A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant.

Apomixis is a naturally occurring mode of asexual reproduction in flowering plants that results in seed formation without the involvement of meiosis or fertilization of the egg. Seeds formed on an apomictic plant contain offspring genetically identical to the maternal plant. Apomixis has significant potential for preserving hybrid vigor from one generation to the next in highly productive crop plant genotypes. Apomictic Pennisetum/Cenchrus species, members of the Poaceae (grass) family, reproduce by apospory. Apospory is characterized by apomeiosis, the formation of unreduced embryo sacs derived from nucellar cells of the ovary and, by parthenogenesis, the development of the unreduced egg into an embryo without fertilization. In Pennisetum squamulatum (L.) R.Br., apospory segregates as a single dominant locus, the apospory-specific genomic region (ASGR). In this study, we demonstrate that the PsASGR-BABY BOOM-like (PsASGR-BBML) gene is expressed in egg cells before fertilization and can induce parthenogenesis and the production of haploid offspring in transgenic sexual pearl millet. A reduction of PsASGR-BBML expression in apomictic F1 RNAi transgenic plants results in fewer visible parthenogenetic embryos and a reduction of embryo cell number compared with controls. Our results endorse a key role for PsASGR-BBML in parthenogenesis and a newly discovered role for a member of the BBM-like clade of APETALA 2 transcription factors. Induction of parthenogenesis by PsASGR-BBML will be valuable for installing parthenogenesis to synthesize apomixis in crops and will have further application for haploid induction to rapidly obtain homozygous lines for breeding.

DNA elimination in embryogenic development of Pennisetum glaucum x Pennisetum purpureum (Poaceae) hybrids.

Interspecific hybridization between Napier grass (Pennisetum purpureum), which is widely grown in Brazil for cattle forage, and pearl millet (Pennisetum glaucum) has been used as a breeding strategy for the development of improved cultivars. However, the hybrid between these two species is sterile due to its triploid condition (2n = 3x = 21 chromosomes), which hinders its use in crop breeding programs. It is known that genomic alterations result from the hybridization process. In order to measure the loss of DNA during embryo development, we used flow cytometry to estimate the nuclear DNA content of triploid and tetraploid embryos produced by interspecific hybridization between Napier grass and pearl millet. The triploid and tetraploid hybrids had a mean DNA content of 4.99-4.87 and 5.25-4.84 pg, at 10 and 30 days after pollination, respectively. The mean reduction in DNA content was higher in the tetraploid hybrids. The flow cytometry results revealed progressive genomic instability in these triploid and tetraploid hybrids, with this instability causing significant alterations in the DNA content of the hybrids.

Effect of salinity on seed germination, accumulation of proline and free amino acid in Pennisetum glaucum (L.) R. Br.

Salinity is a major threat to agriculture, plants exhibits a variety of responses to salt stress that enable them to tolerate and survive in such conditions. Salinity affects physiological and biochemical processes in plants. A short term salt stress induced physiological and biochemical response were observed in P. glaucum. The experiment was conducted to understand the influence of salinity on seed germination, proline and free amino acid accumulation in P. glaucum. It was observed that as the salt concentration increased the germination percentage decreased as compared to control as well as the root/shoot length also decreased. This suggests that salinity greatly influences the germination as well as the plant growth. The levels ofbiochemical components proline and free amino acid were measured during the salt stressed condition. The 14 days old seedlings were subjected to 4 salt treatments (50, 100, 150 and 200 mM NaCI), free proline and free amino acids was calculated at 0, 12, 24, 48, 72 and 96th hour. Proline and free amino acid content in the salt stressed tissues increased with increase in salt concentration as well as with duration of salt stress. This result suggests that proline and free amino acid acids acts as compatible solutes in P. glaucum to protect the cellular macromolecules, maintain the osmotic balance and also scavenge the free radicals under salt stressed condition.

Biolistic-mediated transformation protocols for maize and pearl millet using pre-cultured immature zygotic embryos and embryogenic tissue.

Maize (Zea mays L.) is the most important cereal food crop in sub-Saharan Africa and Latin America, and a key feed crop in Asia, whereas pearl millet (Pennisetum glaucum (L.) R. Br.) is a staple food that supplies a major proportion of calories and protein to large segments of the populations living in the semi-arid tropical regions of Africa and Asia. The limitations of biological gene transfer with Agrobacterium tumefaciens specifically related to recalcitrant cereal crops, led to the development of alternative methods of which high-velocity microprojectiles, biolistic genetic transfer is the most successful and also the most widely employed. Agrobacterium facilitated transformation is the method of choice especially for deregulation of commercial transgenic food crop products, but biolistic-mediated transformation is still valid for proof of concept and functional genomics applications. Biolistic-mediated transformation and the production of transgenic plantlets via somatic embryogenesis of two maize strains viz. Hi-II (a laboratory strain) and M37W (a South African elite white maize genotype) as well as a pearl millet strain (842B) are described in this chapter. The stages described include: (1) proliferation of immature zygotic embryos for biolistic-mediated transformation, (2) induction and maintenance of transgenic embryogenic tissue on selection medium; (3) maturation (both morphological and physiological) of transgenic somatic embryos; and (4) germination of the somatic embryos to putative transgenic primary events. Maize and pearl millet cultures were regenerated via somatic embryogenesis as they are bipolar structures that shoot and root simultaneously. The culture media described in this chapter rarely induced or regenerated plantlets via organogenesis.

Chromosome elimination by wide hybridization between Triticeae or oat plant and pearl millet: pearl millet chromosome dynamics in hybrid embryo cells.

