Rasul Najib; Mohammad Farsi; Amin Mirshamsi Kakhki; Saeid Reza Vessal
Abstract
Introduction: Homozygous doubled haploid lines production through induction of androgenesis is a promising method to accelerate the classical breeding program. However, this technology is relatively under - developed in tomato so that improvements in methodology are required. Tomato (Lycopersicon esculentum ...
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Introduction: Homozygous doubled haploid lines production through induction of androgenesis is a promising method to accelerate the classical breeding program. However, this technology is relatively under - developed in tomato so that improvements in methodology are required. Tomato (Lycopersicon esculentum Mill) is one of the most important vegetables which in addition of it is importance as a food, is utilized as a model plant for cytological and cytogenetic studies. Tomato breeding programs are often based on the production and selection of hybrid plants. To produce hybrid plants and application of features that is needed to breed pure lines with high specific combining abilities, new technologies such as doubled haploid production through induction of androgenesis can be an effective strategy to provide pure lines in tomato. One of the critical factors for induction of androgenesis in tomato is to use of microspores being in appropriate developmental stage. Cytological examination is one of the most accurate methods for determining the correct stage of microspore development. In this study, a number of characteristics were evaluated including the cytological properties of normal microspores development and pollen grains as well as the relationship between length of flower bud and anther length.
Materials and Methods: In this study, four varieties of tomato including Mobil - Netherlands, Baker, U. S. Agriseed and Khoram were chosen. To determine the appropriate stage of microspore development for Anther culture, cytologycal studies were accomplished at different size length of flower buds (2. 0 - 7. 9 mm). Collection of flower buds to conduct experiments was done during 10 - 40 days after flowering for each cultivar. Flower buds collected early in the morning hours and within the containers closed - door ice were transported to the laboratory. To investigate the correlation between the length of flower bud and anther length, randomly selected from within each group of three flower buds, and their length was measurement. Then anthers were removed and anther length was measured for each flower buds. A total of 240 anthers, sixty anthers from each cultivar, were examined by microscope. In order to examine the development stage of microspores and pollen grains, flower buds at different length (5 - 10 mm) were calculated. Flower buds were incubated at 4 oC for 15 minutes and stained in acetocarmin %4 solution and squashed. In order to determine the relative frequency of each stage of the development of microspore and pollen, microspores at least 100 randomly in different parts of prepared slides were counted. Average relative frequency of different stages, meiosis, tetrads, microspores young and old and young and mature pollen grains with a standard deviation was calculated. Cytological studies were accomplished by microscopy research Olympus B X 51 and photographed by a digital camera D P 70. All analysis was conducted using statistical software JMP 8.
Results and Discussion: The time of anthers collection for the induction of haploid is very crucial. In order to determine the appropriate steps to carry out pre - treatment induced changes in the normal development of microspores embryogenesis and cytological properties in various stages of division and development should be monitored. The results showed that there was a significant correlation between the length of flower bud and the anther length (r = 0.8, P
Golnar GhazianTafrishi; Hossein Arouiee; Majid Azizi; Hamidreza Khazaie; Saeid Reza Vessal
Abstract
Introduction: Plants native to tropical and subtropical climates which grown in the temperate climate zone, suffer chilling injury when exposed to non-freezing temperatures for a certain period of time. The optimum growth temperature for cucumber (a tropical plant) is 20 to 25°C. Cucumber is sensitive ...
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Introduction: Plants native to tropical and subtropical climates which grown in the temperate climate zone, suffer chilling injury when exposed to non-freezing temperatures for a certain period of time. The optimum growth temperature for cucumber (a tropical plant) is 20 to 25°C. Cucumber is sensitive to temperatures lower than 10 °C. Cucumber area of production exposes to late spring and early autumn cold weather in Khorasan-e-Razavai, Iran. Studies showed that chilling leads to an alteration in fatty acid composition of membrane lipids and its permeability, changes in photosynthetic pigments content and decrease in photosynthesis. Many researchers pointed to a possible role of polyamine compounds in plant defense against environmental stresses. Exog enous application Spd could prevent the electrolyte and amino acid leakage or recovering the plasma membrane damage in rice and cucumber in response to salinity, chilling and water stressed conditions.
Materials and methods: A factorial experiment, based on completely randomized design was conducted to investigate the effect of short-term chilling on cucumber plantlets which was earlier treated with spermidine. Factors were included two levels of temperature (6 and 12°C) and four levels of spermidine (0, 0.25, 0.5 and .0.75 mg/L). The studied cultivar was ‘Super-Dominus’. In order to determine the extent of chilling injury, plants of each treatment were rated based on visual symptoms. By assigning values of 1, 2, 3, 4, and 5 while 1: no visible symptoms 2:5% of leaf area necrotic, 3: 5-25% of leaf area necrotic, 4: 26-50% of leaf area necrotic but plant still alive, 5: lost, entire plant necrotic and collapsed. Measured traits were root and shoot length, root and shoot dry weight, root and leaf electrical leakage, and leaf chlorophyll content.
