Breeding and Biotechnology of Plant and Flower
Davood Hashemabadi; Behzad Kaviani; Kobra Shakeri Kiasaraei; Rasoul Onsinejad; Mohammad Reza Safari Motlagh
Abstract
Introduction Tulip flower (Tulipa L.) from the family Liliaceae is a bulbous and monocotyledon plant that has the highest level under cultivation among this family group. Tulips can be propagated by seeds and bulbs. Its seeds produce bulbs up to two years after planting and it takes ...
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Introduction Tulip flower (Tulipa L.) from the family Liliaceae is a bulbous and monocotyledon plant that has the highest level under cultivation among this family group. Tulips can be propagated by seeds and bulbs. Its seeds produce bulbs up to two years after planting and it takes six years for the bulbs to reach the flowering stage. In fields, the quality characteristics of flowers can be changed to some extent by changing some planting characteristics such as planting pattern and plant density. Some researchers have reported changes in the quantitative and qualitative characteristics of various crops and orchards, including ornamental plants, with changes in planting pattern and plant density. One effect of changes in planting patterns and plant density is alterations in photosynthesis and plant growth regulators. The purpose of this study was to identify the best planting pattern and determine the optimal planting density, as well as to examine the impact of these factors on the quantitative and qualitative characteristics of tulip (Tulipa L.) cv. 'Spryng'. Materials and MethodsTo evaluate the effect of planting pattern and density on growth and flower characteristics of tulip cv. ʹSpryingʹ, present study study was conducted as a factorial experiment based on completely randomized block design (RCBD) with 3 replications in 27 plots. The first factor was three planting patterns (square, triangle and rectangle) and three planting densities (25, 45 and 65 plant/m2) as the second factor. Morphological and physiological traits such as height, length and diameter of stem, leaf and flower, flowering time, cut flower number, flower longevity, number, diameter and weight of bulb and bulblet, and the content of chlorophyll and carotenoid were measured. Statistical analysis of data was performed with SAS 9 software and mean comparison of the data with LSD test at 5% probability level. Graphs were drawn in Excel. Results and DiscussionResults showed that the maximum number of cut flowers (59.90) was counted in plants cultivated in triangle cultivation design with planting density of 65 plants/m2. The lowest time to start of flowering (69.30 days) and the highest content of leaf chlorophyll (13.57 μg/ml) was obtained in plants cultivated in trianle cultivation design with planting density of 45 plants/m2. The most flower longevity (12.73 day) and the highest content of carotenoid (1.68 μg/ml) was obtained in plants cultivated in square cultivation design with planting density of 45 plants/m2. The height of the flowering stem is one of the important traits for the marketing of cut flowers. The results of the present study showed that the height of the tulip plant was affected by plant density and planting pattern. This result was consistent with the results reported by some researchers. At low plant densities, long plant spacing reduces plant competition for water and nutrient uptake, resulting in larger plant growth and leaf size. Also, long plant distances cause the roots to develop and grow, and the leaves to grow and thicken. Increasing the vegetative competition of adjacent plants at high densities causes photosynthetic organs to be placed in the shade (change in the quantity and composition of the received radiation spectrum in the shade leaves), which has a great effect on the balance of plant growth regulators, resulted in longitudinal and superficial growth of plant organs. It intensifies the longitudinal growth of the petiole and accelerates all the developmental processes of the plant. Plant morphology and angle of leaf deviation can also be effective in increasing leaf size. Uniform distribution of plants and greater absorption of light and nutrients increased leaf length and width. The results revealed that plants compete for light and nutrients, and in these competitive conditions, roots and stems are taller than optimal, and the distance between nodes increases. The effect of planting pattern on flowering process can be related to changes in plant photosynthesis and the availability of photosynthetic materials for the developing reproductive parts. Changes in planting distance or pattern can alter inflorescence characteristics by affecting root growth and modifying the production of plant growth regulators in the roots. These regulators are then transferred to the aerial parts, influencing inflorescence characteristics. Adjusting the planting distance or pattern can also impact various traits of bulbs and bulblets in bulbous plants. Competition for receiving maximum light and photosynthesis is a key factor in changing bulb and bulblet traits. This competition is influenced by planting arrangement and plant density. Some studies have shown that planting pattern and plant density affect the amount of plant pigments such as chlorophyll and carotenoids, the main reason being the difference in light intake.
Breeding and Biotechnology of Plant and Flower
Leila Baghazadeh Daryaii; Davood Samsampoor; Abdoolnabi Bagheri; Jelveh Sohrabipour
Abstract
Introduction
Endophytes have symbiosis life within the plant tissues without causing any obvious negative effects. Seaweeds are one of the large and diverse groups of marine plants that play an essential role in marine and oceans ecosystems. Seaweeds show rich diversity of associated microorganisms ...
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Introduction
Endophytes have symbiosis life within the plant tissues without causing any obvious negative effects. Seaweeds are one of the large and diverse groups of marine plants that play an essential role in marine and oceans ecosystems. Seaweeds show rich diversity of associated microorganisms in compare with the other multicellular organisms. Citrus species, are amongst the most important evergreen fruit trees, cultivated in many countries worldwide. There are several obstacles for citrus production in southern of Iran, limiting continuity of citrus production. Lack of suitable soil, salinity and drought stresses are the main challenges threatening citrus industry in southern of Iran. Similar to other citrus species, the production of Mexican lime is threatened by certain biological stresses (such as pests, plant diseases and weeds) and non-biological stresses (such as salinity, drought, floods, cold and heat stress). Endophytes are advantageous group of microorganisms that protect plants from biotic and abiotic stresses. One of the alternative ways to restore normal plant growth may be to use plant growth to stimulate endophytes. Endophytes can play an important role in plant growth. Endophytes from marine environment are gaining special interest because of their existence in the harsh conditions of marines and ocean ecosystem such as temperature, light availability, high salinity and osmotic stress. Endophytes have already been isolated from various marine habitat, including marine plants, marine invertebrates and vertebrates. Among these organisms, seaweeds are one of the most prevalent sources of marine-derived fungi and bacteria for chemical studies. The purpose of this study was the isolation of associated fungi and bacteria endophytes with seaweed species in Persian Gulf to investigate morphological and molecular characterization by using PCR amplifications ITS1-5.8S-ITS4 regions and 16s rRNA gene respectively. Here, we have evaluated the potential of inoculating Mexican lime seedlings with seaweeds fungi and bacteria endophyte combination, (Aspergillus niger+ Bacillus aquimaris OD14), to improve morphological, biochemical, antioxidant and photosynthesis pigments characterizes of Mexican lime in salinity condition.
Materials and Methods
The main aim of this study was to investigate the role of endophytic fungi (Aspergillus niger) and bacteria (Bacillus aquimaris OD14) in improving the growth of Mexican lime seedlings. The seaweed samples were collected from coastal regions of Bushehr province and Qeshm Island. Fungi and bacteria endophytes were isolated and identified base on morphological and molecular methods. Molecular characterization was investigated using PCR amplification of ITS1-5.8S-ITS4 regions and 16s rRNA gene respectively. Mexican lime seeds were sterilized with 0.5% sodium hypochlorite for 20 minutes and then completely distilled three times with distilled water. Seedlings pots containing autoclaved soil were placed in the greenhouse of the Faculty of Agriculture, Hormozgan University. The experiment was arranged as a factorial experiment based on randomized complete randomize design with three replications. Isolated fungi and bacteria by MT420720 and MT278260 accession numbers were used in eight months old Mexican lime seedlings. The suspension was adjusted to a concentration of 1×106 cell per ml for fungi and 1×108 cell per ml for bacteria inoculums. For better contact of seedlings with endophytes, inoculation was performed three times. After three months, salinity stress was applied. morphological (Leave, Stem and Root dry and fresh weight), biochemical (Protein, MDA and soluble sugars), antioxidant capacity (CAT, POD, SOD, APX and Gr activity) and photosynthesis pigments (Chlorophyll a, Chlorophyll b, Total Chlorophyll and Carotenoids) characteristics in treated Mexican lime seedlings and control were analyzed. Analysis of variance of traits was performed using SAS software version 9.4 and the means were compared using LSD method with a probability level of P≤0.05.
