An in vitro chromosome doubling method for clovers (Trifolium spp.)

Abstract

This research reports a new technique for chromosome doubling of clover (Trifolium sp.) axillary meristems via in vitro colchicine application. Plant material utilized included T. pratense (red clover) cv. Kenstar clones, and three interspecific hybrids: T. ambiguum (kura clover) × T. repens (white clover); T. alpestre × T. pratense; and T. sarosiense × T. pratense. Vegetative axillary meristems were excised from plants, surface sterilized, and trimmed to a length of 0.5–1 mm. Meristems were placed on the surface of a shoot proliferation medium (ML8) containing colchicine (0.1%) for 48 or 72 h and then transferred back to ML8. Alternative treatments were to preculture meristems on ML8 for 7 days prior to colchicine treatment. Plantlets with two or three trifoliolate leaves were induced to root on CR2 or RL rooting media. Preculturing of meristems on ML8 prior to colchicine exposure resulted in the highest chromosome doubling frequencies among the different genotypes, although there was apparent genotype × treatment interaction. Chromosome doubling frequencies were as high as 81 and 44% for initial root tips and mature shoots, respectively. To make rapid assessments of ploidy level of flowering plants, pollen shape was examined. Chromosome doubling increased the pollen stainability of the T. ambiguum × T. repens hybrid from 2.5 to 33.6%, but did not result in fertility in the other two interspecific hybrids.Key words: Trifolium, colchicine, chromosome doubling, interspecific hybrids.

Production of interspecific hybrids between commercial cultivars of the eggplant (Solanum melongena L.) and its wild relative S. torvum

“…Translocations and chromosome pairing could contribute to low hybrid fertility (Kumar et al, 1987;Pickersgill, 1997;Monteiro et al, 2011). This problem can be resolved by chemical treatment with colchicine, which has been successfully applied to double the chromosome number (Anderson et al, 1991). Alternatively, harvesting pollen in different seasons, such as spring time (Kowalczyk et al, 2008), may result in pollen with higher viability to improve pollination and promote advanced generation in subsequent breeding.…”

Production of Trifolium pratense L. and T. hybridum L. tetraploid populations and assessment of their agrobiological characteristics

“…The success of polyploidy induction depends on several variables: plant species, antimitotic agents, explant types, exposure times and concentrations (Dhooghe et al, 2011). Traditionally, colchicine treatment is performed on germinating seeds (Wu et al, 2010) and 7-day old seedlings (Abd El-Naby et al, 2012), apical meristems (Schifino, Morales Fernandes, 1987), apical buds (Wang et al, 2009;Talukdar, 2010), axilary meritems (Anderson et al, 1991), epicotyls (Bliznyuk, Filipovich, 2007) and cotyledons (Stanys et al, 2006).…”

“…Numerous research publications have indicated that colchicine is the most common mitosis inhibitor used for the production of polyploids of legumes (Anderson et al, 1991;Algan, Buyukkartal, 2000;Wang et al, 2009;Wu et al, 2010;Chaisan et al, 2013). One µM colchicine (24 h) was best suited for Brassica napus L. polyploidization.…”

“…After 3-4 weeks, dead plants were counted. When the plants started flowering, plant ploidy level was established according to the shape of dry pollen (Anderson et al, 1991).…”

“…The developed plants were transplanted to soil in pots and were placed in a temperature-controlled growth chamber or a greenhouse. When plants started flowering (C 0 ), ploidy level was determined according to the shape of dry pollen (Anderson et al, 1991). Tetraploid inflorescences were crossed, F 1 population seeds were collected, the number of chromosomes was determined in the preparations of rootlets of emerged seeds.…”