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Optimizing Micropropagation of Apple (Malus × Domestica Borkh) and in Vitro Root Induction By Piriformospora indica

Optimizing Micropropagation of Apple (Malus × Domestica Borkh) and in Vitro Root Induction By... Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 Original paper DOI: 10.2478/agri-2020-0013 Optimizing micrOprOtiagaO p n O f apple (Malus × doMestica BOrkh) and in vitro rOOt inductiOn B y PiriforMosPora indica 1,2 1* 2 Samira maSoudi , am Jaf myar arkhani kermani , ali Soleimani , 3 1 1 haSSan haJnaJari , amin alidadi , Zahra Saadat hoSSeini Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran University of Zanjan, Zanjan, Iran Temperate and Cold Fruits Research Institute (TCFRI), Horticultural Science Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran MASOUDI, S. – KERMANI, M.J. − SOLEIMANI, A. − HAJNAJARI, H. − ALIDADI, A. − HOSSEINI, Z.S.: Optimizing micropropagation of apple (Malus × domestica Borkh) and in vitro root induction by Piriformospora indica. Agriculture (Poľnohospodárstvo), vol. 66, no. 4, pp. 137 – 147. The inoculation of plant species with mycorrhiza fungus Piriformospora indica results in enhancement of growth, increase in yield, and induction of resistance to biotic and abiotic diseases through improvement of the root system. The aim of the present study was to optimize in vitro propagation protocol for three indigenous apples (Malus × domestica) cultivars (ꞌGolbaharꞌ, ꞌSharbatiꞌ, ꞌSoltani Shabestariꞌ) and one commercial cultivar (ꞌGolden Deliciousꞌ). Furthermore, the efficiency of P. indica at rooting stage was investigated on three cultivars (ꞌSharbatiꞌ, ꞌSoltani Shabestariꞌ, ꞌGolden Deliciousꞌ). Establishment and proliferation stages were optimized by collecting explants at different seasons and comparing different culture media respectively. Rooting optimization included six treatments containing different concentrations of auxins in the presence or absence of P. indica. Results showed that at the establishment stage, a maximum percent of survival was observed in explants collected in spring. At the proliferation stage, different media had a divergent effects on distinct cultivars. Although all cultivars reacted favourable to micropropagation in MS (Murashige & Skoog 1962) basal medium, the presence or absence of cytokinin 2ip (N -(2-Isopentenyl) adenine) in the culture media showed significant and incremental improvements in growth indices. In all cultivars highest rooting percent, root length, root thickness, and the number of roots/explant was observed in MS media containing auxins for three weeks followed by a treatment of MS medium containing P. indica for another three weeks. Plantlets treated with P. indica, grow stronger and healthier at the acclimation stage compared to the ones that excluded P. indica. Key words: apple cultivars, culture media, establishment, proliferation Different species or shrubs of the genus Malus zaffarian 1996). The cultivation area of apple in Iran are either found in the wild or cultivated in different is third after grape and pistachio, with 9.1% of the regions of Iran. Among these species, Malus domes- total area under cultivation of orchard products. The tica Borkh has numerous modified breeds and it is area under cultivation is 218,000 hectares with an cultivated as the fruit “apple” in horticulture (Mo- average yield of 16 tons per hectare. This average Samira Masoudi, Maryam Jafarkhani Kermani (*Corresponding author), Amin Alidadi, Zahra Saadat Hosseini, Department of Cell and Tissue Culture, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. E-mail: maryam.j.kermani@gmail.com Samira Masoudi, Ali Soleimani, Department of Horticulture, Faculty of Agriculture, University of Zanjan, Zanjan, Iran Hassan Hajnajari, Temperate and Cold Fruits Research Institute (TCFRI), Horticultural Science Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran © 2020 Authors. This is an open access article licensed under the Creative Commons Attribution-NonComercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/4.0/). 137 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 yield is too far from the leading countries with an was designed to study the effect of symbiont fungi average of 86 tons per hectare in Switzerland (FAO P. indica on the rooting stage of two indigenous 2016). Part of the gap between Iran and developed apple cultivars ('Sharbati', 'Soltani Shabestari') and countries in terms of yield tonnage per hectare is a commercial cultivar ('Golden Delicious'). More- due to the lack of farmers’ access to healthy seed- over, establishment and shoot proliferation stages lings, which has been resulted from the traditional were optimized for the mentioned cultivars and an- propagation of pathogen-infected plants. Thus, pro- other apple cultivar (ˈGolbaharˈ). viding large numbers of healthy high yielding apple seedlings and introducing them to the horticulture industry is quite an important issue. terial ma and methodS In vitro micropropagation of plants is the only method to produce large numbers of pathogen-free Plant material and general procedures plant material in a relatively short period of time. The plant materials were supplied by Temperate There are many reports on successful methods for Fruits Research Center, Horticultural Science Rese- apple micropropagation (Batelja Lodeta et al. 2019; arch Institute of Iran. Stem cutting (15 cm) of three Bhattacharya & Singh 2018; Kaur et al. 2018; Geng indigenous cultivars including 'Golbahar', 'Soltani et al. 2015; Jafarkhani Kermani et al. 2009). Since Shabestari', 'Sharbati' along with a commercial cul- different factors such as genotype and conditions tivar, 'Golden Delicious', were collected during two before and during the regeneration process amongst growing seasons (spring and autumn). The stems plant growth regulators (PGRs), and culture media were cut into 1.5 to 2 cm segments containing at influence stages of in vitro propagation of plants, least one lateral bud. They were disinfected with so- therefore, optimizing different stages of micropropa- dium hypochlorite (2.5%) for 12 – 14 min, followed gation are essential in a successful protocol. by three washes in sterile distilled water. Piriformospora indica is a mycorrhizal endo- In all experiments, media contained 30 g/L su- symbiont of orchid and ericoid roots, which has crose, 7 g/L agar, 100 mg/L PG (Phloroglucinol), been reported to promote the growth of several and pH was adjusted to 5.8. The media were au- plant species, increase yield and product quality, toclaved at 121°C for 15 min. All the cultures were and induce resistance to biotic and abiotic stresses placed in growth chambers