ABSTRACT
Five simple and rapid methods for evaluation of sorghum and pearl millet transgenics resistant to herbicide phosphinothricin (used as selectable marker) were studied. For rapid in vitro selection, three assays (establishment of sensitivity curves for embryogenic calli, determination of lethal doses for seed germination, and a rapid screening of cut young leaves based on the colour change of the medium) were established. For rapid screening of transgenic progeny, effects of in vivo Basta leaf spray and dip tests were studied at three different morphological stages. For all the above assays, LD50, and LD100 values were higher for pearl millet than sorghum. However, in both the crops, genotype effect was not significant. The assays standardized in the study were found to be effective for rapid, economical and mass-scale identification and characterization of transgenic plants of sorghum and pearl millet.
Subject(s)
Aminobutyrates/pharmacology , Biological Assay/economics , Drug Resistance , Germination , Glutamate-Ammonia Ligase/antagonists & inhibitors , Herbicides/pharmacology , Pennisetum/drug effects , Plant Leaves/metabolism , Plants, Genetically Modified , Sorghum/drug effects , Transgenes/drug effectsABSTRACT
A simple, genotype-independent and efficient method for plant regeneration using shoot tip explants of pearl millet (Pennisetum glaucum) was established. Linsmaier and Skoog (LS) medium supplemented with 2,4-dichloro-phenoxyacetic acid (2.5 mg l(-1)) and kinetin (0. 2 mg l(-1)) was used for induction of embryogenic calli. Development of numerous somatic embryos was observed within 10 days after transferring onto Murashige and Skoog (MS) medium supplemented with 6-benzyl aminopurine (2 mg l(-1)) and indole 3-butyric acid (0. 5 mg l(-1)) under light (16 hr photoperiod). Histological observations confirmed the origin of somatic pro-embryoids and globular embryoids. Regenerated plants established in soil, grew normally and produced fertile seeds. RAPD analysis also revealed genetic uniformity of the regenerants. The short duration of time taken for regeneration (30-35 days) and its high frequency (78-87%) makes this system highly suitable for applications such as genetic transformation.