In the post−genomics era, Agrobacterium tumefaciens−mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot−regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co−cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot−regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot−regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non−transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2–1.7%. Screening among T₁ OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T−DNA insertion as a potential source of mutagenesis. The Agrobacterium−mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.

The growth and development of plants are closely tied to growth stages, such as germination, flower bud differentiation, photosynthesis, water and fertilizer use efficiency, stress resistance, etc. Previous studies on the stress resistance of plants with different leaf stages have primarily focused on single-factor environmental conditions. However, there has been a lack of systematic research on the physiology of plant seedlings under combined high-temperature and high-humidity (HH) stress, and the relationship between cucumber growth stages and HH tolerance remains unclear. In this study, we analyzed the phenotype, photosynthetic characteristics, reactive oxygen species content, and antioxidant enzyme activity of cucumber seedlings at 1-, 2-, 3-, and 4-leaf stages under control (25 °C + 80%RH, CK) and HH (42 °C + 95%RH) stress, aiming to clarify the relationship between growth stage and cucumber HH tolerance. The results indicated that the HH tolerance of cucumber seedlings increases with leaf stage. Seedlings at 1-leaf and 2-leaf stages were most sensitive to HH, whereas 4-leaf seedlings showed the greatest tolerance. Under HH stress, the biomass, chlorophyll content, net photosynthetic rate, and photosynthetic electron transfer rate were significantly reduced compared to CK. Simultaneously, there was an increase in reactive oxygen species content and antioxidant enzyme activity. The relative values for dry weight, total chlorophyll content, net photosynthetic rate, Fv/Fm, qP, ETR, and Y (II) in 1-leaf and 2-leaf seedlings were significantly lower, while ROS accumulation and changes in antioxidant enzyme activity were significantly higher compared to 4-leaf seedlings. This lays a foundation for future studies on the growth and physiological response of cucumber plants at different growth stages under varying temperature and humidity combined stresses.

Fruit rind patterns are vital commercial quality traits in melon, in which the spotted or striped fruit rinds contribute to the commercial value of melon and can directly affect the choice of consumers. Although the spotted or non-spotted fruit rind pattern was studied in several cultivated melon accessions, the inheritance and regulating locus of this trait in wild melons are still unknown. Therefore, in this study, the inheritance and regulating loci of the green spotted fruit rind in a wild melon accession XNM020 were explored with F₂ segregating populations derived from crossing between XNM020 and a cultivated melon XNM125. Segregating ratios of phenotypic data indicated that the green spotted fruit rind in XNM020 has a monogenic dominant inheritance. BSA-Seq showed that two potential genomic regions on chromosomes 4 (from 0.00 to 2.97 Mb) and 5 (from 0.00 to 2.34 Mb) regulate the formation of the green spotted fruit rind in wild melon XNM020. According to the annotations of polymorphic SNPs (single-nucleotide polymorphisms) and small InDels (insertions and deletions) in target genomic regions and the predicted gene functions, four genes MELO3C003316, MELO3C003375, MELO3C003388, and MELO3C014660 regulating chloroplast development or chlorophyll biosynthesis may be the best candidate genes. The results of this study enriched the inheritances of spotted fruit rinds in melon and also provided target genomic regions for marker-assisted selection breeding of melon focusing on fruit rinds.