Colchicine Induced Chromosome Doubling in Annual Ryegrass (Lolium multiflorum Lam.) and Identification of Autotetraploid Plants by Flow Cytometer

The production of animal based foods has been threatened in Turkey by insufficient forage production which is one of the long standing problems of the industry. Therefore, alternative forage species for each ecological region of the country must be determined and their higher performing cultivars must be developed. Annual ryegrass (L. multiforum Lam.) is one of the most commonly grown forage grass species in temperate regions of the world. In recent years, its cultivation has been started in Turkey as well, and its acreage has been increasing rapidly. However, except only a few, all of the ryegrass cultivars grown in Turkey have been developed in other countries with different climatic conditions than Turkey. Therefore, it is our own national interest to develop high performing new annual ryegrass cultivars for local conditions. Induced polyploidy by application of mutagens such as colchicine is a well-known technique with a long history. It has been used successfully on many plant species. Induced polyploids generally have different morphological, physiological, and genetic characteristics than their diploid progenitors. As a result of these differences, they have higher biomass yield than their diploid progenitors as they are more tolerant to biotic and abiotic stress. Therefore, induced polyploidy is an important breeding method, especially for forage species. Approximately, 50% of the annual ryegrass cultivars registered in the world are autotetraploids, although it is a natural diploid with 2n=14 chromosome. Based on the results of previous studies, it has been determined that characteristics and performance of autopolyploids depend greatly on their diploid progenitors. Therefore, it has critical importance to evaluate diploid germplasms in the conditions of target regions and select the best performing ones for ploidy induction. However, we have not come across any similar type of study carried out on annual ryegrass in Turkey so far. The objective of this study is to develop an efficient method for our laboratory to induce chromosome doubling in annual ryegrass and identify autotetraploids. Approximately 1200 seeds of a diploid population developed by selecting from an annual ryegrass collection including approximatelly 120 accessions based on their performance in Tekirdağ conditions. Five different doses of colchicine (0.05, 0.1, 0.2, 0.3, 0.4 %) and 3 different periods of application (4, 8 and 24 h) were used in the study. Flow cytometry was used in ploidy analysis after 8 weeks of colchicine application. The highest recovery of autotetraploids (15%) was obtained by treatment of one week old germinating seedlings with 0.2% colchicine for 8 hours. A high incidence of mixoploidy was obtained following colchicine treatment. Hexaploids were also rarely encountered. It was found out that ploidy varied among the tillers of the same plants when ploidy analysis is repeated after 4 months of colchicine applications. Autotetraploid tillers were propagated vegetatively, and used in polycrosses.