The micropropagation of the strawberry cultivar ‘Idea’

International Journal of Agronomy and Agricultural Research (IJAAR) ISSN: 2223-7054 (Print) 2225-3610 (Online) http://www.innspub.net Vol. 4, No. 4, p. 188-194, 2014 OPEN ACCESS RESEARCH PAPER The micropropagation of the strawberry cultivar ‘Idea’ Zoran Nikolic1*, Katerina Nikolic1, Dragan Jankovic1, Ana Krstic2, Goran Pejkovic2 1 Faculty of Agriculture, University of Priština, Lešak, Serbia 2 Primary school Radoje Domanovi, Bošnjace, Serbia Article published on April 29, 2014 Key words: Strawberry, meristem, micropropagation, stolons, multiplication Abstract This work presents the production technology of the strawberry cultivar ‘Idea’ by micropropagation. Meristems of 0.5mm size were isolated in June from the stolons of examined strawberry sorts. They were raised on Murashige and Skoog’s (Murashige and Skoog, 1962) medium in presence of 6-Benzylaminopurine (BAP), Indole-3-butyric acid (IBA) and gibberellic acid (GA3). Fifty days later meristems were organized into a foliate rosette. Multiplication was achieved on Murashige and Skoog’s substratum with BAP and IBA. The plants which came to the size of about 10mm were shifted to the substratum for tree rooting. Tree rooting was achieved on Murashige and Skoog’s substratum with IBA. Sixty days later the plants with strongly developed roots were transplanted into the peaty briquettes. One month later the plants were transplanted into flowerpots and kept in a greenhouse at a temperature of 10-150C. * Corresponding Nikolić et al. Author: Zoran Nikolić  [email protected] Page 188 Introduction a certain sort. Experiments with this promising The district of Jablanica is known for the volume method of reproduction are still ongoing around the production of the berry fruits. The most represented world and are already used in practice. berry fruits are strawberry, raspberry and blackberry. Micropropagation methods can be applied for If during the growing of commercial strawberry obtaining healthy planting materials of strawberry. plants, the runner plants from the production The aim of this research was to determine the plantings are used, diseases and pets are easily production technology of the strawberry cultivar transmitted, contributing mostly to the low yield and ‘Idea’ using the method of micropropagation. poor quality fruit. Therefore, the raise of modern plantation of strawberries gives the emphasis on the Material and methods use of varietal pure, quality, healthy and virus-free Isolation of initial explants planting material. During our three-year research the starting material for examination of the sort ‘Idea’ was taken from the Since the various fungal, bacterial, virus and other mother plantation of strawberries in the social diseases are transmitted using conventional methods company ‘’Porečje Vučje’’. Preparation of the starting of multiplication, and this leads to a yield reduction, material and Murashige and Skoog’s medium was the production of healthy planting material is performed in the biological laboratory ‘’Zdravlje - imposed as a special problem. Actavis’’ Leskovac, whereas the isolation of plant material was performed in the laminar The modern production of strawberry runner plants microbiological consists of the production of selected and virus-free company. The impacts of different phytohormones in strawberry runner plants. Strawberries can be virus- vitro growth conditions were also carried out in these free in vitro growth conditions by using laboratory of the mentioned laboratories. micropropagation method. There are three basic methods of plant regeneration in vitro: micropropagation through axillary bud formation, Sterilization of nutrient medium The nutrient medium is prepared with double- organogenesis through the formation of adventitious distilled water (ddH2O) and after that it is placed into buds the an Erlenmeyer flask or a test tube with cotton wool. formation of somatic embryos (Murashige, 1974). Thus prepared medium is sterilized in autoclave at Micropropagation methods are based on the ability of 120oC for 20 minutes. and somatic embryogenesis through plants to regenerate from single cells, tissues or organs into a new plant. This multiplication is Conditions of growing the cultures provided only under laboratory conditions. The explants were kept at ‘’Zdravlje - Actavis’’ Leskovac in controlled conditions and at a Micropropagation is performed on a special medium, temperature 20 -25 oC, with light intensity of 2000 – which contains a larger number of nutrients, 2500 lx and the length of brightness of 16 hours per especially minerals, sucrose and growth