# Application of Biotechnology for Sunflower Inbred Line Development: Immature Embryo Rescue
## 3 Results
### Survival Rate of Immature Embryos--Multiple Factors Influence  
Multifactor ANOVA analysis demonstrated the significant influence of the three factors considered in the study--the type of genotype, the type of medium used, and the harvesting period of immature embryos (Table 5). According to the results, all these three factors showed a significant effect (\(p<0.001\)), emphasizing the importance of optimizing the techniques for saving immature embryos based on these three factors. The interactions between the factors were significant, which shows that the variability of the embryo rescue rate is influenced by these three factors (\(p<0.01\)) (Table 5). The results demonstrate the fact that the medium without phytohormones (MS1--0:0:0) had the lowest rescue rate of immature embryos. On the other hand, the most effective results were evidenced for the MS5--2:0:0:4:0.4 medium, demonstrating that a balanced combination of phytohormones leads to improved rescue rates of immature embryos. This aspect was observed for all analyzed time intervals, even at the 168 h interval. The interval of 168 h (7 days) after fertilization demonstrated a period with the lowest salvage rates of immature embryos, reflecting the fact that this period is not optimal for the salvage of immature embryos based on the structure and protocol presented in this study (Table 6). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Genotype**} & **Time** ** & **MS1** * & **MS2** * & **MS3** * & **MS4** * & **MS5** * \\ \cline{2-6}  & **hours** & **\%** & **\%** & **\%** & **\%** \\ \hline G1—line & 168 h & \(1\pm 0.5\)d & \(4\pm 1.0\)cd & \(5\pm 1.5\)cd & \(4\pm 1.0\)cd & \(6\pm 1.0\)cd \\ \hline G2—line & 168 h & \(1\pm 0.5\)d & \(1\pm 0.5\)d & \(3\pm 0.5\)cd & \(9\pm 0.5\)abcd & \(10\pm 1.0\)abcd \\ \hline G3—Pop & 168 h & \(1\pm 0.5\)d & \(3\pm 0.5\)cd & \(1\pm 0.5\)d & \(3\pm 0.5\)cd & \(3\pm 0.5\)cdcd \\ \hline G4—Pop & 168 h & \(4\pm 1.0\)cd & \(9\pm 1.5\)abcd & \(9\pm 1.0\)abcd & \(12\pm 1.0\)abc & \(15\pm 0.5\)ab \\ \hline G5—Hib & 168 h & \(9\pm 0.5\)abcd & \(11\pm 0.5\)abc & \(11\pm 1.0\)abc & \(12\pm 1.0\)abc & \(17\pm 1.0\)a \\ \hline G1—line & 216 h & \(5\pm 0.5\)e & \(14\pm 2.0\)de & \(12\pm 1.0\)de & \(14\pm 1.0\)de & \(23\pm 2.5\)cd \\ \hline G2—line & 216 h & \(5\pm 0.5\)e & \(9\pm 1.5\)de & \(15\pm 0.5\)de & \(17\pm 0.5\)de & \(18\pm 2.0\)de \\ \hline G3—Pop & 216 h & \(3\pm 1.5\)e & \(4\pm 1.0\)e & \(9\pm 0.5\)de & \(14\pm 1.0\)de & \(19\pm 0.5\)de \\ \hline G4—Pop & 216 h & \(6\pm 1.0\)e & \(5\pm 0.5\)e & \(9\pm 1.5\)de & \(16\pm 4.0\)de & \(27\pm 0.5\)bcd \\ \hline G5—Hib & 216 h & \(17\pm 2.5\)de & \(41\pm 1.5\)abc & \(47\pm 3.5\)ab & \(52\pm 3.0\)a & \(55\pm 1.5\)a \\ \hline G1—line & 264 h & \(23\pm 1.5\)hi & \(40\pm 1.0\)deefgh & \(45\pm 1.5\)cdefgh & \(44\pm 1.0\)cdefgh & \(49\pm 0.5\)bcefg \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Results of the in vitro conditions regarding the survival rate of immature embryos. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{1}{c}{**Source**} & **Df** & **Sum Sq** & **Mean Sq** & **\(F\)Value** & **Pr (c-F)** \\ \hline Genotype & 4 & 11,530 & 2882 & 91.158 & \textless{}2 \(\times\) 10\({}^{-16}\)*** \\ Time & 3 & 109,268 & 36,423 & 1151.883 & \textless{}2 \(\times\) 10\({}^{-16}\)*** \\ Medium & 4 & 10,210 & 2553 & 80.725 & \textless{}2 \(\times\) 10\({}^{-16}\)*** \\ Genotype–Time & 12 & 4491 & 374 & 11.836 & \(1.66\times\) 10\({}^{-14}\)*** \\ Genotype–Medium & 16 & 957 & 60 & 1.892 & 0.02966 * \\ Time–Medium & 12 & 2049 & 171 & 5.4 & \(5.44\times\) 10\({}^{-7}\)*** \\ Genotype–Time–Medium & 48 & 2810 & 59 & 1.851 & 0.00503 ** \\ Residuals & 100 & 3162 & 32 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 5: ANOVA survival rate (%) of immature embryos—multiple factors influence. The interval of 216 h (9 days) after fertilization demonstrated moderate results and effects on the survival rate of immature embryos. However, for the hybrid genotype (G5), the impact of MS5 resulted in a 55% survival rate.