Assessing the response of indigenous loquat cultivar Mardan to phytohormones for in vitro shoot proliferation and rooting

Assessing the response of indigenous loquat cultivar Mardan to phytohormones for in vitro shoot proliferation and rooting first-author †E-mail:nadeemabbasi65@yahoo.com †E-mail:nadeemabbasi65@yahoo.com nadeemabbasi65@yahoo.com Plant Tissue Culture Laboratory, Department of Horticulture, Pir Mehr Ali Shah-Arid Agriculture University, Rawalpindi 46300, Pakistan Plant Tissue Culture Laboratory, Department of Horticulture, Pir Mehr Ali Shah-Arid Agriculture University, Rawalpindi 46300, Pakistan In vitro cultures of loquat cultivar Mardan were established using shoot apices after treating with NaOCl (5%, 7%, 10%, 12%, 14% (v/v)) for 12 min and HgCl 2 (0.01%, 0.05%, 0.10%, 0.20%, 0.25% (w/v)) for 2 min. A maximum survival rate of 70% was recorded after surface sterilization with 10% NaOCl. Caulogenic response was assessed on Murashige and Skoog (MS) medium fortified with assorted combinations of the cytokinins, benzylaminopurine (BAP), kinetin, and N6-(2-isopentyl)adenine (2iP). Treatment of BAP 1.5 mg/L combined with 2iP 9.0 mg/L and kinetin 1.5 mg/L was found to be optimum for shoot morphogenesis in terms of the number and subsequent growth of shoots, while the highest shoot length was yielded by the combination of BAP 0.5 mg/L, kinetin 0.5 mg/L, and 2iP 3 mg/L. Higher levels of cytokinins induced callogenesis, vitrification and stunted growth to some extent. For rhizogenesis, uniform sized micro-shoots were excised and transferred to half-strength MS medium containing auxins. The best rooting expression was observed with naphthaleneacetic acid (NAA) 1 mg/L combined with indole-3-butyric acid (IBA) 2 mg/L and paclobutrazol (PBZ) 1 mg/L. Eriobotrya japonica Micropropagation Sterilization of loquat Plant growth regulator Shoot proliferation Rhizogenesis Introduction 1 Loquat ( Eriobotrya japonica Lindl.) is an important evergreen fruit crop of subtropical regions. It is grown in more than thirty countries (Feng et al., 2007 2007 ) with China having the leading share (Lin, 2007 2007 ). It is a popular fruit in Pakistan and is available to satisfy consumer needs in spring when no other fruit is available in the market, thus fetching good prices. However, until recently (Hussain et al., 2007 2007 ), it has been neglected in terms of research. In Pakistan, most of the loquat orchards were established using seeds due to the unavailability of certified and true to type planting stock in local nurseries. Consequently, there is a lack of uniformity among plants giving low quality fruits with low yield. Cultivation of superior genotypes of loquat may increase production, thereby increasing availability for the home market and for export (Hussain et al., 2007 2007 ). Loquat cultivar Mardan has large, round, and attractive fruit with an orange yellow skin and sweet tasting pulp (Hussain, 2009 2009 ) which can help it to capture local and export markets. Plant cell, tissue, and organ culture techniques are becoming an integral part of propagation systems for year-round supply of clonal plant material on a mass scale (Al-Sulaiman and Barakat, 2010 2010 ) in a very short time span, which is impossible by conventional approaches (Barakat, 2008 2008 ). Their application will also satisfy sanitary and phytosanitory (SPS) demands in the context of the World Trade Organization (WTO). The current lack of certified germplasm repositories and seasonal fluctuations in supply require immediate application of in vitro techniques to overcome these problems of the fruit industry. Biotechnological tools for mass propagation of different tree species have been introduced recently, particularly in the last decade (Azad et al., 2005 2005 ; George et al., 2008 2008 ), and micropropagation protocols are available for many species and cultivars (Ruzic and Vujovic, 2008 2008 ). However, propagation of woody plants in vitro is an intricate process. Clonal propagation of loquat using in vitro techniques has been carried