Effect of kinetin, IBA, and NAA on shoot multiplication
In the present study, efforts have been made to optimize in vitro propagation protocol for M. stenopetala. Surface sterilization of seeds with 10% NaOCl solution for 25 min was effective to reduce microbial contaminants. In line with this finding, sterilization of Moringa concanensis seeds was effective for 15 min in 10% NaOCl solution . The disparity in time duration it might be due to the nature of seeds in these two different plant species. In this study, there was 100% initiation of the shoot after four weeks of the culture of the shoot tip in shoot initiation media supplemented with BAP alone. This is similar to the finding of  who got 100% initiation of shoots from stem segment explants taken from 10-day old seedlings of M. oleifera.
The present study also revealed that the effect of cytokinin in combination with auxin was compared. Thus, the application of 0.5 mg/L kinetin along with 0.01 mg/L NAA resulted in the maximum mean numbers of shoots (3.43 ± 1.41) with 7.97 ± 4.18 mean number of leaf and 0.67 ± 0.26 cm means shoot length per explant. Besides, there was a continuous decrease in the number of shoots when the concentration of NAA increased from 0.01 to 0.1 mg/L combined with kinetin. Moreover, a high cytokinin to auxin ratio favors shoot formation.
The induction of multiple shoots in M. concanensis has been previously characterized by different growth regulators. Fatima et al.  on nodal explants of M. concanensis obtained 11.00 ± 1.15 mean shoot number with 5.00 ± 1.95 cm mean length on MS medium supplemented with 0.1 mg/L kinetin along with 0.05 mg/L NAA. However, in this study, the maximum mean number of shoots per explant (3.43 ± 1.41) with a mean shoot length of 0.68 ± 0.26 cm was recorded at the concentration of 0.5 mg/L kinetin in along with 0.01 mg/L NAA. Even though the result reported by these authors is greater than the present study, the trend was similar. In both studies relatively at low concentration of NAA and high concentration of kinetin conforms to the best result. The probable difference is due to the genotype difference, source of explants, and the difference in concentration of kinetin and NAA. In addition, the performance of kinetin alone in shoot length is further supported by the sister family (Brassicaceae) of Moringa. The study in Matthiolaincana (Brassicaceae) showed that MS medium supplemented with 2.0 mg/L kinetin without NAA resulted in the best shoot length . In the present study, the medium containing 2.0 mg/L kinetin along with 0.01 NAA produced 1.23 ± 0.73 mean number of the shoot with 0.50 ± 0.21 cm means shoot length. This implies that the combined effect of auxin may inhibit the effectiveness of cytokinin in shoot development. In addition, the difference in genotype constituent and source of explant might result in a difference in the aforementioned studies.
In the present study, a combination effect of kinetin and IBA was also evaluated on shoot multiplication. Thus, shoot explants cultured on MS medium supplemented with 0.5 mg/L kinetin with 0.1 mg/L IBA exhibited both the maximum mean number shoots and leaves of 2.30 ± 1.90 and 7.73 ± 7.57, respectively per explant. The result from the combined effect of IBA and kinetin was found to vary with the concentration of both hormones in the mean number of shoot, leaf, and shoot length. When the concentration of kinetin was greater than that of IBA, relatively better result was recorded. This is due to the effect of cytokinin as it promotes the axilliary branching or axilliary bud proliferation . Although both auxin and cytokinin are usually required for growth or morphogenesis, auxin inhibits cytokinin accumulation  while cytokinin can inhibit at least some of the actions of auxin. There was a continuous decrease in the number of shoots when the concentration of IBA increased from 0.01 to 0.5 mg/L combined with kinetin. However, the combined effect of kinetin and NAA exhibited more effective as compared to the combined effect of IBA and kinetin [25, 26].
Alkhateeb  reported that M. peregrina (Forsk) tested using MS medium supplemented with 1.0 mg/L kinetin resulted in a higher mean shoot number. On the other hand, the above-mentioned author reported a significantly reduced number of leaves in micro-shoot using relatively high levels of kinetin. In this study, when the concentration of kin raised from 0.5 to 2.5 mg/L along with increasing IBA concentration from 0.01 mg/L to 0.5 mg/L lower number of shoot per explant (1.0 ± 0.54) with 0.44 ± 0.42 cm shoot length was obtained for M. stenopetala. In contrast to , the present study revealed that growth regulators free MS medium resulted in an elongated shoot length compared to MS media supplemented with growth regulator hormone. In the present study, the MS medium supplemented with 0.5 mg/L kin along with 0.1 mg/L IBA resulted in the maximum number of the shoot (2.30 ± 1.90), and the mean number of leaves (7.73 ± 7.57). This finding is in agreement with the finding of , who got a simultaneous increase number of nodes from 3.61 to 4.64 when the concentration of kinetin increased from 0.5 to 2.0 mg/l in Matthiola incana. From the aforementioned statements, type and concentration of growth regulators and species genotype seem the most important factors in in vitro shoot multiplication.
The effect of NAA and IBA on rooting
The analysis of variance revealed that the root number and root length varied significantly with half-strength MS medium supplemented with NAA, IBA, and the combination of both. The application of NAA alone exhibited the maximum mean root number per shoot as compared to IBA alone and IBA with NAA. The highest number of mean roots per shoot (1.63 ± 1.03) and mean root length (0.87 ± 1.22 cm) were obtained at 1.0 mg/L NAA and 0.1 mg/L IBA alone, respectively.