with a light intensity of (Franken 2012). P. indica promoted the generation 500 – 3,000 lux, photoperiod of 16/8 h light/dark, and growth of in vivo adventitious roots in pelargo- the relative humidity of 45%, and constant tempe- nium and poinsettia (Druege et al. 2007). Hua et al. rature of 23 ± 1°C. (2017) stated that an important function of P. indica Optimizing the establishment stage is auxin induction and production of its mediators. At the establishment stage, the four mentioned cul- Dong et al. (2013) reported that this fungus plays tivars and two different seasons (spring and autumn) an important role in enhancing the expression of were compared. In each season 60 explants were genes that are effective in phytohormones in barley. collected twice (every time 30 explants). Explants Moreover, the effect of P. indica on gene expression containing at least one lateral bud were surface-ste- of Chinese cabbage and Cymbidium aloifolium has rilized, placed on MS (Murashige & Skoog 1962) been reported by Hua et al. 2017 and Shah et al. basal medium, and incubated in the growth cham- 2019 respectively. Liu et al. (2019) compared the bers. The survival (explants which grew healthy), interaction of P. indica with the roots of seven host elimination (explants that died off), and contamina- plants and showed that root colonization strategies tion (explants that were infected by bacteria or fun- and changes in two phytohormones levels are highly gi) percent were recorded after one month. More- host-specific. over, the survival rate was recorded after three Since the effect of the symbiont fungi P. indica months too and the value change between the two on the rooting stage of micropropagation of apple records was calculated using the following formula has not been reported, the present investigation 138 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 in the Excel program. NAA (1- Naphthalene acetic acid) and 0.5 μmol IBA (Indole-3-butyric acid), whereas other treatments Percentage change = (A – a ) /A 2 1 1 contained relatively high concentrations of auxins (high auxin medium: HAM) which had 2 μmol NAA *a and A are the percent of survival after 1 moth 1 2 and 2 μmol IBA. In the treatments that P. indica (the and 3 months respectively. mycorrhizal Piriformospora indica) was added as Optimizing the shoot proliferation stage a rooting stimulant, 3 g of mycelia was added to At proliferation stage three different media in- every liter of culture media. P indica was supplied cluding; MS (Murashige & Skoog 1962), VS (van by Agricultural Biotechnology research Institute of der Salm et al. 1994) nutrient medium, WPM (Wo- Iran (ABRII). The mycelia growth was achieved by ody Plant Medium, Lloyd & McCown in 1981) and placing them on 8 mm Petri dishes containing Pota- two different concentrations (0 and 5 µmol) of 2iP to Dextrose Agar in incubators at 25°C for 8 days. (N -(2-Isopentenyl) adenine) were compared. The Rooting parameters including rooting percent, root media contained the following growth regulators; length [cm], root thickness [cm], number of roots 5 µmol GA (Gibberellic acid), 0.1 µmol IBA (Indo- per explant were measured after 6 weeks. le-3-butyric acid), 2 µmol BAP (6-Benzylaminopu- Statistical analysis rine); added or not added 2ip. In each experiment 20 Data were analysed in factorial based on a com- replicates (five jars with four explants) were used. pletely randomized design (CRD). A two-factor Growth indices measured after six weeks included; analysis of the genotype × medium variant was car- number of axillary shoots, shoot length [cm], the ried out for proliferation and rooting experiments. number of leaves, fresh and dry weight. The fresh Data were analysed using the statistical program weight of the whole shoot was measured and then SPSS. Statistically significant averages were com- they were dried in an oven (70°C) for 72 hours be- pared using Duncan’s Multiple Range tests. Graphs fore measuring dry weight. were plotted with the Excel program. Differences were regarded as significant at p ≤ 0.05. Investigating the effect of P. indica at rooting stage The shoots were transferred to elongation me- dium (MS + 5 µmol GA + 0.2 µmol IBA) for four RESULTS AND DISCUSSION weeks prior to initiation of rooting. The rooting ex- periment was carried out on three cultivars ('Soltani Comparing the effect of season on the establishment Shabestari', 'Sharbati', 'Golden Delicious') with 15 stage replicates in each treatment. Each explant consisted The results of this study showed that both the ge- of a 4 – 5 cm shoot with three leaves. The treatments netic source and sampling time of explants affected were abbreviated R1 to R6 (Table 1). Some treat- their survival rate as well as contamination caused ments contained low concentrations of auxins (low by bacteria and fungi at the establishment stage (Ta- auxin medium: LAM) which included 0.5 μmol T a b l e 1 Abbreviation for different treatments at rooting stage Treatment Treatment Description r1 MS basal medium (six weeks) r2 LAM (six weeks) r3 HAM (six weeks) R4 MS basal medium (three weeks) + MS basal medium with P. indica (three weeks) R5 LAM (three weeks) + MS basal medium with P. indica (three weeks) R6 HAM (three weeks) + MS basal medium with P. indica (three weeks) Note: MS – Murashigue & Skoog 1962; LAM – low auxin medium; HAM – high auxin medium. 139 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 −147 ble 2). In all cultivars, the percent of contamination Delicious' from the samples collected in autumn. was higher in autumn than in spring season, how- Hohtola (1988) reported that survival and percent ever, the number of eliminated explants due to lack of contaminants in the explants prepared in differ- ent seasons were affected by seasonal changes. The of ability to grow was higher in the explants col- report showed that in pine trees contamination was lected in spring compared to the ones collected in considerably high in winter. Our results also sug- autumn (Figure 1). In all cultivars, the highest per- gested that the contamination rate was significantly cent of survival was observed in explants collected affected by season and it was higher in explants col- in spring. Amongst the cultivars, maximum percent lected in autumn than those collected in the spring. of survival (73.32%) was recorded in 'Golbahar'. Shakya et al. (2013) stated that many factors, such Figure 1 illustrates that the survival rate declined af- as the effect of soil parameters, environmental fac- ter three months in all the cultivars and the decline tors, phenotype and genotype of the host plant, sea- was steeper in explants collected in autumn com- sons and geographic environments were involved