out earlier (Lomtatidze et al., 2009 2009 ), but there is a need to optimize cultivar specific protocols as no trials have been made for indigenous loquat genotypes. Shoot tip culture is highly reliable and is the preferred technique for obtaining disease-free stock (Panjaitan et al., 2007 2007 ), while production of vigorous shoots with a rapid rate of multiplication is one of the prerequisites of reliable micropropagation practice. In vitro root development for subsequent acclimatization is another important factor determining the success of micropropagation. Cytokinins are a class of plant growth regulators (PGRs) involved in many processes of plant growth, including cell division and shoot and root morphogenesis. In particular, PGRs are known to regulate axillary bud growth. Auxins have a cardinal role in coordination of many growth processes in a plant’s life cycle and are essential for plant root development (Nordstrom et al., 2004 2004 ). Considering the importance of these PGRs, this study aimed to evaluate different types and concentrations of cytokinins and auxins for in vitro shoot development and rhizogenesis in loquat. Protocols standardized following this study will be conducive for the micropropagation of elite loquat genotypes on a commercial scale, which will finally uplift the national horticulture industry and export potential. Materials and methods 1 Shoot tips (3–4 cm in size) of loquat cultivar Mardan were collected from the orchard of Tret (Murree, Pakistan) to be used as explants for in vitro culture establishment. After removing leaves, hairs, and dirt, they were placed under running tap water with one drop of Tween 80 and detergent for 30 min to remove any foreign contaminants. After washing, shoot apices were dissected and surface sterilized with different concentrations of NaOCl (5%, 7%, 10%, 12%, 14% (v/v)) for 12 min and HgCl 2 (0.01%, 0.05%, 0.10%, 0.20%, 0.25% (w/v)) for 2 min in a laminar air flow hood. Inoculation was done in culture jars containing Murashige and Skoog (MS) shoot proliferation media (MS macro, micro salts, and vitamins), supplemented with various combinations of cytokinins (BAP, 2iP, and kinetin) (Table 1 1 ). Data were recorded after 28 d for the number of shoots, shoot length (cm), number of leaves, and fresh and dry weights (mg). BAP: benzylaminopurine; 2iP: N6-(2-isopentyl)adenine 2 Treatment 3 Cytokinin (mg/L) BAP 2iP Kinetin T C0 0.0 0.0 0.0 T C1 0.5 3.0 0.5 T C2 1.0 6.0 1.0 T C3 1.5 9.0 1.5 T C4 2.0 12.0 2.0 For root induction, uniform sized (1.5 cm) micro-shoots were transferred to rooting medium (half strength of MS) supplemented with different levels of indole-3-butyric acid (IBA) in combination with naphthaleneacetic acid (NAA) and paclobutrazol (PBZ) (Table 2 2 ). Data were recorded after six weeks for rooting percentage, number of roots, and root length (cm). NAA: naphthaleneacetic acid; IBA: indole-3-butyric acid; PBZ: paclobutrazol 2 Treatment 3 Auxin (mg/L) NAA IBA PBZ T A0 0.00 0.00 0.00 T A1 0.25 0.50 0.25 T A2 0.50 1.00 0.50 T A3 0.75 1.50 0.75 T A4 1.00 2.00 1.00 T A5 1.25 2.50 1.25 The experiment was designed as a single factorial with three replications for each treatment and five plants per replication. Data were statistically analyzed using the analysis of variance (ANOVA) (MSTAT-C software) and means were compared using the least significant difference (LSD) test at 5% α level (Steel et al., 1997 1997 ). Results and discussion 1 Culture establishment 2 Surface sterilization of loquat is a very difficult step in culture initiation due to its hairy nature. Furthermore, exudation of phenolics and browning are other common problems associated with its in vitro culture, as with other members of the Rosaceae family (Bhojwani and Razdan, 1983 1983 ; Amin and Jaiswal, 1987 1987 ). NaOCl is the most suitable disinfectant for a variety of plant species. It plays a significant role in eliminating microorganisms from the surface of explants (Canli and Kazaz, 2009 2009 ). However, the efficacy of surface sterilization can be improved by