stimulators. day The influence of hormones on the plant growth and environmental conditions was carried out in the cell division, and their development into an organized greenhouses of the company under the ‘’mist’’ system. structure, cannot be inferred on the basis of pedigree. After adjustment, the plants were planted on the plots Thus, for example, a certain hormonal treatment for of ‘’Porečje Vučje’’ and one private producer in the the induction, division and the growth of plant cells village of Strojkovce for further research. and 8 hours per night. Adjustment to applied to one sort of strawberries did not show itself as efficient when compared to another sort. A lot of unsolved issues mainly regard the specific features of Nikolić et al. Page 189 Results and discussion sizes of the explants, time of obtaining the explants Factors that determine induction and organogenesis and nutrient medium have an important role. The There are direct and indirect ways of organogenesis. largest number of explants can be developed in a When the callus formation is omitted on the primary short period of time when used with the apical buds explants, it is a direct way of organogenesis. And size of 1-2 mm. In addition, an important role is contrary, when callus is primarily formed on the played by the time of taking the explants, as well as primary of the age of the plants from which explants are taken. organogenesis. The explants multiplicated in vitro do explants, Regarding the strawberries, the largest number of not form callus, if they originate from the apical meristems develops when the explants are taken meristem. It means that this method of multiplication during the period from the second half of May and of organogenesis. June (Nikolić, 2006). At the apical meristem culture, However, when the concentration of IBA hormones is better results are obtained if the explants are taken increased above 1.5 mg/l then it comes to the callus from young plants (Ružić, 2004). strawberries it is manifests an indirect direct way formation, i.e. it comes to the multiplication of strawberries by indirect way of organogenesis The composition of the nutrient medium plays an (Nikolić, 1996). Based on these data, it is evident that important role for the organogenesis and the regeneration of strawberries can be carried out by regeneration. Among the known nutrient medium direct organogenesis. Murashige and Skoog’s medium is most frequently used. The isolated strawberry meristems were Stolons of the runner plants of the strawberry sort cultured on Murashige and Skoog’s mineral solution ‘Idea’ were used for the isolation of meristems. Plant with GA3, BAP and IBA at various concentrations material was collected by the end of May and the (Table 1). In order to identify significant differences, beginning of June from the stolons that were in the LSD test was performed. air. For the first stage, the initiation of culture, the Table 1. The influence of hormone concentrations of initiation of culture of the strawberry cultivar ‘Idea’ Hormones mg/l Number of isolated meristems Uninfected cultures Initiated cultures Success rate No. % No. % % 0.0 30 18 60.00 - - - 0.1BAP+0.1IBA+0.1GA3 30 23 76.67 7 30.43 23.33 0.1BAP 30 20 66.67 2 10.00 6.67 0.5BAP+0.1IBA+0.1GA3 30 25 83.33 12 48.00 40.00 0.5BAP 30 21 70.00 3 14.29 10.00 1BAP+0.1IBA+0.1GA3 30 26 86.67 17 65.38 56.67 1BAP 30 22 73.33 4 18.18 13.33 Using the applied treatments optimal hormone percentage of initiating the cultures of strawberry balance for the culture of primary explants of cultivars ‘Marmolada’ (20%) and ‘Cortina’ (50%). The strawberry cultivar ‘Idea’ is 1.00 mg/l BAP, 0.1 mg/l highest results that they recorded were by the cultivar IBA and 0.1 mg/l GA3 (Table 2). Changes in explants ‘Selena’ (70%) and the lowest by the cultivar ‘Sena’ were already observed after five or six days, and after (10%). High concentration of cytokinins, in this case 1 twenty days formed callus and growth are noticed. mg/l BAP, has a positive effect on the initiation of After 40 days the average number of surviving plants culture. was 36.73% at the optimal concentration of hormones concentration of cytokinins for the cultivar ‘Senga 56.67%. Milosavljević et al. (1999) achieved lower Sengana’ was 81.25% (Petrović and Jaćimović- Nikolić et al. The percentage of initiation at this Page 190 Plavšić, 1990), for the cultivar ‘Selena’ it was 76.67%, optimal combination of hormones survival rate for and the cultivar ‘Senga Sengana’ was 73.33%, ‘Marmolada’ for the ever-bearing strawberry cultivar ‘Hummel’ was 63.33% (Nikolić et al., 2004). At the - 40% and ‘Cortina’ - 60% (Nikolić, 2006). Table 