pared to the ones collected in spring. The highest in bacterial and fungal contamination and hence the percentage change (–48%) was observed in 'Golden Figure 1. The change in survival percent after one month and three months at the establishment stage in explants of different apple cultivars collected in two seasons (The negative percentage change indicates a decrease in survival percent in month three compared to the first month). T a b l e 2 Effect of different seasons on contamination and survival at establishment stage of apple micropropagation Contamination Elimination Survival [%] after Survival [%] after Cultivar Seasons [%] [%] one month three months Spring 10.00 16.68 73.32 71.60 ꞌGolbaharꞌ Autumn 23.34 15.00 61.66 60.00 Spring 20.00 15.00 65.00 61.66 ꞌSharbatiꞌ Autumn 28.33 8.37 62.30 43.50 Spring 6.67 30.00 63.33 55.00 ꞌSoltani Shabestariꞌ Autumn 21.67 13.33 65.00 35.00 Spring 8.33 25.00 66.70 45.00 ꞌGolden Deliciousꞌ Autumn 30.00 10.00 60.00 58.33 140 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 survival percent at the establishment stage of micro- cantly in different cultivars (Table 3). The maxi- propagation. mum number of axillary shoots (3.2) was observed in 'Golbahar' cultured on MS medium without 2ip. Investigating the interactive effect of culture media Whereas, the highest shoot length (5.9 cm) and the and cultivar type at the proliferation stage maximum number of leaves (13.9) were detected in The results of the study indicated that different 'Golden Delicious' cultivated on MS medium con- culture media including Murashige & Skoog 1962 taining 2ip. The maximum amount of fresh weight (MS); van der Salm et al. 1994 (VS); Woody Plant (1,200 mg) was recorded in 'Golden Delicious' and Medium (WPM) with or without 2ip (N -(2-Isopen- 'Golbahar' in WPM and MS media without 2ip re- tenyl) adenine) affected the growth indices signifi- T a b l e 3 Effect of different culture media on indices of explant proliferation in apple cultivars No. of Axillary Mean shoot Mean no. of Fresh weight Dry weight Cultivar Medium shoots length [cm] leaves [mg] [mg] a c def a cd mS 3.2 4.0 9.1 1,200 0.14 ghi i gh gh fgh VS 1.1 2.4 5.8 300 0.08 fgh fg efgh h jkl WPM 1.3 2.8 6.5 200 0.06 'Golbahar' hij gh ab fgh ef MS + 2ip 1.0 2.9 11.5 400 0.10 d efg def gh hijk VS + 2ip 2.0 3.0 9.9 200 0.06 c ghi efg cde efg WPM + 2iP 2.3 2.7 7.3 500 0.09 ef c def fgh de mS 1.5 3.8 7.9 400 0.12 fgh hi h gh kl VS 1.3 2.4 4.5 240 0.05 k fg fgh h kl WPM 0.3 2.9 6.3 200 0.04 'Sharbati' ij fg efgh bc b MS + 2ip 0.9 2.9 6.9 700 0.23 ij efg efgh efgh jkl VS + 2ip 0.9 3.0 6.8 400 0.06 ef ef fgh fgh efg WPM + 2iP 1.4 3.2 6.3 300 0.09 hij ef abcd cde b mS 1.0 3.2 10.4 500 0.02l hij hi gh efgh gj VS 1.0 2.5 5.6 400 0.07 fgh ghi ab fgh jkl WPM 1.2 2.7 11.2 300 0.06 'Soltani Shabestari' fgh ef abc a ef MS + 2ip 1.2 3.2 10.9 1,100 0.11 ghi fg def efgh h-k VS + 2ip 1.1 2.9 8.4 400 0.06 j fg defg efg e-h WPM + 2ip 0.8 2.9 7.5 500 0.08 e de defg efg efghi mS 1.6 3.3 7.5 500 0.08 c cd efg bcd efg VS 2.3 3.7 7.0 600 0.11 cd c bcde a c WPM 2.2 3.9 10.0 1,200 0.17 'Golden Delicious' c a a b c MS + 2ip 2.3 5.9 13.9 800 0.18 c b abc a a VS + 2ip 2.4 5.1 10.7 1100 0.27 b c ab bc c WPM + 2iP 2.8 3.8 11.2 800 0.15 Different letters show significant difference in each column according to Duncan’s Multiple Range Test (P ≤ 0.05). Note: MS – Murashige & Skoog 1962; VS – van der Salm et al. 1994; WPM – Woody Plant Medium; 2ip – N6-(2-Isopen- tenyl) adenine. 141 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 −147 spectively and the highest amount of dry weight has been reported by Monfort et al. (2018). Arab et (0.27 mg) was observed in 'Golbahar' cultured in VS al. (2014) investigated the interaction between me- medium containing 2ip. dia and growth regulators in G × N15 (a hybrid of The recommended medium for each cultivar almond × peach) and reported that the best medium based on the data presented in Table 3 and general was MS supplemented with 1 mg/L BAP. A strong quality of the explants (without data) was as follow: influence of the culture media mineral composition 'Golbahar' (MS), 'Soltani Shabestari' (MS + 2ip), on the micropropagation and the nutritional status 'Sharbati' (MS), 'Golden Delicious' (WPM or MS + of Eucalyptus dunnii cultures was also reported by 2ip). Figure 2 shows the explants at the proliferation Oberschelp and Gonçalves (2016). They stated that stage (different media) tested for the four cultivars. variability introduced by each genotype deterred the The roman numerals highlighted in red and under- detection of additional effects. Hence they were able line, show the best culturemedium for each cultivar to develop a basal media based on the mineral com- The influence of shoot culture media types on the position of tissues of the species and optimized it quality and quantity of growth of Ocimum basilicum for specific clones in order to adjust the nutritional ˈGolbaharˈ I II III IV V VI ˈSharbati I II III IV V VI ˈSoltani Shabestariˈ I II III IV V VI ˈGolden Deliciousˈ I II III IV V VI Figure 2. Effect of different media on explant proliferation of apple cultivars: I = MS, II = VS, III = WPM, IV = MS + 2IP, V = VS + 2IP, VI = WPM + 2IP. The roman numerals highlighted in red and underline show the best culture medium for each cultivar. Note: MS – Murashige & Skoog 1962; VS – van der Salm et al. 1994; WPM – Woody Plant Medium; 2ip – N6-(2-Isopen- tenyl) adenine. 142 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 requirements for each genetic material. Accordin- called the central zone (CZ). Cells moving from the CZ into a region with a higher rate of cell division gly, an interaction effect of various culture media, named the peripheral zone (PZ), give rise to the axi- in terms of their composition, and different apple llary shoots. The OC of the SAM is the site of maxi- cultivars was revealed at current work, though all mal cytokinin activity, rather than of auxin activity. cultivars reacted favorable to micropropagation in They concluded in the SAM cytokinins promote the MS medium. However, the presence or absence of proliferation of undifferentiated cells, while auxins cytokinin 2ip in the culture media showed signifi - act in the PZ to induce cellular differentiation and cant and incremental improvements in growth indi- organ outgrowth. However, the type and concentra- ces. Schaller et al. (2015) reviewed the role of cy- tion of the cytokinin needed for each genotype even tokinin and auxin interaction in plant development within a species is variable, which is in agreement and reported that in shoot apical meristem (SAM) with our results. a group of initial stem cells surrounds a small group of mitotically less active cells, known as