Results and Discussion
The results show that most characterizes were significant compare with control. For example, in 6000 µs/cm water salinity, leave fresh weight (203.49%), root fresh weight (347.41%), stem fresh weight (206.81%) and root dry weight (421.95%) were significantly higher compared with control (P>0.001). Endophytes inoculation can significantly improve photosynthesis pigments such as chlorophyll a (65.21%), chlorophyll b (11.9%), total chlorophyll (28.39%) and carotenoids (59.09%) (P>0.001) compare with control. In antioxidant capacity of seedling, CAT, POD, SOD, Gr and APX were analyzed, Endophytes can increase enzymes activity. For biochemical characterizes, in 6000 µs/cm water salinity, endophytes can significantly increase soluble sugars (17.85%) and decrease MDA (35.18%) in inoculated seedlings compare with control (P>0.001).
Conclusion
The results showed that the use of endophytic fungi and bacteria can increase the growth of Mexican lime seedlings under salinity stress. Thereby it can be used as an effective tool for growing salinity-sensitive plants in saline conditions.
Breeding and Biotechnology of Plant and Flower
Marzieh Ghorbani; Khosro Parvizi; Mohammad Yazdandoost Hamedani; Darab Hassani
Abstract
Introduction
In our country, walnut tree propagation is traditionally done through seed cultivation, often resulting in seed rot and death due to fungal, bacterial, and viral contamination (MC Granahan et al., 1986; Driver & Kenyuki, 1984; Saadat & Henry, 2002). The traditional method, in addition ...
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Introduction
In our country, walnut tree propagation is traditionally done through seed cultivation, often resulting in seed rot and death due to fungal, bacterial, and viral contamination (MC Granahan et al., 1986; Driver & Kenyuki, 1984; Saadat & Henry, 2002). The traditional method, in addition to low multiplication rates, leads to high variation in resulting seedlings, potential loss of seedlings due to contamination, and reduced efficiency in subsequent stages (Unit, 2012; Kaur et al., 2006). Previous research has mainly utilized concentrations of one milligram per liter of benzyl adenine along with small amounts of indole butyric acid for Iranian walnut growth and enrichment (Rodrigues, 1982; Revilla et al., 1989; Penuela, 1988; Mejzadeh et al., 2010, 1997; Amiri & Qaraati, 2012; Riosleal et al., 2012). This research aims to build upon and optimize previous work, evaluating the effectiveness of different concentrations of two growth regulators, benzyl aminopurine and adenine sulfate, on walnut plantlet regeneration and growth traits in tissue culture.
Materials and Methods
This study was conducted to optimize the tissue culture protocol for the "Chandler" cultivar walnut and determine the most suitable culture medium and hormonal composition for micropropagation. Lateral and terminal buds from the current season's branches were sterilized and cultured in DKW medium containing 2 mg/liter of benzyl adenine hormone and 100 mg/liter of indole butyric acid hormone, with polyvinyl pyrrolidine at one g/liter and activated charcoal at 2 g. Two-factorial experiments were used to process and multiply the plant after the establishment phase. The first factor was DKW culture medium containing five levels of adenine sulfate (0, 20, 40, 60, and 80 mg/liter), and the second factor was benzylaminopurine plant growth regulator with five hormonal levels containing 0, 0.5, 1, 1.5, and 2 mg/liter in combination with 0.01 mg/liter of indole butyric acid hormone. DKW base culture medium without any plant growth regulating substances was considered as control. After two months, growth traits including plantlet weight, stem length, number of leaves, number of buds, and number of leaflets per plantlet were measured in different culture media. The resulting data were statistically analyzed using SAS 9.1 software, and means were compared using Duncan's multiple range test with a five percent probability level.
Results and Discussion
The analysis of variance showed that both plant growth regulators, benzyl aminopurine and adenine sulfate, had a very significant effect at 1% probability level on plantlet weight, stem length, number of leaves, number of buds, and number of leaflets. The interaction effect of benzyl aminopurine with adenine sulfate treatment on plantlet weight and stem length was significant at the 1% probability level. However, the interaction effect of benzyl aminopurine with adenine sulfate treatment on the number of leaves, number of buds, and number of leaflets was not significant. The results indicated that an increase in the levels of growth regulators benzyl aminopurine and adenine sulfate led to an increase in plantlet weight. The positive effects of increasing the levels of growth regulating substances in increasing plantlet weight are likely due to their direct effect on nutrient absorption, utilization, and the photosynthesis process. These results align with the research of Hatemzadeh et al. (2017) and Saadat and Henrati (2002). The positive effects of higher concentrations of both growth regulators on the increase in the number of sprouts and the lack of significant difference between the two high concentrations confirm that the use of high levels does not exceed the economic threshold. It can be justified that in excessive and unconventional concentrations, positive effectiveness is not achieved, but it can also impose more costs on the walnut tissue culture program. The appropriate concentration of BAP and adenine sulfate increases the leaf surface through the effect on cell divisions, resulting in receiving more light radiation and increasing the rate of photosynthesis. It seems that the two growth regulating substances in the appropriate concentration intensified each other's effect, affecting the rate of absorption and utilization of materials from photosynthesis, leading to an increase in the fresh and dry weight of the seedling. This, in turn, leads to a decrease in the length of the reproduction period in the resulting seedlings and an increase in the efficiency of the seedling production in walnut tissue culture.
Conclusion
The use of both studied growth regulators significantly increased plantlet weight, stem length, number of leaves, number of buds, and number of leaflets compared to the control treatment. Plantlet growth was achieved with the use of plant growth regulators, whereas no growth was observed in their absence. All assessed traits increased significantly with the addition of plant growth regulators, with the highest trait values obtained through the simultaneous use of benzylaminopurine and adenine sulfate.
Breeding and Biotechnology of Plant and Flower
Farhad Shokouhifar; Mojtaba Mamarabadi; Sahba Toosi
Abstract
Introduction
Melon (Cucumis melo L.) is a diploid plant with (2n = 2x = 24) chromosomes, dicotyledonous and annual, which has been receiving lots of attention for its biological characteristics and economic value for a long time. Iran with production of about 1.6 million tons per year ranks third in ...
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Introduction
Melon (Cucumis melo L.) is a diploid plant with (2n = 2x = 24) chromosomes, dicotyledonous and annual, which has been receiving lots of attention for its biological characteristics and economic value for a long time. Iran with production of about 1.6 million tons per year ranks third in melon production in the world after China and Turkey. Vascular wilt caused by the soil borne fungus Fusarium oxysporum f. sp. melonis is one of the most important diseases causing damage to the melon plant. Due to the survival of this fungus in the form of chlamydospores in the soil and plant debris its control has been a difficult challenge so that, the only way to deal with this disease is to use resistant cultivars. The present study was conducted to characterize morphologically different melon cultivars with varying levels of resistance against Fusarium vascular wilt. Furthermore, the presence pattern of two MRGH genes belonging to the MRGH21 linkage group was tracked in the genome of the melon line, and their variations were defined. Moreover, the potential for using these genes in gene-assisted selection was investigated.
Materials and Methods
Five different varieties of melon named Charentais T, Charentais Fom1, Charentais Fom2, BG-5384 and the local cultivar Khatouni were grown under greenhouse conditions. Different characteristics of the plant, including leaf shape, male flowers, female flowers and normal flowers, and after harvesting the fruits, fruit weight, fruit diameter and length, diameter of flesh and middle cavity. The differentiation of resistant and sensitive cultivars was investigated based on the evaluated morphological traits. Additionally, the presence patterns of resistance genes were examined in the genomic data of the aforementioned melon cultivars. The genomic analysis of melons aimed to locate the MRGH21 linkage group, which carries several resistance genes. The sequence of this linkage group was tracked from two gene bank databases in NCBI and MELONOMICS database.
