Breeding and Biotechnology of Plant and Flower
Gholamhossein Davarynejad; Sajedeh Karimpour
Abstract
Introduction: Pyrus communis L. cv. Natanz is a popular pear cultivar in Iran because of its customer-friendly attribute due to its excellent characteristics. Pear own-rooted plants has better traits such as high vigorous in growth, low levels on tree losses and damaging by insects rather than grafted ...
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Introduction: Pyrus communis L. cv. Natanz is a popular pear cultivar in Iran because of its customer-friendly attribute due to its excellent characteristics. Pear own-rooted plants has better traits such as high vigorous in growth, low levels on tree losses and damaging by insects rather than grafted plants. Meristem culture widely used for micropropagation, in vitro germplasm preservation, and virus eradication purposes in pear. As pear is belonged to difficult-to-root fruit tree cultivars perhaps the rooting stage is the most important stage in propagation process, yet most difficult phase during the in vitro propagation procedure. In vitro rooting of micro-cuts was varied by genotypes (cultivars), type and concentration of auxin, the method of root induction and formation, different additional materials such as PVP, polyamines, and so on. This study was aimed to investigate the effect of different levels of BAP and Fe-EDDHA on shoot proliferation, BAP and GA3 on meristem establishment, and IBA and NAA on micro-cut rooting of pear cv. Natanz in in vitro condition.
Materials and Methods: Vegetative buds were taken from current growth shoots of Pyrus communis cv. Natanz from Pear collection orchard (25.36 E, 58.54 N, and ASL altitude 1380 m) of Agricultural and Natural Resources Research and Education Centre of Semnan Province (Shahrood city). In the first experiment, new shoots of active buds after 4 weeks grown in PMI medium (MS ×1.5 CaCl2. 2H2O, KH2PO4 and MgSO4. 7H2O) + 1 mg.l-1 BAP were transferred to PMI medium containing different levels of BAP (0.5, 1, 1.5 mg.l-1) and Fe-EDDHA (0, 100, 150 and 200 mg.l-1). In the second experiment, meristems (containing two newest leaf primordia) was excited from in vitro shoots and incubation on MS media containing BAP (0.5, 1, and 1.5 mg.l-1) and GA3 (0.1 and 0.5 mg.l-1) + 0.1 mg.l-1 IBA. Meristems were kept in dark for 4 days then were transferred to growth chamber with photoperiod 16/8 hrs. light/dark. Different concentrations and combinations of two auxins were used for root induction of micro-cuts in third experiment. 1000, 2000, 3000, and 4000 mg.l-1 of IBA or NAA and two combination solutions of them (1000 IBA+1000 NAA, and 2000 IBA+2000 NAA, mg.l-1). Shoots were immersing dip in solutions for 5 seconds then transfer to PGRs-free PMI medium and kept them to growth chamber. Data of all experiments were analyzed according by completely randomized design (CRD) with five replications. BAP (3 levels) and Fe-EDDHA (4 levels) for experiment 1; BAP (3 levels) and GA3 (2 levels) for experiment 2 were considered as factorial. SAS (v. 9.1) was used for analysis and means were compared with LSD test at 5% of probability level.