the organi- Optimizing rooting stage using mycorrhiza P. indica zing center (OC). The cells of the OC are located at The results of the study indicated that rooting per- the base of a region with a low rate of cell division cent, the number of roots/explant, total root length/ bc bc bc bc r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 r1 r2 r3 R4 R5 R6 r1 r2 r3 R4 R5 R6 r1 r2 r3 R4 R5 R6 Sharbati Soltani Shabestari Golden Sharbati Soltani Shabestari Golden Delicious Delicious 1.6 1.4 1.2 0.8 0.6 0.4 0.2 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 0 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 Sharbati Soltani Shabestari Golden Sharbati Soltani Shabestari Golden Delicious Delicious Figure 3. Comparison of the interaction between cultivar and type of medium on A) rooting percent, B) average number of roots/explant, C) total length of roots /explant [cm], D) average root thickness [cm] of different apple cultivars. R1: MS, R2: LAM, R3: HAM, R4: MS + P. indica, R5: LAM + P. indica, R6: HAM + P. indica. Note: MS – Murashigue & Skoog 1962; LAM – low auxin medium; HAM – high auxin medium. Rooting percent Total length of roots/explant [cm] Average number of roots/explant Average root tickness [cm] Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 −147 explant, and thickness of the roots in the three cul- Shabestari' (10.75) was attained in R6 (Figure 3B). The highest total length of roots/explant in 'Sharbati' tivars ('Sharbati', 'Soltani Shabestari', and 'Golden (18.75 cm), ꞌSoltani Shabestariꞌ (6.7 cm) and 'Gold- Delicious') were significantly affected by rooting en Delicious' (11 cm) was observed in treatment R5 treatments. In all cultivars, MS medium containing (Figure 3C). The greatest root thickness in 'Shar- either low or high auxin concentrations (LAM & bati' (1.35 cm) and 'Soltani Shabestari' (1.17 cm) HAM) for three weeks followed by a treatment of were detected in R5, but in ˈGolden Deliciousˈ MS medium containing P. indica for another three maximum root thickness (1.3 cm) was observed weeks (treatments R5 & R6) increased the quality in R2 (Figure 3D). Figure 4 and Figure 5 illustrate and quantity of root production. Figure 3A illus- the prototype plantlets at rooting and acclimating trates that maximum rooting percent in 'Sharbati' stages of micropropagation respectively. The fig- (40%) and 'Soltani Shabestari' (80%) were obtained ures indicate that in 'Sharbati' and 'Golden Deli- in treatments R5 and R6 respectively. Whereas, the cious' using fungus P. indica in combination with highest percent of rooting (80%) in 'Golden Deli- auxins resulted in strong plantlets with substantial cious' was attained in R5. The maximum number amounts of roots. However, in 'Soltani Shabestari' of roots/explant in ꞌSharbatiꞌ (3.5) and ꞌGolden De- although MS basal medium also resulted in the liciousꞌ (8.87) were observed in treatment R5, but emergence of roots the plantlets from treatments the maximum number of roots/explants in 'Soltani ˈSharbatiˈ LAM MS MS+ P. indica HAM HAM + P. indica LAM + P. indica ˈSoltani Shabestariˈ LAM MS + P. indica HAM MS HAM + P. indica LAM + P. indica ˈGolden Deliciousˈ LAM MS MS + P. indica HAM HAM + P. indica LAM + P. indica Figure 4. Effect of different treatments at rooting stage of three cultivars ('Sharbati', 'Soltani Shabestari' and 'Golden Delicious'). Note: MS – Murashigue & Skoog 1962; LAM – low auxin medium; HAM – high auxin medium. 144 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 containing P. indica grew stronger and healthi- host plants such as; growth promotion, develop- er compared to the ones that excluded P. indica. ment of the root systems, and increases tolerance In this investigation, a combination of two types to abiotic stresses (Lahrmann & Zuccaro 2012). of auxins (IBA and NAA) rather than one type of aux- Shah et al. (2019) investigated the growth pattern in was used at the rooting stage. Yusnita et al. 2018 (number and length of roots and shoots) and vari- showed that in Malay apple cuttings, a combination ous metabolites of in vitro propagated Cymbidium of IBA and NAA was more effective on rooting than aloifolium plantlets that interacted with the P. indi- NAA or IBA alone. They reported that application ca. They identified growth regulators such as auxin, of IBA + NAA, caused greater root length and the ascorbic acid, andrographolide, hexadecanoic acid, adventitious root morphologies were more branched and DL-proline and reported P. indica-colonized and more fibrous, compared to the unbranched cut- plantlets showed improved growth rate compared to tings treated with NAA or IBA. Moreover, Yusnita et un-colonized control plantlets. In another study, P. al. 2018 illustrated that combination of the two aux- indica plugs (P-1) and a P. indica chlamydospore ins (IBA and NAA) increased shoot sprouting per- suspension (P-2) were co-cultivated with banana cent, shoot length, and number of leaves per shoot. seedlings at the rooting stage and the results indi- The endophytic fungus P. indica is a symbiot- cated that P-2 treatment was more effective than the ic microorganism that has beneficial effects on the P-1 treatment on plant height and root length in the ˈSharbatiˈ HAM + P. indica LAM + P. indica LAM MS MS + P. indica HAM ˈSoltani Shabestariˈ ˈSoltani Shabestariˈ MS HAM MS + P. indica LAM LAM + P. indica HAM + P. indica MS ˈGolden Deliciousˈ MS + P. indica HAM LAM LAM + P. indica HAM + P. indica ˈGolden Deliciousˈ MS + P. indica MS HAM HAM + P. indica LAM LAM + P. indica Figure 5. Effect of different treatments at rooting stage on the acclimation stage of three cultivars ('Sharbati', 'Soltani Shabestari' and 'Golden Delicious'). Note: see Figure 4. 145 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 first 3 weeks (Li et al. 2019). The strong potential of REFERENCES P. indica as bio-fertilizer for ornamental plant culti- ARAB, M.M. ‒ YADOLLAHI, A. ‒ SHOJAEIYAN, A. ‒ vation has been demonstrated by Lin et al. (2019). SHOKRI, S. ‒ GHOJAH, S.M. 2014. Effects of nutrient When biochemical analysis of the base biosyn- media, different cytokinin types and their concentrations on in vitro multiplication of G × N15 (hybrid of almond thesis pathways for the production of auxin was × peach) vegetative rootstock. In Journal of Genetic Engi- carried out the results indicated that P. indica can neering and Biotechnology, vol. 12, no. 2, pp. 81 ‒ 87. 