Characterization of terminal flowering cowpea (Vigna unguiculata (L.) Walp.) mutants obtained by induced mutagenesis digs out the loss-of-function of phosphatidylethanolamine-binding protein.

Cowpea (Vigna unguiculata (L.) Walp) is one of the major food legume crops grown extensively in arid and semi-arid regions of the world. The determinate habit of cowpea has many advantages over the indeterminate and is well adapted to modern farming systems. Mutation breeding is an active research area to develop the determinate habit of cowpea. The present study aimed to develop new determinate habit mutants with terminal flowering (TFL) in locally well-adapted genetic backgrounds. Consequently, the seeds of popular cowpea cv P152 were irradiated with doses of gamma rays (200, 250, and, 300 Gy), and the M1 populations were grown. The M2 populations were produced from the M1 progenies and selected determinate mutants (TFLCM-1 and TFLCM-2) from the M2 generation (200 Gy) were forwarded up to the M5 generation to characterize the mutants and simultaneously they were crossed with P152 to develop a MutMap population. In the M5 generation, determinate mutants (80-81 days) were characterized by evaluating the TFL growth habit, longer peduncles (30.75-31.45 cm), erect pods (160°- 200°), number of pods per cluster (4-5 nos.), and early maturity. Further, sequencing analysis of the VuTFL1 gene in the determinate mutants and MutMap population revealed a single nucleotide transversion (A-T at 1196 bp) in the fourth exon and asparagine (N) to tyrosine (Y) amino acid change at the 143rd position of phosphatidylethanolamine-binding protein (PEBP). Notably, the loss of function PEPB with a higher confidence level modification of anti-parallel beta-sheets and destabilization of the protein secondary structure was observed in the mutant lines. Quantitative real-time PCR (qRT-PCR) analysis showed that the VuTFL1 gene was downregulated at the flowering stage in TFL mutants. Collectively, the insights garnered from this study affirm the effectiveness of induced mutation in modifying the plant's ideotype. The TFL mutants developed during this investigation have the potential to serve as a valuable resource for fostering determinate traits in future cowpea breeding programs and pave the way for mechanical harvesting.

Development of ß-carotene, lysine, and tryptophan-rich maize (Zea mays) inbreds through marker-assisted gene pyramiding.

Maize (Zea mays L.) is the leading cereal crop and staple food in many parts of the world. This study aims to develop nutrient-rich maize genotypes by incorporating crtRB1 and o2 genes associated with increased ß-carotene, lysine, and tryptophan levels. UMI1200 and UMI1230, high quality maize inbreds, are well-adapted to tropical and semi-arid regions in India. However, they are deficient in ß-carotene, lysine, and tryptophan. We used the concurrent stepwise transfer of genes by marker-assisted backcross breeding (MABB) scheme to introgress crtRB1 and o2 genes. In each generation (from F1, BC1F1-BC3F1, and ICF1-ICF3), foreground and background selections were carried out using gene-linked (crtRB1 3'TE and umc1066) and genome-wide simple sequence repeats (SSR) markers. Four independent BC3F1 lines of UMI1200 × CE477 (Cross-1), UMI1200 × VQL1 (Cross-2), UMI1230 × CE477 (Cross-3), and UMI1230 × VQL1 (Cross-4) having crtRB1 and o2 genes and 87.45-88.41% of recurrent parent genome recovery (RPGR) were intercrossed to generate the ICF1-ICF3 generations. Further, these gene pyramided lines were examined for agronomic performance and the ß-carotene, lysine, and tryptophan contents. Six ICF3 lines (DBT-IC-ß1σ4-4-8-8, DBT-IC-ß1σ4-9-21-21, DBT-IC-ß1σ4-10-1-1, DBT-IC-ß2σ5-9-51-51, DBT-IC-ß2σ5-9-52-52 and DBT-IC-ß2σ5-9-53-53) possessing crtRB1 and o2 genes showed better agronomic performance (77.78-99.31% for DBT-IC-ß1σ4 population and 85.71-99.51% for DBT-IC-ß2σ5 population) like the recurrent parents and ß-carotene (14.21-14.35 µg/g for DBT-IC-ß1σ4 and 13.28-13.62 µg/g for DBT-IC-ß2σ5), lysine (0.31-0.33% for DBT-IC-ß1σ4 and 0.31-0.34% for DBT-IC-ß2σ5), and tryptophan (0.079-0.082% for DBT-IC-ß1σ4 and 0.078-0.083% for DBT-IC-ß2σ5) levels on par with that of the donor parents. In the future, these improved lines could be developed as a cultivar for various agro-climatic zones and also as good genetic materials for maize nutritional breeding programs.

