Multiple Shoot Regeneration from Shoot Tip and Nodal Explants of Rotala rotundifolia ( Buch-Ham . ex Roxb ) Koehne

Rotala rotundifolia (Buch-Ham. ex Roxb) Koehne, an aquatic plant belonging to the family Lythraceae, is used for the treatment of some diseases due to its medical and anti-microbial properties. This study presents multiple shoot regeneration from shoot tip and nodal explants of R. rotundifolia cultured on Murashige and Skoog (MS) nutrient medium containing 0.051.25 mg/L Kinetin (KIN) and 0.25 mg/L Gibberellic acid (GA3) combinations for eight weeks. At the end of the second week, shoot formations began to be observed on the explants. High shoot regeneration frequencies were determined for both explants in the culture medium. The maximum number of shoots per explant was obtained from shoot tip (38.66) and nodal (30.77) explants cultured on MS medium containing 0.25 mg/L KIN + 0.25 mg/L GA3. Whereas the minimum number of shoots per explant was determined on MS medium containing 1.25 mg/L KIN + 0.25 mg/L GA3 for both explant types. The highest shoot lengths for shoot tip (1.87 cm) and nodal (1.79 cm) explants were obtained on MS culture medium containing 0.75 mg/L KIN + 0.25 mg/L GA3 and 0.50 mg/L KIN + 0.25 mg/L GA3, respectively. For in vitro rooting of the regenerated shoots, 2 cm long cut shoots were transferred to MS medium containing 0.25 mg/L indole-3-butyric acid (IBA). The rooted shoots were then successfully acclimatized to external conditions in the aquarium environment.

Rotala rotundifolia (Buch-Ham.ex Roxb) Koehne (Lythraceae family) is an aquatic and amphibian plant of South and Southeast Asia, Japan, Africa, Australia, China, India and North America (Tan et al., 2009;Bhowmik et al., 2012).R. rotundifolia is reputed of antipyretic, detoxication, anti-swelling and diuresis properties and useful in treatments of cirrhosis ascetic fluids, gonorrhea, menstrual cramps and piles in the south of China (Zhang et al., 2011).The plant has also been used for the treatment of carbuncle, furuncle, rheumatism, and arthralgia (Tan et al., 2009).In a study to determine antioxidant and total phenolic content of 31 wetland plants, aqueous extracts of R. rotundifolia have been reported to have the highest antioxidant capacity (Ho et al., 2012).
The aim of this study is to investigate the efficient and rapid propagation from shoot tip and nodal explants of R. rotundifolia cultured on MS nutrient medium containing 0.05-1.25 mg/L KIN and 0.25 mg/L GA 3 combinations.This study may help to use protocol for isolation of pharmacologically useful components from the plant and may offer an alternative method for the mass production of R. rotundifolia in the aquarium trade industry.

Materials and Method
The plants of R. rotundifolia were obtained from the local aquarium of Konya province of Turkey.After taxonomic studies, 3-5 cm long twigs were washed under tap water for 10 minutes.Surface sterilization was performed with 20% hydrogen peroxide (H 2 O 2 ) for 10 min followed by rinsed thrice with sterilized distilled water by continuous stirring for 5 min each.After sterilization, shoot tip and nodal explants were isolated under sterile conditions and cultured on Murashige and Skoog (1962) medium (MS) devoid of growth variants (Table 1) for 2 weeks to obtain contamination free explants.Thereafter, the explants were cultured on MS medium supplemented with 3% sucrose, different concentrations (0.05, 0.25, 0.50, 0.75, 1.00 and 1.25 mg/L) of Kinetin (KIN) and 0.25 mg/L Gibberellic Acid (GA 3 ) in Magenta GA 7 vessels (Table 3).The pH of the culture media was adjusted to 5.7±1 before the autoclaving (1.2 atmospheric pressure, 120 o C for 20 min).All cultures were incubated under 16 h light photoperiod.After 8 weeks of culture, the experiment was terminated and the data were recorded for shoot regeneration and analyzed.
The regenerated shoots were rooted on agar-solidified MS rooting medium containing 0.25 mg/L indole-3-butyric acid (IBA) in Magenta GA 7 vessels.After 4 weeks of culture, the agar medium was removed carefully from the rooted plantlets without damaging the roots by washing under running tap water.Thereafter, the plants were transferred to an aquarium containing tap water and sand for acclimatization (23°C with 16 h light photoperiod).
The experiment was replicated 6 times.Statistical analysis was performed as One Way ANOVA using SPSS 16 for Windows and post hoc tests were performed using Duncan.Data given in percentages were subjected to arcsine transformation (Snedecor and Cocharan, 1967) before statistical analysis

