BACKGROUND: Traditional breeding methods have long been employed worldwide for the evaluation and development of pepper cultivars. However, these methods necessitate multiple generations of screening, line development, evaluation, recognition, and crossing to obtain highly homozygous lines. In contrast, in vitro anther-derived microspore culture represents a rapid method to generate homozygous lines within a single generation. In the present study, we have optimized a protocol for microspore embryogenesis from anther cultures of pepper hybrids Orobelle and Bomby. RESULTS: We achieved early and successful embryo formation from both genotypes by subjecting the buds to a cold pretreatment at 4 °C for 4 days. Our optimized culture medium, comprised of MS medium supplemented with 4 mg/L NAA, 1 mg/L BAP, 0.25% activated charcoal, 2.6 g/L gelrite, 30 g/L sucrose, and 15 mg/L silver nitrate, exhibited the highest efficiency in embryo formation (1.85% and 1.46%) for Orobelle and Bomby, respectively. Furthermore, successful plant regeneration from the anther derived microspore embryos was accomplished using half-strength MS medium fortified with 2% sucrose and 0.1 mg/L 6-benzylaminopurine (BA), solidified with 2.6 g/L gelrite. The ploidy status of the microspore-derived plantlets was analyzed using flow cytometry technique. Notably, the haploid plants exhibited distinct characteristics such as reduced plant height, leaf length, leaf width, and shorter internode length when compared to their diploid counterparts derived from seeds. CONCLUSION: Our findings highlight the potential of anther culture and microspore embryogenesis as an advanced method for accelerating pepper breeding programs, enabling the rapid production of superior homozygous lines.
The haploid and doubled haploid plants serve as valuable tools for breeders due to their ability to expedite the mapping of genes of agronomic importance, as well as accelerate the breeding cycle for generation of novel hybrids and improved homogenous varieties. Successful anther/microspore culture largely depends on the use of microspores at appropriate developmental stages at the time of culture, which can be specific for each plant species and genotype. In the present study, we described the visible morphological characteristics of flower buds and anthers at different developmental stages to identify the optimal microspore stage within the anther/buds of two pepper hybrids, Indra and Lakshmi. This information enabled us to predict the suitable microspore stage for successful haploid production. To enhance the visualization of nuclei in the pepper microspores, different concentrations of FeCl3 were employed as a mordant to Carnoy's fixative I, followed by DAPI staining. A clear and distinct nucleus was observed using DAPI staining procedures in the pepper microspores when fixed in Carnoy's solution containing ferric chloride (40-90 µl) as mordant. The use of mordant thus facilitated the efficient cytological analysis of the pepper microspores. Present results indicate that, to achieve efficient haploid production, flower buds with an average length of 4.4 to 5.02 mm for the hybrid Indra and 5.15 to 5.40 mm for the hybrid Lakshmi should be utilized. Additionally, these buds should have a calyx covering approximately 80-90% of the total bud length. We observed that in such buds, microspores are in the late-uninucleate and early binucleate stage which has been reported to be the most conducive stage for androgenesis induction in pepper.
Gametogenesis, Plant , Indoles , Plant Breeding , Fixatives , Genotype , Haploidy
To address the complex challenges faced by our planet such as rapidly changing climate patterns, food and nutritional insecurities, and the escalating world population, the development of hybrid vegetable crops is imperative. Vegetable hybrids could effectively mitigate the above-mentioned fundamental challenges in numerous countries. Utilizing genetic mechanisms to create hybrids not only reduces costs but also holds significant practical implications, particularly in streamlining hybrid seed production. These mechanisms encompass self-incompatibility (SI), male sterility, and gynoecism. The present comprehensive review is primarily focused on the elucidation of fundamental processes associated with floral characteristics, the genetic regulation of floral traits, pollen biology, and development. Specific attention is given to the mechanisms for masculinizing and feminizing cucurbits to facilitate hybrid seed production as well as the hybridization approaches used in the biofortification of vegetable crops. Furthermore, this review provides valuable insights into recent biotechnological advancements and their future utilization for developing the genetic systems of major vegetable crops.
[This corrects the article DOI: 10.3389/fnut.2023.1098320.].
