# Application of Biotechnology for Sunflower Inbred Line Development: Immature Embryo Rescue
## 3 Results
### Survival Rate of Immature Embryos--Time of Maturation After Accumulation Factor  
A key factor in the rescue of immature embryos is their developmental stage or level of maturity. The timing of harvesting after fertilization has a significant impact on the survival rate of immature embryos. (\(p<0.001\)) (Table 4).  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Source** & **Df** & **Sum Sq** & **Mean Sq** & **\(F\) Value** & **Pr (\textgreater{}F)** \\ \hline Medium Type & 4 & 10,210 & 2552.5 & 3.707 & 0.00622 ** \\ \hline Residuals & 195 & 134,267 & 688.5 & & \\ \hline \hline \end{tabular}  
Significance levels: **\(p<0.01\)**.  
\end{table}
Table 3: ANOVA survival rate (%) of immature embryos—medium type factor.  
Figure 5: Results of the rescue rate of immature embryos—medium type factor (BAP:NAA:GA\({}_{3}\)-(mg/L): MS1—0:0:0 (Control), MS2—0.5:0.05:0.05, MS3—1.0:0.1:0.1, MS4—2.0:0.2:0.2, and MS5—2.0:0.4:0.4. Tukey test was performed on all five types of medium. The highest values are denoted starting with the letter “a”. Similarity between superscript letters indicates non-significant differences between the values obtained (\(p>0.05\)): LSD\({}_{0.05}=11.57\), LSD\({}_{0.01}=15.20\), and LSD\({}_{0.001}=19.42\).  
Significant influences were observed for all genotype types based on the harvesting time of the embryos after fertilization, with a high level of significance (\(p\) < 0.05). The lowest survival rates (%) were recorded at 168 h (1-15%), followed by an increase at 216 h (3-55%). Survival rates continued to rise at 264 h, reaching values between 11 and 79%, while the highest values, ranging between 37 and 81%, were observed at 312 h.  
Additionally, the survival rate was significantly influenced by the type of medium used, emphasizing the importance of optimizing both the harvesting time and culture conditions (Figure 6).  
The results indicate that embryo survival rates are strongly influenced by their developmental stage and harvest timing, with mature embryos (312 h) showing the highest survival rates. This suggests that longer maturation periods allow embryos to accumulate nutrients and develop structural integrity, enhancing their ability to withstand in vitro culture conditions.  
However, the lower survival rates observed at 168 h highlight the vulnerability of immature embryos during the early developmental stages. These findings emphasize the importance of optimizing harvest time to balance developmental maturity with regeneration potential, particularly for genotypes requiring early rescue to preserve genetic material. These insights are critical for advancing embryo rescue techniques and improving the efficiency of in vitro breeding programs, especially for genotypes prone to reproductive barriers or delayed maturation.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Source** & **Df** & **Sum Sq** & **Mean Sq** & **F Value** & **Pr (>F)** \\ \hline Time Influence & 3 & 109,268 & 36,423 & 202.8 & \textless{}2\times 10^{-16}\)*** \\ \hline Residuals & 196 & 35,209 & 180 & \\ \hline Significance levels: ***\(p\) < 0.001. & & & & \\ \end{tabular}
\end{table}
Table 4: ANOVA survival rate (%) of immature embryos—time of maturation after foundation factor.  
Figure 6: The rescue rate of immature embryos across three genotype categories: inbred lines (G1 and G2), populations (G3 and G4), and hybrids (G5). Tukey test was performed on all intervals used. 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): LSD\({}_{0.05}\) = 5.28, LSD\({}_{0.01}\) = 6.94, and LSD\({}_{0.001}\) = 8.87.