2. The least significant difference of the influence of hormones concentration for the initiation of culture of the strawberry cultivar ‘Idea’ Hormone concentration mg/l x x-2 x-3 x-4 x-7 x-12 1BAP+0.1IBA+0.1GA3 17 15** 14** 13** 10** 5** 0.5BAP+0.1IBA+0.1GA3 12 10** 9** 8** 5** 0.1BAP+0.1IBA+0.1GA3 7 5** 4* 4* 1BAP 4 2 1 0.5BAP 3 1 0.1BAP 2 LSD (0.05) =3.41; LSD (0.01) =4.78; *significant difference at the level 0.05; **significant difference at the level 0.01 Multiplication and elongation of scions of reproduction. Multiplication of isolated buds was The main aim of this stage was to produce a maximum achieved at Murashige and Skoog’s medium with number of units for reproduction. At this stage, the phytohormones BAP and IBA (Table 3). The highest apical meristem is organized into the leaf rosette. The multiplication index is achieved by using hormones at most abundant and genetically stable method is concentrations of 1 mg/l BAP and 1 mg/l IBA. multiplication by axillary buds (Boxus, 1974) Without the presence of BAP hormones in growth medium multiplication did not exist, and the After 40 days of cultivation in a culture, formed omission of hormones IBA has led to a reduction in scions size 10-20mm were transferred to the medium the multiplication index. Table 3. The influence of different concentrations of BAP hormones on multiplication of the strawberry cultivar ‘Idea’ Hormone combination mg/l Number of plants/cultures Number of produced plants/passage Multiplication index 0.0 BAP+1.0 IBA 20 - - 0.1 BAP+1.0 IBA 20 31 1.55 0.5 BAP+1.0 IBA 20 48 2.40 1.0 BAP+1.0 IBA 17 96 5.65 1.5 BAP+1.0 IBA 20 105 5.25 2.0 BAP+1.0 IBA 20 101 5.05 1.0 BAP+0.0 IBA 20 98 4.90 Cytokinins are used at the stage of multiplication to the examination of multiplication of some strawberry overcome the dominance of scions and to increase cultivars by the method of meristem culture, branching of the lateral buds from the leaf axils. In Milosavljević et al. (1999) achieved the multiplication their researches, Nikolić et al. (2004) reached the index 4.0 for the cultivar ‘Sena’ and 6.0 for the lowest index of multiplication with the cultivar cultivar ‘Careca’. ‘Marmolada’ (5.42), and the highest index was with the cultivar ‘Senga Sengana’ (8.77). Petrović and If the hormone concentration is increased above 1 Jaćimović-Plavšić (1990) reached the multiplication mg/l BAP, there is a higher percentage of callus index 8.67 with the cultivar ‘Senga Sengana’. During formation of 30% (15% for combinations of 1mg/l Nikolić et al. Page 191 BAP, 45% of 1.5 mg/ BAP). In order to obtain plants The significance of the influence of BAP hormones on without mutations, it is recommended to remove this multiplication was determined by LSD test (Table 4). callus. The combination of 1 mg/l BAP is favored, due Between the optimal concentration of hormones and to the fact that it provides a lower percentage of other hormones combinations, there are significant calluses (Nikolić, 1996). differences at 0.05 and 0.01 levels. Table 4. The least significant difference of the influence of different concentrations of BAP hormones on multiplication of the strawberry cultivar ‘Idea’ Hormone combination mg/l x x-1.55 x-2.40 x-5.05 x-5.05 x-5.25 1.0 BAP+1.0 IBA 5.65 4.10 ** 3.25** 0.75* 0.60* 0.40* 1.5 BAP+1.0 IBA 5.25 3.70 ** 2.85** 0.35 0.20 2.0 BAP+1.0 IBA 5.05 3.50** 2.65** 0.15 1.0 BAP+0.0 IBA 4.90 3.35** 1.55** 0.5 BAP+1.0 IBA 2.40 0.85* 0.1 BAP+1.0 IBA 1.55 LSD (0.05) =0.659; LSD (0.01) =0.959; *significant difference at the level 0.05; **significant difference at the level 0.01 The presence of BAP hormones in nutrient medium micropropagation methods can achieve rapid and effectively prevented rooting of strawberries. At the mass production of strawberries. optimal concentration of hormones, multiplication index was higher than 1:5 and it was approximately Rooting 1:5-7 (in some passages even 8). Omission of IBA Strawberry hormones leads to the reduction the multiplication, multiplication and elongation, are distinguished by but not to the omission of multiplication. The the absence of rootlets. Therefore, when the plantlets biological potential of starting material is not reached a height of 10-20 mm, they were transferred significantly reduced by the three-year cultivation, at the rooting medium. The composition of this and it ranges within the boundaries of the calculated medium includes macro and micro salts, sucrose, and multiplication index value. High value of the agar inositol, and regarding the hormones, hormone multiplication factor indicates that the use of IBA at different concentrations (Table 5). scions obtained in the phase of Table 