The intake of diets with higher sodium (Na) and lower potassium (K) has been considered a leading factor for the development of hypertension (HTN). Majority of junk, processed and packaged food have higher Na contents. To counter the effects of diet on HTN, the identification of high K/Na ratio plant-based food is needed. Among fruits and vegetables, onion could be the ideal option since it contains high K content. Keeping this in mind, 45 commercially well adapted short day Indian onion cultivars were evaluated for K and Na content and their ratio to isolate suitable cultivars to prevent HTN in the Indian population. The data suggested wide variation among the genotypes for K, Na, and K/Na ratio ranging from 490.2 ± 17.0 to 9160.0 ± 96.7 mg/kg on dry matter basis, 52.7 ± 3.0 to 458.2 ± 61.7 mg/kg on dry matter basis and 3.1 ± 0.7 to 109.5 ± 17.3, respectively. The K content was recorded as significantly highest in the yellow-coloured bulb variety "Arka Pitamber" (9160.1 ± 96.7) followed by Pusa Sona (7933.2 ± 292.8). On the other hand, minimal K was assessed in the white-coloured bulb variety "Agrifound White" (490.3 ± 17.0) followed by Udaipur Local (732.9 ± 93.4). Twelve cultivars exhibited > 7000 mg K content, while nine cultivars recorded < 1500 mg. On the contrary, Na was recorded as significantly highest in the dark-red-coloured bulbs and the lowest in white bulbs. Furthermore, it was determined that there was a more than 35-fold difference observed between the highest (109.5) and lowest (3.1) K/Na ratio in the bulbs of tested cultivars. Cluster analysis revealed three major groups comprising of 23, 13 and 9 genotypes. This information could form the base for public health, food and onion researchers to design suitable cultivars to prevent HTN as a population-wide approach. The next century is going to be food-based for the amelioration of human diseases in a sustainable way without any after-effects on the human body.
Stemphylium leaf blight, caused by Stemphylium vesicarium, is a very important fungal disease in onions since its epidemics are able to affect both the bulb yield and the seed quality. The aim of this study was to screen onion genotypes at stage I (seed to bulb) and further screen the identified resistant and susceptible genotypes at stage II (bulb to seed). One hundred and fifty-seven genotypes were screened against SLB under artificially inoculated field conditions. Results revealed a significant variation among the morphological and biochemical traits studied. Correlation studies revealed a significant and negative correlation between percent disease incidence (PDI), pseudostem width, neck thickness, and dry matter. Fifteen genotypes were identified as moderately resistant, and the rest were categorized as susceptible. Bulbs of the genotypes, identified as moderately resistant, were again screened for resistance in stage II. All the genotypes were categorized as moderately susceptible. Biochemical analysis revealed that total foliar phenol content, pyruvic acid, catalase, and peroxidase increased up to 20 days after inoculation (DAI) and thereafter declined. Protein content was highest in the initial stage and declined at 10, 20, and 30 DAI. The higher biochemical activity was observed in moderately resistant category genotypes compared with the susceptible ones. Correlation analysis showed a highly significant and negative correlation of PDI with total foliar phenol content (TFPC), pyruvic acid, catalase, peroxidase, and protein content. To conclude, it was observed that screening against SLB should be done at both the stages (stage I and Stage II) to identify resistant onion genotypes. Direction selection for genotypes with high dry matter, higher phenols, and enzymes may be an alternative pathway to select genotypes for a robust resistance breeding program.