Unlocking the genetic diversity of Indian turmeric (Curcuma longa L.) germplasm based on rhizome yield traits and curcuminoids.

Turmeric is an important commercial crop widely grown in Asia due to its pharmacological and nutritional value. India is the centre of turmeric diversity and many turmeric accessions have good rhizome yield, varying curcuminoids content and are well-adapted to various agro-climatic zones. In the present study, we unravel the diversity among 200 Indian turmeric accessions based on rhizome yield traits and curcuminoids content. Clustering and correlation studies were also performed to group the turmeric accessions and to observe the relationship between the traits. Results revealed the presence of large variability among turmeric accessions including the major traits such as yield (24.77 g p-1 to 667.63 g p-1), dry recovery percentage (13.42% to 29.18%), curcumin (0.41% to 2.17%), demethoxycurcumin (0.38% to 1.45%), bisdemethoxycurcumin (0.37% to 1.24%) and total curcuminoid content (1.26% to 4.55%). The superior germplasm identified for curcuminoids content were as follows; curcumin (CL 157 - 2.17% and CL 272 - 2.13%), demethoxycurcumin (CL 253 - 1.45% and CL 157 - 1.31%), bisdemethoxycurcumin (CL 216 - 1.24% and CL 57 - 1.11%) and total curcuminoid content (CL 157 - 4.55% and CL 272 - 4.37%). Clustering based on dendrogram, grouped 200 accessions into seven clusters. Among seven clusters, the maximum number of accessions were grouped into cluster II while cluster VII showed maximum mean value for majority of the traits. Correlation analysis revealed a significant relationship between the traits where the total curcuminoid content is significantly and positively correlated with the primary rhizome core diameter and length of the secondary rhizome. The selection of these particular traits may result in the identification of germplasm with high total curcuminoid content. Taken together, it is the first report on the large screening of turmeric accessions for variation in the rhizome yield traits and curcuminoids content. The genetic diversity revealed in this study could be useful for further crop improvement programs in turmeric to develop new varieties with high rhizome yield coupled with high curcuminoids content.

Improvement of a Yairipok Chujak Maize Landrace from North Eastern Himalayan Region for ß-Carotene Content through Molecular Marker-Assisted Backcross Breeding.

In the North Eastern Himalayan region (NEHR) of India, maize is an important food crop. The local people cultivate the maize landraces and consume them as food. However, these landraces are deficient in ß-carotene content. Thus, we aimed to incorporate the crtRB1 gene from UMI285ß+ into the genetic background of the NEHR maize landrace, Yairipok Chujak (CAUM66), and thereby enhance the ß-carotene content through marker-assisted backcrossing (MABC). In this regard, we backcrossed and screened BC1F1 and BC2F1 plants possessing the heterozygous allele for crtRB1 and then screened with 106 polymorphic simple sequence repeat (SSR) markers. The plants having maximum recurrent parent genome recovery (RPGR) were selected in each generation and selfed to produce BC2F2 seeds. In the BC2F2 generation, four plants (CAUM66-54-9-12-2, CAUM66-54-9-12-11, CAUM66-54-9-12-13, and CAUM66-54-9-12-24) having homozygous crtRB1-favorable allele with maximum RPGR (86.74-90.16%) were selected and advanced to BC2F3. The four selected plants were selfed to produce BC2F3 and then evaluated for agronomic traits and ß-carotene content. The agronomic performance of the four lines was similar (78.83-99.44%) to that of the recurrent parent, and ß-carotene content (7.541-8.711 µg/g) was on par with the donor parent. Our study is the first to improve the ß-carotene content in NEHR maize landrace through MABC. The newly developed lines could serve as potential resources to further develop nutrition-rich maize lines and could provide genetic stock for use in breeding programs.