Wide crossing is one of a number of practical methods that can be used to expand genetic variation in common wheat (Triticum aestivum). However, in crosses between wheat and distantly related species such as maize (Zea mays) and pearl millet (Pennisetum glaucum), non-wheat chromosomes are often eliminated from the hybrid during embryogenesis. In this study, we used pearl millet pollen to pollinate the pistils of a range of plants in the tribe Triticeae, as well as oat. Seven days after pollination, the dynamics of the pearl millet chromosomes in the embryos were observed using in situ hybridization, probing both the pearl millet genomic DNA and its centromere-specific repeats. In embryos from the crosses with oat, all seven of the pearl millet chromosomes were retained. However, in hybrids with the Triticeae species, chromosome elimination occurred during embryogenesis. Pearl millet chromosome showed chromosome rearrangements and non-disjunction together with micronuclei. These rearranged chromosomes and micronuclei derived from the breakage of bridges and retention of acentric fragments in anaphase, respectively. The cause of the chromosome elimination of wheat-pearl millet hybrid is not malfunction of the kinetochores binding to the spindles but the malfunction of the sister chromatids segregation at anaphase especially of chromosome arm.

Importance of seed-borne fungi of sorghum and pearl millet in Burkina Faso and their control using plant extracts.

Seed-borne fungi of sorghum and pearl millet in Burkina Faso were surveyed. A total of 188 seed samples from various locations, collected in 1989 (42) and 2002 (146), were tested, using the blotter, dry inspection and washing methods. Infection experiments were carried out with the major fungi recorded on each crop by the blotter test. Six essential oils of plants were investigated for their inhibitory activity against eight pathogenic fungi. Thirty four and 27 fungal species were found in seed samples of sorghum and pearl millet, respectively. Phoma sp. and Fusarium moniliforme infected 95 to 100% of the seed samples of both sorghum and pearl millet. Sphacelotheca sorghi and Tolyposporium ehrenbergii were encountered in respectively, 75 and 33% of seed samples of sorghum. T. penicillariae, Sclerospora graminicola and Claviceps fusiformis were present in 88, 41 and 32% of seed samples of pearl millet, respectively. Seeds inoculated with Acremonium strictum, Curvularia oryzae, F. equiseti, F. moniliforme and F. subglutinans and sown in sterilized soil, showed considerable mortality of the seedlings. Three essential oils inhibited in vitro the mycelial growth of all the fungi used by 85 to 100% and reduced significantly sorghum and pearl millet seed infection rates of Phoma sp., Fusarium sp., Curvularia sp., Colletotrichum graminicola and Exserohilum sp. Presence of many pathogenic fungi in considerable number of seed samples indicates the need of field surveys for these and other pathogens. Development of plant extracts for the control of seed-borne pathogens and public awareness on seed-borne diseases management measures for maintaining quality seed should be increased.

Uniparental chromosome elimination at mitosis and interphase in wheat and pearl millet crosses involves micronucleus formation, progressive heterochromatinization, and DNA fragmentation.

Complete uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanisms underlying selective elimination of the paternal chromosomes during the development of wheat (Triticum aestivum) x pearl millet (Pennisetum glaucum) hybrid embryos. All pearl millet chromosomes were eliminated in a random sequence between 6 and 23 d after pollination. Parental genomes were spatially separated within the hybrid nucleus, and pearl millet chromatin destined for elimination occupied peripheral interphase positions. Structural reorganization of the paternal chromosomes occurred, and mitotic behavior differed between the parental chromosomes. We provide evidence for a novel chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei is different, and heterochromatinization and DNA fragmentation of micronucleated pearl millet chromatin is the final step during haploidization.

Fertile transgenic pearl millet [ Pennisetum glaucum (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues.

Pearl millet [ Pennisetum glaucum (L.) R. Br.] is a drought-tolerant cereal crop used for grain and forage. Novel traits from outside of the gene pool could be introduced provided a reliable gene-transfer method were available. We have obtained herbicide-resistant transgenic pearl millet plants by microprojectile bombardment of embryogenic tissues with the bar gene. Embryogenic tissues derived from immature embryos, inflorescences and apical meristems from diploid and tetraploid pearl millet genotypes were used as target tissues. Transformed cells were selected in the dark on Murashige and Skoog medium supplemented with 2 mg/l 2,4-D and 15 mg/l phosphinothricin (PPT). After 3-10 weeks in the dark, herbicide-resistant somatic embryos were induced to germinate on MS medium containing 0.1 mg/l thidiazuron and 0.1 mg/l 6-benzylaminopurine. Plants were transferred to the greenhouse after they were rooted in the presence of PPT and had passed a chlorophenol red assay (the medium turned from red to yellow). Transgenic plants were recovered from bombardments using intact pAHC25 plasmid DNA, a gel-purified bar fragment, or a mixture of pAHC25 plasmid or bar fragment and a plasmid containing the enhanced green fluorescent protein ( gfp) gene (p524EGFP.1). Analyses by the polymerase chain reaction, Southern blot hybridization, GFP expression, resistance to herbicide application, and segregation of the bar and gfp genes confirmed the presence and stable integration of the foreign DNA. Transformed plants were recovered from all three explants, although transformation conditions were optimized using only the tetraploid inflorescence. Time from culture initiation to rooted transgenic plant using the tetraploid inflorescence ranged from 3-4 months. Seven independent DNA/gold precipitations were used to bombard 52 plates, 29 of which produced an average of 5.5 herbicide-resistant plants per plate. The number of herbicide-resistant plants recovered per successful bombardment ranged from one to 28 and the frequency of co-transformation with gfp ranged from 5% to 85%.