standardizing the most appropriate combination of dose and exposure time of disinfectants (Yildiz and Er, 2002 2002 ). Effects of NaOCl and HgCl 2 on in vitro culture establishment of loquat cultivar Mardan 2 Data regarding the effect of disinfectants on surface sterilization of loquat shoot tips after 28 d are presented in Fig. 1 1 . Treatment with 10% NaOCl showed significant results with an increased survival percentage (70%). Treatment with 14% NaOCl resulted in minimum survival (2%) and maximum necrosis (90%) (Fig. 1a 1a ). Generally, necrosis increased as the concentration of NaOCl increased which shows that higher concentrations of NaOCl damage young tissues. Fungal and bacterial contamination was high (86.66%) after treatment with 5% NaOCl, but absent after treatment with 14% NaOCl. The highest NaOCl concentration badly damaged the explants and decreased the survival rate. HgCl 2 significantly reduced contamination (to 3.33%) after treatment of 0.25% HgCl 2 but increased mortality (to 53.3%). Percentage survival was highest (63.33%) after treatment of 0.10% HgCl 2 followed by 0.20% HgCl 2 (43.33%) (Fig. 1b 1b ). The percentage survival after treatments of 0 and 0.05% HgCl 2 was not significantly different (40%). Increases in the concentration of HgCl 2 showed increasing necrosis and mortality, but a decline in contamination. HgCl 2 is one of the strongest disinfectants used in culture establishment. The action of HgCl 2 may be through lysis of cells of microorganisms or it may act on thiol groups in microbial enzymes. Like NaOCl, HgCl 2 is known to be a powerful antimicrobial agent (Russell and Chopra, 1990 1990 ). Treatment with 0.10% HgCl 2 for 5–7 min is used commonly for surface sterilization of many plant species (Skirvin et al., 1986 1986 ). El-Zaher ( 2008 2008 ) obtained good results with 0.30% HgCl 2 for 5 min in jackfruit. Our results are in line with the findings of Chavan et al. ( 1996 1996 ) who stated that HgCl 2 at 0.20% for 4 min or at 0.10% for 10 min gave good sterilization results in shoot tip cultures of jackfruit. Comparison of the two sterilizing agents showed that NaOCl gave a maximum survival rate of 70% and a minimum mortality rate of 10% after treatment of 10% NaOCl, while HgCl 2 gave a maximum survival rate of 63.33% after treatment of 0.10% HgCl 2 and a minimum mortality rate of 6.66% after treatment of 0.01% HgCl 2 . From the above results, we infer that treatment with NaOCl at 10% performed better than treatment with HgCl 2 . NaOCl is also a readily available and economical sterilant. Effects of cytokinins on shoot development 2 Number of shoots 3 Different combinations of cytokinins (BAP+2iP+kinetin) interacted significantly in terms of the number of shoots (Fig. 2 2 ). Treatment T C3 (BAP 1.5+2iP 9.0+kinetin 1.5 mg/L) yielded the maximum number of shoots (4.84) followed by T C4 (BAP 2.0+2iP 12.0+kinetin 2.0 mg/L) at 3.25 (Fig. 3a 3a ). Increasing BAP doses in combination with 2iP+kinetin had an increasing effect up to a certain level (T C3 ), after which a declining trend was observed in shoot growth. Our findings are in accordance with the results of Al-Maarri and Al-Ghamdi ( 1995 1995 ), who reported that application of BAP and 2iP produced a good response for initiation and multiplication of shoot tip cultures in date palm. Histological studies showed that the inclusion of BAP in shoot proliferation media enhanced the growth of axial shoots and promoted the multiplication of shoots from the basal tissues of explants (Ohki and Sawaki, 1999 1999 ). The successful use of BAP for shoot production may be accredited to the capability of plant tissues to metabolize BAP more efficiently than other synthetic PGRs or to the capacity of BAP to encourage the production of natural hormones, such as zeatin, within the tissues (Malik et al., 2005 2005 ). Kukreja et al. ( 1990 1990 ) and Vincent et al. ( 1992 1992 ) also reported a decline in the number of shoots with higher BAP levels. Waseem et al. ( 2009 2009 ) showed that the use of higher than optimal concentrations of