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In vitro plant regeneration through somatic embryogenesis and direct shoot organogenesis in Pennisetum glaucum (L.) R. Br

In vitro plant regeneration through somatic embryogenesis and direct shoot organogenesis in Pennisetum glaucum (L.) R. Br,10.1007/s11627-009-9198-6,in

In vitro plant regeneration through somatic embryogenesis and direct shoot organogenesis in Pennisetum glaucum (L.) R. Br   (Citations: 2)
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An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.
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    • ...We have developed an efficient in vitro plant regeneration protocol through somatic embryogenesis (from seeds, shoot apices and immature inflorescences explants) and direct shoot organogenesis (from shoot apex explant) for pearl millet (Jha et al. 2009)...
    • ...Emerging shoot apices consisting of shoot apical meristem and a part of mesocotyl were excised from 3 to 4 days old seedlings and cultured on MS medium ? 17.6 lM BAP ? 30 g/l sucrose ? 8 g/l agar (pH 5.8) for multiple shoot induction (Jha et al. 2009)...
    • ...The co-cultivated shoot apices were first washed with cefotaxime solution (250 mg/l) for 5 min, then washed four times with sterile distilled water and finally blot dried and transferred onto recovery medium [MS ? 17.6 lM BA ? 30 g/l sucrose ? 8 g/l agar ? 500 mg/l cefotaxime (pH 5.8) (modified from Jha et al. 2009)] for 7 days to inhibit the growth of Agrobacterium...
    • ...(modified from Jha et al. 2009)] for 30 days, with 15 days subculture intervals, to stimulate the production of transgenic shoots...
    • ...Surviving hygromycin-resistant shoots were transferred onto pre-regeneration medium [MS ? 17.6 lM BA ? 30 g/l sucrose ? 8 g/l agar ? 250 mg/l cefotaxime (pH 5.8) (modified from Jha et al. 2009)] for 4‐6 weeks...
    • ...Surviving shoots were regenerated on regeneration medium [MS ? 30 g/l sucrose ? 8 g/l agar ? 30 mg/l hygromycin (pH 5.8) (modified from Jha et al. 2009)] for 2‐3 weeks and were rooted on rooting medium [ MS ? 0.4% w/v activated charcoal ? 15 g/l sucrose ? 8 g/l agar ? 30 mg/l hygromycin (pH 5.8) (modified from Jha et al. 2009)]...
    • ...Surviving shoots were regenerated on regeneration medium [MS ? 30 g/l sucrose ? 8 g/l agar ? 30 mg/l hygromycin (pH 5.8) (modified from Jha et al. 2009)] for 2‐3 weeks and were rooted on rooting medium [ MS ? 0.4% w/v activated charcoal ? 15 g/l sucrose ? 8 g/l agar ? 30 mg/l hygromycin (pH 5.8) (modified from Jha et al. 2009)]...
    • ...In pearl millet, direct shoot organogenesis without visible intervening callus phase was developed by us (Jha et al. 2009) using shoot apices as explants, which was a rapid and reproducible regeneration protocol...
    • ...As reported previously, a maximum of 22 shoots formed in 9 weeks on MS ? 17.6 lM BA (Jha et al. 2009)...
    • ...This kind of meristematic shoot culture resulting in the formation of multiple shoots by differentiating axillary and adventitious buds by the use of BA have been described for maize (Zhong et al. 1992), oat (Zhang et al. 1996), barley (Zhang et al. 1998), sorghum (Zhong et al. 1998), pearl millet (Devi et al. 2000; Jha et al. 2009), finger millet (Kumar et al. 2001) and wheat (Ahmad et al. 2002)...
    • ...Kinetin was also reported to induce multiple shoots in finger millet (Kumar et al. 2001) and pearl millet (Jha et al. 2009)...

    Pooja Jhaet al. Efficient Agrobacterium -mediated transformation of Pennisetum glaucum...

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