Results
Based on the obtained results, although it was possible to differentiate melon cultivars based on morphological traits, but since the study of these traits in the evaluation of a large number of samples in selective studies is a very time-consuming and costly task. Therefore, the presence pattern of resistance genes were analyzed in the genomic data of different melon cultivars. The sequence between two genes MRGH12 and MRGH13 including MRGH21 linkage group as one of the linkage groups carrying a number of resistance genes on Ch09 chromosome was retrieved form two gene bank databases in NCBI and MELONOMICS. Due to the presence of multiple point mutations in the genomic data, the MRGH13 gene sequence was selected for investigation in melon cultivars. Specific primers, PSh21-F/R, were designed to track part of this gene's sequence. The tracking results showed that a single specific band, corresponding to the expected size, could be detected in the cultivars Charentais Fom1 and BG-5384. Sequence analysis using the InterPro network tool confirmed the possible role of the protein coded by the MRGH13 gene. It was identified as a member of the protein family carrying leucine-rich repeat sequences, including the TIR, NB-ARC, and LRR domains.
Discussion
Attaining suitable markers to distinguish melon cultivars resistant to Fusarium wilt disease can support the development of breeding programs with higher accuracy and speed. The results of the present study showed that based on the morphological traits such as leaf shape, the presence of full flowers, and the number of petals, some differences can be observed between different melon cultivars, but the noteworthy point is that in selection programs searching for these morphological traits will be a very time-consuming and expensive task due to the large number of investigated samples. Therefore, if molecular markers related to the resistance trait are available, the efficiency of breeding programs is expected to increase significantly. In the present study MRGH13 gene was selected to be investigated for tracking in melon cultivars and specific primers were designed to track part of the sequence of this gene. The tracking results showed that a single specific band could be detected in the cultivars Charentais Fom1 and BG-5384. Biological processes related to MRGH13 protein in the QuickGO network tool showed its relevance in the signaling pathway that regulates immune responses. In future studies, it is suggested to evaluate the ability to distinguish resistant cultivars based on resistance genes, including the MRGH13 gene, in a larger number of samples. Moreover, considering to the predicted functions of MRGH13 protein, more investigation on its interaction with other resistance proteins as well as proteins of pathogenic agents can be useful for identification of its functional role in resistance.
Breeding and Biotechnology of Plant and Flower
Letafat Moradi; Seyyed Najmodin Mortazavi; Mitra Alaei
Abstract
IntroductionSuccess in tissue culture technique, especially in bulbous plants, depends on the microbial contamination control during in vitro culture. Decontamination is considered as a fundamental challenge in the technique of cell, tissue and plant organ culture. Although there are various methods ...
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IntroductionSuccess in tissue culture technique, especially in bulbous plants, depends on the microbial contamination control during in vitro culture. Decontamination is considered as a fundamental challenge in the technique of cell, tissue and plant organ culture. Although there are various methods for this purpose, the development of disinfection methods specific to each species is considered an important factor in the establishment and success of the tissue culture system. Applying different treatments can control the microbial contamination and consequently increase the percentage of explant survival. This plant is among the poisonous plants of pastures and the chemical compounds present in it have important medicinal effects, so that the existence of alkaloid and glycosidic compounds has been reported for it. These chemical compounds are used to treat rheumatic pains, lymphatic system diseases and also as liver cleansers. This ornamental plant also has the ability to produce pots and its export is possible in pots . Currently, in order to overcome the issues of the usual methods of propagation of this ornamental plant and to produce pollution-free plants, the use of in vitro cultivation methods in this plant has become very important. bulbes is one of the most common explants of inverted tulip tissue culture, but it often faces very high internal and external fungal and bacterial contamination. In the in vitro culture of onion plants, the existence of fungal and bacterial contaminations are one of the main problems that can affect the efficiency of this type of propagation methods. Fungal and bacterial contaminations are one of the most important problematic factors in different in vitro culture methods. Internal contamination usually appears several weeks after planting. These contaminations may not be seen externally, but they affect the growth, division and greening of small samples. Mercuric chloride is mainly used as a surface disinfectant along with sodium hypochlorite and it controls the spectrum of bacteria and fungi and increases the disinfection efficiency. The results of the study showed that mercuric chloride relatively controls the bacterial and fungal contamination of the samples, but it reduces the survival percentage of the samples in high concentration. It seems that some plants are sensitive to mercuric chloride and determining the appropriate amount of this substance plays a very important role in preparing a healthy and living specimen. Determining the duration of disinfectant treatment was also considered in various researches, so that sometimes carbendazim 1% fungicide for 20 minutes, 70% alcohol for 60 seconds, sodium hypochlorite 2.5% for 10 minutes. And 0.1% mercury chloride was used for 7 minutes to sterilize aloe vera sample. Considering the importance of plant tissue culture techniques and the need to create new protocols to reduce contamination and increase the number of healthy cultures, the main goal of this study is to create an efficient protocol for the disinfection of Fritillaria spp. onion explants in order to increase the efficiency of in vitro cultures. Materials and Methods This study aimed to investigate the effect of time and different concentrations of sodium hypochlorite, carbendazim fungicide and mercuric chloride in inhibiting the contamination of inverted tulip bulbs (Fritillaria spp.) in tissue culture environment. So, an experiment was done as a completely randomized design at four replications in the biotechnology laboratory of Zanjan University during 2023. The experimental treatments consisted of 0.1% fungicide at different times (30, 25, 35 and 40 minutes), 5 levels of sodium hypochlorite (0,1, 1.5, 2, 2.5 and 3%) at different times (7, 9, 10, 12 and 15 minutes), 70% Ethanol at two different times (0,60 and 90 seconds) and mercury chloride (0,0.1 and 0.2%). Bulbs that collected from nature were transferred to the Biotechnology Laboratory of the Faculty of Agriculture, Department of Horticulture, Zanjan University and kept them in the refrigerator at 4°C for two weeks. After this period of time, the bulbs were washed with detergent and then remained in water for 30 minutes. Then they were disinfected by using the above-mentioned treatments: It should be noted that at the end of each step, Fritillaria bulbs were washed with sterile distilled water. Also, after the end of the disinfection treatments, the desired explants were cultured in MS basic culture medium. The statistical analysis of this experiment was carried out using SAS software, version 9.1. The differences between mean values were compared using Duncan’s multiple range test method at the 5% significant level (p < 0.05). Results and Discussion The control of fungal and bacterial contamination, especially in relation to bulbous and bulbous plants that are in direct contact with the culture medium, is one of the basic steps in in vitro culture conditions. Applying different treatments including the use of fungicides, alcohol, sodium hypochlorite and mercuric chloride in the cultivation environment can help control fungal and bacterial contamination in the Fritillaria spp. plant. However, the application of such treatments can have a negative effect on the regeneration potential of the explants, so it is important to choose the appropriate treatment in a way that controls the fungal and bacterial contamination and on the other hand preserves the regeneration potential of the explants. This study investigated the effectiveness of various disinfection protocols on Laleh-Avgagun onion explants. While the lowest contamination (0%) was achieved using 0.1% fungicide for 40 minutes, 70% alcohol for 90 seconds, 3% sodium hypochlorite for 15 minutes, and 0.1% mercury chloride, this treatment significantly reduced explant survival and regeneration, causing browning. Conversely, applying 0.1% fungicide for 30 minutes, 70% alcohol for 60 seconds, 2% or 2.5% sodium hypochlorite for 12 or 10 minutes respectively, and 0.1% mercury chloride effectively reduced contamination while maintaining explant survival. This optimized protocol improved the growth and regeneration ability of the explants.Therefore, in the conditions of in vitro cultivation, the use of the mentioned treatments is recommended to control contamination and optimal reproduction of the bulbs of Fritillaria spp. plant.