Production of many crops, including onion, under salinity is lagging due to limited information on the physiological, biochemical and molecular mechanisms of salt stress tolerance in plants. Hence, the present study was conducted to identify salt-tolerant onion genotypes based on physiological and biochemical mechanisms associated with their differential responses. Thirty-six accessions were evaluated under control and salt stress conditions, and based on growth and bulb yield. Results revealed that plant height (6.07%), number of leaves per plant (3.07%), bulb diameter (11.38%), bulb yield per plant (31.24%), and total soluble solids (8.34%) were reduced significantly compared to control. Based on percent bulb yield reduction, seven varieties were classified as salt tolerant (with <20% yield reduction), seven as salt-sensitive (with >40% yield reduction) and the remaining as moderately tolerant (with 20 to 40% yield reduction). Finally, seven salt-tolerant and seven salt-sensitive accessions were selected for detailed study of their physiological and biochemical traits and their differential responses under salinity. High relative water content (RWC), membrane stability index (MSI), proline content (PRO), and better antioxidants such as super oxide dismutase (SOD), peroxidase (POX), catalase (CAT), and ascorbate peroxidase (APX) were observed in tolerant accessions, viz. POS35, NHRDF Red (L-28), GWO 1, POS36, NHRDF Red-4 (L-744), POS37, and POS38. Conversely, increased malondialdehyde (MDA) and hydrogen peroxide (H2O2) content, reduced activity of antioxidants, more membrane injury, and high Na+/K+ ratio were observed in sensitive accessions, viz. ALR, GJWO 3, Kalyanpur Red Round, NHRDF Red-3 (L-652), Agrifound White, and NHRDF (L-920). Stepwise regression analysis identified bulb diameter), plant height, APX, stomatal conductance (gS), POX, CAT, MDA, MSI, and bulb Na+/K+ ratio as predictor traits accounting for maximum variation in bulb yield under salinity. The identified seven salt-tolerant varieties can be used in future onion breeding programs for developing tolerant genotypes for salt-prone areas.
BACKGROUND: The cytoplasmic-genic male-sterility system has been extensively employed for the production of onion hybrids. Molecular marker-assisted characterization of the cytotypes and genotyping at the restorer-of male-fertility (Ms) locus is important for the accelerated breeding of onion hybrids. Indian onion breeding has focussed more on open-pollinated varieties than hybrids. To accelerate the breeding efforts, marker-assisted selection (MAS) plays a pivotal role. METHODS AND RESULTS: This study aimed to characterize the Indian breeding lines, varieties, hybrids, and exotic accessions for cytotype and Ms locus. For cytoplasm, cytotype markers, accD, and MKFR and for Ms locus identification, PCR markers AcPMS1 and AcSKP1 were employed. Bulk strategy to identify cytoplasm and Ms locus was tested. Sequencing of PCR products amplified by accD was also tried. Both the accD and MKFR were synonymous in cytoplasm identification except in T821 where T cytoplasm was identified. AcPMS1 was more reliable than AcSKP1 for Ms locus identification. Sequencing proved that N and T cytoplasm are identical. Bulking strategy can be used for cytotype identification but not for Ms locus. CONCLUSIONS: Indian onions have a predominance of normal (N) cytoplasm and homozygous recessive (msms) locus. This might be beneficial for hybrid development. S cytoplasm was identified in exotic varieties. For the first time, T cytoplasm has been reported from India. These findings will assist Indian onion breeders to develop MAS strategies for accelerating hybrid development programs. And for the release of onion hybrids with high productivity and uniformity.
Infertility, Male , Onions , Cytoplasm/genetics , Humans , India , Male , Plant Breeding
Bulb onion is cultivated throughout the world for consumption as vegetable and processed products. Although having high global demand and economic significance, information about genetic diversity and genomic resources is limited. This study investigated the variability of 96 accessions representing seventeen countries. Out of 145 SSR markers, 62 SSRs amplified and 15 SSRs gave consistent polymorphic bands. Fifty three alleles were detected with an average of 3.533 alleles per locus. PIC value ranged from 0.219 (ACM463) to 0.715 (ACM091). Structure and cluster analysis grouped the onion accessions into two clusters. Discriminant analysis of principal components, a tool that maximizes variation between groups while minimizing that within groups, assorted accessions into five clusters. Analysis of molecular variance revealed maximum variation within the populations than among the populations. Highest genetic differentiation (FST = 0.11045; p < 0.001) was observed between Europe and Japan populations whereas the lowest genetic differentiation (FST = 0.05714; p < 0.001) was recorded between India and Japan. Principal component analysis of morphological traits suggested two principal components cumulatively accounting for 74.4% of the total variance. First component (PC1) was positively and strongly correlated with bulbing whereas second component (PC2) had leaf colour with the highest coefficient. Clustering was not on the basis of bulb colour, bulb formation, or flowering but on the basis of geographical origin. Based on clustering, crossing of distantly related accessions can provide an insight about the hybrid vigour of these diverse accessions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01101-3.