PGRs may lead to poor performance. Rapid growth and multiplication of shoots are based on the quantity and quality of cytokinins and auxins in media as well as on their endogenous levels in plants, which vary from species to species and between growth phases (Panjaitan et al., 2007 2007 ). BAP manipulates shoot production by stimulating rapid cell division to induce multiple shoot formation (Roy and Banerjee, 2003 2003 ; Ronzhina, 2003 2003 ). Lane ( 1992 1992 ) reported that shoot production was improved in response to BAP application. In olive, 2iP at 10 mg/L produced a high number of shoots (Agrawal et al., 1991 1991 ). Similarly, Al-Sulaiman and Barakat ( 2010 2010 ) documented that the presence of 2iP in the medium at 10.0 mg/L had a positive effect on the production of lateral shoots and other growth parameters of Ziziphus spinachristi . Increases in shoot number might be related to the application of an appropriate dose of cytokinin which promotes bud growth and overcomes apical dominance (Asaad et al., 2009 2009 ). Shoot length 3 Data presented in Fig. 2 2 show that the shoot length was markedly affected by various combinations of cytokinins. Statistically, longer shoots (1.97 and 1.92 cm) were obtained with treatments T C2 (BAP 1.0+2iP 6.0+kinetin 1.0 mg/L) and T C1 (BAP 0.5+2iP 3.0+kinetin 0.5 mg/L), respectively, followed by T C3 (BAP 1.5+2iP 9.0+kinetin 1.5 mg/L) (1.74 cm) (Fig. 3b 3b ). After treatment T C4 (BAP 2.0+2iP 12.0+kinetin 2.0 mg/L) with elevated levels of cytokinins, shoot length decreased to 1.56 cm, demonstrating the negative impact of increased doses of PGRs as reported by Carelli and Echeverrigary ( 2002 2002 ). Cytokinins release apical dominance, which hastens axillary bud formation and reduces the length of explants (Huetteman and Preece, 1993 1993 ). Increased BAP levels in shoot multiplication media suppress shoot growth due to the regeneration of multiple shoots (Waseem et al., 2009 2009 ). Singh and Arora ( 1995 1995 ) similarly reported that increasing BAP concentrations increased the number of shoots but depressed the shoot length in chrysanthemum. At minimum levels, cytokinins produce longer shoots, which may be due to their reduction in number. Baig et al. ( 2011 2011 ) also found that BAP is a strong cytokinin which reduces shoot length and increases the number of side shoots. Exposure of cultures to higher concentrations of BAP during regeneration of shoots may lead to an increase in cytokinins, which inhibits further shoot growth (Malik et al., 2005 2005 ). In woody plants, BAP is paramount for growth compared to kinetin (Fráguas et al., 2004 2004 ). The frequency of shoot multiplication and the length and number of shoots depends mainly on the optimal level of BAP, after which response declines (Waseem et al., 2009 2009 ). Higher than optimal concentrations of BAP enhance ethylene production in the base of plants which may block the transport of exogenous auxin in the basal portion of explants and result in minimum shoot growth and development (Ahmad et al., 2003 2003 ). Yakimova et al. ( 2000 2000 ) stated that cultured plants producing large numbers of shoot buds exhibit minimum shoot length because all the nutrients are utilized for the formation of lateral shoots. The best shoot elongation was observed with 0.6 mg BAP in olive (Ahmad et al., 2003 2003 ). BAP is preferable to other cytokinins for promoting shoot production and proliferation (Siddique and Anis, 2009 2009 ). BAP is degraded very slowly in culture medium which makes it available to plant tissues for a longer period of time (Hussain et al., 2011 2011 ). Number of leaves 3 The maximum number of leaves (4.26) was observed after treatment T C3 (BAP 1.5+2iP 9.0+kinetin 1.5 mg/L) followed by T C4 (BAP 2.0+2iP 12.0+kinetin 2.0 mg/L) (Fig. 2 2 ). We infer from the results that the combination of cytokinins in treatment T C3 is optimum for better performance, as the shoot number, and therefore the number of leaves, increased with this treatment. In contrast, the leaf count was meager (2.16) of the control treatment, confirming the significance of these phytohormones for leaf growth. 