Document Type : Research Paper
Authors
1
Former M. Sc. student, Biotechnology Department, Faculty of Agriculture and Natural Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2
Researcher, Temperate Fruits Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran.
3
Assistant Professor, Tissue Culture and Gene Transformation Department, Agricultural Biotechnology Research Institute of Iran, Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran.
4
Associate Professor, Temperate Fruits Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran.
5
Associate Professor, Genomics Department, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education and Extension Organization, Karaj, Iran.
6
Assistant Professor, Biotechnology Department, Faculty of Agriculture and Natural Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.
10.22092/spj.2026.367421.1450
Abstract
In vitro propagation of quince tree is more difficult than other pome fruit trees, and as a result, it is necessary to optimize tissue culture methods for cultivars and genotypes of this tree with various goals, such as producing virus-free mother stocks. The aim of this study was to optimize proliferation, in vitro growth and rooting conditions, for three selected quince genotypes of Isfahan region including KVD4, KM1 and cv. Behta. Effects of MS and QL culture media were evaluated on in vitro proliferation traits. The results showed that QL in comparison with compared with MS, significantly improved shoot proliferation and was selected for further experiments. In the second experiment, the effects of different concentrations of cytokinins including 2iP and BAP at the 0.5, 1.0 and 2.0 mgl-1 and TDZ at the 0.22 and 0.022 mgl-1 levels were examined on growth and proliferation. The results indicated that the best proliferation rate, shoot length, shootlet number, leaflet number shootlet-1 and growth quality index were obtained in culture media containing 1.0 mgl-1 2iP, 2.0 mgl-1 BAP and 0.22 mgl-l TDZ. The third experiment was conducted to study the effects of different concentrations of IBA, NAA and IBA+NAA on rooting of these genotypes. The highest rooting was observed using 1.0 mgl-l IBA. QL media containing 2.0 mgl-l BAP, 0.5 mgl-l 2iP and 0.22 mgl-l TDZ for shoot growth and proliferation, and the same media enriched by 1.0 mgl-l IBA for rooting of quince genotypes can be recommended.
Keywords: Quince, auxin, proliferation, rooting, cytokinin, meristem culture.
Introduction
The quince tree (Cydonia oblonga Mill.) is a pome fruit tree native to temperate regions. Despite all progresses in quince cultivation and growth, the large-scale commercial propagation of virus-free and healthy rootstocks of this tree has not been materialized yet (Abdollahi et al., 2005). Early investigations into the application of tissue culture for quince trees included those by Al Maarri et al. (1986). Al Maarri et al. (1986) focused on in vitro propagation of quince rootstocks, examining the effects of different BAP and NAA concentrations on micro-cutting proliferation and rooting.
Considering the quince tree as an indigenous plant in Iran and an invaluable genetic resources for the country, the production of virus-free and uniform seedlings from newly released cultivars is important in the nurseries and mother orchards. Furthermore, the development of biotechnology-based breeding programs for applied research can be accelerated through tissue culture methods.
Therefore, this study aimed to develop an optimized in vitro culture protocol for three selected quince genotypes from Isfahan region of Iran, with the ultimate goal of using this protocol for subsequent production of virus-free primary quince rootstocks.
Materials and Methods
The plant materials used in this study included two promising genotypes; KVD4, KM1, and cv. Behta. In vitro establishment of plant materials was achieved by shoot tip culture after surface sterilization. Meristems were transferred to square 25-well Petri dishes under a photoperiod of 16 hours light provided by white fluorescent lamps (Sylvania, Germany) at an intensity of 40 µmol m-2s-1 and a constant temperature of 23 ± 1°C.
Evaluation of cytokinin effects was performed by using three cytokinins, 2iP, BAP, and TDZ in separate experiment. The plant growth regulators 2iP and BAP were applied at the concentrations of 0.5, 1.0, and 2.0 mgl-l, while due strong effects of TDZ this cytokinin was used at 0.022 and 0.22 mgl-1 concentrations.
Similarly, the optimization of rooting conditions was established by two types of auxins, IBA and NAA, IBA+NAA in separate experiments. All rooting experiments were conducted using QL basal medium supplemented with 30 gl-1 sucrose as the carbon source. Five auxin concentrations, including 0.25, 0.5, 1.0, 2.0, and 4.0 mg-l were applied in two separate experiments for both IBA and NAA. In IBA+NAA treatments, three concentrations of IBA (0.5, 1, and 2 mg1-1) were applied along with a constant concentration of 0.5 mgl-1 NAA, and two concentrations of NAA (1.0 and 2.0 mgl-1) were used with a constant 0.5 mgl-1 IBA.
