Twenty-four years lucerne (Medicago sativa L.) breeder seed production in India: a retrospective study.

Lucerne (Medicago sativa L.) is the second most significant winter leguminous fodder crop after berseem in India. Breeder seed (BS) is the first stage of the seed production chain, as it is the base material for producing foundation and certified seeds. In India, lucerne BS demand has been reduced by 85.58% during the last 24 years (1998-1999 to 2021-2022), declining from 2150 kg to 310 kg. Out of 14 varieties released and notified so far, only nine varieties entered the seed chain since 1998-1999. It shows narrow varietal diversification and, hence, needs robust breeding programs towards enriching genetic variability and varietal development. The present study also highlights the disparity in BS demand and production over the years and puts forth the possible reasons behind the reduction in BS demand and production in the country. Out of the nine varieties, the BS demand of Anand-2 (53.11%) was highest, followed by Type-9 (19.44%) and RL-88 (13.60%). Varietal replacement rate (VRR) was found to be moderate, i.e., 23.67% for the varieties having <5 years old age in the last 3 years (2019-2020 to 2021-2022). It has also been estimated that BS produced (233 kg) during 2021-2022 can cover the approximate area of 6,300 ha at farmers' fields in 2024-2025 if the seed chain functions 100%, effectively. The present study provides a holistic overview of lucerne BS demand and production, challenges in BS production, and the way forward to develop more varieties and surplus BS production in the country.

First report of Curvularia penniseti causing leaf blight of Bajra Napier hybrid grass in India.

Bajra Napier hybrid (Pennisetum glaucum x Pennisetum purpureum) is a perennial, high yielding grass and is widely grown for fodder in India. During August-2021, Bajra Napier hybrid germplasm line (NBN 15-2) showed severe leaf blight symptoms at ICAR-Indian Grassland and fodder research institute, Jhansi (25.527890 N, 78.5451400 E). Symptoms were initial irregular yellow spots on the leaf lamina, which later became brownish, coalesced and gave blighted appearance to the leaf surface. Disease severity recorded was 55 to 60 percent. To isolate the pathogen, 10 symptomatic leaf samples were cut into small pieces (~4 mm2), surface-sterilized with 70% ethanol for 30 seconds and rinsed with sterile water. Sterilized leaf pieces were transferred to potato dextrose agar (PDA) and incubated at 28°C for 7 days. Four similar fungal isolates (BNHCP-1 to BNHCP-4) were obtained from the affected portions. The colonies were grayish-brown with dark brown margins. Conidia were mostly clavate, elongated, straight or bent at the terminal cell, with 2-3 septa with dimensions of 17.5 to 30 µm × 10 to 12.5 µm (avg. 24 µm × 12 µm; n=40). The third cell from the base was broader and darker. These morphological characteristics were consistent with previous descriptions of Curvularia penniseti (Mitra) Boedijn (Ellis, 1971). To confirm the species, BNHCP-1 was chosen as representative isolate for further studies. Internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of isolate BNHCP-1 was amplified with primers ITS1F/ITS4R (White et al. 1990) and GDF/GDR (Templeton et al. 1992), and sequenced. The sequences were deposited in GenBank (ITS: OM073980; GAPDH: OM103702.2). BLASTn analysis showed 99.6% and 98% similarity of ITS and GAPDH gene respectively with GenBank accession numbers MH859833.1 (548 bp/550 bp) and MN688838.1 (130 bp/133 bp) of C. penniseti. A maximum-likelihood phylogenetic analysis based on concatenated sequences of ITS and GAPDH gene using MEGA X placed the isolate BNHCP-1 within a clade comprising C. penniseti. Pure culture of BNHCP-1 was deposited in National Agriculturally Important Microbial Culture Collection (NAIMCC), Maunath Bhanjan (Uttar Pradesh) with accession number NAIMCC-F-04251. For pathogenicity, root slips of Bajra Napier hybrid germplasm line NBN 15-2 were transplanted in pots (6 pots; 2 root slips per pot) and kept for fresh growth in a growth chamber at 25 0C for 21 days. Bajra-Napier hybrid plants were sprayed until runoff with conidial suspension (5 × 105 conidia/ml) made from 2-week old fungal colony grown on PDA petri dish. The pots were covered with plastic bag for 48 h to maintain humidity. Inoculated plants displayed small, brown, oval-shaped lesions within seven days on the lamina and edges of the leaf which later enlarged and gave blighted appearance to the leaf. Control plants were asymptomatic. The pathogen was re-isolated from the inoculated leaves and confirmed morphologically, fulfilling Koch's postulates. C. penniseti has been reported earlier from Pennisetum americanum, P. clandestinum, Sorghum and Triticum sp. from different parts of the world (Sivanesan, 1987). However, there is no report of C. penniseti in Bajra Napier hybrid. Thus, to the best of our knowledge, this is the first report of C. penniseti from Bajra-Napier hybrid grass in India. Further studies on economic impact of this disease on Bajra-Napier hybrid production and its presence on commercial cultivars are needed.

Phenotype study of multifoliolate leaf formation in Trifolium alexandrinum L.

BACKGROUND: The genus Trifolium is characterized by typical trifoliolate leaves. Alterations in leaf formats from trifoliolate to multifoliolate, i.e., individual plants bearing trifoliolate, quadrifoliolate, pentafoliolate or more leaflets, were previously reported among many species of the genus. The study is an attempt to develop pure pentafoliolate plants of T. alexandrinum and to understand its genetic control. METHODS: The experimental material consisted of two populations of T. alexandrinum with multifoliolate leaf expression, i.e.,interspecific hybrid progenies of T. alexandrinum with T. apertum, and T. alexandrinum genotype Penta-1. Penetrance of the multifoliolate trait was observed among multifoliolate and trifoliolate plant progenies. In vitro culture and regeneration of plantlets from the axillary buds from different plant sources was also attempted. RESULTS: The inheritance among a large number of plant progenies together with in vitro micro-propagation results did not establish a definite pattern. The multifoliolate leaf formation was of chimeric nature, i.e., more than one leaf format appearing on individual branches. Reversal to normal trifoliolate from multifoliolate was also quite common. Penetrance and expression of multifoliolate leaf formation was higher among the plants raised from multifoliolate plants. Multifoliolate and pure pentafoliolate plants were observed in the progenies of pure trifoliolate plants and vice-versa. There was an apparent increase in the pentafoliolate leaf formation frequency over the years due to targeted selection. A few progenies of the complete pentafoliolate plants in the first year were true breeding in the second year. Frequency of plantlets with multifoliolate leaf formation was also higher in in vitro axillary bud multiplication when the explant bud was excised from the multifoliolate leaf node. CONCLUSION: Number of leaflets being a discrete variable, occurrence of multifoliolate leaves on individual branches, reversal of leaf formats on branches and developing true breeding pentafoliolates were the factors leading to a hypothesis beyond normal Mendelian inheritance. Transposable elements (TEs) involved in leaf development in combination with epigenetics were probably responsible for alterations in the expression of leaflet number. Putative TE's movement owing to chromosomal rearrangements possibly resulted in homozygous pentafoliolate trait with evolutionary significance. The hypothesis provides a new insight into understanding the genetic control of this trait in T. alexandrinum and may also be useful in other Trifolium species where such observations are reported.

Partitioning Apomixis Components to Understand and Utilize Gametophytic Apomixis.

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.

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.

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.