutrient media used are evaluated at the end of 1 -3 subcultures. These periods are not sufficient to find out the abnormalities in the plants. Therefore, in articles belonging to similar studies, the continuous use of all three polyamines together is reported to be successful. In the method that is the subject of the invention, the results in plants that were subcultured for two years were used as a starting point. Switching to the medium containing only putrescine as polyamine after a few (2-3) subcultures in the medium containing all three polyamines, healthy development and new shoots are obtained in the materials and the number of plants to be taken to the rooting stage increases rapidly. The rooting percentage in the rooting method in the detailed description of the invention is determined as 100%. The technique and liquid medium used in this method are effective in the success achieved. Rooting is easier in hybrid hazelnuts. There is no other study in the state of the art that reports the rooting of Corylus avellana L. as 100%.

[0014] Another aim of the invention is to ensure continuous and healthy plant formation in the hazelnut micropropagation method. The continuous and healthy plant formation is ensured by using the polyamines used in the propagation stage, which is the 2ndstage in the hazelnut micropropagation method that is the subject of the invention, in a specific order, that is, firstly, three together and then only putrescine. The main effect in propagation is made by polyamines. However, using them first as a combination of three, then as just putrescine ensures continuous and healthy plant formation. Detailed Description of the Invention

[0015] The invention relates to a micropropagation method for rapid propagation of hazelnut (Corylus avellana L.) plants by tissue culture, comprising special nutrient media developed for the propagation stage and a technique for rooting.

[0016] Micropropagation method that comprises a technique applied for rooting with special nutrient media developed for the propagation stage in order to ensure rapid propagation of the hazelnut plant by tissue culture method comprises the process steps of: i. applying surface sterilisation to 15-20 cm long branches cut from 2-5 year old seedlings grown in the greenhouse, ii. placing the parts whose surface sterilisation has been completed on sterile filter papers in the sterile cabinet and leaving them to dry for 30 minutes, iii. adjusting the pH of the nutrient medium to 5.75 with 1 N sodium hydroxide (NaOH) or 1 N hydrochloric acid (HCI) before adding agar to the nutrient media and sterilizing it in an autoclave (1.2 atm pressure - 121 °C - 20 minutes), iv. at the end of this period, placing the bud pieces one by one in tubes containing 1 / 2 MS (2.2 g / l) nutrient medium, 30 g / l sucrose, 1 .5 mg / l 6-benzyl amino purine (BAP), 6 g / l agar, v. culturing the tubes in a growth chamber at 4°C for 45 days to ensure that the buds complete their vernalisation, vi. at the end of this period, transferring the branch pieces to new tubes containing the same nutrient medium (subculture) and placing them in a growth chamber with conditions of 24°C, 16-hour photoperiod, and 45 pmol.m-2s-1daylight, vii. obtaining fresh shoots from all the buds that sprout within a month, viii. when they reach 5-6 cm in length in the tubes, separating the shoots from the branch pieces and transferring them to the nutrient medium to be used in 3 stages into the jar, ix. performing 1 or 2 subculturing steps to the freshly sprouted shoots in the nutrient medium in the 1ststage over a period of 30 days, x. at the end of the 1ststage, waiting for the plants to produce 1 -2 new shoots, xi. in the 2ndstage, performing 2-3 subculturing steps in 45-day periods in a medium containing 3 different polyamines, xii. in the 3rdstage, continuing subculturing of plant clusters in the medium containing only putrescine as polyamine, xiii. separating the new shoots that are 2.5-3 cm long as the number of shoots increases and planting them in a new medium with the same content, xiv. taking the shoots that have reached 4-5 cm in length in the third medium one by one into sterile empty tubes, xv. adding 1 ml of liquid rooting medium containing 1 / 2 MS (2.2 mg / l) nutrient medium without agar, 20 g / l sucrose, 1 mg / l indole-3-butyric acid (IBA) to the tubes, xvi. taking the rooted shoots out of the tubes and placing them in a container of tap water, xvii. planting the plants in pots containing a slightly moist 2:1 peat:perlite mixture prepared in advance, xviii. wetting the leaves by spraying them with water again, placing the plants in transparent storage containers, keeping the lid of the container completely closed for 1 week, lifting it every 2-3 days and completing the acclimatisation to external conditions in the growth chamber by opening it completely after approximately 15 days, and xix. continuing to grow seedlings in the greenhouse.

