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 ...
Read More
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
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 ...
Read More
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
