Fig 1.
Transformation and regeneration of wheat plants from immature embryo explants.
a) Spikes were collected from wheat 14-21 days after anthesis. b) Immature embryos were dissected for use in transformation. c) Immature embryos were immersed in Agrobacterium for transformation in 50 mL centrifuge tubes. d) Coculture of immature embryos with Agrobacterium. e) After one month on callus induction medium (CI), the calli were transferred to selection medium (SM) supplemented with 0.2 mg/L IAA, where shoots and small roots appeared within 3–5 days. f) Surviving shoots were selected on 15 mg/L hygromycin. g) subsequently transferred to rooting medium. h) Regenerated plantlets were acclimatized in soil under high-humidity conditions. i) well-developed plants were maintained until seed harvest. Bar = 0.5 cm in a-b, 1 cm in c-h, and 5 cm in i.
Fig 2.
Transformation and regeneration of wheat plants from callus derived from mature embryos.
a) Callus was induced from mature embryos placed on culture medium. b) Subsequently used for transformation, the callus surface was covered with Agrobacterium suspension during transformation. c) Calli were transferred to selection medium (SM) supplemented with 0.2 mg/L IAA d) First shoots appeared within five days. e) Survived plantlets after four weeks of selection f) Surviving plantlets e were transferred to rooting medium. g) Well-developed plants in rooting medium. h) Acclimatized wheat in soil, they were maintained until seed harvest. Scale bar = 1 cm.
Fig 3.
General workflow of in planta transformation.
a, b) Germinated seedlings were injected with Agrobacterium into the plumule part. c) Following transformation, plants were maintained in the dark for at least two days to allow bacterial interaction Bar = 1 cm.
Fig 4.
Comparison of yield parameters among mutant lines generated by immature embryos, callus-derived, and in planta transformation, alongside the non-edited variety.
a) Representative spikes of mutant and non-edited plants are shown. Box plots illustrate differences in b) the number of grains per spike, c) spike length, d) grain length, and e) thousand-grain weight. The box plots were generated using ten measurements for each line, both non-edited and edited, for each phenotype. Statistical significance was determined by one-way ANOVA followed by Tukey’s HSD test (p = 0.01). Bars sharing the same letter are not significantly different, while those with different letters differ significantly. All measurements are available in S6 Table.
Fig 5.
Bar chart comparing survival and transformation efficiency across different methods.
a) represent hygromycin selection outcomes for immature embryo transformation depends on different parameters, b) for callus (from mature embryo) transformation, and c) show PCR screening results for in planta transformation. Statistical significance was determined at p = 0.01 (one-way ANOVA followed by Tukey’s HSD test). Bar charts were generated using three replicates of each optimization parameter. The raw data is available in S7 Table.
Fig 6.
Effects of hormones on regeneration efficiency and molecular screening of T-DNA integration and mutation types.
a) Bar graph comparing the effect of 0.2 mg/l IAA and 1 mg/l zeatin on regeneration from immature and mature embryo–derived calluses (three replicates) b) Number of plants screened and confirmed as transgenic using the optimized transformation methods. c) Number of plants carrying CRISPR/Cas9-induced mutations. Bars with the same letter are not significantly different from each other, while bars with different letters are significantly different according to Tukey’s HSD (p = 0.01). The data is available in S8 Table) Mutation types identified in selected T0 plants. e) Mutation types identified in selected T1 plants.
Table 1.
Comparison of optimized wheat transformation methods for gene-editing across different aspects.