P. Kaushal

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.

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.

Genetic improvement of fodder legumes especially dual purpose pulses

Cultivated forage legumes and range legumes are crucial for the nutritional security for mankind as they are integral component for increased availability of animal protein and product which has higher biological value than the plant proteins. Genetic improvement programme in forages in general and forage legumes in particular could not gain due impetus probably due to the nature of the crop and also because the economically important plant part is vegetative biomass. Although crop specific objectives need to be taken care of, forage legumes have some common problems and possible strategies. In the present article, common issues as well as crop specific improvement issues are discussed. The common improvement objectives include utilization of plant genetic resource and broadening genetic base; enhancing digestive quality; tolerance to abiotic and biotic stress; quality seed production in cultivated species while specific features such as increased persistence, tolerance to abiotic stresses, easy establishment, good seeding ability and grazing tolerance among range species. Developing terminal drought tolerant, early maturing, pest tolerant and erect type Cowpea; drought tolerant dual purpose Guar; moisture stress tolerance with standing ability and harvestability, disease/pest resistance and tolerance to herbicide in field pea and forage pea; short duration, resistance to biotic stress and low β-ODAP content in grass pea; drought and biotic stress tolerance in addition to increased biomass yield in Rice bean, perennial soybean, Vicia sativa, Faba bean; improved drought tolerance, anthracnose resistance, nutritional parameters such as high methionine and regenerative potential in Stylosanthes; high tillering quick regeneration, cold and drought tolerance, diseases and pest resistance in Lucerne and broadening genetic base, improved first cut yield, root rot and stem rot resistance and dry matter content in Egyptian clover could be some of the objective in major crops.

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.

Development of an ISSR-derived SCAR marker linked to apospory in buffel grass (Cenchrus ciliaris L.)

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.