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SIRRENBERG, A. – GOBEL, C. – GROND, S. – CZEMPINS- tmonfor , l.e.f . – BERTOLUCCI, S.K.V. – lima, a.f . – ki, n. – RATZINGER, A. – KARLOVSKY, P. – SoS, ant DE CARVALHO, A.A. – mohammed, a. – Blank, P. – feuSSner, i. – PAWLOWSKI, K. 2007. Piriformo- a.f . – PINTO, J.E.B.P. 2018. Effects of plant growth regu- spora indica affects plant growth by auxin production. In lators, different culture media and strength MS on produc- Physiologia Plantarum, vol. 131, no. 4, pp. 581 – 589. tion of volatile fraction composition in shoot cultures of yuSa, nit y . – Jamaludin, J. – AGUSTIANSYAH, A. Ocimum basilicum. In Industrial Crops and Products, vol. – HAPSORO, D. 2017. A combination of IBA and NAA 116, pp. 231 – 239. resulted in better rooting and shoot sprouting than single MOZAFFARIAN, V.A. 1996. Dictionary of Iranian plant auxin on malay apple [Syzygium malaccense (L.) Merr. & names (Latin, English, Persian). Farhang Moaser Publlish- Perry] stem cuttings. In AGRIVITA, Journal of Agricultural ers. ISBN 964-5545-40-4. Science, vol. 40, no. 1, pp. 80 – 90. OBERSCHELP, G.P.J. – GONÇALVES, A.N. 2016. Assessing Received: June 28, 2020 the effects of basal media on the in vitro propagation and Accepted: December 14, 2020 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Agriculture de Gruyter

Optimizing Micropropagation of Apple (Malus × Domestica Borkh) and in Vitro Root Induction By Piriformospora indica

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Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 Original paper DOI: 10.2478/agri-2020-0013 Optimizing micrOprOtiagaO p n O f apple (Malus × doMestica BOrkh) and in vitro rOOt inductiOn B y PiriforMosPora indica 1,2 1* 2 Samira maSoudi , am Jaf myar arkhani kermani , ali Soleimani , 3 1 1 haSSan haJnaJari , amin alidadi , Zahra Saadat hoSSeini Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran University of Zanjan, Zanjan, Iran Temperate and Cold Fruits Research Institute (TCFRI), Horticultural Science Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran MASOUDI, S. – KERMANI, M.J. − SOLEIMANI, A. − HAJNAJARI, H. − ALIDADI, A. − HOSSEINI, Z.S.: Optimizing micropropagation of apple (Malus × domestica Borkh) and in vitro root induction by Piriformospora indica. Agriculture (Poľnohospodárstvo), vol. 66, no. 4, pp. 137 – 147. The inoculation of plant species with mycorrhiza fungus Piriformospora indica results in enhancement of growth, increase in yield, and induction of resistance to biotic and abiotic diseases through improvement of the root system. The aim of the present study was to optimize in vitro propagation protocol for three indigenous apples (Malus × domestica) cultivars (ꞌGolbaharꞌ, ꞌSharbatiꞌ, ꞌSoltani Shabestariꞌ) and one commercial cultivar (ꞌGolden Deliciousꞌ). Furthermore, the efficiency of P. indica at rooting stage was investigated on three cultivars (ꞌSharbatiꞌ, ꞌSoltani Shabestariꞌ, ꞌGolden Deliciousꞌ). Establishment and proliferation stages were optimized by collecting explants at different seasons and comparing different culture media respectively. Rooting optimization included six treatments containing different concentrations of auxins in the presence or absence of P. indica. Results showed that at the establishment stage, a maximum percent of survival was observed in explants collected in spring. At the proliferation stage, different media had a divergent effects on distinct cultivars. Although all cultivars reacted favourable to micropropagation in MS (Murashige & Skoog 1962) basal medium, the presence or absence of cytokinin 2ip (N -(2-Isopentenyl) adenine) in the culture media showed significant and incremental improvements in growth indices. In all cultivars highest rooting percent, root length, root thickness, and the number of roots/explant was observed in MS media containing auxins for three weeks followed by a treatment of MS medium containing P. indica for another three weeks. Plantlets treated with P. indica, grow stronger and healthier at the acclimation stage compared to the ones that excluded P. indica. Key words: apple cultivars, culture media, establishment, proliferation Different species or shrubs of the genus Malus zaffarian 1996). The cultivation area of apple in Iran are either found in the wild or cultivated in different is third after grape and pistachio, with 9.1% of the regions of Iran. Among these species, Malus domes- total area under cultivation of orchard products. The tica Borkh has numerous modified breeds and it is area under cultivation is 218,000 hectares with an cultivated as the fruit “apple” in horticulture (Mo- average yield of 16 tons per hectare. This average Samira Masoudi, Maryam Jafarkhani Kermani (*Corresponding author), Amin Alidadi, Zahra Saadat Hosseini, Department of Cell and Tissue Culture, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. E-mail: maryam.j.kermani@gmail.com Samira Masoudi, Ali Soleimani, Department of Horticulture, Faculty of Agriculture, University of Zanjan, Zanjan, Iran Hassan Hajnajari, Temperate and Cold Fruits Research Institute (TCFRI), Horticultural Science Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran © 2020 Authors. This is an open access article licensed under the Creative Commons Attribution-NonComercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/4.0/). 137 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 yield is too far from the leading countries with an was designed to study the effect of symbiont fungi average of 86 tons per hectare in Switzerland (FAO P. indica on the rooting stage of two indigenous 2016). Part of the gap between Iran and developed apple cultivars ('Sharbati', 'Soltani Shabestari') and countries in terms of yield tonnage per hectare is a commercial cultivar ('Golden Delicious'). More- due to the lack of farmers’ access to healthy seed- over, establishment and shoot proliferation stages lings, which has been resulted from the traditional were optimized for the mentioned cultivars and an- propagation of pathogen-infected plants. Thus, pro- other apple cultivar (ˈGolbaharˈ). viding large numbers of healthy high yielding apple seedlings and introducing them to the horticulture industry is quite an important issue. terial ma and methodS In vitro micropropagation of plants is the only method to produce large numbers of pathogen-free Plant material and general procedures plant material in a relatively short period of time. The plant materials were supplied by Temperate There are many reports on successful methods for Fruits Research Center, Horticultural Science Rese- apple micropropagation (Batelja Lodeta et al. 2019; arch Institute of Iran. Stem cutting (15 cm) of three Bhattacharya & Singh 2018; Kaur et al. 2018; Geng indigenous cultivars including 'Golbahar', 'Soltani et al. 2015; Jafarkhani Kermani et al. 2009). Since Shabestari', 'Sharbati' along with a commercial cul- different factors such as genotype and conditions tivar, 'Golden Delicious', were collected during two before and during