Ak Roy

Reproductive pathways of seed development in apomictic guinea grass (Panicum maximum Jacq.) reveal uncoupling of apomixis components

One hundred and sixty accessions representing global germplasm of guinea grass (Panicum maximum Jacq.), an important apomictic (aposporous) fodder crop, were subjected to study on reproductive diversity in apomictic seed development utilizing ovule clearing and flow cytometric seed screen (FCSS). Single seed FCSS of selected 14 tetraploid and five hexaploid lines demonstrated uncoupling between the three apomixis components, viz. apomeiosis, parthenogenesis and functional endosperm development, in natural as well as experimental populations, though it differed across ploidy levels and genotypes. Reconstruction of reproductive pathways yielded a total of eight different pathways of seed development, arising by uncoupling/recombination between apomixis components. Amongst these, two pathways involving modifications in embryo-sac (ES) (presence of two polar nuclei in aposporous ES that fuse prior to fertilization) and fertilization process (fusion of only one polar nucleus in a sexual ES) have been reported for the first time. Some of the combinations, such as MI (haploids arising from parthenogenetic development of reduced egg cell), were found viable only in hexaploid background. Germplasm lines with higher expression of individual components were also identified. These components (including autonomous endosperm development) were also experimentally partitioned in hexaploid progenies (derived from a tetraploid parent viz. accession IG 04-164) that showed segregation in their reproductive capacities, and are reported for the first time. Occurrence of several apomixis recombinants (phenotypic) in guinea grass lines suggested their hybrid origin, favors a multigene model for apomixis, with their penetrance affected by modifiers and epigenetic mechanisms, in contrast to earlier reports of single locus control. Implications of partitioning components on apomixis research are discussed.

Isozyme banding pattern and estimation of genetic diversity among Guinea grass germplasm

Isozyme banding pattern was studied in Guinea grass (Panicum maximum Jacq.), a widely cultivated grass having good fodder value. Similarity among 63 accessions collected from diverse sources was worked out using five enzyme systems (SOD, GOT, ACP, Esterase and Peroxidase) following horizontal starch gel electrophoresis. Biochemical markers such as isozymes are useful supplements in identifying the genetic variation present in any crop. A total of 35 clear and unambiguous bands were used for analysis of which 8 bands were monomorphic. Polymorphism exhibited by 27 bands from all five enzyme systems indicate presence of considerable diversity in this species. The dendrogram generated after UPGMA and SAHN cluster analysis using Jaccard genetic distance showed that 63 accessions from diverse geographical locations could be grouped in three main clusters, of which two could further be divided into sub-clusters. Although the clusters comprised members from different locations, most of the accessions from similar geographical locations tended to cluster in same group.

Genetic similarity among Trifolium species based on isozyme banding pattern

The study deals with similarity among 25 species of the genus Trifolium represented by 134 accessions. Clustering of the species based on isozyme banding pattern of five enzymes revealed that T. repens was distinctly different from other species. T. repens and T. retusum formed independent clusters. The group of species comprising of T. pratense, T. cherleri, T. spumosum, T. subterraneum, T. resupinatum, T. alexandrinum, T. echinatum, T. constantinopolitanum and T. tembense exhibited considerable similarity to the second cluster. This group joined another group of five species, i.e. T. nigrescens, T. glomeratum, T. apertum, T. alpestre and T. hybridum with nearly 50% similarity. T. purpureum, T. hirtum, T. campestre, T. incarnatum, and T. argutum grouped separately. There was no marked difference for banding pattern among T. alexandrinum genotypes. T. alexandrinum showed close affinity with T. subterraneum and T. resupinatum.T. lappaceum, T. diffusum, T. campestre, T. incarnatum and T. argutum showed only 44.8% similarity with other Trifolium species. Grouping together of accessions belonging to individual species indicated that incompatibility among species under study had restricted interspecific hybridization. Species belonging to subgenus Lotoidea clustered with species of subgenus Trifolium. Chonosemium species T. campestre formed one cluster with two Trifolium species T. hirtum and T. incarnatum. T. nigrescens was placed quite apart from the T. repens.

