Bacillus thuringiensis (Bt) is a gram negative soil bacterium. This bacterium secretes various proteins during different growth phases with an insecticidal potential against many economically important crop pests. One of the important families of Bt proteins is vegetative insecticidal proteins (Vip), which are secreted into the growth medium during vegetative growth. There are three subfamilies of Vip proteins. Vip1 and Vip2 heterodimer toxins have an insecticidal activity against many Coleopteran and Hemipteran pests. Vip3, the most extensively studied family of Vip toxins, is effective against Lepidopteron. Vip proteins do not share homology in sequence and binding sites with Cry proteins, but share similarities at some points in their mechanism of action. Vip3 proteins are expressed as pyramids alongside Cry proteins in crops like maize and cotton, so as to control resistant pests and delay the evolution of resistance. Biotechnological- and in silico-based analyses are promising for the generation of mutant Vip proteins with an enhanced insecticidal activity and broader spectrum of target insects.

Cotton is an important economic crop affected by different abiotic stresses at different developmental stages. Salinity limits the growth and productivity of crops worldwide. Na⁺/H⁺ antiporters play a key role during the plant development and in its tolerance to salt stress. The aim of the present study was a genome-wide characterization and expression pattern analysis under the salinity stress of the sodium-proton antiporter (NHX) of Gossypium barbadense in comparison with Gossypium hirsutum. In G. barbadense, 25 NHX genes were identified on the basis of the Na⁺_H⁺ exchanger domain. All except one of the G. barbadense NHX transporters have an Amiloride motif that is a known inhibitor of Na⁺ ions in plants. A phylogenetic analysis inferred three classes of GbNHX genes—viz., Vac (GbNHX1, 2 and 4), Endo (GbNHX6), and PM (GbNHX7). A high number of the stress-related cis-acting elements observed in promoters show their role in tolerance against abiotic stresses. The Ka/Ks values show that the majority of GbNHX genes are subjected to strong purifying selection under the course of evolution. To study the functional divergence of G. barbadense NHX transporters, the real-time gene expression was analyzed under salt stress in the root, stem, and leaf tissues. In G. barbadense, the expression was higher in the stem, while in G. hirsutum the leaf and root showed a high expression. Moreover, our results revealed that NHX2 homologues in both species have a high expression under salinity stress at higher time intervals, followed by NHX7. The protein-protein prediction study revealed that GbNHX7 is involved in the CBL-CIPK protein interaction pathway. Our study also provided valuable information explaining the molecular mechanism of Na⁺ transport for the further functional study of Gossypium NHX genes.

Summary: Hybrid crop varieties have been repeatedly demonstrated to produce significantly higher yields than their parental lines; however, the low efficiency and high cost of hybrid seed production has limited the broad exploitation of heterosis for cotton production. One option for increasing the yield of hybrid seed is to improve pollination efficiency by insect pollinators. Here, we report the molecular cloning and characterization of a semidominant gene, Beauty Mark (BM), which controls purple spot formation at the base of flower petals in the cultivated tetraploid cotton species Gossypium barbadense. BM encodes an R2R3 MYB113 transcription factor, and we demonstrate that GbBM directly targets the promoter of four flavonoid biosynthesis genes to positively regulate petal spot development. Introgression of a GbBM allele into G. hirsutum by marker‐assisted selection restored petal spot formation, which significantly increased the frequency of honeybee visits in G. hirsutum. Moreover, field tests confirmed that cotton seed yield was significantly improved in a three‐line hybrid production system that incorporated the GbBM allele. Our study thus provides a basis for the potentially broad application of this gene in improving the long‐standing problem of low seed production in elite cotton hybrid lines.

Protoplasts, which lack cell walls, are ideal research materials for genetic engineering. They are commonly employed in fusion (they can be used for more distant somatic cell fusion to obtain somatic hybrids), genetic transformation, plant regeneration, and other applications. Cotton is grown throughout the world and is the most economically important crop globally. It is therefore critical to study successful extraction and transformation efficiency of cotton protoplasts. In the present study, a cotton callus protoplast extraction method was tested to optimize the ratio of enzymes (cellulase, pectinase, macerozyme R-10, and hemicellulase) used in the procedure. The optimized ratio significantly increased the quantity and activity of protoplasts extracted. We showed that when enzyme concentrations of 1.5% cellulase and 1.5% pectinase, and either 1.5% or 0.5% macerozyme and 0.5% hemicellulase were used, one can obtain increasingly stable protoplasts. We successfully obtained fluorescent protoplasts by transiently expressing fluorescent proteins in the isolated protoplasts. The protoplasts were determined to be suitable for use in further experimental studies. We also studied the influence of plasmid concentration and transformation time on protoplast transformation efficiency. When the plasmid concentration reaches 16 µg and the transformation time is controlled within 12–16 h, the best transformation efficiency can be obtained. In summary, this study presents efficient extraction and transformation techniques for cotton protoplasts.