the regeneration process amongst growing seasons (spring and autumn). The stems plant growth regulators (PGRs), and culture media were cut into 1.5 to 2 cm segments containing at influence stages of in vitro propagation of plants, least one lateral bud. They were disinfected with so- therefore, optimizing different stages of micropropa- dium hypochlorite (2.5%) for 12 – 14 min, followed gation are essential in a successful protocol. by three washes in sterile distilled water. Piriformospora indica is a mycorrhizal endo- In all experiments, media contained 30 g/L su- symbiont of orchid and ericoid roots, which has crose, 7 g/L agar, 100 mg/L PG (Phloroglucinol), been reported to promote the growth of several and pH was adjusted to 5.8. The media were au- plant species, increase yield and product quality, toclaved at 121°C for 15 min. All the cultures were and induce resistance to biotic and abiotic stresses placed in growth chambers with a light intensity of (Franken 2012). P. indica promoted the generation 500 – 3,000 lux, photoperiod of 16/8 h light/dark, and growth of in vivo adventitious roots in pelargo- the relative humidity of 45%, and constant tempe- nium and poinsettia (Druege et al. 2007). Hua et al. rature of 23 ± 1°C. (2017) stated that an important function of P. indica Optimizing the establishment stage is auxin induction and production of its mediators. At the establishment stage, the four mentioned cul- Dong et al. (2013) reported that this fungus plays tivars and two different seasons (spring and autumn) an important role in enhancing the expression of were compared. In each season 60 explants were genes that are effective in phytohormones in barley. collected twice (every time 30 explants). Explants Moreover, the effect of P. indica on gene expression containing at least one lateral bud were surface-ste- of Chinese cabbage and Cymbidium aloifolium has rilized, placed on MS (Murashige & Skoog 1962) been reported by Hua et al. 2017 and Shah et al. basal medium, and incubated in the growth cham- 2019 respectively. Liu et al. (2019) compared the bers. The survival (explants which grew healthy), interaction of P. indica with the roots of seven host elimination (explants that died off), and contamina- plants and showed that root colonization strategies tion (explants that were infected by bacteria or fun- and changes in two phytohormones levels are highly gi) percent were recorded after one month. More- host-specific. over, the survival rate was recorded after three Since the effect of the symbiont fungi P. indica months too and the value change between the two on the rooting stage of micropropagation of apple records was calculated using the following formula has not been reported, the present investigation 138 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 in the Excel program. NAA (1- Naphthalene acetic acid) and 0.5 μmol IBA (Indole-3-butyric acid), whereas other treatments Percentage change = (A – a ) /A 2 1 1 contained relatively high concentrations of auxins (high auxin medium: HAM) which had 2 μmol NAA *a and A are the percent of survival after 1 moth 1 2 and 2 μmol IBA. In the treatments that P. indica (the and 3 months respectively. mycorrhizal Piriformospora indica) was added as Optimizing the shoot proliferation stage a rooting stimulant, 3 g of mycelia was added to At proliferation stage three different media in- every liter of culture media. P indica was supplied cluding; MS (Murashige & Skoog 1962), VS (van by Agricultural Biotechnology research Institute of der Salm et al. 1994) nutrient medium, WPM (Wo- Iran (ABRII). The mycelia growth was achieved by ody Plant Medium, Lloyd & McCown in 1981) and placing them on 8 mm Petri dishes containing Pota- two different concentrations (0 and 5 µmol) of 2iP to Dextrose Agar in incubators at 25°C for 8 days. (N -(2-Isopentenyl) adenine) were compared. The Rooting parameters including rooting percent, root media contained the following growth regulators; length [cm], root thickness [cm], number of roots 5 µmol GA (Gibberellic acid), 0.1 µmol IBA (Indo- per explant were measured after 6 weeks. le-3-butyric acid), 2 µmol BAP (6-Benzylaminopu- Statistical analysis rine); added or not added 2ip. In each experiment 20 Data were analysed in factorial based on a com- replicates (five jars with four explants) were used. pletely randomized design (CRD). A two-factor Growth indices measured after six weeks included; analysis of the genotype × medium variant was car- number of axillary shoots, shoot length [cm], the ried out for proliferation and rooting experiments. number of leaves, fresh and dry weight. The fresh Data were analysed using the statistical program weight of the whole shoot was measured and then SPSS. Statistically significant averages were com- they were dried in an oven (70°C) for 72 hours be- pared using Duncan’s Multiple Range tests. Graphs fore measuring dry weight. were plotted with the Excel program. Differences were regarded as significant at p ≤ 0.05. Investigating the effect of P. indica at rooting stage The shoots were transferred to elongation me- dium (MS + 5 µmol GA + 0.2 µmol IBA) for four RESULTS AND DISCUSSION weeks prior to initiation of rooting. The rooting ex- periment was carried out on three cultivars ('Soltani Comparing the effect of season on the establishment Shabestari', 'Sharbati', 'Golden Delicious') with 15 stage replicates in each treatment. Each explant consisted The results of this study showed that both the ge- of a 4 – 5 cm shoot with three leaves. The treatments netic source and sampling time of explants affected were abbreviated R1 to R6 (Table 1). Some treat- their survival rate as well as contamination caused ments contained low concentrations of auxins (low by bacteria and fungi at the establishment stage (Ta- auxin medium: LAM) which included 0.5 μmol T a b l e 1 Abbreviation for different treatments at rooting stage Treatment Treatment Description r1 MS basal medium (six weeks) r2 LAM (six weeks) r3 HAM (six weeks) R4 MS basal medium (three weeks) + MS basal medium with P. indica (three weeks) R5 LAM (three weeks) + MS basal medium with P. indica (three weeks) R6 HAM (three weeks) + MS basal medium with P. indica (three weeks) Note: MS – Murashigue & Skoog 1962; LAM – low auxin medium; HAM – high auxin medium. 