Breeding and Biotechnology of Plant and Flower
Reza Shafiee; Mohammad Reza Abdollahi; Asghar Mirzaie Asl; Seyyed Saeid Moosavi; Hassan Sarikhani
Abstract
Introduction
Given the economic importance of growing flowers and plants in the world, the use of new technologies and methods in the improvement of ornamental plants in order to market them can play a significant role in marketing of these products and their trade in the international markets. Marigold ...
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Introduction
Given the economic importance of growing flowers and plants in the world, the use of new technologies and methods in the improvement of ornamental plants in order to market them can play a significant role in marketing of these products and their trade in the international markets. Marigold (Tagetes spp.), is an annual and essential plant that is cultivated as an ornamental plant in many parts of the world (Neher, 1968). But in recent years, marigolds have been used as a commercial source of essential oils, biological compounds, pigments and cosmetics (Anonymous, 1976), control of malaria mosquitoes (Wanzala and Ogoma, 2013), antihypertensive, anti-inflammatory, antidepressant, antimicrobial, antimicrobial (Senatore and Feo, 1999), and control of nematode (Prasad et al., 1992). Anther or microspore culture is known to be an effective method for producing haploid plants (Henry and De Baizer, 1980). Hybrid seed production requires pure line (as a parent), and the double haploid method can reduce the production period of pure lines to 5-6 years. Production of hybrid seeds in this valuable plant is of great importance, and the double haploid method can be important in this regard.
Materials and Methods
In the anther culture of marigold, a culture medium containing 0.2 mg/l of Naphthalene acetic acid and 1 mg /l of 6-Benzylaminopurine was used. In this study, the effect of genotype, bud size and mannitol on androgenesis induction of marigold anthers was evaluated during two separate experiments. A factorial experiment was conducted in a completely randomized design with two factors including genotype at 5 levels (T1, T2, T3, T4, T5) and bud size at three levels (3-5, 5-10 and 10-15 mm) for first experiment. The second experiment was also performed as a factorial in a completely randomized design with 3 replications. In the recent experiment, the first factor included the method of mannitol application and the second factor included the different concentrations of mannitol (0.1, 0.2, 0.3, 0.4 and 0.5 M). The factor of application method was in 2 levels: 1: adding different concentrations of mannitol to the solid culture medium and 2: pre-treating the anthers with the liquid culture medium containing different concentrations of mannitol for 24 hours. The T4 genotype was used in the second experiment.
Results
In the first experiment, the effect of different bud sizes and 5 different genotypes on callus formation, mean number of shoot per anther, mean number of shoot per callus and percentage of complete plant regeneration in anther culture of marigold were studied. The results of this experiment showed that buds with the length of 5-10 mm have anthers which their microspores are at the proper growth and development stage for callogenesis and shoot production. The T3 and T4 genotypes, (both of them belonging to French species, Tagetes patula), produced the highest percentage of plant regeneration among the various cultivars. In the second experiment, we explored the impact of mannitol treatment on androgenic traits in marigold anther culture. Specifically, we examined two concentrations: 0.1 M and 0.2 M mannitol, both applied in the form of solid culture medium. Additionally, we investigated two concentrations, 0.0 M and 0.2 M mannitol, when applied as a pre-treatment in a liquid medium containing mannitol. These treatments yielded the highest percentage of callus formation. While the pre-treatment of anthers with a liquid culture medium containing 0.5 M mannitol led to the highest mean number of shoot per anther and the mean number of shoots per callus. Also, the pre-treatment with liquid medium containing 0.2 M mannitol showed the highest percentage of complete plant regeneration.
Conclusion
Results showed that in marigold, buds with the size of 5-10 mm contained microspores with mid-uninucleate stage to early bi-nucleate stage showed the highest response to the induction of androgenesis. Also, T3 and T4 genotypes belong to the French species showed the highest response to the regeneration. In another experiment, the pre-treatment of anthers with 0.2 and 0.5 M mannitol by using mannitol in a liquid culture medium for 24 hours, respectively showed the highest percentage of complete plant regeneration and the highest mean number of shoot per callus and anther. Chromosome counting results showed that 3 out of 5 examined plants were dihaploid and had 24 chromosomes in their root tip cells, while examined mother plants were tetraploid and showed 48 chromosomes in their root tip cells.
Breeding and Biotechnology of Plant and Flower
Fatemeh Keykha Akhar; Abdolreza Bagheri; Nasrin Moshtaghi; Masud Fakhrfeshani
Abstract
Introduction
Flower color is one of the most significant characteristics in ornamental plant breeding. New varieties of various plants in relation to their flower color have been obtained by monitoring the expression levels of genes involved or regulating the flavonoid and anthocyanin biosynthesis ...
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Introduction
Flower color is one of the most significant characteristics in ornamental plant breeding. New varieties of various plants in relation to their flower color have been obtained by monitoring the expression levels of genes involved or regulating the flavonoid and anthocyanin biosynthesis pathway. Flavonoids possess significant and diverse biological functions. They are the major pigments for flowers, fruits, seeds, and leaves. They are natural products that contain a C6-C3-C6 carbon framework and are synthesized by a branched pathway that yields both colored and colorless compounds. The gene encoding chalcone isomerase (CHI) is among the genes and enzymes identified in the flavonoid pathway. This enzyme catalyzes the isomerization of naringenin chalcone into the corresponding flavanone. CHI enzyme belongs to the family of isomerases, specifically the class of intramolecular lyases. Chalcone isomerase has a core 2-layer alpha/beta structure and has attracted much attention recently due to its role in stress response and pigment production. One of the most effective methods of genetic engineering is the reduction of flower pigments by suppression of required enzymes for their biosynthesis. RNA interference (RNAi) has provided the tool for the investigation of genes involved in the production of flower color. Silencing of any gene in the anthocyanin biosynthetic pathway can result in reduced or inhibited anthocyanin production. RNAi technology is an effective gene silencing method and a powerful tool for studying gene function and development of new traits by transformation of viral RNA or hairpin RNA (hpRNA) constructs into plants. The processing of dsRNA into 21-23-nt small interfering RNAs (siRNAs), and the mediators of RNAi, triggers cognate mRNA degradation. The hpRNAi methodology simply requires a transgene construct containing an inversely-repeated sequence of the target gene flanked with a promoter and terminator which effectively function in plants.
Material and Methods
In this research, with the design and construction of chiRNAi, the transformation of the RNAi construct was carried out of Petunia plants. Potted plants of P. hybrida were grown under standard greenhouse conditions (16-17°C night temperature and 21-24°C day temperature and photoperiod 16/8 (light/dark)). The RNAi construct including the 530 bp cds of the chalcone isomerase (chi) gene and 741 bp of pdk gene as intron between chi sense and antisense were used for transient RNAi-induced silencing. The pBI121-chi530 plasmids were introduced into A. tumefaciens strain LBA4404 by electroporation method. Colonies of A. tumefaciens carrying the desired plasmid were screened by PCR with specific primers for chi gene. RNAi construct co-cultured with petunia’s leave. Samples was kept in dark condition for 3 days and then transferred to branch induction media. Samples were investigated for phenotypical changes and chi gene expression by qRT-PCR.
Results and Discussion
Transgenic lines showed a reduced number of pigments and a faded flower color. So that, in purple petunia, was shown 5 phenotypical groups. These groups was indicated different levels of chi gene silencing. In pink petunia was seen two groups of phenotypical changes. In these plants, chi-RNAi construct was reduced pigment production and so, these plants had faded colors in petals. Also, the chi gene expression was reduced in all transgenic lines. Generally, the results of this research showed that RNAi can be used as an efficient method for gene silencing. The application of gene silencing can indicate the gene’s function in biosynthesis pathways of various components such as anthocyanins. In addition, the chalcone isomerase gene was identified as one of the effective genes in anthocyanin biosynthesis pathway in Petunia plants that could be involved in the production of color in these plants; hence, chi gene silencing resulted in clear phenotypic alterations in this plant.