[0017] The process steps numbered ii-xiii applied in the hazelnut micropropagation method, which is the subject of the invention, comprise the propagation stage of the plants.

[0018] The process steps numbered xiv-xv applied in the hazelnut micropropagation method, which is the subject of the invention, comprise the rooting stage of the plants.

[0019] The process steps numbered xvi-xix applied in the hazelnut micropropagation method, which is the subject of the invention, comprise the acclimatisation stage of the plants to external conditions. In one embodiment of the method according to the invention, surface sterilisation described in process step (i) comprises the process steps of:

[0020] - cutting 15-20 cm long branches from 2-5 year old seedlings grown in the greenhouse and bringing them to the laboratory,

[0021] - cutting 2-3 cm long pieces from the branches in a way to include the buds in the leaf axils using garden shears,

[0022] - rinsing these parts by hand for 5 minutes in water containing antibacterial soap,

[0023] - applying surface sterilisation process to the parts in a sterile cabinet,

[0024] - shaking the cut pieces with 70% ethyl alcohol (C2H6O) for 1 minute after placing them in a sterile jar, and rinsing them with sterile distilled water for 1 minute,

[0025] - adding 20% commercial sodium hypochlorite (NaCIO) and a few drops of nonionic detergent (C58H114O26) to the jar and leaving it on the magnetic stirrer for 20 minutes, and

[0026] - rinsing the branch pieces with sterile pure water 3 times for 5 minutes.

[0027] In one embodiment of the invention, the hazelnut mentioned is the species Corylus avellana L.

[0028] In the method that is the subject of the invention, micropropagation begins with the sterilisation of leaf buds on the branches of the plants and their cultivation in a nutrient medium in glass tubes. Propagation continues with rooting and adaptation to external conditions stages. By means of the arrangements made in the nutrient medium and the techniques applied in this invention, propagation continues in each subculture (2- 3 shoots), and rooting is provided at a rate of 100% in healthy plants. Thus, mass production of hazelnut plants is provided in a short time. The special nutrient medium used in the method of the invention is shaped on the NRM basic nutrient medium. The amount of FeEDDHA (iron chelate), which was originally 100 mg / l in the medium, was increased to 250 mg / l as a result of observations made in subcultures and a- Tocopherol (vitamin E) was not used.

[0029] NRM nutrient medium was used as the basic nutrient medium (basal medium) in the creation of the hazelnut micropropagation method. The content of the NRM nutrient medium used is explained in Table 1. In the preparation of 1 litre medium, the macro / micro elements and organic substances in the table were used in the specified amounts. 30 g / l sucrose, 6 g agar and necessary growth regulators and polyamines were added according to the stages. Before adding agar to the nutrient medium, the pH was adjusted to 5.75 with 1 N sodium hydroxide (NaOH) or 1 N hydrochloric acid (HCI) and the media were sterilised in an autoclave (1.2 atm pressure-121 °C-20 minutes).

[0030] At the beginning of the culture stage, 15-20 cm long branches are cut from 2-5 year old seedlings grown in the greenhouse and brought to the laboratory. 2-3 cm long pieces are cut from the branches with pruning shears, including the buds in the leaf axils. These pieces are rinsed by hand for 5 minutes in water containing a few drops of antibacterial soap. After this stage, surface sterilisation processes are started in a sterile cabinet. After the cut pieces are placed in a sterile jar, they are shaken with 70% ethyl alcohol (C2H6O) for 1 minute and rinsed with sterile pure water for 1 minute. 20% commercial sodium hypochlorite and a few drops of non-ionic detergent are added to the jar and kept on the magnetic stirrer for 20 minutes. The branch pieces are then shaken with sterile pure water 3 times for 5 minutes. The pieces, whose surface sterilisation is completed, are placed on sterile filter papers in the sterile cabinet and left to dry for 30 minutes. At the end of this period, the bud pieces are placed one by one in tubes containing nutrient medium. The nutrient medium contains 1 / 2 MS nutrient medium (2.2 g / l) , 20 g / l sucrose, 1 .5 mg / l BAP, 6 g / l agar. The tubes are cultured in a growth chamber at 4°C for 45 days, allowing the buds to complete their cooling. At the end of this period, the branch pieces were transferred to new tubes containing the same nutrient medium (subculture) and are placed in a growth chamber with conditions of 24°C, 16-hour photoperiod, and 45 pmol.m-2s-1daylight. Within a month, all buds sprouted and fresh shoots are obtained.

