Abstract
As a pioneer tropical pasture legume, stylo (Stylosanthes guianensis) is well adapted to growth-limiting factors in acid soils. Considering the importance of stylo, there is a need to improve Agrobacterium-mediated genetic transformation to enable development of elite cultivars. In this study, S. guianensis cv. RY5 was used to systematically optimize Agrobacterium-mediated transformation based on its plant regeneration. Results showed that Murashige and Skoog (MS) medium containing 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/L 6-benzylaminopurine (6-BA) was the optimal callus induction medium. MS medium supplemented with 2 mg/L 6-BA was suitable for shoot regeneration from cotyledon-derived calluses, and 0.5 mg/L indole-3-acetic acid (IAA) and 0.5 mg/L indole-3-butyric acid (IBA) applications were beneficial for rooting. The highest transformation efficiency (67%) was obtained at an Agrobacterium concentration of optical density = 0.6 combined with an infection time of 15 min and 3 days of co-cultivation. Furthermore, 200 mg/mL carbenicillin (Carb) and 0.6 mg/L Basta® supplements were effective in eliminating excess bacterial growth and selecting transgenic plants, respectively. Subsequent polymerase chain reaction (PCR) analysis confirmed that the β-glucuronidase (GUS) and BAR genes were successfully integrated into the stylo genome. Wider testing of this improved protocol as a means of enhancing genetic improvement and gene function analysis of stylo seems warranted.
Highlights
Species of Stylosanthes, referred to as stylos, are important pasture legumes and widely distributed across the tropical and subtropical areas of the Americas, Africa and Asia
The Agrobacterium-mediated genetic transformation approach is widely used for the development of transgenic plants (Cheng et al 2004)
Despite the importance of the Stylosanthes genus for pasture, only a few transgenic plants have been generated and used for gene function analysis of Stylosanthes species (Wang et al 2008; Bao et al 2016), suggesting that the existing protocols might not be efficient for the development of transgenic stylo plants
Summary
Species of Stylosanthes, referred to as stylos, are important pasture legumes and widely distributed across the tropical and subtropical areas of the Americas, Africa and Asia. The genus includes more than 40 species, such as S. guianensis (common stylo), S. hamata (Caribbean stylo), S. scabra (shrubby stylo), S. viscosa (sticky stylo) and S. seabrana (Caatinga stylo) (Chandra 2013). Considering the importance of this pasture legume, it was introduced to China from Colombia and Australia in the 1960s (Tang et al 2009). RY5, a single plant isolated from the population of its parent CIAT 184, exhibits the traits of earlier flowering, higher seed production, anthracnose resistance and cold tolerance compared with its parent, and is considered an elite stylo cultivar that is widely grown in South China (Liu et al 2001; Tang et al 2009)
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