139 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 −147 ble 2). In all cultivars, the percent of contamination Delicious' from the samples collected in autumn. was higher in autumn than in spring season, how- Hohtola (1988) reported that survival and percent ever, the number of eliminated explants due to lack of contaminants in the explants prepared in differ- ent seasons were affected by seasonal changes. The of ability to grow was higher in the explants col- report showed that in pine trees contamination was lected in spring compared to the ones collected in considerably high in winter. Our results also sug- autumn (Figure 1). In all cultivars, the highest per- gested that the contamination rate was significantly cent of survival was observed in explants collected affected by season and it was higher in explants col- in spring. Amongst the cultivars, maximum percent lected in autumn than those collected in the spring. of survival (73.32%) was recorded in 'Golbahar'. Shakya et al. (2013) stated that many factors, such Figure 1 illustrates that the survival rate declined af- as the effect of soil parameters, environmental fac- ter three months in all the cultivars and the decline tors, phenotype and genotype of the host plant, sea- was steeper in explants collected in autumn com- sons and geographic environments were involved pared to the ones collected in spring. The highest in bacterial and fungal contamination and hence the percentage change (–48%) was observed in 'Golden Figure 1. The change in survival percent after one month and three months at the establishment stage in explants of different apple cultivars collected in two seasons (The negative percentage change indicates a decrease in survival percent in month three compared to the first month). T a b l e 2 Effect of different seasons on contamination and survival at establishment stage of apple micropropagation Contamination Elimination Survival [%] after Survival [%] after Cultivar Seasons [%] [%] one month three months Spring 10.00 16.68 73.32 71.60 ꞌGolbaharꞌ Autumn 23.34 15.00 61.66 60.00 Spring 20.00 15.00 65.00 61.66 ꞌSharbatiꞌ Autumn 28.33 8.37 62.30 43.50 Spring 6.67 30.00 63.33 55.00 ꞌSoltani Shabestariꞌ Autumn 21.67 13.33 65.00 35.00 Spring 8.33 25.00 66.70 45.00 ꞌGolden Deliciousꞌ Autumn 30.00 10.00 60.00 58.33 140 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 survival percent at the establishment stage of micro- cantly in different cultivars (Table 3). The maxi- propagation. mum number of axillary shoots (3.2) was observed in 'Golbahar' cultured on MS medium without 2ip. Investigating the interactive effect of culture media Whereas, the highest shoot length (5.9 cm) and the and cultivar type at the proliferation stage maximum number of leaves (13.9) were detected in The results of the study indicated that different 'Golden Delicious' cultivated on MS medium con- culture media including Murashige & Skoog 1962 taining 2ip. The maximum amount of fresh weight (MS); van der Salm et al. 1994 (VS); Woody Plant (1,200 mg) was recorded in 'Golden Delicious' and Medium (WPM) with or without 2ip (N -(2-Isopen- 'Golbahar' in WPM and MS media without 2ip re- tenyl) adenine) affected the growth indices signifi- T a b l e 3 Effect of different culture media on indices of explant proliferation in apple cultivars No. of Axillary Mean shoot Mean no. of Fresh weight Dry weight Cultivar Medium shoots length [cm] leaves [mg] [mg] a c def a cd mS 3.2 4.0 9.1 1,200 0.14 ghi i gh gh fgh VS 1.1 2.4 5.8 300 0.08 fgh fg efgh h jkl WPM 1.3 2.8 6.5 200 0.06 'Golbahar' hij gh ab fgh ef MS + 2ip 1.0 2.9 11.5 400 0.10 d efg def gh hijk VS + 2ip 2.0 3.0 9.9 200 0.06 c ghi efg cde efg WPM + 2iP 2.3 2.7 7.3 500 0.09 ef c def fgh de mS 1.5 3.8 7.9 400 0.12 fgh hi h gh kl VS 1.3 2.4 4.5 240 0.05 k fg fgh h kl WPM 0.3 2.9 6.3 200 0.04 'Sharbati' ij fg efgh bc b MS + 2ip 0.9 2.9 6.9 700 0.23 ij efg efgh efgh jkl VS + 2ip 0.9 3.0 6.8 400 0.06 ef ef fgh fgh efg WPM + 2iP 1.4 3.2 6.3 300 0.09 hij ef abcd cde b mS 1.0 3.2 10.4 500 0.02l hij hi gh efgh gj VS 1.0 2.5 5.6 400 0.07 fgh ghi ab fgh jkl WPM 1.2 2.7 11.2 300 0.06 'Soltani Shabestari' fgh ef abc a ef MS + 2ip 1.2 3.2 10.9 1,100 0.11 ghi fg def efgh h-k VS + 2ip 1.1 2.9 8.4 400 0.06 j fg defg efg e-h WPM + 2ip 0.8 2.9 7.5 500 0.08 e de defg efg efghi mS 1.6 3.3 7.5 500 0.08 c cd efg bcd efg VS 2.3 3.7 7.0 600 0.11 cd c bcde a c WPM 2.2 3.9 10.0 1,200 0.17 'Golden Delicious' c a a b c MS + 2ip 2.3 5.9 13.9 800 0.18 c b abc a a VS + 2ip 2.4 5.1 10.7 1100 0.27 b c ab bc c WPM + 2iP 2.8 3.8 11.2 800 0.15 Different letters show significant difference in each column according to Duncan’s Multiple Range Test (P ≤ 0.05). Note: MS – Murashige & Skoog 1962; VS – van der Salm et al. 1994; WPM – Woody Plant Medium; 2ip – N6-(2-Isopen- tenyl) adenine. 141 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 −147 spectively and the highest amount of dry weight has been reported by Monfort et al. (2018). Arab et (0.27 mg) was observed in 'Golbahar' cultured in VS al. (2014) investigated the interaction between me- medium containing 2ip. dia and growth regulators in G × N15 (a hybrid of The recommended medium for each cultivar almond × peach) and reported that the best medium based on the data presented in Table 3 and general was MS supplemented with 1 mg/L BAP. A strong quality of the explants (without data) was as follow: influence of the culture media mineral composition 'Golbahar' (MS), 'Soltani Shabestari' (MS + 2ip), on the micropropagation and the nutritional status 'Sharbati' (MS), 'Golden Delicious' (WPM or MS + of Eucalyptus dunnii cultures was also reported by 2ip). Figure 2 shows the explants at the proliferation Oberschelp and Gonçalves (2016). They stated that stage (different media) tested for the four cultivars. variability introduced by each genotype deterred the The roman numerals highlighted in red and under- detection of additional effects. Hence they were able line, show the best culturemedium for each cultivar to develop a basal media based on the mineral com- The influence of shoot culture media types on the position of tissues of the species and optimized it quality and quantity of growth of Ocimum basilicum for specific clones in order to adjust the nutritional ˈGolbaharˈ I II III IV V VI ˈSharbati I II III IV V VI ˈSoltani Shabestariˈ I II III IV V VI ˈGolden Deliciousˈ I II III IV V VI Figure 2. Effect of different media on explant proliferation of apple cultivars: I = MS, II = VS, III = WPM, IV = MS + 2IP, V = VS + 2IP, VI = WPM + 2IP. The roman numerals highlighted in red and underline show the best culture medium for each cultivar. Note: MS – Murashige & Skoog 1962; VS – van der Salm et al. 1994; WPM – Woody Plant Medium; 2ip – N6-(2-Isopen- tenyl) adenine. 