[0031] During the propagation stage, when the shoots in the tubes reach 5-6 cm in length, they are separated from the woody pieces with the help of a scalpel and forceps and transferred to the nutrient medium in the jar. NRM nutrient medium is used during the propagation stage. This stage is carried out with 3 different media, in order.

[0032] The nutrient medium in the 1ststage contains NRM nutrient medium, 30 g / l sucrose, 1 .5 mg / l BAP, 0.1 mg / l IBA, 250 mg / l Fe-EDDHA, and 6 g / l agar. The nutrient medium in the 2ndstage contains NRM nutrient medium, 30 g / l sucrose, 4 mg / l BAP, 1 mg / l 6-(Y,Y-dimethyl allyl amino purine (2-iP), 0.1 mg / l gibberellic acid (GA3), 0.03 mg / l IBA, 64.42 mg / l putrescine, 58.10 mg / l spermidine, 20.20 mg / l spermine, 250 mg / l Fe-EDDHA, and 6 g / l agar.

[0033] Nutrient medium in 3rdstage contains NRM nutrient medium, 30 g / l sucrose, 4 mg / l BAP, 1 mg / l 2-iP, 0.1 mg / l GA3, 0.03 mg / l IBA, 64.42 mg / l putrescine, 250 mg / l Fe- EDDHA, and 6 g / l agar.

[0034] Continuous and healthy plant formation is ensured by using the polyamines used in the propagation stage, which is the 2ndstage in the hazelnut micropropagation method that is the subject of the invention, in a special order, namely first three being together and then only putrescine.

[0035] Freshly sprouted shoots are subcultured in 1 or 2 times for 30 days in the 1stmedium. The development capacity of each bud varies according to its position on the branch. At the end of this period, it is expected that the plants will produce 1 -2 new shoots. 2- 3 subcultures are performed in 45-day periods in the 2ndmedium and in this medium, new shoots propagate in the plants and clustering begins. In the 3rdmedium, the subculture of plant clusters is continued in the medium containing only putrescine as polyamine. The new shoots formed from the 1stmedium are not separated from the mother plant and are transferred as a cluster. The propagation continues in the 2ndand 3rdmediums without separating the shoots. As the number of shoots increases, the new shoots that are 2.5-3 cm long are separated and planted in a new medium with the same content. The factors that ensure regular and continuous propagation at this stage are the 3rdnutrient medium (only putrescine addition) and the transfer of the shoots as a cluster.