2iP and BAP are important PGRs for bud break and shoot differentiation due to their role in cell multiplication and the breakdown of apical dominance (Casimiro et al., 2001 2001 ). Multiplication of shoots depends mainly on the number of buds and leaves on an explant. Specific plant hormones may arrest or hasten the growth and development of leaves. Previously, it was indicated that BAP had a strong effect on promoting the growth of leaves and bud initials, causing a rise in the number of bud primordia in chrysanthemum (Karim et al., 2002 2002 ; 2003 2003 ). Kapchina-Toteva and Yakimova ( 1997 1997 ) reported that peroxidase activities increased following cytokinin application, leading to the sprouting of axillary buds which subsequently developed into leaves. Various scientists have studied the role of cytokinins in bud formation and leaf growth (Mok, 1994 1994 ). Fresh and dry weights 3 Results showed that the maximum fresh weight (200.55 mg) was obtained after treatment T C3 (BAP 1.5+2iP 9.0+kinetin 1.5 mg/L) followed by treatments T C4 (198.88 mg, BAP 2.0+2iP 12.0+kinetin 2.0 mg/L), and T C2 (190.12 mg, BAP 1.0+2iP 6.0+kinetin 1.0 mg/L). The control treatment produced the minimum fresh weight (171.51 mg) (Fig. 4 4 ). The combination of BAP, 2iP, and kinetin at 1.5, 9.0, and 1.5 mg/L, respectively, proved to be optimum for the fresh weight of shoots, which is consistent with the results for other parameters (i.e., the number of shoots per explant and the number of leaves). Fresh weight is correlated with the quality of shoots. After treatment T C3 , the shoots were quite healthy and had an excellent leaf:stem ratio but after treatment T C4 some vitrified plants were observed (Fig. 5 5 ), which reduced the total fresh weight of the plants. Moreover, the decrease in fresh weight after treatment T C4 was due to reduced shoot and leaf number at this level. Vitrified plants are deficient in chlorophyll content and show hyperhydricity, hypolignification, and changes in enzyme activity and protein synthesis, which can cause alterations in normal plant metabolic processes (Ziv, 1992 1992 ). These results support those of Li et al. ( 2003 2003 ) who proposed that the combination of high concentrations of cytokinins with a high water potential in the medium is one of the major causes of vitrification. Dry weight showed synergism with fresh weight. The maximum accumulation of dry weight (40.04 mg) was after treatment T C3 (BAP 1.5+2iP 9.0+kinetin 1.5 mg/L) followed by T C4 (BAP 2.0+2iP 12.0+kinetin 2.0 mg/L) (39.13 mg). A declining trend was observed in dry matter as the number of shoots and leaves decreased, as for fresh weight. The highest dry weight was found after treatments T C3 and T C4 , which we attribute to there being the same amounts of shoots, leaves, and biomass accumulation. Barberaki and Kintzios ( 2002 2002 ) reported that the addition of macronutrients in plant tissues is significantly affected by the addition of PGRs. Kinetin and BAP enhance leaf area and maximum dry weight accumulation (de Oliveira et al., 2010 2010 ). Increases in cytokinin levels lead to increases in the numbers of leaves and shoots, and in the rooting percentage by triggering cell division and elongation (Schmulling et al., 2003 2003 ). Baig et al. ( 2011 2011 ) studied the role of BAP in dry matter accumulation and reported that it may enhance physiological vigor, leading to the conversion of more sugars into dry matter in different regions of explants. Significant increase in dry weight may be related to sucrose concentrations in media. Sucrose is incorporated into structural carbohydrates in cell walls, which form the major part of a plant’s dry weight (Gollagunta et al., 2004 2004 ). Moreover, dry weight is affected by an increase in enzyme activities coupled with the production of more shoots. An enhancement of peroxidase activity in response to cytokinins and in association with an increased number of open axillary buds was reported