Enhancing ß-Carotene Concentration in Parental Lines of CO6 Maize Hybrid Through Marker-Assisted Backcross Breeding (MABB).

Vitamin A deficiency (VAD) is a global health problem; many people around the world, especially children and pregnant women, are VAD deficient or insufficient. Maize is known as an important source of provitamin A for humans. Hence, enhancement of provitamin A carotenoids (pVAC) in maize varieties through breeding or biofortification is a good option for alleviating VAD in developing countries, especially India. So far, numerous maize hybrids have been developed in India. Among them, CO6, derived from UMI1200 × UMI1230, is a popular maize hybrid and adapted to different agro-climatic zones of India, especially Tamil Nadu, a southern state of India. However, CO6 is deficient for pVAC carotenoid ß-carotene. Thus, the objectives of this study were to increase the ß-carotene concentration in UMI1200 and UMI1230 and generate the ß-carotene enriched hybrids through marker-assisted backcross breeding (MABB). For this purpose, the maize genotype HP467-15 was used as the donor for transferring the ß-carotene gene, crtRB1, into UMI1200 and UMI1230. In the MABB scheme, we used one gene-specific marker (crtRB1 3'TE) and 214 simples sequence repeat (SSR) markers for foreground and background selection, respectively. As a result, six improved lines with recurrent parent genome recovery (RPGR) ranging from 90.24 to 92.42%, along with good agronomic performance, were generated. The ß-carotene concentration of the improved lines ranged from 7.056 to 9.232 µg/g. Furthermore, five hybrid combinations were generated using improved lines and evaluated in a comparative yield trial (CYT) and multi-location trials (MLT) along with the original hybrid CO6 and commercial hybrids. It was revealed that ACM-M13-002 was a superior hybrid with a 7.3-fold increase in ß-carotene concentration and with a comparable yield to CO6. In summary, the improved maize inbreds can be used as possible donors for the development of ß-carotene-rich cultivars in maize breeding programs and the ß-carotene enriched hybrid developed in this study will hold great promise for food and nutritional security.

Marker aided introgression of opaque 2 (o2) allele improving lysine and tryptophan in maize (Zea mays L.).

Maize is the predominant food source for the world population, but lack of lysine and tryptophan in maize endosperm cannot fulfill the nutritional requirements of humans. Hence, the improvement of lysine and tryptophan content is the ultimate goal of maize biofortification programs. In the present study, the marker-assisted backcross (MABC) breeding strategy was used to enhance the lysine and tryptophan content of the elite maize inbred line UMI1230 by introgressing opaque 2 (o2) gene from the VQL1. During the transfer of the gene into UMI1230, SSR marker umc1066 tightly associated with o2 used for foreground selection. Background recovery was estimated using 168 SSR markers. Phenotype screening for morphological traits was adopted to choose plants parallel to UMI1230. As a result, four BC2F3 improved lines (DBT5-1-14/25-5/25-8/25-8/25, DBT5-1-14/25-5/25-8/25-7/25, DBT5-1-14/25-5/25-8/25-10/25 and DBT5-1-14/25-5/25-8/25-12/25) with o2 were developed. The improved line's background genome recovery varied between 90.60 and 94.80%. Also, the improved lines had better agronomic performance along with increased lysine (0.311-0.331%) and tryptophan (0.040-0.048%) contents. In summary, the MABC breeding strategy has successfully improved the levels of lysine and tryptophan in UMI1230 without affecting agronomic performance. The improved line's hold great potential as donors in biofortification programs in maize.