Conclusion
In general the concentration of the target mRNA in a particular tissue could be a factor that influences silencing efficiency. At very low levels of gene expression, small amounts of the silencing target, mRNA, could be completely degraded by the RNA-induced silencing complex (RISC), whereas the presence of higher amounts of the target mRNA may result in incomplete silencing, allowing some residual functional mRNA to be translated into the corresponding protein. This research demonstrated the hpRNA construct has been successfully established for floral tissues of P. hybrida. The hpRNA construct was developed for chi-RNAi silencing of one of the key genes in the anthocyanin biosynthetic pathway in Petunia flowers. The silencing of the chi gene is a prototype for the modification of the anthocyanin biosynthetic pathway in Petunia through gene suppression. This strategy could also be useful for rapid functional analysis of other genes involved in flower development.
Breeding and Biotechnology of Plant and Flower
Mohammad Esmaeil Naddaf; Ebrahim Ganji Moghadam; Gholmreza Rabiei; Abdolrahman Mohammadkhani
Abstract
Introduction Sweet Cherry (Prunus avium) belongs to the Rosaceae family, which due to vegetative propagation problems, in vitro propagation is recommended to increase mass and disease-free production. Micropropagation has many advantages over other vegetative methods. Although the most suitable ...
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Introduction Sweet Cherry (Prunus avium) belongs to the Rosaceae family, which due to vegetative propagation problems, in vitro propagation is recommended to increase mass and disease-free production. Micropropagation has many advantages over other vegetative methods. Although the most suitable organ that preserves the genetic characteristics of the cultivar is bud meristem, plant regeneration from meristem culture is difficult in many species of woody plants, so micro-grafting is a suitable technique to overcome these problems. The aim of this study was to investigate the effect of scion size and origin of commercial sweet cherry cultivars interact with micrografting on the vegetative rootstocks.Materials and Methods In this study, factorial experiment was used as a test unit in a completely randomized design (CRD) with two factors in five replications and ten seedlings per replication. The first factor was cultivar in seven levels (B: Bing, D: Dovomres, H: Haj Yousefi, P: Pishres, S: Siah- Mashhad, T: Takdaneh, Z: Zard) and the second factor was scion type in four levels (M1R1: 2 mm in vivo explant, M2R1: 5 mm in vivo explant , M1R2: 2 mm in vitro explant and M2R2: 5 mm in vitro explant). To prepare the scion, 1.5 to 2 cm long explants were isolated from shoot tips and then disinfected with 75% ethanol and 20% Sodium hypochlorite. After rinsing with distilled water, the shoot tips with 2 and 5 mm length were extracted for in vivo explants. In vitro explants were obtained from shoo tips that was previously established in MS culture medium with supplement of 1 mg.l-1 of BAP. The meristems were prepared in 2 and 5 mm and used as in vitro explant. 5 cm length in vitro shoots of sweet cherry ‘Gisella 6’ was used as rootstock. Micro-grafting was performed according to the standard method for sweet cherries. Micro-grafted plantlets were transferred to MS medium supplemented with 1 mg.l- l BAP, and kept under low light (100 lux) condition for one week, then transferred to growth chamber at 27.1 °C photoperiods 16/8 hrs light/darkness (1500 lux). In order to induce root, grafted plantlets were transferred to Perlite: MS medium supplement with 1 mg.l- l IBA. After rooting, plants were placed in polyethylene pots containing perlite: peatmoss (1:1) for acclimation. Micro-grafting success indices were recorded in each of the micro-grafted plantlets. The data were analyzed by SAS statistical software (9.1) and the means were compared by Duncan's multiple range test (1 and 5 % of probability levels).Results and Discussion The results showed that in all indices there was a significant difference between scion types and cultivar scion type interactions except grafting time, but there was no difference between cultivars (except longitudinal growth of scion). Among the scion types, the 5 mm in vitro scion (M2R2) had the highest micro-grafting success rate (42%), number of leaves (3.7), longitudinal growth (6.3 cm) and taken grafting time (two days). Finally, in successful micro-grafted plants, ‘Pishres’ cultivar had better results in rooting (32.8%) and ‘Zard’ cultivar in acclimation (3.4%) traits. Probably the presence of leaves led to better nutrient supply and surface contact, so it mostly improved the success of micrografting technique. In this study, micro-grafting success indices were lower than previous reports using seedling rootstocks. This might be due to difficult grafting operations, poor rootstock-scion communication, low physiological activity, and high in vitro oxidase activity. In the type of scion, micro-grafting success rate of 5 mm in vitro scions (include leaf primordia), was better than 2 mm scions (without leaf primordia). These results were consistent with most reports in sweet cherries and other stone fruit that were more successful in micro-grafting using larger in vitro explant.Conclusion Based on our results, it can be concluded that the micro-grafting method in sweet cherry micro-propagation is a fast practical method with high potential for production and regeneration of healthy orchards, which is also possible for other cultivars. In micro-grafting success, in vitro explants are preferable to explants taken directly from in vivo mother trees, and the use of larger explants for scion is recommended due to the presence of leaf primordia in micro-grafting success. However, smaller-size explants are more likely to produce healthy plants.
Breeding and Biotechnology of Plant and Flower
Leila Baghazadeh Daryaii; Davood Samsampoor; Abdolnabi Bagheri; Jelveh Sohrabipour
Abstract
Introduction
Fungal Endophytes have symbiosis life within the plant tissues without causing any obvious negative effects. Seaweeds are one of the large and diverse groups of marine plants that play an essential role in marine and oceans ecosystems. Seaweeds show rich diversity of ...
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Introduction
Fungal Endophytes have symbiosis life within the plant tissues without causing any obvious negative effects. Seaweeds are one of the large and diverse groups of marine plants that play an essential role in marine and oceans ecosystems. Seaweeds show rich diversity of associated microorganisms compare with the other multicellular organisms. Citrus species, are amongst the most important evergreen fruit trees, cultivated in many countries worldwide. There are several obstacles for citrus production in southern of Iran that limiting continuity of citrus production. Lack of suitable soil, is one of the main challenges threatening citrus industry in southern of Iran. Similar to other citrus species, the production of Mexican lime is threatened by certain biological stresses (such as pests, plant diseases and weeds) and non-biological stresses (such as salinity, drought, floods, cold and heat stress). Here, we have evaluated the potential of inoculating Mexican lime seedlings with seaweeds fungi endophyte, Aspergillus niger, to improve morphological, biochemical, antioxidant and photosynthesis pigments characterizes. Endophytes are advantageous group of microorganisms that protect plants from biotic and abiotic stresses. One of the alternative ways to restore normal plant growth may be to use plant growth to stimulate endophytes. Endophytes can play an important role in plant growth. Endophytes from marine environment are gaining special interest because of their existence in the harsh conditions of marines and ocean ecosystem such as temperature, light availability, high salinity and osmotic stress. Fungi have already been isolated from various marine habitat, including marine plants, marine invertebrates and vertebrates. Among these organisms, seaweeds are one of the most prevalent sources of marine-derived fungi for chemical studies. The purpose of this study was the isolation of associated fungi with seaweed species in Persian Gulf to investigate morphological and molecular characterization by using PCR amplifications ITS1-5.8S-ITS4 regions and primitive assessment of their potential as bio-fertilizer.
Materials and Methods
The main aim of this study was investigation the role of endophytic fungi (Aspergillus niger), in improving the growth of Mexican lime seedlings. Cladophoropsis membranacea, green seaweed, was collected from coastal region of Bushehr province. Fungal endophytes were isolated and identified based on morphological and molecular methods. Molecular characteristic was investigated using PCR amplification of ITS1-5.8S-ITS4 regions. Mexican lime seeds were sterilized with 0.5% sodium hypochlorite for 20 minutes and then completely distilled three times with distilled water. Seedlings pots containing autoclaved soil were placed in the greenhouse of the Faculty of Agriculture, Hormozan University. Isolated fungi by MT420720 accession number was used as bio-fertilizing agents in eight months old Mexican lime seedlings. The suspension was adjusted to a concentration of 1×106 cell per ml. For better contact of seedlings with fungi, inoculation was performed three times. After three months, morphological (trunk diameter, stem length, root length and width, leaf and branch number, leaf, stem and root dry and fresh weight), biochemical (Protein, MDA and SPAD), antioxidant (CAT, POD, SOD, APX and Gr enzymes activity) and photosynthesis pigments (Chlorophyll a, Chlorophyll b, Total Chlorophyll and Carotenoids) characterizes in treated Mexican lime seedlings were analyzed. The experiment was arranged in randomized complete design with three replications. Analysis of variance of traits was performed using SAS software version 9.4 and the means were compared using LSD method with a probability level of P≤0.05.
