Determining Cytological Developments of Microspore in Four Varieties of Tomato (Lycopersicum esculentum Mill)

Document Type : Research Article


1 Ferdowsi University of Mashhad

2 Research Center For Plant Sciences, Ferdowsi University of Mashhad


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


1- Bal U., and Abak K. 2005. Induction of symmetrical nucleus division and nucleus multicellular structures from the isolated microspores of tomato (Lycopersicumesculentum L.). Biotechnololgy and Biotechnological Equipement, 19(1):35-42.
2- Bal U., and Abak K. 2007. Haploidy in tomato (Lycopersiconesculentum Mill.). Euphytica, 158:1–9.
3- EnayatiShariatpanahi M., and Emami Meybodi D. 2009. Microspore: a haploid cell with various applications in genetics and plant breeding. Modern Genetics Journal, 4(3):5-16. (in Persian(
4- EnayatiShariatpanahi M., and Herbele-Bros E. 2009. Induction of embryogenesis in microspores of Tomato (Lycopersicumesculrntum Mill.) cv. Microtom. Seed and Plant Production Journal, 25-2(3):315-328. (in Persian(
5- Forster B.P., and Thomas W.T. 2003. Doubled haploids in genetic mapping and genomics. Kluwer Academic Publishers, Dordrecht, the Netherlands.
6- Goralski G., Rozier F., and Matthys E. 2004. Cytological features of various microspore derivatives appearing during culture of isolated maize microspores. ActaBiologicaCracoviensia Series Botanica 47(1):75–83.
7- Jain S. M., Sopory S. K., and Villeux R. E. (eds). 1997. In vitro haploid production in higher plants. Vol. 1, kluwer Academic Publishers, Dordrecht, The Netherlands.
8- Jain S. M., Sopory S. K., and Villeux R. E. (eds). 1997. In vitro haploid production in higher plants. Vol. 5, kluwer Academic Publishers, Dordrecht, The Netherlands.
9- Kasperbauer M. J., and Wilson H. M. 1979. Haploid plant production and use. In: Durbin R. D. (eds). Nicotiana procedures for experimental use. Washington, United States Department Of Agriculture Technical Bulletin, 1586:33-39.
10- Lauxen M., Kaltchuk-Santos E., Ching-yeh H., and Callegari-Jacques S. M. 2003. Associaion between floral bud size and developmental stage in soybean microspores. Brazilian Archives Of Biology And Technology 46:515-520.
11- Marina I., Tsvetova L., and Elkonin A. 2013. Cytological investigation of pollen development in sorghum line with male sterility induced by sodium ascorbate in tissue culture. American Journal of Plant Sciences 4:11-18.
12- Perera P. I. P. 2003. Cytological examination of microspore development for microspore and anther culture of coconut (Cocosnucifera L). Printed in Sri Lanka, Cocos, 15:53-59.
13- Summers W. L., J. Jaramillo T. B. 1992. Microspore developmental stage and anther length influence the induction of tomato anther callus. Horticultural Science 27:838-840.
14- Segui-Simarro J. M., and Nuez F. 2008. Pathways to doubled haploidy: chromosome doubling during androgenesis. Cytogenet GenomeResearch 120:358–369.
15- Segui-Simarro J.M., and Martinez P. 2011. Androgenesis in recalcitrant solanaceous crops (a Review). Plant Cell Reports 30:765-778.
16- Tomasi P., Dierig D. A.; Backhaus R. A. and Pigg K. B. 1999. Floral bud and mean petal length as morphological predictors of microspore cytological stage in Lesquerella. Horticulture Science, 34:1269-1270.
17- Touraev A., and Heberle-Bors E. 2005. Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH.Planta 219:141–146.
18- Touraev A., Brian P., and Mohan S. (eds). 2009. Advances in Haploid Production in Higher Plants. Springer Science.
19- Zagorska N. A., Shtereva A., and Dimitrov B. D. 1998. Induced androgenesis in tomato (Lycopersicon esculentum Mill.): II. Factors affecting induction of androgenesis. Plant Cell Reports 18:312-317.
20- Zhijun L. I., Yanrong Z., and Chunyan L. I. 2009. Cytological observation of the microspore development of chinese kale and false pakchoi. Agricultural China 3(1):24–28.
21- Zhao D. X., Yang X., Yan-Chen H. and Sheng W.H. 2013. Cytological investigation of anther developmentin DGMS line shaan-GMS in (Brassica napus L). Czech J. Genetics Plant Breeding, 49:16–23.