The effects of different levels of growth regulators × genotype interactions were assessed in a factorial arrangements in completely randomized design. Traits measured included the number of roots explant-1, root length, root number explant-1, and visual quality index of growth. The collected data were analyzed using SAS software, and mean comparisons were performed using Duncan’s Multiple Range Test.
Results and Discussion
The results indicated that the main effect of culture media was significant (p≤0.01) on the shoot number, leaflets number explant-1, and visual growth quality, but not on shoot length. Genotype had significant effect on all evaluated traits. Genotype × culture media interaction was not significant for the number of shootlets explant-1. Mean comparisons showed that use of QL basal medium led to better results in all traits on in vitro quince shootlets.
The QL medium not only exhibited a higher proliferation rate as 2.73 shoots explant-1, compared to 1.73 shoots explant-1 in the MS medium, but also enhanced the mean shoot length and of leaflet number explant-1. These results of the present study is in accordance with those reported by Abdollahi et al. (2005) on pear rootstocks. However, it contradicts the findings of Ciccotti et al. (2008), who recommended MS medium for apple proliferation.
Mean comparisons of different concentrations of 2iP showed that 1.0 mgl-1 was the most effective, and yielded significantly higher proliferation than 2.0 mgl-1. At 1.0 mgl-1 of 2iP, higher proliferation rates (2 shoots explant-1) was observed, which was significantly different from the other concentrations. This concentration also produced longer shootlets and higher leaflet number explant-1, as well as better overall shoot quality.
Although increasing 2iP concentration from 0.5 to 1.0 mgl-1 improved all traits, this improvement was remarkably more substantial than the differences between 1.0 and 2.0 mgl-1. Khodaee Chegenee et al. (2011) reported concentration of 0.5 mgl-1 2iP for pear rootstocks. The genotype effect was significant for all traits at the 1% probability level. Among the evaluated quince genotypes, the promising genotype KVD4 demonstrated superior results in comparison with others in in vitro conditions.
The rooting experiments showed that media supplemented with IBA were more effective than those with NAA or IBA+NAA. The effect of IBA concentration, genotype, and their interaction were all significant (p≤0.01) for all evaluated root traits. Genotypes responded differently to the various IBA concentrations. At the 1.0 mgl-1 IBA, the highest rooting rates were obtained for cv. Behta (87.50%) and promising genotype KVD4 (81.25%), while promising genotype KM1 showed the highest rooting rate (75%) at the 2.0 mgl-1 IBA. Similarly, the highest mean root number micro-cutting-1 was observed under the same conditions. Cv. Behta produced the longest roots (mean of 55.31 mm) at the 1.0 mgl-1 IBA. The best quality of micro-cuttings was recorded for the promising genotype KVD4 and cv. Behta at the 1.0 mgl-1 IBA and for promising genotype KM1 at the 2.0 mgl-1 IBA. These findings are in agreement with those of Khodaee Chegenee et al. (2011), who also reported the 1.0 mgl-1 IBA for pear cultivars.
References
Abdollahi H., Muleo, R. and Rugini, E. 2005. Study of basal growth media, growth regulators and pectin effects on micropropagation of pear (Pyrus communis L.) cultivars. Seed and Plant Journal, 21, pp.373-384 (in Persian).
Al Maarri, K., Arnaud, K. and Miginiac, E. 1986. In vitro micropropagation of quince (Cydonia oblonga). Scientia Horticulturae, 28, pp.315–321. DOI: 10.1016/0304-4238(86)90105-6
Ciccotti, A.M., Bisognin, C., Battocletti, I., Salvadori, A., Herdemertens, M. and Jarausch, W. 2008. Micropropagation of apple proliferation-resistant apomictic Malus sieboldii genotypes. Agronomy Research, 6, pp.445-458.
Khodaee Chegenee F., Abdollahi H., Ershadee A. and Esna Ashari M. 2011. Determination of micropropagation protocol for OH×F333 and OH×F69 pear clonal rootstock. Seed and Plant Journal, 27-2, pp.297-312 (in Persian). DOI: 10.22092/sppj.2017.110439
Keywords
)