# Application of Biotechnology for Sunflower Inbred Line Development: Immature Embryo Rescue
## 4 Discussion  
Immutance embryo saving offers substantial advantages for sunflower breeding programs and indirectly to farmers who have use of hybrids adapted to both abiotic and biotic conditions. Enhancing the combinatory ability and exploiting heterosis through the introduction of inbred lines represent a crucial aspect of sunflower breeding programs [54; 55]. However, given the increasing number of new lines and populations in the breeding programs, sustaining them economically while ensuring feasibility has become increasingly challenging. Whenever a new technology is introduced into breeding programs, it is essential to evaluate whether it can be scaled up effectively or if it should remain a specific tool for improving the breeding process [56]. Our results regarding the rate of survival of the immature embryos show a higher survival rate and root generation rate compared to the hormone-free MS1 medium (free of phytohormones). However, medium MS5--2.0:0.4:0.4 (BAP:NAA:GA3) demonstrated a stable rate of embryo saving for all the genotypes at the 312 h (13 days) harvesting interval. Thus, differences were identified both in terms of the phenotypic characteristics of the medium used and based on the time since pollination and the maturation stage of the immature embryos. Although the embryo rescue rate at 168 h (7 days) after fertilization was not significant in our study, other studies achieved immature embryo saving at 5 days using the following two-stage approach: a growth medium for the immature embryo followed by a germination medium achieving an overall rate of 32.6%, depending on the genotype [49]. Immature embryos at 5 days after fertilization were first cultured on a solid growth medium (B5 macronutrients, vitamins, amino acids, NAA, sucrose, and agar) for complete maturation. In the second step, after 1-2 weeks, embryos were transferred to a germination medium with modified sucrose (10 g/L) and agar. After 3-5 weeks, the germinated embryos with well-developed roots were acclimatized under controlled conditions [49]. However, our results indicate that it is possible to have significant results that can compensate for the elaborate procedure in only one stage, only saving embryos at 13 (312 h) days at MS5--2.0:0.4:0.4 (BAP:NAA:GA3). Other reports also show a significant result in a one stage embryo rescue procedure, in embryos of 10 days [29; 34]. Other studies by other authors demonstrate that the rescue of immature embryos was possible 10 days after fertilization in the MS medium supplemented with 20 g/L sucrose, at a pH range of 5.6-5.7, achieving notable results in inbred lines during the CMS conversion process [40]. Additionally, herbicide evaluation and immature embryo rescue were demonstrated in the MS medium, free of hormones, with 20 g/L sucrose and a pH of 6.00, according to [PERSON], [PERSON] et al. [34]. Genotypically, the results indicate that the phenomena of inbreeding (such as G1 and G2) and heterosis (such as G5) are expressed in the ability to influence the immature embryos saving rate and their normal development. This aspect may influence the selection process of immature embryos within the population (such as G3 and G4), as genotypes with a high degree of inbreeding, closer to the final breeding objectives in developing inbred lines, may sometimes be excluded in favor of better-developed embryos or more vigorously growing immature embryos. However, more vigorously growing immature embryos could also exhibit a relatively higher level of heterosis than homozygosity. This approach could drive selection over time, considering the risk of excessive heterosis in F2 generations. Although there is minimal risk, this highlights the need to alternate embryo rescue in the greenhouse with field evaluation when developing populations of new B-lines and Rf-lines. This approach ensures a better understanding of the stage of inbred line development, as well as the uniformity in flowering and the overall morphology of the plants.