بررسي قابليت آندروژنز و كالوس‌زايي در چهار رقم گوجه‌فرنگي (Lycopersicon esculentum Mill) به‌وسيله كشت بساك

Study of Androgenesis Ability and Callus Induction in Four Varieties of Tomato (Lycopersicon esculentum Mill) by Anther Culture

به منظور بررسی پاسخ به آندروژنز و القاء كالوس از كشت بساك گوجه‌فرنگی، چهار رقم به نام‌های موبیل‌ هلند، بیكر، U. S. Agriseed و خرم انتخاب شدند. جهت تعیین مرحله مناسب توسعه میكروسپور برای كشت بساك بررسی‌های سیتولوژیكی در اندازه‌های مختلف طولی 9/7-2 میلی‌متری جوانه‌های گل انجام شد. برمبنای مطالعات سیتولوژیكی ‌در هر چهار رقم مورد آزمایش، جوانه‌های‌گل با اندازه طولی 9/4-4 میلی‌متر بدلیل داشتن بالاترین فراوانی میكروسپورهای مرحله میوز تا اواسط مرحله تك‌هسته‌ای، جهت كشت انتخاب شدند. جهت پیش‌تیمار از محلول كلشی‌سین 025/0 درصد در 4 درجه سانتی‌گراد به مدت 48 ساعت استفاده شد. سپس بساك‌ها در محیط‌ كشت‌ MS حاوی 2 میلی‌گرم بر لیتر IAA و 1 میلی‌گرم بر لیتر 2ip كشت شدند. روند تغییرات در فراوانی القاء كالوس و ضخامت كالوس‌ها در طی هشت هفته ثبت شد. نتایج حاصل از آزمایش نشان داد كه ژنوتیپ‌های مورد مطالعه از نظر فراوانی نسبی القاء كالوس با هم اختلاف معنی‌داری دارند (0001/0 P <). دو رقم موبیل‌ هلند و خرم پتانسیل نسبتا بالایی برای پاسخ به آندروژنز از طریق كشت بساك نشان دادند. به‌نظر می‌رسد با بهبود شرایط فیزیولوژیكی جهت رشد گیاه مادری و بهینه‌سازی تركیبات محیط كشت در آزمایشات بعدی بتوان در زمینه تولید گیاهان هاپلویید در این دو رقم گوجه‌فرنگی به پیشرفت‌هایی دست پیدا كرد.

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 <0.0001). Two varieties of Mobil-Netherlands and Khoram had a relatively higher potential for androgenic response through anther culture compared to other lines. Conclusions: The results showed that the and rogenes is in tomato is influenced by genotype. Two varieties including Mobil-Netherlands and Khoram showeda relatively high potential for induction of callus. It seems that by improving medium components and physiological conditions of donor plant in next experiments, there would be possibly more success in these two varieties in terms of haploid production.