Results and Discussion
The genera of Aspergillus was the most frequent isolates of the isolated fungi. The results show that most traits were significant compared with control. For example, leaf number (144.42%), root fresh weight (144.13%), stem fresh weight (94.85%) and root width (105.55%) were significantly higher compared with control (P>0.001). Fungal inoculation can significantly improve the photosynthesis pigments such as chlorophyll a (10.98%) and carotenoids (40.62%) (P>0.001) compared with control. In antioxidant capacity of seedling, CAT, POD, SOD, Gr and APX enzymes were analyzed. Fungal inoculation can increase the enzymes activity. For biochemical traits, fungal inoculation can significantly increase SPAD number and decrease MDA in inoculated seedlings compare with control (P>0.001).
Conclusion
The results showed that the use of entophytic fungi increased the growth of Mexican lime seedlings. Thereby it can be used as an effective tool for growing salinity-sensitive plants such as Mexican lime in saline conditions.
Breeding and Biotechnology of Plant and Flower
Nima Javaheri; Behzad Kaviani
Abstract
Introduction
Lily, a member of the genus Lilium, belonging to the Liliaceae family is one of the most important commercial pot and cut flower species and one of the three major bulb crops in the commercial market because of its large, colorful and fascinating flowers. Lily hybrids are the most ...
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Introduction
Lily, a member of the genus Lilium, belonging to the Liliaceae family is one of the most important commercial pot and cut flower species and one of the three major bulb crops in the commercial market because of its large, colorful and fascinating flowers. Lily hybrids are the most economically important plants with varied flowers. Hybrid Eastern lily (Lilium oriental hybrid ‘Casablanca’) is a perennial bulbous plant that its propagation by bulb in natural condition is time-consuming, so produces 1–2 bulblets per bulb scale in one years’ time which is not sufficient for large scale cultivation of this plant. One of the most important and best methods for vegetative propagation and breeding of lilies is in vitro bulb scale culture. In vitro adventitious bud regeneration from scales of Lilium rely on many factors like cytokinin and auxin concentrations such as BA and NAA. The successful use of tissue culture techniques for rapid propagation of some species of the genus Lilium including L. ledebourii, L. orientalis, L. longiflorum, L. japonicum, L. speciosum, L. concolor, L. nepalense, L. regale, L. oriental hybrid, L. Asiatic hybrid has been reported. The purpose of the current study was to evaluate the effect of different concentrations of BA and NAA on in vitro proliferation of Lilium oriental hybrid ‘Casablanca’ using bulb scale as explant to establish a suitable protocol.
Materials and Methods
Effect of various concentrations of 6-benzyle adenine (BA; 0, 0.5, 1 and 2 mg l–1) and ɑ-naphtaleneacetic acid (NAA; 0, 0.1, 0.2 and 0.4 mg l–1) were evaluated on in vitro proliferation of L. ‘Oriental’. Bulb scale as explant and MS basal medium as culture medium were used. Activated charcoal was applied to inhibit the browning of the culture medium and explant. The experiments were conducted in completely randomized design (CRD). The 16 treatments were applied, each treatment had 4 replications and each replication had 4 individuals. Therefore, in these experiments, a total of 192 bulbs were used. Traits including total plantlets fresh weight, leaf length, leaf number, bulblet weight, bulblet diameter, bulblet number, survival percentage, root length and root number related to in vitro proliferation were measured. All the statistical analyses were done by using SAS and Tukey’s test. Arcsin software was used for changing percent data.
Results and Discussion
The interaction effect of BA and NAA was significant for all measured traits. Results showed that the maximum number of bulblet (8.66) and root (5.36) were obtained in culture medium enriched with 0.5 mg l–1 BA together with 0.4 mg l–1 NAA. Culture media supplemented with 0.5 mg l–1 BA together with 0.2 mg l–1 NAA and 1 mg l–1 BA together with 0.1 mg l–1 NAA with induction of 7.33 bulblets per explant were suitable media. The largest number of leaf (4.33) was measured in culture medium containing 1 mg l–1 BA together with 0.1 mg l–1 NAA. The highest bulblet weight was measured in culture medium supplemented with 1 mg l–1 BA along with 0.2 mg l–1 NAA. The greatest survival rate (100%) was observed in medium enriched with 0.5 mg l–1 BA together with 0.1 mg l–1 NAA. Survival rate (90%) in explants treated with 2 mg l–1 BA along with 0.4 mg l–1 NAA was high. Obtained results revealed that the presence of both BA and NAA in culture media for enhancement of most traits is necessary and critical. Plantlets were transferred to a growing medium containing cocopeat, peat moss and perlite in identical proportion for acclimatization following proliferation. Approximately, 90% of regenerated plantlets survived and were morphologically similar to the mother stocks. This study will help the producers and breeders for commercial and improvement purposes. The effective role of the simultaneous presence of both auxin and cytokinin in the culture medium in effective organogenesis was shown in the present study. Similar findings were reported for some lilies such as L. ledebourii (Baker) Bioss., L. longiflorum and L. regale. Auxin was effective in stimulating bulb production and growth of the aerial part of the eastern lily, and its presence along with cytokinin is essential for leaf induction. Some studies have reported similar results. The type and optimal concentration of plant growth regulators (PGRs) in the culture medium for suitable in vitro propagation varies in different species. Genetic variations (species type), differences in the amount of endogenous production of PGRs and their interaction with each other are among some reasons for this difference. The proper ratio of auxin and cytokinin in the culture medium is effective for inducing cell division, cell differentiation, organogenesis and finally for achieving a complete plant. Root production with appropriate quantity and quality leads to the suitable survival of seedlings resulting from the growth of cultured explants under in vitro conditions and adapted plants. Current study showed that the presence of both BA and NAA is better than the presence of one of these two PGRs for induction and growth of root. Some similar findings were reported, however in most studies, the presence of auxin as individual PGR has been found to be more suitable for root induction.
Breeding and Biotechnology of Plant and Flower
Fatemeh Bidarnamani; Seyyed Najmodin Mortazavi; Yasob Shiri
Abstract
Introduction
Phalaenopsis is one of the beautiful and commercial species of orchid family that there is little research on its medicinal properties. Phalaenopsis orchids can be grown outdoors in warm, humid conditions that are damp but not wet, in a location that is shady but bright. Since the ...
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Introduction
Phalaenopsis is one of the beautiful and commercial species of orchid family that there is little research on its medicinal properties. Phalaenopsis orchids can be grown outdoors in warm, humid conditions that are damp but not wet, in a location that is shady but bright. Since the introduction of orchid meristem culture in 1960, branching in micro-propagation methods develop based on using two clearly different culture schemes. It have been used from medicinal properties of orchids for anti-rheumatic, anti-inflammatory, anti-viral, anti-cancer, anti-seizure, diuretic, nutrient protection, relaxation, anti-aging, wound healing, hypoglycemia, anti-tumor, antimicrobial, antibacterial, anti-oxidants and anti-diarrhea.