5. The influence of IBA hormone concentration on rooting strawberry cultivar ‘Idea’ after 30 days Hormone combination IBA mg/l Number of plants in a culture Number of rooted plants Percentage of rooted plants The average number of roots 0.0 30 13 43.33 2.84 0.1 30 19 63.33 4.79 0.5 30 22 73.33 5.82 1.0 30 16 53.33 4.07 The optimal concentration of IBA hormones for the A high percentage of rooting was achieved with the rooting of the cultivar ‘Idea’ is 0.5 mg/l. At a higher strawberry cultivar ‘Senga Sengana’ - 80% (Petrović and concentration of IBA hormones the larger mass of Jaćimović-Plavšić, 1990); cultivar ‘Cortina’ - 90% callus was formed at the basal part of the scion, but at cultivar ‘Marmolada’ - 83.33% (Nikolić, 2006). In the a lower concentration of hormones, the number of experiments with a large number of strawberry cultivars, rooted scions was lower. the highest rooting percentage is achieved with the cultivar ‘Cortina’ (85%), while other cultivars (‘Sena’, Nikolić et al. Page 192 ‘Selena’) have a lower percentage and it ranges from The significance of the influence of BAP hormones on 18.87% to 29.63% (Milosavljević et al., 1999). rooting was determined by LSD test (Table 6). Table 6. The least significant difference of the influence of different concentrations of IBA hormone on rooting strawberry cultivar ‘Idea’ after 30 days Hormone combination IBA mg/l x x-13 x-16 x-19 0,5 22 9** 6** 3 0,1 19 6** 3 1,0 16 3 0,0 13 LSD (0.05) =3.98; LSD (0.01) =5.58; *significant difference at the level 0.05; **significant difference at the level 0.01 Transferring to non-sterile conditions achieved when the explants are taken in May or June After rooting, in vitro regenerated plants are from stolons that are in the air. transferred to non-sterile conditions. The success of transplantation and survival of plantlets greatly The optimal balance of the hormones concentration depends on the quality of root (Pons et al., 1983). for the initiation of cultures is: 1 mg/l BAP, 0.1 mg/l IBA and 0.1 mg/l GA3. The best multiplication of the The most convenient moment for transplanting the strawberry scions is achieved by a combination of obtained plantlets after rooting is 60 days after phytohormones: 1.0 mg / l BAP and 1.0 mg / l IBA. incubation of scions on agrarian 1/2 Murashige and On the medium without BAP hormones, despite the Skoog’s medium, when the rootlets have reached the presence of IBA hormones, multiplication does not length of 30-50 mm. Then the roots of young plants occur. Phytohormone BAP improves reproduction were carefully separated from medium, followed by and reduces apical dominance strawberries. The rinsing with water to remove the agar, in order to optimal concentration of IBA hormones for the avoid harmful settled microflora. Thus washed, young rooting of the cultivar ‘Idea’ is 0.5 mg/l. During the plants were placed into sterile plastic dishes with a adaptation to external conditions the number of mixture of vermiculite, peat, and sand 1:1:1. 1/2 adapted plants for the cultivar ‘Idea’ is 72.50%. Murashige and Skoog’s mineral solution was added to this mixture. The courts were placed in plastic tubs The results presented in this study indicate that with water (2-3 cm) and covered with jar. After that, micropropagation can significantly speed up the the plants were kept for a month under plastic sheets process of obtaining high-quality and healthy plant or uncovered, and then transplanted into pots with material. soil and taken out. By the end of the critical period of 30 days, the plants were transplanted to the open field. Percentage of adapted plants for this strawberry cultivar is 72.50%. Conclusion Considering the conducted analyses, we can conclude that multiplication of the strawberry sort ‘Idea’ is possible using the micropropagation method. For the strawberry cultivar ‘Idea’, good organogenesis References Boxus Ph. 1974. The production of strawberry plants by in vitro micropropagation. Journal of Horticultural Science and Biotechnology 49, 209-210. Milosavljević S, Zečević A, Vitošević I, Đorđević G, Marinković D. 1999. Razmnožavanje novih sorti jagoda metodom kulture meristema in vitro. Zbornik naučnih radova Instituta PKB Agroekonomik 20(1), 11-18. and regeneration in vitro is achieved when apical buds are used as initial explants. The best results are Nikolić et al. Page 193 Murashige T. 1974. Plant propagation through Nikolić Z, Nikolić K, Selamovska A. 2004. tissue culture. Annual Review of Plant Physiology Meristemskoto 25, 135-166. dobivanje na zdrav saden materijal kaj neki sorti razmnožuvanje kako metod za jagoda. 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