A study on the formation and development of Panax bipinnatifidus Seem. adventitious root

This study aimed to generate adventitious roots from root and stem-derived callus of Panax bipinnatifidus Seem. Callus formation was observed best (83.3%) in MS media supplemented with 3.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/L indolebutyric acid (IBA). Callus generated adventitious roots in MS media supplemented with 2.0 mg/L IBA at high frequency (66.7%). The optimal culture condition for the development of adventitious roots was liquid MS media supplemented with 40 mg/L sucrose and maintained at pH 6.0.

Adventitious roots are non-transgenic, proliferating through plant growth regulators supplemented in the media.Adventitious roots were rapidly proliferated and accumulated high concentrations of secondary metabolites (Hahn et al., 2003;Yu et al., 2005).This method could be used to increase the accumulation of secondary  metabolites in plants.In this research, we have generated adventitious roots from callus of P. bipinnatifidus Seem.and investigated effects culture conditions on development of adventitious roots.

Induction of adventitious roots
The roots and stems of P. bipinnatifidus were collected in Sapa, Laocai, Vietnam and were taxonomically identified by botanist Ngo Van Trai (Institute of Medicinal Materials, Hanoi, Vietnam).Explants were washed under running water and soap, then washed again with sterilized distilled water.Explants were then treated with Hypochlorite Na 50% (v/v) solution for 25 min and washed with sterilized distilled water.Explants were then soaked in cefotaxim (250 ppm) for 24 h, and washed with sterilized distilled water.Explants were cut into pieces and cultured on MS media (Murashige and Skoog, 1962).Explants were cut into 0.1-0.3cm length and cultured on MS medium suplemented with 30 g/L sucrose, 8 g/L agar, pH 5.8-5.9 and 2,4-dichlorophenoxyacetic acid (2,4-D) (2 and 3 mg/L), indolebutyric acid (IBA) (0.1, 0.3, 0.5 mg/L), NAA (0.1, 0.3 and 0.5 mg/L).The callus induction frequency and percentage of callus were recorded after 8 weeks of culture.Callus were isolated and subcultured on MS media supplemented with 30 g/l sucrose, 8 g/l agar and IBA (0.5, 1.0, 1.5, 2.5 mg/L), NAA (0.5, 1, 1.5, 2, 3 mg/L), with pH adjusted to 5.8.Observations were made after 8 weeks of culture by percentage of explants producing adventitious roots.Cultures were maintained at 25±2°C in the dark.

Effects of culture conditions on the adventitious root
Adventitious roots were harvested from callus cultures and cut into 1 g and was cultured on different media including MS, Chu (N6) Medium and Gamborg's B5; supplemented with 30 g/l sucrose, 8 g/l agar, pH 5.8-5.9 and 2.0 mg/L IBA.Adventitious roots were culture selected media at three status: liquid (0 g/l agar, shaking at 80 rpm, semi-solid (4 g/L agar) and solid (8 g/L agar).Fresh weight and dry weight of cultured roots were recorded after four to eight weeks culture.Cultures were maintained at 25± 2°C in the dark.

Effects of pH, sugar content and mineral proportions on the adventitious root
Adventitious roots was cultured on media with different pH (5.5; 6.0; 6.5; 7.0 and 7.5), sucrose content (20, 30, 40, 50, 60 and 70 g/l), mineral proportions (25, 50, 75 and 100% when compared with the basic media).Fresh weight and dry weight of cultured roots were recorded after 4-8 weeks culture.The experiments were carried out in dark condition, at 25± 2°C, shaking at 80 rpm for liquid media.

Statistics
Experiments were repeated three times.Experimental data were processed by Duncan test (p<0.05,for experiment ≥ 6 treatments) or t test (for experiment with less than 6 treatments) by SAS program (ver.6.12).

Callus induction
After sterilization, explants were cut into small segments and cultured on MS media without plant growth regulators.The percentage of successfully sterilized explants was 65.2% after 14 days of culture (Figures 1 and 2).
Sterilized P. bipinnatifidus explants were cut into 0.1-0.3cm segments and cultured on media for callus induction.Results are shown in Table 1, Figures 3 and 4.
Results from ANOVA table show that there is no significant difference between the effects of IBA and NAA on the callus induction efficiency (P>0.001);there was a significant difference of the D treatment (2,4-D) (F=127.6 với p<0,001), indicating that 2,4 D played a main role in callus induction capacity of P. bipinnatifidus, as in Panax ginseng roots (Thành and Yoeup, 2008).
Results indicate that the treatment C4 (2,4-D 3.0 and IBA 0.1 mg/l) induced the highest number of 83.3%.

Adventitious root induction from callus
The results showed (Figure 5 and Table 2), RI 4 and RI5 adventitious rooting sample rates are the same, RI 5 treatment had higher adventitious rooting average number       and Paek, 2008).