[0036] When the rooting stage is reached; the shoots that are 4-5 cm long in the third medium are taken one by one into sterile empty tubes. 1 ml of agar-free liquid rooting medium is added to the tubes. This medium contains 1 / 2 MS, 20 g / l sucrose, and 1 mg / l IBA. Rooting begins within 15 days. After the rooting stage, in order to get used to external conditions, the rooted shoots are removed from the tubes and placed in tap water in a container to prevent sudden moisture loss from the leaves. Plants are planted in pre- prepared vials, containing slightly moist 2:1 peat:perlite mixture. The leaves are wetted by spraying with water again and placed in transparent storage containers. In order to acclimatize the plants, which are kept in a fully closed storage container for 1 week, to the humidity level of the outside environment, the lid of the container is raised at 2-3 day intervals and after approximately 15 days, it is completely opened and the acclimatisation to the outside conditions is completed in the growth room. As the roots of the plants develop, they are transferred to larger vials and seedling bags. The growth of the seedlings continues in the greenhouse. In this method, in order to ensure effective reproduction in common hazelnut varieties, putrescine, spermine and spermidine polyamines were added to the NRM nutrient medium in different combinations, and their positive effects on reproduction and growth were observed. In the development of samples whose subculture continued for a longer period in the medium containing all three polyamines, problems such as deformities in the leaves, bushy structures at the tips of newly developing shoots, and failure to develop and darkening of new shoot buds formed at the bottom of the plant clusters were encountered. In the medium with only putrescine, buds coming from the bottom developed rapidly, regular reproduction and healthy growth were achieved in the plants. Growth regulators added to the NRM nutrient medium during the propagation stage (4 mg / BAP (6-benzyl amino purine) +1 mg / l 2-iP (6-(y,y-Dimethyl allyl amino purine), 0.1 mg / l GA3 (gibberellic acid) + 0.03 mg / l IBA (indole-3-butyric acid) cannot provide normal development and regular propagation after several subcultures without polyamines. The same level of growth and development cannot be achieved in plants obtained from buds arranged from the tip to the bottom of a branch. This situation is related to the developmental physiology in fruit trees. Some buds age earlier in subcultures than others. At this stage, polyamines added to the medium (putrescine, spermidine and spermine) provide rejuvenation and shoots begin to form in the plants again. However, in this medium, after 3-4 subcultures, the propagation in the plants slows down and the morphological appearance of the newly developing shoots changes. Adding all three polyamines to the nutrient medium together disrupts the hormonal order in the plant in the subsequent subcultures. In the natural life cycle of plants, spermidine and spermine, among these polyamines, are synthesised from putrescine. When a nutrient medium containing both high doses of cytokinin (4 mg / l BAP+1 mg / l 2-iP) and 3 separate polyamines is continuously applied to the plants, the interactions between these growth regulators either negatively affect or stop healthy and continuous propagation. In said rooting method, the rooting percentage was determined as 100%. The technique and liquid medium used in this method are effective in the success achieved. Rooting is easier in hybrid hazelnuts. There is no other study reporting 100% rooting of Corylus avellana L. in the state of the art.

[0037] Table 1. Content of NRM nutrient medium REFERENCES

[0038] [1] Murashige, G. and Skoog, F. (1962). A Revised Medium for Rapid Growthand Bioassays With Tobacco Tissue Culture. Physiologia Plantarum, 15(3), 473-497.

[0039] [2] Driver, J. A. and Kuniyuki, A. H. (1984). In vitro propagation of Paradox Walnut Rootstock. Hortscience, 19(4). 507-509.

[0040] [3] Nas, M.N. and Read, P.E. (2004). A hypothesis for the development of a defined tissue culture medium of higher plants and micropropagation of hazelnuts. Scientia Horticulturae, 101 (1 -2), pp.189-200.

[0041] [4] Liu, Jian-feng*, Cheng, Yun-qing,Chen, Zhi-wen,and Duan, Ai-Ping. (2009). Studies on tissue culture and rapid propagation of hybrid hazelnut. Acta Horticulturae, 36(3), 409-414.