in cultured Rosa hybrida in vitro (Yakimova et al., 2000 2000 ). The activity of enzymes is induced by high cytokinin concentrations, due to an elevation of the RNA levels from a subset of the genes involved (Taiz and Zeiger, 2002 2002 ). According to Vuylsteker et al. ( 1997 1997 ), the fresh weight of a plant depends on the number of shoots and therefore it may decline due to a reduced shoot number. Bennet et al. ( 1994 1994 ) reported that a decrease in fresh weight might arise because kinetin is less effective than BAP in prompting the enzyme activity involved in promoting vegetative growth. High concentrations of cytokinins cause senescence of tissues, possibly due to the production of ethylene, which consequently gives lower fresh weight (George et al., 2008 2008 ). Effects of auxins on root development 2 Number of roots 3 Different combinations of auxins significantly affected the number of roots. The maximum number (12.06) was obtained after treatment T A4 (NAA 1.00+IBA 2.00+PBZ 1.00 mg/L) (Fig. 6b 6b ) followed by T A5 (NAA 1.25+IBA 2.50+PBZ 1.25 mg/L) (10.34) (Fig. 7 7 ). An increasing trend was observed with increasing concentrations of auxins up to the level of treatment T A4 but after that there was a slight reduction in root number with T A5 . Rooting response was highly impoverished in the control treatment (T A0 ). Auxins play a vital role in regulating the growth and multiplication of plant organs (Woodward and Bartel, 2005 2005 ; Teale et al., 2006 2006 ) and in lateral root formation (Ruegger et al., 1997 1997 ; Rogg et al., 2001 2001 ). IBA is the most common auxin, and is the strongest, most highly stable and least toxic rooting plant hormone among all auxins (Weisman et al., 1988 1988 ; Hartmann et al., 2007 2007 ). George et al. ( 2008 2008 ) also found that development and growth of roots is accelerated by auxins, which enhance cell division in the root zone. Root initiation is dependent on the interaction between external and internal factors, such as the type and level of endogenous PGRs. However, auxin is one of the important factors (Asaad et al., 2009 2009 ). The application of IBA causes changes in the synthesis of protein and the production of RNA. These changes lead to more rapid cell growth and division and, as a result, the number of roots increases (Husen and Pal, 2007 2007 ). PBZ belongs to the triazole family which includes hormones that act as plant multiprotectants against stresses (Gilley and Fletcher, 1997 1997 ). The role of PBZ in root initiation is still unknown, but it acts as an antigibberellin in plants (Kefford, 1973 1973 ). Various scientists have reported that application of PBZ increases antigibberellin compounds, which may promote root production (Kefford, 1973 1973 ; Tim et al., 1985 1985 ). In the present study, we observed that high levels of auxins induced callogenesis in the basal part of shoots and exerted an inhibiting effect on root growth and development. IBA at higher levels enhances degradative metabolism in tissues and inhibits root initiation and development (Baker and Wetzstein, 1994 1994 ). The findings of Baker and Wetzstein ( 1994 1994 ) also suggest that auxins at higher than optimal concentrations may lead to reductions in the number of roots and in the rooting percentage. Root length 3 Root length of the loquat genotypes was influenced by the various types and combinations of auxins (Fig. 7 7 ). Maximum root length (13.07 cm) was obtained after treatment T A4 (NAA 1.00+IBA 2.00+PBZ 1.00 mg/L) (Fig. 6a 6a ) followed by T A5 (NAA 1.25+IBA 2.50+PBZ 1.25 mg/L) (12.33 cm). With increasing concentrations, auxins greatly promoted root length but at the highest concentrations of NAA, IBA, and PBZ, root length decreased. Auxin concentration is most important for root elongation which may be inhibited by increasing levels of exogenous auxins in the rooting media (Baker and Wetzstein, 1994 1994 ). Cell multiplication and proliferation are responsible for fresh tissues in the root multiplication zone that direct the regeneration of roots