Onion yellow dwarf virus (OYDV) belonging to the genus Potyvirus, family Potyviridae, is one of the widely distributed viruses of Allium species worldwide. It causes dwarfing, yellow striping, crinkling and flaccidity of the leaves of onion and garlic. To see the occurrence and incidence of OYDV on Allium crop, an attempt was made to develop antibody based diagnostic assay which would be useful for routine indexing and screening of the germplasm. The total RNA was isolated from the symptomatic leaves of onion and the gene encoding coat protein (CP) was cloned. The nucleotide sequencing analysis of the cloned RT-PCR product revealed ~ 774 bp amplicon (OYDV CP) and it was further cloned in pET-28a ( +) expression vector which yielded ~ 30 kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was primarily checked on SDS-PAGE and further confirmed by Western blot. The His6BP-OYDV-CP was obtained in soluble state after purification and was used to immunize New Zealand white rabbit for the production of polyclonal antibody (PAb). The produced PAb against the purified fusion protein successfully detected OYDV from onion and garlic samples at 1:2000 dilutions in indirect-enzyme linked immunosorbent assay (DAC-ELISA). Thus, this study presents first report that Histidine tag (His6BP) fusion OYDV-CP based antibody production and its successful application in identification of virus free onion and garlic genotypes.
The characterization of garlic germplasm improves its utility, despite the fact that garlic hasn't been used much in the past. Garlic has an untapped genetic pool of immense economic and medicinal value in India. Hence, using heuristic core collection approach, a core set of 46 accessions were selected from 625 Indian garlic accessions based on 13 quantitative and five qualitative traits. The statistical measures (CV per cent, CR per cent, VR per cent) were used to sort the core set using Shannon-Wiener diversity index and the Nei diversity index. In addition, the variation within the core set was tested for 18 agro-morphological and six biochemical characteristics (allicin, phenol content, pyruvic acid, protein, allyl methyl thiosulfinate (AMTHS), and methyl allyl thiosulfinate (MATHS)). Further study of the core set's molecular diversity was performed using sequence related amplified polymorphism (SRAP) markers, which revealed a wide range of diversity among the core set's accessions, with an average polymorphism efficiency (PE) of 80.59 percent, polymorphism information content (PIC) of 0.29, effective multiplex ratio (EMR) of 3.51, and marker index (MI) of 0.99. The findings of this study will be useful in identifying high-yielding, elite garlic germplasm lines with the trait of interest. Since this core set is indicative of total germplasm, these selected breeding lines will be used for genetic improvement of garlic in the future.
The Poales (includes the grasses) and Asparagales [includes onion (Allium cepa L.) and asparagus (Asparagus officinalis L.)] are the two most economically important monocot orders. The Poales are a member of the commelinoid monocots, a group of orders sister to the Asparagales. Comparative genomic analyses have revealed a high degree of synteny among the grasses; however, it is not known if this synteny extends to other major monocot groups such as the Asparagales. Although we previously reported no evidence for synteny at the recombinational level between onion and rice, microsynteny may exist across shorter genomic regions in the grasses and Asparagales. We sequenced nine asparagus BACs to reveal physically linked genic-like sequences and determined their most similar positions in the onion and rice genomes. Four of the asparagus BACs were selected using molecular markers tightly linked to the sex-determining M locus on chromosome 5 of asparagus. These BACs possessed only two putative coding regions and had long tracts of degenerated retroviral elements and transposons. Five asparagus BACs were selected after hybridization of three onion cDNAs that mapped to three different onion chromosomes. Genic-like sequences that were physically linked on the cDNA-selected BACs or genetically linked on the M-linked BACs showed significant similarities (e < -20) to expressed sequences on different rice chromosomes, revealing no evidence for microsynteny between asparagus and rice across these regions. Genic-like sequences that were linked in asparagus were used to identify highly similar (e < -20) expressed sequence tags (ESTs) of onion. These onion ESTs mapped to different onion chromosomes and no relationship was observed between physical or genetic linkages in asparagus and genetic linkages in onion. These results further indicate that synteny among grass genomes does not extend to a sister order in the monocots and that asparagus may not be an appropriate smaller genome model for plants in the Asparagales with enormous nuclear genomes.
Asparagus Plant/genetics , Onions/genetics , Oryza/genetics , Synteny , Chromosome Mapping , Chromosomes, Artificial, Bacterial , DNA, Complementary , DNA, Plant/genetics , Genome, Plant , Genomics , Sequence Analysis, DNA