Claims

CLAIMS1. Micropropagation method for rapid propagation of hazelnut (Corylus avellana L.) plants by tissue culture, comprising special nutrient media developed for the propagation stage and a technique for rooting, comprising the process steps of: i. applying surface sterilisation to 15-20 cm long branches cut from 2-5 year old seedlings grown in the greenhouse, ii. placing the parts whose surface sterilisation has been completed on sterile filter papers in the sterile cabinet and leaving them to dry for 30 minutes, iii. adjusting the pH of the nutrient medium to 5.75 with 1 N sodium hydroxide (NaOH) or 1 N hydrochloric acid (HCI) before adding agar to the nutrient media and sterilizing it in an autoclave (1.2 atm pressure - 121 °C - 20 minutes),, iv. at the end of this period, placing the bud pieces one by one in tubes containing 1 / 2 MS (2.2 g / l) nutrient medium, 20 g / l sucrose, 1 .5 mg / l 6-benzyl amino purine (BAP), 6 g / l agar, v. culturing the tubes in a growth chamber at 4°C for 45 days to ensure that the buds complete their vernalisation, vi. at the end of this period, transferring the branch pieces to new tubes containing the same nutrient medium (subculture) and placing them in a growth chamber with conditions of 24°C, 16-hour photoperiod, and 45 pmol.m-2s-1daylight, vii. obtaining fresh shoots from all the buds that sprout within a month, viii. when they reach 5-6 cm in length in the tubes, separating the shoots from the branch pieces and transferring them to the nutrient medium of 3 stages into the jar, ix. performing 1 or 2 subculturing steps to the freshly sprouted shoots in the nutrient medium in the 1ststage over a period of 30 days, x. at the end of the first stage, expecting the plants to produce 1 -2 new shoots. xi. in the 2ndstage, performing 2 subculturing steps in 45-day periods, xii. in the 3rdstage, continuing subculturing of plant clusters in the medium containing only putrescine as polyamine, xiii. separating the new shoots that are 2.5-3 cm long as the number of shoots increases and placing them in a fresh medium, xiv. taking the shoots that have reached 4-5 cm in length in the third environment one by one into sterile empty tubes,v. adding 1 ml of liquid rooting medium containing 1 / 2 MS (2.2 mg / l) nutrient medium without agar, 20 g / l sucrose, 1 mg / l indole-3-butyric acid (IBA) to the tubes, xvi. taking the rooted shoots out of the tubes and placing them in a container of tap water, xvii. planting the plants in pots containing a slightly moist 2:1 peat:perlite mixture prepared in advance, xviii. wetting the leaves by spraying them with water again, placing the plants in transparent storage containers, keeping the lid of the container completely closed for 1 week, lifting it every 2-3 days and completing the acclimatisation to external conditions in the growth chamber by opening it completely after approximately 15 days, and xix. continuing to grow seedlings in the greenhouse.

2. A micropropagation method according to Claim 1 , wherein the surface sterilisation as described in process step (i) comprises the process steps of:- cutting 15-20 cm long branches from 2-5 year old seedlings grown in the greenhouse and bringing them to the laboratory,- cutting 2-3 cm long pieces from the branches in a way to include the buds in the leaf axils using pruning shears,- rinsing these parts by hand for 5 minutes in water containing antibacterial soap,- applying surface sterilisation process to the parts in a sterile cabinet,- shaking the cut pieces with 70% ethyl alcohol (C2H6O) for 1 minute after placing them in a sterile jar, and rinsing them with sterile distilled water for 1 minute,- adding 20% commercial sodium hypochlorite (NaCIO) and a few drops of non-ionic detergent (C58H114O26) to the jar and leaving it on the magnetic stirrer for 20 minutes, and- rinsing the branch pieces with sterile distilled water 3 times for 5 minutes.

3. A micropropagation method according to Claims 1 or 2, wherein the process steps ii-xiii include the propagation stage of the plants.

4. A micropropagation method according to Claims 1 or 2, wherein the process steps xiv-xv include the rooting stage of the plants.

5. A micropropagation method according to Claims 1 or 2, wherein the process steps xvi-xix include the acclimatisation of the plants to external conditions.

6. A micropropagation method according to Claims 1 or 2, wherein said hazelnut plant is the Corylus avellana L. species.

7. A micropropagation method according to any of the Claims 1 to 6, wherein the nutrient medium in the first stage described in the process step (x) contains NRM (Nas and Read Medium, 2004) nutrient medium, 30 g / l sucrose, 1.5 mg / l BAP, 0.1 mg / l IBA, 250 mg / l Fe-EDDHA, and 6 g / l agar.

8. A micropropagation method according to any of the Claims 1 to 6, wherein the nutrient medium in the 2ndstage described in the process step (xi) contains NRM nutrient medium, 30 g / l sucrose, 4 mg / l BAP, 1 mg / l 6-(y,y-dimethyl allyl amino purine (2-iP), 0.1 mg / l gibberellic acid (GA3), 0.03 mg / l IBA, 64.42 mg / l putrescine, 58.10 mg / l spermidine, 20.20 mg / l spermine, 250 mg / l Fe-EDDHA, and 6 g / l agar.

9. A micropropagation method according to any of the Claims 1 to 6, wherein the nutrient medium in the 3rdstage described in the process step (xii) contains NRM nutrient medium, 30 g / l sucrose, 4 mg / l BAP, 1 mg / l 2-iP, 0.1 mg / l GA3, 0.03 mg / l IBA, 64.42 mg / l putrescine, 250 mg / l Fe-EDDHA, and 6 g / l agar

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