Furthermore, increasing NAA from 0.0 to 1.0 mg/L at 0.0 mg/l IBA concentration showed a significant increase in the mean number of roots per shoot from 0.26 ± 0.57 to 1.63 ± 1.03, and mean root length from 0.12 ± 0.21 to 0.84 ± 0.54 cm. However, a further increase in the concentration of NAA from 1.0 to 2.0 mg/L showed a reduction in the mean root number per shoot and mean root length from 1.63 ± 1.03 to 0.27 ± 0.45 and 0.84 ± 0.54 to 0.14 ± 0.24 cm, respectively. The same trend was observed in both treatments: concentration of IBA alone and with NAA. Increasing IBA from 0.0 to 1.0 mg/L increased both the number of roots per shoot from 0.26 ± 0.57 to 1.50 ± 0.38 and mean root length from 0.12 ± 0.21 to 0.67 ± 0.28 cm. Further increase in the concentration of IBA from 1.0 mg/L to 2.0 mg/L, mean root number was reduced from1.50 ± 0.38 to 0.07 ± 0.25 and mean root length from 0.67 ± 0.28 to 0.05 ± 0.20 cm, respectively. In agreement with the present finding,  reported the decreasing number of the root when the concentration of IBA is greater than 2.0 mg/L. Moreover, the above-mentioned authors indicated that the inhibitory effect of a high concentration of auxin on root formation is caused for such decreasing. In addition to concentration variations, the difference in the genotype of the explant can determine root formation. Therefore, based on the present finding, the optimum concentration is between1.0 and 2.0 mg/L for root formation in M. stenopetala.
The combined effect of NAA and IBA on root induction confirmed a medium effect than the effect rendered by each hormone. Increasing NAA from 0.1 to 1.0 mg/L at 0.5 mg/L IBA showed a significant increase from 0.50 ± 0.97 to 1.20 ± 1.16 in mean number of roots per shoot and from 0.13 ± 0.21 cm to 0.43 ± 0.40 cm mean root length. However, a further increase in the concentration of NAA above 1.0 mg/L by keeping IBA concentration at 0.5 mg/L reduced the mean root number from 1.20 ± 1.16 to 0.27 ± 0.58.
In the combination of 1.0 mg/L NAA with 0.5 mg/L IBA, the mean number of roots and mean root length was 1.20 ± 1.16 and 0.43 ± 0.40 cm, respectively. On the other hand, the number of roots and mean root length was 0.27 ± 0.58 and 0.09 ± 0.19 cm, respectively at 2.0 mg/L NAA and 0.5 mg/L IBA.
Stephenson and Fahey  obtained 4.7 roots per explant in 1/2 strength MS medium supplemented with 0.5 mg/L NAA in M. oleifera. In contrast, Islam et al.  reported that the hormone-free medium was ideal for rooting. In the present study, less mean root numbers (1.23 ± 1.19) was obtained at 0.5 mg/L concentration and growth regulator free medium (0.26 ± 0.57) as compared to the result reported by the aforementioned authors. In the present study, the highest mean number of root and mean root length per explant was 1.63 ± 1.03 and 0.87 ± 1.22 cm in MS medium containing 1.0 mg/L NAA and 0.1 mg/L IBA, respectively. Islam et al.  stated that medium supplemented with 1.0 and 2.0 mg/L NAA was not inducing rooting at all. In addition, these authors also stated that the medium supplemented with 0.05, 1.0 and 2.0 mg/L IBA produced 0.0, 2.0, and 2.8 mean numbers of roots per explant respectively. In this study, the first, second, and least mean root numbers were produced at concentrations of 1.0 mg/L NAA, 1.0 mg/L IBA, and 2.0 mg/L IBA, respectively. When the concentrations of IBA increased from 0.05 to 2.0 mg/L, the mean number of roots per explant also increased. The trend reported by  agreed with the present study particularly in the effect of IBA. The result of the present study indicated that when the concentration of IBA increased from 0.1 to 1.0 mg/L, the mean number of roots per explant continuously increased but further increasing of IBA concentration beyond 1.0 mg/L led to a decreasing mean number of roots per explant. In contrast with the present finding, Islam et al.  found the highest number of roots per explant at 2.0 mg/L IBA.
In agreement with the finding of  on M. peregrina (Forsk) who reported the maximum number of roots in MS medium supplemented with 1.0 mg/L IBA and MS medium containing 0.5 mg/L IBA produced longer roots than control or medium supplemented with different levels of NAA. Here, the medium supplemented with1.0 mg/L IBA alone showed the highest rate of root induction (1.50 ± 0.38) with longer roots at 0.1 mg/L IBA (0.87 ± 1.22) compared to the control medium. As results showed, using lower levels of auxin (NAA or IBA) favors higher levels of root induction and elongation. This implies that a higher concentration of growth regulators inhibits root elongation . This is might be related to ethylene deposition and poor vascular connection of the root with the stem because of callus intervention .
In an experiment for in vitro rooting of M. oleifera, when micro-shoots were cultured on a medium containing 0.5 mg/L IAA with IBA at 1.0 mg/L resulted in the highest number of root induction . These variable responses could be due to genetic differences, differences in the explant source, the concentration difference of growth regulators, and the type and/or age of explants used to establish the cultures .
About 76% of acclimatized plantlets were survived in the greenhouse. This survival rate is similar to the result of [31, 32] on M. oleifera. In a shaded greenhouse experiment, 80% of M. oleifera was survived . Saini et al.  also obtained the same result as , provided that the potted plantlets were covered with clear polythene bags and kept in a shaded greenhouse for 15 days before exposure to ambient conditions.