Effects of different media on P. bipinnatifidus on adventitious root development
Adventitious root was cultured on three different media (MS, N6, B5) supplemented with 2 mg/L IBA.Fresh weight and dry weight of root cultures were recorded after 6 weeks culture (Table 3 and Figure 6).The experiments indicated that MS media gave the best results for adventitious root development.Similar results were obtained when culturing Astragalus membranaceus adventitious roots (Thwe et al., 2012), Vernonia amygdalina (Khalafalla et al., 2009) and Bupleurum falcatum (Kusakari et al., 2000).According to   The average score with different letters are significantly different at p = 0.01 level.Letters a, b, c, d, e and f in the same column represents the differences among treatments by t tests (LSD).Amzallag et al. (1992), the mineral content affected root development through the metabolism of plant growth regulators.

Effects of the physiscal states of the media on P. bipinnatifidus adventitious root development
Adventitious root was cultured on MS media at different physical states: solid (8 g/L agar), semi-solid (4 g/L agar) and liquid (0 g/L agar; shaked at 80 rpm), supplemented with 2 mg/L IBA.Fresh weight and dry weight of cultured roots were recorded after six weeks of culture (Table 4 and Figure 7).Results show there was an effect from selected media on the adventitious root development of P. bipinnatifidus, particularly adventitious roots cultured on media without agar performed better as compared to those cultured on media supplemented with agar according to its fresh and dry weight (Table 4).The good contact of roots in the liquid medium could be the reason.

Effects of pH on P. bipinnatifidus adventitious root development
Adventitious roots (1 g/75 ml media) were cultured on liquid MS with pH 5.5, 6.0, 6.5, 7.0 or 7.5, supplemened with 2 mg/l IBA, in dark condition, 25±2°C, shaked at 80 rpm.Fresh weight and dry weight were recorded after 6 weeks of culture (Table 5 and Figure 8).
Results from   the root development through an ion exchange process through the membrane.According to "developing hypotheses about the acid", the size of cells were manipulated by its environment's pH, where it decreases with decreased pH (Cosgrove, 1999).Winch and Pritchard (1999), Evans (1976) and Edwards and Scott (1974) concluded that environemtal pH induced root elongation.The effects of pH on the development of adventitious roots were proved by Ling et al. ( 2009) while culturing Orthosiphon stamineus roots.pH 6.0 was the best among pH 4.0; 5.0; 5.8 and 7.0 and pH 6.0 was also appropriate for the adventitious roots development and saponin accumulation in P. ginseng (Kim et al., 2005).

Effects of sugar content on P. bipinnatifidus adventitious root development
Adventitious roots was cultured on liquid MS media, with different sucrose concentration (20, 30, 40, 50, 60 and 70 g/L), supplemented with 2 mg/L IBA at pH 6.0 in dark condition, 25±2°C and shaked at 80 rpm.Fresh weight and dry weight of root cultures were recorded after 6 weeks of culture (Table 6 and Figure 9).The results (Table 6) show that culture media supplemented with 40 g/L sucrose were optimal for P. bipinnatifidus adventitious roots development.Carbohydrate was essential for plant metabolism in vitro, therefore sugar content in the media had a crucial impact on the root induction and development.Sugar plays an important role in the regulation and expression of the transcription of photosynthesis genes (Sheen, 1990) and also in signals of abscisic acid and ethylene (Leon and Sheen, 2003).Cheng et al. (1992) reported sucrose concentration at 2-3% positively affected the root induction of Eucalyptus sideroxylon.Moreover, sugar concentration was reported to have effects on adventitious roots induction of O. stamineus and Scopoliaparviflora at concentration of 30 and 50%, respec-tively (Ling et al., 2009;Min, 2007).Echinacea angustifolia adventitious roots were cultured on bioreactor on MS media supplemented with 2 mg/l IBA and 50 g/L sucrose.The initial sugar concentration (50 g/L) gave the best result in the mass development and saponin content of P. ginseng adventitious roots (Thành and Yoeup, 2008).

Conclusion
This study showed the possibilities of P. bipinnatifidus adventitious root development from callus.Results indicated variable effects on root development, forming the basis for further investigations of culturing P. bipinnatifidus adventitious roots to collect valuable metabolites.
The average score with different letters are significantly different at p = 0.01 level.Letters a, b, c, d, e and f in the same column represent the differences among treatments by Duncan test.

Figure 3 .
Figure 3. Callus induced on C 4 and C 10 medium.

Figure 6 .
Figure 6.The adventitious root development of P. Bipinnatifidus on different media.

Figure 7 .
Figure 7. Effects of the physical states of media on P. Bipinnatifidus adventitious root development.

Figure 8 .
Figure 8. Effects of pH on the development of P. bipinnatifidus adventitious roots.

Figure 9 .
Figure 9. Effects of the sugar content on the root development of P. bipinnatifidus.

Table 1 .
Effects of plant growth regulators on callus induction.

Table 2 .
Effects of plant growth regulators on callus induction.

Roots that induced callus (%) Number of roots/explant Root length (cm)
abcThe average score with different letters are significantly different at p = 0.01 level.Letters a, b, c, d, e and f in the same column represent the differences among treatments by Duncan test.

Table 3 .
Effects of different media on adventitious root development.

Table 5 .
Effects of pH.sucrose on P. bipinnatifidus adventitious root development.
Table 5 indicate that P. bipinnatifidus adventitious root developed best at pH 6.0.pH affected