Results and Discussion
For in vitro shoot regeneration, shoot and nodal explants of R. rotundifolia were cultured on MS medium containing combinations of 0.05-1.25 mg/L KIN and 0.25 mg/L GA 3 .At the end of two weeks, the first shoot formations were started to be observed.In the fifth week, red colorations were observed on the ends and leaves of some regenerated shoots.After eight weeks, in vitro shoot regeneration from shoot tip (Figure 1a) and nodal (Figure 1b) explants of R. rotundifolia was successfully achieved and then variance analysis was performed for shoot regeneration frequency, the mean number of shoots per explant, shoot length, and root regeneration frequency (Table 2).Similarly, the use of shoot tip or nodal explants for multiple shoot regeneration has been reported for some aquatic plants such as Mentha viridis L. (Raja and Arockiasamy, 2008), Veronica anagallis-aquatica L. (Shahzada et. al., 2011), Ludwigia palustris (L.) Ell.(Fontanili et al., 2015) Ceratophyllum demersum L. (Karatas et al., 2015;Emsen et al., 2016;Dogan et al., 2017) and Pogostemon erectus (Dalzell) Kuntze (Dogan et al., 2016).
As seen in Table 2, while there was no statistically significant difference in terms of root regeneration frequency, there was a statistically significant difference in shoot regeneration frequency, the mean number of shoots per explant and shoot length for shoot tip explants (p<0.05).In the analysis of variance of the nodal explants, a statistically significant difference was not detected for root regeneration frequency and shoot regeneration frequency, but a statistically significant difference was found in the 99% confidence interval for mean number of shoots per explant and shoot length (p<0.01).Duncan test was performed to determine the significance level of these differences (Table 3).
Mean number of shoots per explants of shoot tip and nodal explants was recorded 13.17-38.66and 13.44-30.77,respectively (Table 3).The maximum number of 38.66 and 30.77shoots per explant were obtained from shoot tip and nodal explants on MS medium supplemented with   0.25 mg/L KIN + 0.25 mg/L GA 3 , respectively.On the other hand, minimum number of shoots per explants for both explant types was determined on MS medium with 1.25 mg/L KIN + 0.25 mg/L GA 3 .The results revealed that the increase in the KIN + GA 3 combination in the MS medium had a negative effect on the number of shoots per explant.These results are in line with Bhattacharyya and Bhattacharya (2001) who cultured the shoot tip explants of Phyllanthus amarus Schum.&Thom.on MS medium containing 0.05-5.0mg/L KIN and reported a decrease in the number of shoots per explant with an increase in KIN ratio.Banerjee and Shrivastava, (2008) obtained the minimum number of 8 ± 1.86 shoots per explant of B. monnieri cultured on MS medium with 2.0 mg/L KIN.
Shoot lengths ranged from 1.03 to 1.87 cm for the shoot tip explant and from 1.13 to 1.79 cm for the nodal explant (Table 3).The highest shoot length of shoot tip (1.87 cm) was obtained on MS medium containing 0.75 mg/L KIN + 0.25 mg/L GA 3 , whereas the highest shoot length of nodal explant (1.79 cm) was obtained on MS medium supplemented with 0.50 mg/L KIN + 0.25 mg/L GA 3 .In both explant types, the shortest shoots were determined in MS medium containing 1.25 mg/L KIN + 0.25 mg/L GA 3 .Kaviani et al. (2013) reported that the longest shoots (1.20 cm) were obtained from the shoot tip explants of Matthiola incana on MS medium supplemented with 2 mg/L KIN.
Root occurrences with KIN effect were recorded on in vitro propagation medium.For both types of explants, 100% root formation was obtained on MS medium containing 0.05-0.75mg/L KIN + 0.25 mg/L GA 3 .It has been determined that high KIN doses have an adverse effect on root formation.
In spite of root formation on the propagation medium, in vitro rooting studies of regenerated shoots were carried out in MS medium containing 0.25 mg/L IBA for four weeks.In vitro rooted plantlets were successfully acclimatized to external conditions in the aquarium environment.Similarly, successful acclimatization of in vitro regenerated aquatic plants had been reported for Cryptocoryne wendtii and Cryptocoryne beckettii (Stanly et al., 2011), A. sessilis (Gnanaraj et al., 2011), and C. demersum (Dogan et al., 2015).

Figure 1 .
Figure 1.In vitro shoot regeneration of R. rotundifolia.Multiple shoot regeneration from shoot tip (a) and nodal (b) explants on MS medium containing 0.25 mg/L KIN + 0.25 mg/L GA3 after 8 weeks of culture

Table 2 .
Analysis of variance of shoot tip and nodal explants of R. rotundifolia in MS medium containing differentKIN and GA3

Source of variance Degree of freedom Shoot regeneration frequency (%) Mean number of shoots per explant Shoot length (cm) Root regeneration frequency (%)
**Significant at p <0.01 level; is: Insignificant

Table 3 .
Effect of different combinations of KIN and GA3 on multiple shoot regeneration from shoot tip and nodal explants of R. rotundifolia after eight weeks of culture Values followed by different small letters in the same column differ significantly at p < 0.01 **Values followed by different small letters in the same column differ significantly at p < 0.05 is: Insignificant *