Abstract
An efficient and reproducible protocol for Agrobacterium tumefaciens mediated genetic transformation was developed for kodo millet (Paspalum scrobiculatum L.) by optimizing various parameters. Agrobacterium strains EHA 105 and LBA 4404 harboring plasmids pCNL 56 and pCAMBIA 2300, respectively, provided the highest transformation efficiency. Addition of acetosyringone (AS) in infection medium (200 µM-EHA 105, 250 µM-LBA 4404) and co-cultivation medium (50 µM) increased the transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of transformed plants, respectively. The best GUS response was obtained by pretreatment of callus with an antinecrotic mixture (10 mg/L Cys + 5 mg/L Ag + 2.5 mg/L As) at infection time of 20 min followed by co-cultivation for 3 days (EHA 105) and 5 days (LBA 4404) in dark. Regenerated transgenic plants were obtained after 8 to 10 weeks of selection on callus induction medium (NAA 0.5 mg/L, BAP 1 mg/L) containing 50 mg/L Kan + 250 mg/L Cef and were rooted for 2 weeks on MS medium containing PAA (1 mg/L) and phytagel. The plantlets established in greenhouse showed normal growth. Therefore, the protocol developed in the present study can be used for development of improved varieties of kodo millet.
Highlights
They are often treated as nutri-cereals due to their rich protein and mineral profile along with high vitamin, calcium, non-starchy polysaccharide and fiber contents and a lower glycine index [6,7] and are considered as ideal components in different food formulations intended for specific target groups [8,9]
5 different varieties of Paspalum scrobiculatum L. were screened for their callus regeneration potential
Callus induction from immature seeds was observed on MS medium supplemented with 3 mg/L 2,4-D (Figure 2a)
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Millet is a generic term used for unrelated small seeded forage grass cereals with coarse grains consisting of 10 genera, and are used for food, feed and forage worldwide [1,2]. Millets serve as excellent alternatives for cereals due to having an equivalent nutritional level, with the advantage of growing with minimum inputs [4,5] They are often treated as nutri-cereals due to their rich protein and mineral profile along with high vitamin, calcium, non-starchy polysaccharide and fiber contents and a lower glycine index [6,7] and are considered as ideal components in different food formulations intended for specific target groups [8,9]. A successful and reproducible protocol for in vitro regeneration and multiplication of Paspalum scrobiculatum has already been reported in our previous study [3] In this current study, an efficient protocol for Agrobacterium mediated genetic transformation in kodo millet was developed. The present study is the first report of genetic transformation of such an important millet
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