142 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 requirements for each genetic material. Accordin- called the central zone (CZ). Cells moving from the CZ into a region with a higher rate of cell division gly, an interaction effect of various culture media, named the peripheral zone (PZ), give rise to the axi- in terms of their composition, and different apple llary shoots. The OC of the SAM is the site of maxi- cultivars was revealed at current work, though all mal cytokinin activity, rather than of auxin activity. cultivars reacted favorable to micropropagation in They concluded in the SAM cytokinins promote the MS medium. However, the presence or absence of proliferation of undifferentiated cells, while auxins cytokinin 2ip in the culture media showed signifi - act in the PZ to induce cellular differentiation and cant and incremental improvements in growth indi- organ outgrowth. However, the type and concentra- ces. Schaller et al. (2015) reviewed the role of cy- tion of the cytokinin needed for each genotype even tokinin and auxin interaction in plant development within a species is variable, which is in agreement and reported that in shoot apical meristem (SAM) with our results. a group of initial stem cells surrounds a small group of mitotically less active cells, known as the organi- Optimizing rooting stage using mycorrhiza P. indica zing center (OC). The cells of the OC are located at The results of the study indicated that rooting per- the base of a region with a low rate of cell division cent, the number of roots/explant, total root length/ bc bc bc bc r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 r1 r2 r3 R4 R5 R6 r1 r2 r3 R4 R5 R6 r1 r2 r3 R4 R5 R6 Sharbati Soltani Shabestari Golden Sharbati Soltani Shabestari Golden Delicious Delicious 1.6 1.4 1.2 0.8 0.6 0.4 0.2 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 0 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 r1 r2 r3 R4R5R6 Sharbati Soltani Shabestari Golden Sharbati Soltani Shabestari Golden Delicious Delicious Figure 3. Comparison of the interaction between cultivar and type of medium on A) rooting percent, B) average number of roots/explant, C) total length of roots /explant [cm], D) average root thickness [cm] of different apple cultivars. R1: MS, R2: LAM, R3: HAM, R4: MS + P. indica, R5: LAM + P. indica, R6: HAM + P. indica. Note: MS – Murashigue & Skoog 1962; LAM – low auxin medium; HAM – high auxin medium. Rooting percent Total length of roots/explant [cm] Average number of roots/explant Average root tickness [cm] Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 −147 explant, and thickness of the roots in the three cul- Shabestari' (10.75) was attained in R6 (Figure 3B). The highest total length of roots/explant in 'Sharbati' tivars ('Sharbati', 'Soltani Shabestari', and 'Golden (18.75 cm), ꞌSoltani Shabestariꞌ (6.7 cm) and 'Gold- Delicious') were significantly affected by rooting en Delicious' (11 cm) was observed in treatment R5 treatments. In all cultivars, MS medium containing (Figure 3C). The greatest root thickness in 'Shar- either low or high auxin concentrations (LAM & bati' (1.35 cm) and 'Soltani Shabestari' (1.17 cm) HAM) for three weeks followed by a treatment of were detected in R5, but in ˈGolden Deliciousˈ MS medium containing P. indica for another three maximum root thickness (1.3 cm) was observed weeks (treatments R5 & R6) increased the quality in R2 (Figure 3D). Figure 4 and Figure 5 illustrate and quantity of root production. Figure 3A illus- the prototype plantlets at rooting and acclimating trates that maximum rooting percent in 'Sharbati' stages of micropropagation respectively. The fig- (40%) and 'Soltani Shabestari' (80%) were obtained ures indicate that in 'Sharbati' and 'Golden Deli- in treatments R5 and R6 respectively. Whereas, the cious' using fungus P. indica in combination with highest percent of rooting (80%) in 'Golden Deli- auxins resulted in strong plantlets with substantial cious' was attained in R5. The maximum number amounts of roots. However, in 'Soltani Shabestari' of roots/explant in ꞌSharbatiꞌ (3.5) and ꞌGolden De- although MS basal medium also resulted in the liciousꞌ (8.87) were observed in treatment R5, but emergence of roots the plantlets from treatments the maximum number of roots/explants in 'Soltani ˈSharbatiˈ LAM MS MS+ P. indica HAM HAM + P. indica LAM + P. indica ˈSoltani Shabestariˈ LAM MS + P. indica HAM MS HAM + P. indica LAM + P. indica ˈGolden Deliciousˈ LAM MS MS + P. indica HAM HAM + P. indica LAM + P. indica Figure 4. Effect of different treatments at rooting stage of three cultivars ('Sharbati', 'Soltani Shabestari' and 'Golden Delicious'). Note: MS – Murashigue & Skoog 1962; LAM – low auxin medium; HAM – high auxin medium. 144 Agriculture (Poľnohospodárstvo), 66, 2020 (4): 137 − 147 containing P. indica grew stronger and healthi- host plants such as; growth promotion, develop- er compared to the ones that excluded P. indica. ment of the root systems, and increases tolerance In this investigation, a combination of two types to abiotic stresses (Lahrmann & Zuccaro 2012). of auxins (IBA and NAA) rather than one type of aux- Shah et al. (2019) investigated the growth pattern in was used at the rooting stage. Yusnita et al. 2018 (number and length of roots and shoots) and vari- showed that in Malay apple cuttings, a combination ous metabolites of in vitro propagated Cymbidium of IBA and NAA was more effective on rooting than aloifolium plantlets that interacted with the P. indi- NAA or IBA alone. They reported that application ca. They identified growth regulators such as auxin, of IBA + NAA, caused greater root length and the ascorbic acid, andrographolide, hexadecanoic acid, adventitious root morphologies were more branched and DL-proline and reported P. indica-colonized and more fibrous, compared to the unbranched cut- plantlets showed improved growth rate compared to tings treated with NAA or IBA. Moreover, Yusnita et un-colonized control plantlets. In another study, P. al. 2018 illustrated that combination of the two aux- indica plugs (P-1) and a P. indica chlamydospore ins (IBA and NAA) increased shoot sprouting per- suspension (P-2) were co-cultivated with banana cent, shoot length, and number of leaves per shoot. seedlings at the rooting stage and the results indi- The endophytic fungus P. indica is a symbiot- cated that P-2 treatment was more effective than the ic microorganism that has beneficial effects on the P-1 treatment on plant height and root length in the ˈSharbatiˈ HAM + P. indica LAM + P. indica LAM MS MS + P. indica HAM ˈSoltani Shabestariˈ ˈSoltani Shabestariˈ MS HAM MS + P. indica LAM LAM + P. indica HAM + P. indica MS ˈGolden Deliciousˈ MS + P. indica HAM LAM LAM + P. indica HAM + P. indica ˈGolden Deliciousˈ MS + P. indica MS HAM HAM + P. indica LAM LAM + P. indica Figure 5. 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Journal

Agriculturede Gruyter

Published: Dec 1, 2020

Keywords: apple cultivars; culture media; establishment; proliferation

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