Material and Methods
The purpose of this study was to evaluate the canonical correlation between morphological and phytochemical characteristics of five phalaenopsis orchid varieties: Nottingham, Bucharest, Andorra, Memphis, and Dubrovnik. Micro-propagation of orchid seeds was performed after sterilization of capsules by bleach and ethanol. Leaf and root organs of 5 orchid varieties were harvested from Chen medium in Institute of Agricultural Research, University of Zabol, on 2020. The sliced samples were dried in an oven at 50 °C for 72 hours, after separating the leaves and roots of each variety. The powdered leaves and roots of each variety (5 g) were separately immersed in 50 ml of ethanol for 24 hours at room temperature. Then the extracts were placed on a shaker at room temperature at 120 rpm.
Result and Discussion
The results showed that the first two canonical variables were significant and third variable was not significant. The first canonical variable has the highest special value of 65.83 and the second and third canonical variables have 4.59 and 0.10 special values, respectively. According to this research the first canonical variable with the highest special values, square of canonical correlation and the most cumulative percentages should be studied in all morphological and phytochemical characteristics. Significant canonical correlation in morphological characteristics fit 94% of the total variance of physiological variables, while significant canonical variables in phytochemical characteristics fit 100% of the variance of phytochemical characteristics. Due to the fact that phalaenopsis orchid flower has many leaf and root wastes, the findings of this study show that due to the medicinal properties of this plant, we can reduce the waste of this plant in greenhouses and production centers of this flower. In this study, the amount of phenol, flavonoid and antioxidants were determined. Also the relationship between morphological and phytochemical traits showed that this plant has medicinal properties. The lowest number of leaves belongs to Memphis variety and the highest number of leaves belongs to Nottingham variety, among the studied varieties. Therefore, it can be concluded that the roots are longer and shorter in orchid varieties with white (Nottingham) and purple (Memphis) flower respectively. Also, the second focal variable of root trait with the highest coefficient had a negative correlation with the number of leaves and root length, which shows that in the second correlation of the number of leaves and root length, the number of roots will decrease. In the second focal variable, the number of roots had a positive focal correlation with leaf length. That is, the closer we get to the second center of correlations between morphological traits, the more direct and positive becomes the relationship between root number and leaf length traits.
Conclusion
According to the results and high percentage of fitness in phytochemical characteristics, it can be concluded that phytochemical traits can more precisely express the correlation between the traits. Also it showed significance of canonical correlation between two variable groups, the relationship between phytochemical and morphological traits of five varieties of phalaenopsis orchid. In general, the results of this study showed orchid varieties have high phenolic and flavonoid compounds and have the highest restraint power of free radical in canonical correlation, and they can be used as medicine. In this study, it was found that Phalaenopsis orchid varieties had phenolic content and good antioxidant capacity relatively and there was a high positive correlation between morphological and phytochemical traits and enhanced with adding the plant's length or number of leaves containing phytochemical compounds. Also, according to the results of this study and similar findings in other research, it was found that the production of secondary metabolites of orchids and other plants under the influence of genetic and environmental conditions can affect the production and amounts of chemical compounds and medicinal performance of plants.
Breeding and Biotechnology of Plant and Flower
Masumeh Vakili-Gartavol; Naser Mahna
Abstract
Introduction: Red-fleshed apples (Malus spp.) are one of the rarest apple genotypes in the world and the accumulation of a high amount of anthocyanin, is the main cause of the redness of their fruit flesh. Anthocyanins are among important flavonoids and due to antioxidative activity, scavenge reactive ...
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Introduction: Red-fleshed apples (Malus spp.) are one of the rarest apple genotypes in the world and the accumulation of a high amount of anthocyanin, is the main cause of the redness of their fruit flesh. Anthocyanins are among important flavonoids and due to antioxidative activity, scavenge reactive oxygen species, and hence, are considered as one of the health-promoting nutraceuticals in the human diet. The amount of anthocyanins depends upon the expression of the transcription factors that are regulating their metabolic pathway. Among these transcription factors are the members of the MYB gene family. MdMYB10, belonging to this gene family in apple, has been shown to have a significant role in controlling the amount of anthocyanin production and redness in fruit flesh. The expression of MdMYB10 and consequently, the production of MdMYB10 proteins has positive feedback on its own expression. This happens due to a 23 bp microsatellite tandemly repeated 5 times in its promoter region (called allele R6) which is a target sequence for MdMYB10 acting as a positive regulator. This structure invokes the overexpression of MdMYB10 which in turn increases the expression of anthocyanin producing enzymes and finally the amount of anthocyanin in all organs of the apple plant including fruit flesh. The apple Malus pumila var. Niedzwetzkyana and its derivatives have been reported to have such a structure in the promoter region of the MdMYB10 gene. The length of the R6 allele is 496 bp, while the R1 allele is only 392 bp long.
However, in some cases, a locus linked to the S3 allele of the S-RNase gene has been proposed to be responsible for the redness of the fruit flesh in some genotypes. It has been reported that even the offspring of these plants have had red-fleshed fruits.
Materials and Methods: To study the mechanism of the redness of the fruit flesh in some local genotypes, genomic DNA was extracted using the CTAB method from the leaf samples obtained from 9 red- and white-fleshed apple genotypes including Red Delicious, Golden Delicious, Miandoab, Makamik (Khalatpoushan), Bud 9, Varzighan, and Ivand. Then the allelotype of the promoter region of the MdMYB10 gene as well as the existence of S3 allele at S-RNase locus was investigated using polymerase chain reaction. For amplification of the target sequences, MdMYB10 and S3 specific primers were exploited and 1% agarose gel electrophoresis of the amplified fragments was used for observing and scoring the bands. All steps were repeated seven times.
Results and Discussion: The results in this research showed that the white-fleshed genotypes (Red Delicious, Golden Delicious, and Granny Smith) were lacking any R6 allele at the promoter region of the MdMYB10 gene and were R1R1 homozygotes, while the red-fleshed genotypes (Miandoab, Makamik (Khalatpoushan), Bud 9, and Varzighan) had at least one R6 allele at the mentioned promoter region as well as a S3 allele in the self-incompatibility locus S-RNase. These results were in accordance with the previous reports. Therefore, these samples could be traced back to Malus pumila var. Niedzwetzkyana. Evaluating the S-RNase locus in these genotypes illustrated that Granny Smith (as positive control), Golden Delicious (as positive control), Makamik (Khalatpoushan), Miandoab, Varzeghan, Bud 9 and tissue culture sample, showing a band around 500 bp (smaller) had S3 allele, while for Ivand and Red Delicious (as negative control) no S3 band was obtained. For the tissue culture sample which was R1R1 at the promoter region and S3 at S-RNase locus, it was postulated that flesh-redness may be due to the locus linked to the S3 allele. We also got an unknown R band for the Ivand genotype when analyzing for the MdMYB10 promoter region. The sequencing of in the future studies, may help to unravel the mechanism by which shoot-redness happens in this genotype.
Conclusion: The development of highly potent and novel cultivars for the fast-evolving market is indispensable in the plant breeding field. In this way, breeding apple plant, as an important temperate fruit with a long postharvest life, for redness of fruit flesh can be considered as a noticeable case. We could confirm in this research that in the endemic, red-fleshed apples, R6 may be responsible for their high anthocyanin production. However, the S3-RNase-linked locus should also be considered in marker-assisted breeding methods for this trait. Therefore, these red-fleshed genotypes are highly recommended to be employed in the national breeding programs for increasing the anthocyanin content of apple fruits.
Breeding and Biotechnology of Plant and Flower
Mehdi Rezaei; Mitra Rahmati; Abdolreza Kavand; Morteza Hemati; Seyyed Reza Kazemi
Abstract
Introduction: Apricot (Prunus armeniaca L.) as an important fruit crop belongs to the Amygdaloideae in the Rosacea family and is grown in regions with Mediterranean climates in the world. Apricot species were classified into six eco-geographical groups including: Central Asian, East Chinese, North Chinese, ...