Results and discussion: Plants which exposed to low temperature showed chilling injury symptoms (5-25% leaf area necrotic). The symptoms reduced (less than 5% leaf area necrotic) by using 0.25 and 0.5 mg/L spermidine. The symptoms enhanced by 50% by applying 0.75 mg/L spermidine at 6°C. Analysis of variance showed that there was significant difference between temperature levels, spermidine levels and interaction between them in respect to root length, shoot length, shoot dry weight and root and leaf electrical leakage. Root dry weight, root to shoot ratio and chlorophyll content just affected by temperature and spermidines levels but not by interaction between them. Root and shoot length and dry weight decreased by low temperature. At cold stress condition growth decreased due to a reduction in photosynthesis and carbohydrate metabolism .Root and shoot length decreased more than 79% at 6°C compare with 12°. Root to shoot ratio increased at cold condition which was the result of lower root weight loss in response to cold temperature compared with shoot weight losses. Electrical leakage (EL) enhanced in leaf and root cells at chilling temperature, but the enhancement was significantly more at root cells. Electrical leakage enhanced more than 52% in root cells at 6°C compared with 35% in leaf cells. EL suppressed, using 0.25 and 0.5 mg/L spermidine while an increase observed in El at 0.75 mg/L spermidine. The lowest EL percentage observed for leaf samples treated with 0.25 and 0.5 mg/L spermidine at 12°C. The highest EL percentage belonged to root samples treated with 0.75 mg/L Spd at 6°C .Chlorophyll content (ChlC) decreased at cold condition. ChlC was 52% at12°C compared with 37% at 6°C. High significant correlation observed between chlorophyll content and shoot dry matter (r2= 0.96**). Root and shoot length and dry weight and leaf chlorophyll content enhanced using 0.25 and 0.5 mg/L spermidine at both chilling and control temperatures. A decrease observed in measured traits applying 0.75mg/L spermidine. There was no significant difference between 0.25 and 0.5 mg/L spermidine levels in respect of measured traits expect for shoot dry weight. Spermidine enhances chilling tolerance in cucumber by prohibiting the activity of NADPH oxidase. The capacity of PAs to enhance the tolerance of cucumber to chilling injury is attributed to the scavenging of H2O2 production under chilling condition.
Conclusion: Results showed that root and shoot length and weight, root and leaf electrical leakage and chlorophyll content of leaf adversely affected by chilling stress. Using 0.25 mg/L spermidine modulates plant responses to chilling stress. There was no significant difference between 0.25 and 0.5 mg/L spermidine in respect of measured traits. But all measured traits adversely affected using 0.75 mg/L spermidine at both 6 and 12°C.
Rasul Najib; Mohammad Farsi; Amin Mirshamsi Kakhki; Saeid Reza Vessal
Abstract
Introduction Tomato (Lycopersicon esculentum Mill) is one of the most important vegetables which in addition of its importance as a food, is utilized as a model plant for cytological and cytogenetic studies. Tomato breeding programs are often based on the production and selection of hybrid plants. Producing ...
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Introduction Tomato (Lycopersicon esculentum Mill) is one of the most important vegetables which in addition of its importance as a food, is utilized as a model plant for cytological and cytogenetic studies. Tomato breeding programs are often based on the production and selection of hybrid plants. Producing hybrid plants and application of features that is needed to breed pure lines with high specific combining abilities, is highly required.New technologies such as doubled haploid can be an effective strategy to provide pure lines in tomato. Generation of homozygous doubled haploid lines through induction of androgenesis is a promising alternative method to the classical breeding programs. However, this technology is poorly developed in tomato so that some improvements in methodology are required. Genotype and stages of microspore development are critical factors for induction of androgenesis in tomato. Among them, the genotype is more important than other factors. The purpose of this study was to investigate the possibility of callus induction from anthers in some tomato genotypes.
Materials and Methods: In order to investigate the androgenic response and callus induction through anther culture in tomato, four varieties including Mobil-Netherlands, Baker, U. S. Agriseed and Khoram were chosen. To determine the appropriate stage of microspore development for anther culture, cytologycal studies were accomplished at different size length of flower buds (2-7.9 mm). Flower buds were incubated at 4oC for 15 minutes and stained in acetocarmin %4 solution. Based on cytological studies in four tested cultivars, flower buds with size length 4-4.9 mm were chosen, as they had the highest frequency of meiotic microspores to microspores mid uninucleate. Pretreatments were colchicine solution (250 mgr/L) at 4 °C for 48 h. The anthers were cultured on MS medium containing 2 mgr/L IAA and 1 mgr/L 2ip. All changes in frequency of callus induction and diameter of callus were recorded for eightweeks. Diameter of callus was measured using a microscope equipped with a camera and Dino Capture 2.0 software version 4.1. Cytological studies were accomplished by microscopy research Olympus BX51 and photographed by a digital camera DP70. To determine the presence or absence of a significant difference between the observed proportions a chi-square test was used. All analysiswas conducted using statistical software JMP 8. Charts were providedusing Excel software.
Results and Discussion: Anther development stage is one of the factors determining the success of anther culture in the production of embryos. The results of most studies showed that the stage between meiosis and mid-stage of unicellular microspores is optimum to androgenesis response in tomatoes. Since microspores in the anthers are at various stages of development, to determine the appropriate size of flower buds, the relative frequency of each of the stages of development should be understood. Based on the obtained results, in all study cultivars, flower buds with a length of 4-4.9 mm (Containing anthers with an approximate length of 3-4 mm), due to having the highest frequency of meiotic and unicellular microspores, can be used for anther culture. Study of deformation and induced callus in this experiment showed that both the Baker and U. S. Agriseeds did not show callus induction. Anthers of varieties over three weeks after culture gradually became yellowish-brown and in the fourth week of the increased frequency of haploid were brown. After six weeks of culture, all anthers in both became brown and died. The anthers of the varieties, Mobil-Netherlands and Khoram, inflated at the second to fourth week, anther wall was eventually broken and callus was observed. At third week the frequency of deformed anthers were gradually increased. Four weeks after culture, the frequency of callus induction reduced and after five weeks of callus induction no change in frequencyof callus induction was observed. The results showed that frequency of callus induction was significantly different among genotypes (P
