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Apomixis is a method of reproduction to generate clonal seeds and offers tremendous potential to fix heterozygosity and hybrid vigor. The process of apomictic seed development is complex and comprises three distinct components, viz., apomeiosis (leading to formation of unreduced egg cell), parthenogenesis (development of embryo without fertilization) and functional endosperm development. Recently, in many crops, these three components are reported to be uncoupled leading to their partitioning. This review provides insight into the recent status of our understanding surrounding partitioning apomixis components in gametophytic apomictic plants and research avenues that it offers to help understand the biology of apomixis. Possible consequences leading to diversity in seed developmental pathways, resources to understand apomixis, inheritance and identification of candidate gene(s) for partitioned components, as well as contribution towards creation of variability are all discussed. The potential of Panicum maximum, an aposporous crop, is also discussed as a model crop to study partitioning principle and effects. Modifications in cytogenetic status, as well as endosperm imprinting effects arising due to partitioning effects, opens up new opportunities to understand and utilize apomixis components, especially towards synthesizing apomixis in crops.

Panicum maximum (guinea grass) is a model crop for apomixis and polyploidy studies. It is predominantly tetraploid (2n = 32) and is characterized by gametophytic apomixis, Panicum-type apospory and pseudogamous endosperm development. The three components of apomixis, viz. apomeiosis, parthenogenesis and functional endosperm development, can be uncoupled in this crop. An exhaustive single progenitor-derived ploidy series comprising of 32 accessions representing 3x, 4x, 5x, 6x, 7x, 8x, 9x and 11x cytotypes was utilized in present study to understand ploidy effects on expression of apospory as well on uncoupled components in two phases of progeny formation i.e. in matured ovules (using embryo-sac analysis) and in matured self-pollinated seeds (using Flow Cytometric Seed Screen method). Rise in ploidy enhanced the formation of sexual embryo-sacs (ES) thereby increasing the frequency of facultative accessions at higher ploidy level. Our results suggested that the eventual phenotype depends on relative doses of apospory and sexual factors in the genome. Ploidy level was also found affecting the penetrance and expressivity of uncoupled apomixis components. Formation of BIII hybrids (3n) appeared to be more stabilised and less affected by the ploidy change, however, formation of M1 (1n) progenies increased with the rise in ploidy. Ploidy effects on traits such as occurrence of multiple ES, autonomous endosperm development, and twin embryos were also studied. Flexibility of guinea grass to tolerate excessive genome burden and successful formation of seeds overcoming endosperm balance number and endosperm imprinting constraints is also discussed.

†BackgroundandAims During seed fill in cereals, nutrients are symplasmically unloaded to vascular parenchyma in ovules, but thereafter nutrient transport is less certain. In Zea mays, two mechanisms of nutrient passage through the chalaza and nucellus have been hypothesized, apoplasmic and symplasmic. In a recent study, nutrients first passed non-selectively to the chalazal apoplasm and were then selectively absorbed by the nucellus before being released to the endosperm apoplasm. This study reports that the promoter of OUTER CELL LAYER3 (PSbOCL3 )f romSorghum bicolor (sorghum) directs gene expression to chalazal cells where the apoplasmic barrier is thought to form. The aims were to elucidate PSbOCL3 expression patterns in sorghum and relate them to processes of nutrient pathway development in kernels and to recognized functions of the homeodomain-leucine zipper (HD-Zip) IV transcription factor family to which the promoter belongs. †Methods PSbOCL3 was cloned and transformed into sorghum as a promoter‐GUS (b-glucuronidase) construct. Plant tissues from control and transformed plants were then stained for GUS, and kernels were cleared and characterized using differential interference contrast microscopy. †KeyResults A symplasmic disconnect between the chalaza and nucellus during seed fill is inferred by the combination of two phenomena: differentiation of a distinct nucellar epidermis adjacent to the chalaza, and lysis of GUSstained chalazal cells immediately proximal to the nucellar epidermis. Compression of the GUS-stained chalazal cells during kernel maturation produced the kernel abscission zone (closing layer). †Conclusions The results suggest that the HD-Zip IV transcription factor SbOCL3 regulates kernel nutrition and abscission. The latter is consistent with evidence that members of this transcription factor group regulate silique abscission and dehiscence in Arabidopsis thaliana. Collectively, the findings suggest that processes of floral organ abscission are conserved among angiosperms and may in some respects differ from processes of leaf abscission.

Apomixis is a mode of asexual reproduction through seeds. The apomictic process bypasses both meiosis and egg cell fertilization, producing offspring that are exact genetic replicas of the mother plant. Cenchrus ciliaris (buffelgrass) is a valuable forage grass, which reproduces through aposporous apomeiosis. We identified one sexual plant in this obligate apomictic population. The objective of this work was to identify molecular markers linked to apospory trait through bulked segregant analysis. Facultative apomictic F1 plants obtained from open pollinated sexual plant were selfed to derive F2 population. Individual F2 plants were classified through embryo sac analysis as sexual, facultative apomictic or obligate apomictic. Two bulks namely, obligate apomictic and sexual were prepared by pooling DNA of individual identified plants and used in PCR analysis. Out of 208 primers tested, 20 were found to be polymorphic. A 600 bp RAPD amplicon specific to apomictic bulk, obtained with OPF-08 primer was cloned and sequenced. Based on the sequence information, a pair of SCAR (sequence characterized amplified region) primers was designed which generated a 600 bp amplicon (named OPF08-600) only in the apomictic plants. Southern hybridization of genomic DNA of apomictic and sexual plants using the OPF08-600 fragment as probe indicated the presence of two copies of the SCAR sequence in only the apomictic plants. Gel elution of restricted DNA fragments and PCR analysis confirmed the presence of duplicated copies of the SCAR sequence in the apomictic plants.

