# Application of Biotechnology for Sunflower Inbred Line Development: Immature Embryo Rescue
## 4 Discussion
Minimizing risks in developing inbred lines emphasizes the importance of incorporating immature embryo rescue technology into the breeding program. Converting maintainer lines (B-lines) into male-sterile lines (A-CMS) does not pose a significant risk. Therefore, the application of CMS conversion through embryo rescue could be considered and optimized in the greenhouse. In vitro conditions and harvesting intervals influence the rate of the embryos saved, in combination with the stage of the development of embryos, reaching a significative increase in the 264 h and 312 h harvest intervals for all genotypes. Our results revealed that the rescue of immature embryos was also possible without the addition of phytohormones, with notable results after 264 h and 312 h of plant fecundation. However, the research shows a decrease in the rate of embryo rescue in the MS medium supplemented with BAP and NAA when the embryo age reached 18-30 days, with the success rate dropping below 20% for embryos at 30 days after fertilization [49]. Therefore, dormancy played a crucial role in embryo germination at this stage. However, our results indicate an increasing rescue rate, suggesting that potential dormancy can be effectively overcome through the incorporation of GA\({}_{3}\) into the MS medium [49; 56; 57]. This finding underscores the significance of GA\({}_{3}\) in enhancing embryo rescue efficiency in sunflowers [56; 57]. The combination of cytokins, auxins, and gibberellins significantly influenced the embryo rescue rate compared to the control MS medium in our experiments. On the other hand, the embryo rescue rate at 168 h (7 days) after fecundation did not demonstrate a significant success rate, with rates below 20% in all genotypes used. However, studies reflect the procedure for rescuing five-day-old immature embryos at different stages of embryonic development, successfully achieved by initially placing them in growth medium (high sucrose content--120 g/L, amino acids, vitamins, and NAA for growth stimulation, using [PERSON]'s B5 macronutrients), followed by transfer to germination medium (lower sucrose content--10-20 g/L, B5 salts, pH buffer (MES), and solidified with agar), resulting in a germination rate of 50% in some cases [49; 56]. Differences in sucrose concentrations at various stages of embryo development were also successfully managed in a medium containing 90 g/L sucrose for immature embryos aged 4-21 days [39]. Also, the source of carbohydrates influences the embryo rescue process. As reported in scientific studies, the best regeneration results were achieved with 120 g/L sucrose (46.8%), followed by 90-120 g/L glucose (33.1%). Medium supplementation with maltose was found to be inhibitory, and embryos did not respond positively [53]. Our results show that the rescue of immature embryos can be successfully implemented in breeding programs for the development of inbred lines. This approach represents faster technology, with better-established protocols, which can be effectively integrated into sunflower breeding programs with results of a 71-81% rate of immature embryo saving, allowing for 4 to 5 cycles depending on the maturity of the genotype [58]. Other biotechnological methods used in developing complete homozygous sunflower inbred lines for breeders include gynogenesis and androgenesis, which involve the production of haploid plants with a single set of chromosomes (\(n\)) [59]. Consequently, although these technologies are functional, their limited economic benefits have hindered their large-scale application in sunflower breeding. Research shows that androgenesis was achieved using a simple technique based on an MS medium supplemented with 0.5 mg/L IAA and 0.5 mg/L BAP for callus induction from anthers. Shoots were obtained using the L\({}_{4}\)M culture medium containing 30 g/L sucrose and a pH of 5.7. Callus formation was successful in over 80% of cases. For shoot formation, the calluses derived from pollen were transferred to the MS medium without hormones [59; 60].