around the periphery of the quiescent region, consequently enhancing root length (Hopkins, 1995 1995 ). Hu and Wang ( 1983 1983 ) reported that the root elongation stage is very responsive to auxin concentration and may be inhibited by higher concentrations. PBZ application, for in vitro rooting of plants, increases resistance against desiccation (Roberts and Matthews, 1995 1995 ). Auxins intensify adventitious root initiation due to production of endogenous enzymes, which may hasten cell division, differentiation, and multiplication (Husen and Pal, 2007 2007 ). High concentrations of auxin (IBA) reduce root length and number below optimal levels. Ozel and Arslan ( 2006 2006 ) reported that they also raise the endogenous auxin levels in plants providing protection against catabolism and increasing interactions with growth inhibitors by conjugation. Plants with long roots and higher root numbers are produced, which is probably due to effect of endogenous hormones (George et al., 2008 2008 ). Taiz and Zeiger ( 2002 2002 ) also found that lower concentrations of auxin promote root growth but higher levels reduce growth due to the production of ethylene in the root zone, which acts as a growth inhibitor. Baker and Wetzstein ( 1994 1994 ) observed that the production of degradative metabolites increases with increasing concentrations of IBA, which blocks the root regeneration progress (Rai et al., 2009 2009 ). Rooting percentage 3 Significant differences ( P < 0.05) were observed among different auxin treatments in terms of rooting percentage (Fig. 7 7 ). The highest rooting percentage (99.71%) was recorded after treatment T A3 (NAA 0.75+IBA 1.50+PBZ 0.75 mg/L) followed by treatments T A4 (NAA 1.00+IBA 2.00+PBZ 1.00 mg/L) (74.930%) and T A5 (NAA 1.25+IBA 2.50+PBZ 1.25 mg/L) (67.667%). However, the minimum rooting percentage (16.66%) was recorded in the control treatment. Data showed that IBA at 1.50 mg/L with NAA and PBZ each at 0.75 mg/L was the optimum combination. The rooting percentage showed a decreasing trend with higher concentrations of auxins. Auxins are effective PGRs which enhance the process of root growth and multiplication through the differentiation of vascular tissues (Asghar et al., 2011 2011 ). Among auxins, IBA is known to stimulate rooting more efficiently due to its weak toxicity and greater stability for root induction (Han et al., 2009 2009 ). The rooting percentage is increased by increasing the auxin levels, but at higher concentrations the number of roots is decreased (Asghar et al., 2011 2011 ). Auxins encourage rooting through biochemical changes in the plant system (Henrique et al., 2006 2006 ). Bhatt and Tomar ( 2010 2010 ) found that IBA at a minimum concentration produced the best results. Differentiation of root primordia due to an optimum concentration of IBA, enhances cambial development at the base of explants, which increases the maximum rooting percentage (Haq et al., 2009 2009 ). Efficient changes in IBA genes can promote auxin biosynthesis and then alter root development, while PBZ can reduce stem growth due to an antigibberellin effect (Rademacher et al., 1984 1984 ; Graebe, 1987 1987 ). PBZ inhibits gibberellin synthesis and ethylene production, and increases cytokinin production in roots (Kamountsis and Chronopoulou-Sereli, 1999 1999 ). Auxin stimulates the production of growth substances for root development and elongation (Han et al., 2009 2009 ). Conclusions 1 The current study documents the protocol optimization for Loquat micropropgation. 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Morphogenic control of plants micropropagated in bioreactor cultures and its possible impact on accilimatization Compliance with ethics guidelines Nadeem Akhtar ABBASI, Tariq PERVAIZ, Ishfaq Ahmed HAFIZ, Mehwish YASEEN, and Azhar HUSSAIN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1200277 Project (No. 20-1540/R&D/09) supported by the Higher Education Commission (HEC) of Pakistan 2012-10-11 2013-08-13 2013-03-11 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 14