An alloplasmic cytoplasmic male sterile (CMS) line of Brassica juncea was developed by repeated backcrossing of the sexual hybrid (Diplotaxis erucoides [multiplication] Brassica rapa) with B. juncea cv. 'Pusa Bold'. The CMS line is comparable to the B. juncea nuclear donor line for all morphological traits except male fertility. Southern and cleaved amplified polymorphic sequence (CAPS) analyses showed that the CMS line carries organellar genomes from the wild species that differ from the organellar genomes of B. juncea. Northern analysis with three mitochondrial gene probes revealed altered atp[alpha] transcription pattern associated with male sterility. The restorer of CMS (Moricandia arvensis) B. juncea was found to restore male fertility to the new CMS system. The mode of fertility restoration was found to be monogenic and gametophytic. The study identified CAPS markers for the psbB gene that could be useful in quick identification of the erucoides CMS line. Broad similarities and important differences observed at the molecular level between M. arvensis and D. erucoides CMS systems of B. juncea are discussed.

Cytoplasmic male sterility (CMS) in higher plants is encoded by the mitochondrial genome. Several evidences support the view that organelle genomes of eukaryotes are derived from ancestral prokaryotic symbioants. Hence, it is expected that techniques suitable for distinguishing prokaryotes will be useful for distinguishing organelle genomes. Repetitive sequence based polymerase chain reaction (rep-PCR) technique is widely employed to identify different bacterial strains. We, therefore, tested the utility of rep-PCR for distinguishing different CMS lines of Brassica juncea. Using just three sets of rep-PCR primers, all the six Brassica lines could be unambiguously identified. Cloning and sequencing of a unique 1.5 kb amplicon obtained with Box1AR primer and template DNA of CMS catholica (W), indicated that the amplicon is indeed derived from mitochondrial genome. Further, based on sequence information of the 1.5 kb clone, a sequence characterized amplified region (SCAR) primer capable of distinguishing CMS catholica (W) from other lines was identified. The advantages of rep-PCR over other methods for distinguishing CMS lines are discussed.

Though many tissue-specific promoters have been identified, few have been associated specifically with the angiospermous megasporangium (nucellus). In the present study the 2000-bp regulatory region upstream to the watermelon, Citrullus lanatus (Thunb.) Matsum & Nakai, gene WM403 (GenBank accession no. AF008925), which shows nucellus-specific expression, was cloned from watermelon gDNA and fused to the β-glucuronidase reporter gene (GUS). The resulting plasmid, WM403 Prom::GUS(+), which also contained NPTII, was transformed into Arabidopsis thaliana ecotype Co1-0. Seedlings were selected on kanamycin-containing medium, and transformants were confirmed by PCR. GUS assays of T(3) transformants revealed weak promoter activation in epidermal layers of the placenta and locule septum during premeiotic ovule development but strong activation in the nucellus, embryo sac and early embryo, from early embryo sac formation to early globular embryo formation. Expression in seeds was absent thereafter. These results indicate that the WM403 promoter may be useful in driving nucellus-specific gene expression in plants including candidate genes for important nucellus-specific traits such as apospory or adventitious embryony.

Cenchrus ciliaris (buffel grass) is a valuable forage grass, reproducing through aposporous apomixis. Inter Simple Sequence Repeat (ISSR) markers converted to SCAR marker linked to apospory in this crop is reported. Out of twenty-five ISSR primers tested in DNA bulks from apomictic and sexual progenies, five primers detected polymorphism. The 1.1 kb amplicon derived from the primer ISSR-856 showed co-segregation with apomictic mode of reproduction. Accordingly, a pair of SCAR (Sequence Characterized Amplified Region) primers were designed which also generated a 1.1kb amplicon (Apo-856) only in the apomictic plants. Importance of such molecular markers in genetics and breeding of C. ciliaris is discussed.

Background Genetically unreduced (2n) embryo sacs (ES) form in ovules of gametophytic apomicts, the 2n eggs of which develop into embryos parthenogenetically. In many apomicts, 2n ES form precociously during ovule development. Whether meiosis and sexual ES formation also occur precociously in facultative apomicts (capable of apomictic and sexual reproduction) has not been studied. We determined onset timing of meiosis and sexual ES formation for 569 Sorghum bicolor genotypes, many of which produced 2n ES facultatively. Results Genotype differences for onset timing of meiosis and sexual ES formation, relative to ovule development, were highly significant. A major source of variation in timing of sexual germline development was presence or absence of apomictic ES, which formed from nucellar cells (apospory) in some genotypes. Genotypes that produced these aposporous ES underwent meiosis and sexual ES formation precociously. Aposporous ES formation was most prevalent in subsp. verticilliflorum and in breeding lines of subsp. bicolor. It was uncommon in land races. Conclusions The present study adds meiosis and sexual ES formation to floral induction, apomictic ES formation, and parthenogenesis as processes observed to occur precociously in apomictic plants. The temporally diverse nature of these events suggests that an epigenetic memory of the plants' apomixis status exists throughout its life cycle, which triggers, during multiple life cycle phases, temporally distinct processes that accelerate reproduction.