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Introduction: Apricot (Prunus armeniaca L.) as an important fruit crop belongs to the Amygdaloideae in the Rosacea family and is grown in regions with Mediterranean climates in the world. Apricot species were classified into six eco-geographical groups including: Central Asian, East Chinese, North Chinese, Dzhungar-Zailij, Irano-Caucasian and European. Iranian genotypes which belong to the Irano-Caucasian group are mostly self-incompatible with low chill requirement. The high level of genetic diversity in Iranian apricots is due to sexual reproduction by seeds during the years. In Iran, many of apricot local varieties have been relocated between provinces and subsequently, in some cases their names, have been changed over the years. Hence, to determine the genetically different cultivars and detection of synonyms, screening of apricot germplasm seems necessary in Iran.Materials and Methods: Thirty eight commercial genotypes of apricot with five biological replications were collected from 14 nurseries in West Azarbaijan, East Azarbaijan, Esfahan, Semnan, Alborz, and Tehran Provinces in Iran. Additionally Orang Red apricot (Porteghali) included in the study as an outgroup sample. Also DNA sample of previously registered apricots in national list were used in this study. Young and healthy leaves of each cultivar were sampled and stored at -70 °C. Samples were powdered using mortar and pestle in presence of liquid nitrogen. CTAB extraction buffer was used for nucleic acid extraction. Quantity and quality of extracted DNA were measured by spectrophotometry and agarose gel electrophoresis.Thermal cycles were done in Eppendorf thermocycler and the cycling program were set on one cycle of 94°C for 4 minute, 30 cycles of 94°C for 30 seconds, annealing temperature of each primer for 30 seconds and 72°C for 30 seconds followed by one cycle of 72°C for 5 minutes. PCR products were resolved on 10% polyacrylamide gels in 1x TBE buffer. GelRed (Biotium) was applied for gel staining and amplified bands were revealed by UV (300 nm). Eight SSR markers which showed more diversity were selected and scored. Polymorphic alleles were scored as one for presence and zero for absence. For detection of off types, samples were classified by Paired Group method and Euclidean algorithm in PAST software. Then the data of off-types were removed from the dataset and samples were reclassified by the method. Principal Coordinate Analysis (PCoA) was carried out using PAST software. Genetic diversity indices were evaluated in Popgene 32 software. Results and Discussion: Eight SSR loci produced 124 alleles with the average 15.5 allele per locus. Nei’s gene diversity and Shannon’s information index were 0.32 and 0.48, respectively which showed high level of diversity in this collection. Distance matrix based on Nei’s gene diversity showed that the most genetic distance (0.74) was between Askar Abadi and Zodras, Mahali Goushti Zodras and Nasiri cultivars. Clustering of samples indicated that some samples including 19 (Shahroudi), 59 (Nakhjavan), 107 (Shahroudi), 127 (Soltani), 137 (Ghavami), 144 (Tabraze) and 156 (Daneshkade) were off-types.For identification of synonyms the off-type samples were disregarded. Cluster analysis illustrated that some local cultivars with different names had same genetic backgrounds. Thus, the names of these samples should be unified in the germplasm. Depicted graph based on first and second coordinates in PCoA demonstrated that the 38 collected groups of apricots are genetically 26 distinct cultivars and there are some duplicates in the germplasm. Results showed that three loci including UDP98-021, UDP98-409 and UDP98-411 were able to distinguish all 26 genotypes. To check the genetic identity of saplings with the same name, some cultivars including Jahangiri, Askar Abadi, Shamlou, Saltanati, Shahroudi, Shams, Tabarze and Rajab Ali were collected from two different nurseries. Surprisingly, the results showed that Nasiri, Tabraze and Shahroudi which were sampled twice from distinct nurseries and provinces, despite of identical names, had different genetic backgrounds.Conclusion: Detected off-types among five biological replications of local varieties propagated asexually by nurseries showed that there was not sufficient attention in the selection of propagating material in the nurseries. In this context, establishment of foundation blocks by public sector and mother orchards by private sector of economy from Iranian apricot local varieties can be an effective solution so that nursery operators can provide certified propagating material for production of certified nursery stocks. Moreover, seed and plant certification and registration institute should made inspection and testing procedures in mother orchards and nurseries to ensure that propagated trees are healthy, genetically uniform and original.
Breeding and Biotechnology of Plant and Flower
Ahmad Sharifi; Maryam Moradiyan; Nasim Safari; Azadeh Khadem; Seyyedeh Mahdiyeh Kharrazi
Abstract
Introduction: Ornamental foliage plants are commonly used for beautifying indoor spaces. Consequently, determining the best method of mass propagation in a short time, is necessary for these plants. For this purpose, an experiment was designed and performed to micropropagate the Singonium ornamental ...
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Introduction: Ornamental foliage plants are commonly used for beautifying indoor spaces. Consequently, determining the best method of mass propagation in a short time, is necessary for these plants. For this purpose, an experiment was designed and performed to micropropagate the Singonium ornamental plant.Materials and Methods: For the first stage, the effect of different plant growth plant growth regulators on the regeneration of nodule explants was evaluated. In this experiment, 1 cm nodal explants were placed in semi-solid MS culture medium containing different concentrations of BA and Kin (0, 1, 2, 3 and 4 mg/l) in combination with 0.2 mg/l IAA. This experiment was performed as a factorial based on a completely randomized design with four replications. The first factor included different types of cytokinin (BA and Kin) and the second factor included different concentrations (0, 1, 2, 3 and 4 mg/l). In the second stage, the effect of different types of culture medium and its different concentrations on plantlet proliferation was investigated. This study was performed as a factorial experiment based on a completely randomized design with five replications. The first factor included the type of culture medium (MS and B5) and the second factor was the different concentrations of culture medium (0.5 and 1). In the third stage of the experiment, the acclimation of in vitro plantlet was investigated. This experiment was performed as a completely randomized design with three replications. The experimental treatments included different culture media: vermiculite, peat moss, vermiculite + peat moss, perlite + peat moss, perlite, coco peat + vermiculite, coco peat + perlite, coco peat and rock wool.Results and Discussion: According to the results of the first experiment, it was found that the use of cytokinin in MS culture medium is effective on plant regeneration. The highest number of regenerated plantlet was observed in culture medium containing 4 mg/l BA with 0.2 mg/l IAA. It is noteworthy that with increasing BA concentration in the culture medium, the plant height decreased, but in contrast, the number of produced plantlets increased. Benzyl aminopurine (BAP) has been introduced as the most important and effective cytokinin in inducing and increasing branching in plants. Research has shown that the use of external benzyl adenine affects plant growth by affecting plant cells or by controlling the accumulation of a number of cytokinin compounds. As a result, the use of cytokinins in culture medium under in vitro culture conditions is necessary to induce and increase cell division. According to the second experimental results, produced plantlets in MS culture medium had higher height, number of roots and root length compared to B5 culture medium. Results also demonstrated that the use of ½ MS in the propagation stage of this plant is appropriate due to the improvement of growth traits such as plantlet height and root length. The use of optimal and suitable culture medium is very effective in the success of plant micropropagation. In the present study, the use of MS culture medium showed better performance compared to B5 culture medium. The appropriate amount of components, better ion strength and more minerals in this culture medium compared to B5 culture medium are probably the factors influencing its superiority in in vitro culture. In the acclimation stage, the use of vermiculite + peat moss culture medium led to 100% adaptation of plants and improvement of growth traits in the plant.Conclusion: The results showed that the type and concentration of cytokinin had a significant effect on the most of the evaluated parameters. With increasing the concentration of cytokinin in the culture medium, the number of regenerated plantlet was increased, but on the other hand, the height of regenerated plantlet also decreased. The use of culture medium containing BA compared to KIN had a more pronounced effect on increasing the number of regenerated plantlets. Application of MS medium compared to B5 medium was more effective in increasing plantlet height, root length, number of roots and number of produced plantlets. Also, half-strength MS medium, increased plantlet height and root length. Therefore, at this stage of propagation, the application of ½ MS culture medium is recommended. In the acclimation stage, it can be stated that the use of vermiculite substrate in combination with peat moss is a suitable option considering 100% compatibility of syngonium plantlets and improvement of growth traits in comparison with other substrates.
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.