Results and Discussion: Proliferated shoot number was affected by BAP (p≤0.01) and Fe-EDDHA (p≤0.05) concentrations and also interaction of them (p≤0.05), while BAP (p≤0.01) was caused elongation of proliferated shoots and Fe-EDDHA had no effect. BAP (p≤0.05), Fe-EDDHA (p≤0.01) concentrations and BAP×Fe-EDDHA (p≤0.01) interaction had significant effect on leaf production. Shoot tip necrosis was shown in shoots grown in all media based on BAP concentration with different intensities (p≤0.05). Vegetative growth was counted as a power index of medium that in our experiment was under influence of BAP concentrations (p≤0.01), Fe-EDDHA (p≤0.05) and BAP×Fe-EDDHA interaction (p≤0.05). Shoots were proliferated (5.50 shoot.explant-1) and elongated in PMI medium containing 1.5 mg.l-1 BAP with no Fe-NaEDDHA while the lower concentrations of both BAP and Fe-NaEDDHA caused the higher mature leaf production. PMI media containing 1 mg.l-1 BAP + 150 mg.l-1 Fe-NaEDDHA is recommended for Natanz shoot proliferation because of the highest vegetative growth and highest quality in proliferated shoots. MS medium with 0.5 mg.l-1 BAP+ 0.5 mg.l-1 GA3 (81%) and 1 mg.l-1 BAP + 0.1 mg.l-1 GA3 (63%) had the highest meristem establishment, respectively. The established meristems naturally grown in medium supplement with 0.5 mg.l-1 BAP + 0.5 mg.l-1 GA3+0.1 mg.l-1 IBA. Different types of auxin and their concentrations had significantly effect on Natanz pear cultivar micro-cut rooting (p≤0.05). NAA induced rooting in lower concentrations while IBA had positive effect on rooting with concentration increasing. Micro-cuts were rooted via quick dip in 1000+1000 mg.l-1 (IBA+NAA) solution followed by incubation in PMI medium. The rooted shoots well adapted to environmental condition.
Conclusion: Important steps of in vitro propagation of pear is optimized in this experiment. MS medium containing 0.5 mg.l-1 BAP+0.5 mg.l-1 GA3+0.1 mg.l-1 IBA had suitable for meristem establishment. To produce in vitro healthy proliferated shoots of pear cv. Natanz using PMI medium supplement with 1 mg.l-1 BAP+150 mg.l-1 Fe-NaEDDHA is recommended. Micro-cuts were rooted easily by quick immersion of the end of micro-cuts in 1000+1000 mg.l-1 (IBA+NAA) solution for 5 seconds then incubation in PGRs-free medium.
Sajedeh Karimpour; Gholamhossein Davarynejad; Mohammad ZakiAghl; Mohammad Reza Safarnejad
Abstract
Introduction: Micropropagation is important for both multiplication and preservation of a wide range of nursery plants, including many fruit crops. A number of studies exist on optimization of growth in in vitro condition for one or two cultivars, but often these results cannot be used for the other ...
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Introduction: Micropropagation is important for both multiplication and preservation of a wide range of nursery plants, including many fruit crops. A number of studies exist on optimization of growth in in vitro condition for one or two cultivars, but often these results cannot be used for the other genotypes because individual cultivars may differ greatly in their requirements. Therefore, genotype-specific medium are usually empirically developed for many plants including pear. Pear cultivars and species are often recalcitrant to tissue culture manipulations and Murashige and Skoog (23) (MS) basal nutrient medium at full or half strength or with slight modifications is the most media were used. The QL, DKW, and WPM media are also used and they differ mostly in types or amounts of calcium and nitrogen in compared with MS. Developing growth media for specific and unique cultivars is complex and time-consuming. Currently, improved experimental design and using statistical softwares allow much more efficient approaches to be utilized for the improvement of micropropagation media and conditions. Improving of growth medium for in vitro propagation of plants depends on type and quantities of mineral nutrients and plant growth regulators as important ones. The existence of statistical softwares to manage effective factors is very needed to access an optimized growth medium for in vitro propagation of plants. Design Expert is used as auxiliary software to identify essential factors in in vitro culture. Since the in vitro proliferation parameters of Pyrus communis cv. ʽShekariʼ need to optimize for growth better, we were designed and performed a multifactor surface response experiment by Design Expert software to following two goals. First, to find optimized amount of some elements in medium and second, to show the response surface method can be useful for improving in vitro culture.
