# Application of Biotechnology for Sunflower Inbred Line Development: Immature Embryo Rescue
## 3 Results  
### Survival Rate of Immature Embryos--Genotype Factor  
The rescue of immature embryos was successfully achieved for all genotypes, including inbred lines (G1 and G2), populations (G3 and G4), and the hybrids. Regarding the influence of the genetic factor, a significant effect (\(p<0.01\)) was observed on the regeneration capacity of plants from immature embryos (Table 2).  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
\begin{tabular}{c} 168 h, 216 h, \\ 264 h, 312 h \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table}
Table 1: Experimental design of the immature embryo rescue optimization study.  
Regarding the survival rates of immature embryos, the lowest values were recorded for the inbred lines (G1 and G2). Intermediate values were observed for the populations derived from the F2 generation (G3 and G4). In contrast, the highest values were obtained for the hybrid genotype, showing a significant difference compared to the survival rates of the other genotypes (Figure 4).  
The impact of genetic variability on the rate of immature embryo rescue highlights the importance of the genotypic factor in optimizing protocols for immature embryo rescue. The lowest survival rates observed in the inbred lines could be attributed to their higher levels of homozygosity, which are associated with a reduction in vigor and, consequently, a decrease in survival rate (Figure 4). Furthermore, hybrids exhibited a higher survival rate, most likely due to the heterosis effect, which enhances growth, vigor, and tolerance to various stresses during the in vitro culture procedures (Figure 4).  
Additionally, partial heterozygosity provides a higher survival rate compared to inbred lines, which are affected by the inbreeding effect. This could pose challenges in selection, as embryos with higher heterozygosity--and thus greater vigor--are more likely to be promoted to the next generation, potentially conflicting with the objective of inbreeding and the development of pure lines. However, the influence of the F2 population was not significantly higher than that of the inbred lines, nor significantly lower than the F1 hybrids, suggesting that this may not represent a major issue, but rather an aspect to consider when applying embryo culture techniques at different stages of sunflower breeding programs. To mitigate risks, it may be more advantageous to apply embryo culture techniques in later generations, such as F3 or F4, where uniformity, flowering period, and other morphological traits can be further evaluated under field conditions, ensuring a correlation with homozygous progenies. Nonetheless, a safer and more direct approach with minimal risk in sunflower breeding programs could be the application of embryo rescue techniques specifically for CMS conversion of B-lines.  
Figure 4: Results of the rescue rate of immature embryos—genotype factor: inbred lines (G1 and G2), populations (G3 and G4), and hybrids (G5). Tukey test was performed on all five genotypes The highest values are denoted starting with the letter “a”. Similarity between superscript letters indicates non-significant differences between the values obtained (\(p>0.05\)): LSD0.05 = 11.51, LSD0.01 = 15.13, and LSD0.001 = 19.33.