Polymorphism and Genotype-specific Markers for Dichanthium Identified by Random Amplified Polymorphic DNA

One hundred decamer arbitrary primers were tested for PCR based amplification of seven genotypes (IG2208-S-1, IG2177, IG2180, IG2178, IG2165-S-1-1, IG2165-1 and Local-1) of an apomictic grass, Dichanthium annulatum, with the aim of screening polymorphic primers and genotype-specific markers. Out of 100 decamer primers tested, 42 produced no amplification or smeared non scorable bands, 12 amplified only single band and 46 yielded more than one polymorphic bands. Thirty-two primers out of 46 selected showed high level of polymorphism, producing 3–15 reproducible bands each for the seven Dichanthium genotypes examined. Among the total of 307 amplified fragments 222 were polymorphic, 53 bands were unique to the genotypes and 32 were monomorphic. Thus, with selected primers sufficient polymorphism could be detected to allow identification of individual genotypes. Genetic similarities of RAPD profiles generated were estimated via a coefficient of DICE and then the data were processed by cluster analysis (UPGMA). The maximum similarities between two genotypes (IG2180 and IG2178) was 58% and these two made a cluster with genotype IG2177 having similarity of only 54%. It clearly corroborated existence of high levels of polymorphism in this grass though being apomictic in nature. Primers like OPE-16, OPG-02, OPG-18, OPH-05, OPH-09, OPH-16, OPI-07 and OPF-06 found most informative as they produced specific bands pertaining to five out of seven genotypes. Polymerase chain reaction (PCR) offers a substantially simple, rapid and reliable method for identification of large number of Dichanthium genotypes once enough number of reproducible and suitable primers is screened.

Transcriptome Analysis of Differentially Expressed Genes During Embryo Sac Development in Apomeiotic Non-Parthenogenetic Interspecific Hybrid of Pennisetum glaucum

Apomixis results in the production of genetically uniform progeny, derived from the fertilization independent development (parthenogenesis) of an unreduced egg cell (apomeiosis). To identify genes involved in the apomeiosis, a comparative transcriptome analysis of differentially expressed genes during embryo sac (ES) development in a sexual Pennisetum glaucum (genotype 81A1) and its apomeiotic (aposporic) non-parthenogenetic interspecific hybrid (BC1GO) was investigated. BC1GO exhibited the partitioned apomeiosis component, whereby the second apomixis component viz., parthenogenesis was completely lacking. A total of 96 non-redundant transcripts were recovered using suppression subtractive hybridization and classified into 11 different categories according to their putative functions. Amongst the identified transcripts, many of them belonged to unknown function (40%) followed by those involved in protein metabolism, stress response, pollen/ovule/embryo development, and translation/protein modification process. A data search of transcriptional profiling in other apomictic species revealed that 75% of the differentially expressed transcripts have not been reported in previous studies. By macroarray analysis, we identified differential expression pattern of 96 transcripts, 45 (47%) of which showed ≥2-fold induction in apomeiotic BC1GO. Further, the obtained results were validated by quantitative real-time polymerase chain reaction to have a comparative expression profiling of eight selected up-regulated transcripts (≥2.5-fold) between BC1GO and 81A1 at different phases of ovule development. In silico mapping demonstrated that 13 transcripts were located onto rice chromosome 2, region syntenic with the apospory locus as reported in Brachiaria brizantha and Paspalum notatum. The expression patterns of these transcripts showed a significant difference at differentiating megaspore mother cell and gametogenesis stages thereby suggesting their involvement in floral development during apomeiotic (Panicum-type aposporous) ES development.

Estimation of Variability of Five Enzyme Systems Among Wild and Cultivated Species of Trifolium

The genus Trifolium comprises of 290 annual and perennial species of which the species such as T. repens, T. hybridum, T. pratense, T. ambiguum, T. resupinatum, T. alexandrinum are economically important. Boundaries between species in many cases have been difficult to define because of wide range of diversity caused by primary polymorphism. Hence, inter- and intraspecies variation in Trifolium, for zymogram pattern of five enzyme system was made to work out estimate of variability for isozymic banding pattern and get an insight into the species relationship. A total of 25 species represented by 134 accessions were compared for 5 enzymes viz. peroxidase, esterase, superoxide dismutase, acid phosphatase, and glutamate oxalo acetate transaminase using starch gel electrophoresis. Forty-six types of zymograms for Est isozyme pattern were observed amounting to 4.38 estimate of variability. The estimates of variability revealed maximum variation in T. resupinatum (4.24) followed with 3.02 in T. nigrescens. Estimate of variability for superoxide dismutase ranged from 0.46 to 2.67 among species amounting to 1.08 total variability across species. A total of 28 types of glutamate oxalo acetate transaminase zymograms were observed accounting for 2.48 estimate of variability. All but one band attributed to an estimate of variability of 1.43 in the genus for ACP and 16 different types of ACP zymograms were noticed. Highest variation for ACP was observed in T. resupinatum (4.53). Estimate of variation for peroxidase was 4.83 and 51 types of zymogram were observed. The species differed markedly for zymograms. The species distributed both in temperate and tropical parts like T. resupinatum had more variability as compared to cultivated species like T. alexandrinum and T. pratense. The rich variability present among these species can provide good source of gene transfer from wild to cultivated species which otherwise have no specific zymogram and exhibit low variability. The species sharing zymogram pattern for one or more enzymes with cultivated species were considered to have affinity and can further be utilized in attempting interspecific hybridization.