Materials and Methods: One experiment was designed by Design Expert software and was performed to improve in vitro proliferated shoots of Pyrus communis cv. ʽShekariʼ. Shoots were grown in a modified MS medium (supplement with 1 mg l-1 of N6-benzyladenine) were used for this experiment. The experiment was included 20 model points randomly based on three nutritional factors: NH4NO3 (0.5-1.5×), Fe (0.5-1.5×) and micro nutrients (1-2×) in different concentration of their MS amounts. Media enriched with sucrose (30 g l-1) and agar (8 g l-1) after pH adjustment at 5.7. Cultures were grown at 25°C under a 16-h photoperiod with 70–90 μM m-2 s-1 irradiance provided by a combination of cool- and warm-white fluorescent bulbs and were transferred to new medium every 3 weeks. Several responses were recorded after two months: Proliferated shoot number, proliferated shoot length (cm), total leaf number, leaf chlorophyll a (mg g-1), leaf carotenoids (mg g-1), and vegetative growth (cm). Responses for each point were the mean of 5 replicates. Experimental design, model evaluation, and analysis were done by Design-Expert® 8 (2010) software and the highest-order polynomial model that was significant for each response was used for ANOVA.
Results and Discussion: Factors statistically were significant for responses according to ANOVA in linear, 2FI and quadratic models. Reduced NH4NO3 (×0.5) and enhanced Fe (×1.5) induced the higher number of proliferated shoots up to 4.43 folds of control according a quadratic model. NH4NO3 and Fe×Micro had negative liner relationships with shoot length, while leaf number negatively was affected by micros. Fe and NH4NO3 were effective factors on leaf chlorophyll a and carotenoids contents. Increasing Fe (×1.5) and decreasing NH4NO3 (×0.5) led to 2 folds higher production of chlorophyll a and carotenoids. Vegetative growth of Pyrus communis cv. ʽShekariʼ in a quadratic-order method (negatively controlled by NH4NO3 and micros) increased by high values of proliferated shoot number and shoot length induced by reduced NH4NO3 (×0.5). Optimized amount of three studied factors based on two important responses, maximum amount of proliferated shoot number and length, were 0.9, 1 and 0.5× for Fe, micro and NH4NO3 in MS medium, respectively.
Conclusions: Design Expert software and response surface method were used successfully for in vitro optimizing of Pyrus communis cv. ʽShekariʼ regenerated shoots. Fe, Micro and NH4NO3, were the effective factors for shoot regeneration responses in linear, 2FI and quadratic models. The multifactor investigation in surface response design will enable us to predict an optimal medium for several effective factors and estimate suitable responses. Outputs of these types of experiments provide a suitable background to increase optimization accuracy for future experiments.
Hajar Neamati Darbandi; Majid Azizi; Samane Mohammadi; Sajede Karimpour
Abstract
To investigate the effect of spraying with different concentrations of Vermiwash solution on the morphological traits, percentage and yield of essential oil of lemon balm (Melissa officinalis L.), this experiment was conducted as Randomized Complete Block Design (RCBD) with four replications in experimental ...
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To investigate the effect of spraying with different concentrations of Vermiwash solution on the morphological traits, percentage and yield of essential oil of lemon balm (Melissa officinalis L.), this experiment was conducted as Randomized Complete Block Design (RCBD) with four replications in experimental field of Agricultural Faculty of Ferdowsi University of Mashhad (FUM). The treatments were included 4 different concentrations of Vermiwash solution (0, 3000. 6000 and 12000 ppm). At the flowering stage, morphological characteristics include of plant height, number of nod and internodes length, as well as total fresh weight, leaf area, dry weight of plant, yield and percentage of essential oil were determined. Results showed that there was a significant difference between different treatments in terms of plant height, number of node, internodes length, leaf area, and dry weight of plant and essential oil yield. The highest height (84.91 cm), number of node (23.20), leaf area (8853.52 cm2) and plant dry weight (174.31 g) related to vermiwash concentration of 3000 ppm and the longest internodes (7.02 cm) and essential oil yield (6 g/m2) related to concentration of 1200 ppm. In conclusion, application of 3000 ppm vermiwash was the superior among the all studied treatments that confirmed activity of plant growth regulators in low concentration of vermiwash and availability of nutrient in the solution.