Marker-Assisted Selection to Pyramid the Opaque-2 (O2) and ß-Carotene (crtRB1) Genes in Maize.

Maize is an excellent nutritional source and is consumed as a staple food in different parts of the world, including India. Developing a maize genotype with a combination of higher lysine and tryptophan, along with ß-carotene, can help alleviate the problem of protein-energy malnutrition (PEM) and vitamin A deficiency (VAD). This study is aimed at improving lysine and tryptophan content by transferring opaque-2 (o2) gene from donor HKI163 to ß-carotene-rich inbred lines viz., UMI1200ß+ and UMI1230ß+. For this purpose, F1, BC1F1, BC2F1, BC2F2, and BC2F3 plants were developed using an o2 line HKI163 and two ß-carotene-rich inbred lines, UMI1200ß+ and UMI1230ß+, as the parents. Foreground selection using the associated marker umc1066 for the o2 gene and the marker crtRB1 3'TE for the crtRB1 gene was used to select the target genes. A total of 236 simple sequence repeat (SSR) markers distributed evenly across the maize genome were employed for the background selection. To fix the crtRB1 allele in the BC1F1 stage, individual plants homozygous at the crtRB1 locus and heterozygous at the o2 locus were selected and used for backcrossing to produce BC2F1 plants. Furthermore, the selected heterozygous BC2F1 plants from both crosses were selfed to obtain the BC2F2 plants, which were then selected for the target gene and selfed to generate the BC2F3 lines. From each cross, five improved lines with homozygous marker alleles for the crtRB1 and o2 genes with a recurrent parent genome (RPG) recovery ranging from 86.75 to 91.21% in UMI1200ß+×HKI163 and 80.00 to 90.08% in UMI1230ß+×HKI163 were identified. The improved lines had good agronomic performance and possessed high lysine (0.294-0.332%), tryptophan (0.073-0.081%), and ß-carotene (6.12-7.38 µg/g) content. These improved lines can be used as genetic resources for maize improvement.

Characterization of Soybean mosaic virus resistance derived from inverted repeat-SMV-HC-Pro genes in multiple soybean cultivars.

KEY MESSAGE: Soybean mosaic virus resistance was significantly improved in multiple soybean cultivars through genetic transformation induced by inverted repeat-SMV- HC - Pro genes based on RNAi and post-transcriptional gene silencing. Here, we demonstrate Soybean mosaic virus (SMV) resistance in transgenic soybean plants. Transformation of five soybean genotypes with a construct containing inverted repeat-SMV-HC-Pro genes-induced high-level SMV resistance. Through leaf-painting assays, polymerase chain reaction (PCR) verification and LibertyLink(®) strip detection, 105 T0 and 1059 T1 plants were confirmed as transgene-positive. Southern blotting confirmed insertion of the T-DNA into the genomic DNA and revealed a low-copy integration pattern. Most T0 plants were fertile and transmitted the exogenous genes to their progenies (ratios of 3:1 or 15:1). In the T1 generation, virus resistance was evaluated visually after inoculation with SMV (strain SC3) and 441 plants were highly resistant (HR). SMV disease rating was classified on a scale with 0 = symptomless and 4 = mosaic symptoms with severe leaf curl. In the positive T1 plants, the disease rating on average was 1.42 (range 0.45-2.14) versus 3.2 (range 2-4) for the nontransformed plants. With the T2 generation, 75 transgene-positive plants were inoculated with SC3, and 57 HR plants were identified. Virus-induced seed coat mottling was eliminated in the resistant lines. Analysis of SMV levels in the plants was performed using quantitative real-time PCR and double-antibody sandwich enzyme-linked immunosorbent assays; the results revealed no virus or a gradual reduction over time in the viral content, thereby supporting the visual examination results. This is the first report demonstrating pathogen-derived resistance to SMV induced by inverted repeat-SMV-HC-Pro genes in multiple soybean cultivars. Our findings contribute positively to the study of transgenic SMV-resistance using RNA interference.