Generation of Interspecific Hybrids of Trifolium Using Embryo Rescue Techniques

The genus Trifolium Leguminosae (Fabaceae), commonly called clovers, includes 237-290 annual and perennial species, of which about 20 are important as cultivated and pasture crops. Taxonomic distribution supported by molecular analysis indicates that Mediterranean region is one of the main centers of distribution of the genus and also a center of domestication and breeding. Self-incompatibility is prevalent in the genus, controlled by a single, multiallelic gene expressed gametophytically in the pollen. It was suggested that hybridity did not play a major role in the evolution of the genus due to the poor crossability of the species under natural conditions. Interspecific hybridization in the genus Trifolium by conventional crossing techniques has been largely unsuccessful. Post-zygotic barriers appear to be a primary cause of the reproductive isolation, associated with endosperm disintegration and consequent abnormal differentiation and starvation of the hybrid embryo. As hybridization using conventional techniques has almost failed in Trifolium, embryo culture technique was used by breeders to obtain new combinations of interspecific hybrids. Embryo culture has been effectively used in developing interspecific hybrids in Trifolium ambiguum, T. pratense, T. montanum, T. occidentale, T. isthomocarpum, T. repens, T. nigrescens, T. uniflorum, T. sarosiense, T. alexandrinum, T. apertum, T. resupinatum, T. constantinopolitanum, T. rubens, and T. alpestre in various combinations. The successful embryo -rescue and development of hybrid plantlets requires skilled techniques of tissue culture and field practices. It includes hybridization in field; excision of hybrid embryos at appropriate stage; disinfection and culture in suitable culture media to allow maturation of embryo, multiplication of shoots, and rooting; hardening of the plantlets; inoculation with suitable Rhizobium culture; and transfer to field.

Exploiting EST databases for the development and characterisation of 3425 gene-tagged CISP markers in biofuel crop sugarcane and their transferability in cereals and orphan tropical grasses

BackgroundSugarcane is an important cash crop, providing 70% of the global raw sugar as well as raw material for biofuel production. Genetic analysis is hindered in sugarcane because of its large and complex polyploid genome and lack of sufficiently informative gene-tagged markers. Modern genomics has produced large amount of ESTs, which can be exploited to develop molecular markers based on comparative analysis with EST datasets of related crops and whole rice genome sequence, and accentuate their cross-technical functionality in orphan crops like tropical grasses.FindingsUtilising 246,180 Saccharum officinarum EST sequences vis-à-vis its comparative analysis with ESTs of sorghum and barley and the whole rice genome sequence, we have developed 3425 novel gene-tagged markers — namely, conserved-intron scanning primers (CISP) — using the web program GeMprospector. Rice orthologue annotation results indicated homology of 1096 sequences with expressed proteins, 491 with hypothetical proteins. The remaining 1838 were miscellaneous in nature. A total of 367 primer-pairs were tested in diverse panel of samples. The data indicate amplification of 41% polymorphic bands leading to 0.52 PIC and 3.50 MI with a set of sugarcane varieties and Saccharum species. In addition, a moderate technical functionality of a set of such markers with orphan tropical grasses (22%) and fodder cum cereal oat (33%) is observed.ConclusionsDeveloped gene-tagged CISP markers exhibited considerable technical functionality with varieties of sugarcane and unexplored species of tropical grasses. These markers would thus be particularly useful in identifying the economical traits in sugarcane and developing conservation strategies for orphan tropical grasses.

In vitro regeneration of Trifolium glomeratum

In vitro regeneration of Trifolium glomeratum, a leguminous forage species, was attempted through leaf, petiole, cotyledon, hypocotyl, collar and root explants and two media combinations. Root and collar explants showed no callus induction. Medium with 0.05 mg dm−3 α-naphthaleneacetic acid (NAA) and 0.10 mg dm−3 N6-benzyladenine (BA) was more effective for hypocotyl explant whereas cotyledon and petiole explant were more responsive to 5.0 mg dm−3 NAA and 1.0 mg dm−3 BA. Friable, green calli obtained from petiole explant on this medium showed organogenetic potential. Modified root-inducing medium having 0.21 mg dm−3 indole-3-acetic acid and 2.5 % sucrose was successful for root induction and plantlets were successfully transferred to field after hardening and Rhizobium inoculation.

Status of the linac booster for NSC pelletron

This paper reviews the progress made in the development of the linac booster for Nuclear Science Centre (NSC). The prototype resonator for the booster has surpassed design accelerating field. A cryostat has been fabricated to house this prototype for in beam tests. The cryogenic system has been installed and several rf modules have been constructed.