C. lanceolata is a perennial vine plant belonging to the family of Campanulaceae. It is widely distributed in Korea, Japan, China, and other regions and its root is used as food or medicine. The root of C. lanceolata has been widely used among people for cough control, cough syrup, tonic, and others (Ichikawa et al., 2009). Recent reports have demonstrated that Saponin of C. lanceolata root is effective for the improvement of PADAM-like symptoms (Ushijima et al., 2007), acute colitis (Joh et al., 2009), anti-inflammation, and decrease in alcoholic fatty liver (Shirota et al., 2008). Therefore, its value as natural cure has been increasing (Shirota et al., 2008).

Although the propagation of C. lanceolata is achieved via seeds, fixed trait cannot be identified, thereby there is no self-fertilization in the case of seed propagation. Also, regarding mass propagation using somatic embryogenesis, it is difficult to pass down the trait of species due to heterogeneous mutation of embryogenesis. Therefore, in order to overcome the genetic trait of C. lanceolata, it is best to apply propagation through tissue culture (Kim et al., 2009). Tissue culture has been used as one of the potential methods to achieve mass production of homogeneous plant from various tissues in short period of time (Kim & Kim, 1999; Suh et al. 2000). Such tissue culture can be used as effective method in agricultural product improvement and rapid propagation as adventitious bud of multicellular origin may form depending on the fragment of plant used and somatic embryogenesis of single-cell origin may from through either in-vitro callus formation or direct formation from tissue fragment for regeneration into plant (Hussey & Stacey, 1981; Kim & Kim, 1997). Also, ever since tissue culture technology has been applied to various field through callus induction, cell culture, and others, various methods are being used for in-vitro regeneration of plant (Amirouche et al., 1985; Kim, et al., 2003; Park et al. 2013).

During the past decade, a few studies have investigated that tetraploid plants have larger organs or stems (Cockerham and Galletta, 1976; Lapins, 1975) with thicker and longer stems and larger leaves and flowers. As a result of polyploidization, contents of secondary metabolites such as saccharide in sugar canes, vitamin C in tomato and apple, and nicotine in tobacco leaf has been changed, and sometimes, physiological characteristics such as virus resistance in radish (Hahn, 1969) and freezing resistance in mulberry tree have attempted to improve notably (Park, 1994).

Therefore, the purpose of this study was to examine the effects of culture medium composition on organogenesis from in-vitro cultured diploid and tetraploid C. lanceolata with the purpose of acquiring basic data for in-vitro mass propagation of superior species of C. lanceolata.

MATERIALS & METHODS

Diploid and tetraploid C. lanceolata (Fig. 1) seeds were selected as testing materials that were grown in-vitro aseptically. Leaf explant (1 cm²) and stem segment including node of in-vitro cultured diploid and tetraploid C. lanceolata were plated to the culture medium. In regards to optimum concentration experiment (2 MS, MS, 1/2 MS, and 1/ 4 MS culture medium) of MS medium (Murashige & Skoog, 1962) composition among culture medium compositions, agar 0.8% was added after controlling sucrose and pH as 3% and 5.8 respectively. For the sucrose experiment (1, 3, 5, 7%), agar (0.8%) was added after adjusting the pH of MS culture medium at 5.8. For pH (3.8, 4.8, 5.8, 6.8, 7.8) and agar concentration experiment (0.4, 0.6, 0.8, 1.0 1.2%), 1/2 MS culture medium with added sucrose 5% and 3% respectively to leaf explant and stem segment of diploid and tetraploid C. lanceolata was selected as reference culture medium and it was plated based on the findings of MS medium compositions and sucrose concentration experiment. Then, it was cultivated for 6 weeks. For all experiments, it was carried out with 6 segments per petridish and comparison and analysis were conducted after 10 replications of experiment based on completely random design. Culture condition was 16 hours of lighting under the light of 25 ± 1°C, 40 μmol·m⁻²·s⁻¹ and number, length, and others of shoot and adventitious root were examined after 6 weeks of cultivation.

RESULTS & DISCUSSION

Effects of various Concentration of culture medium compositions

Better result was observed for organogenesis from in-vitro cultured C. lanceolata when culture was conducted with stem segment rather than leaf explant as material regardless of type and concentration of culture medium compositions and polyploidy of C. lanceolata. In case of leaf explant, no organogenesis response was observed towards all concentrations of medium (data not shown). Such result coincided with the report which suggested that there was favorable organogenesis and growth when culture of stem rather than leaf segment in most experiments conducted to examine the influence of culture medium compositions on organogenesis of Platycodon grandiflorum (Kwon et al., 2003).

Results observed from diploid and tetraploid C. lanceolata regarding various concentration of MS medium compositions are presented in Fig. 2. In the case of diploid C. lanceolata, shoot formation was inhibited at higher mineral salt concentration. Notably, the highest shoot growth was obtained at the concentration of 1/2 MS medium and Diploid shoot growth of deodeok has decreased as the highest concentration of salt. This trend has showed the same trend in the adventitious root. The highest growth (4.9 cm) was observed from 1/2 MS medium. Adventitious root formation also showed similar inhibition with that of shoot formation. This inhibition was observed at higher mineral salt concentration. The highest formation (3.8) was found from 1/2MS medium. In the case of adventitious root growth, there was dramatic inhibition of growth (0.3 cm) in 2 MS medium at highest mineral salt concentration, while active growth (3.2 cm) was observed in 1/ 2MS medium. For shoot formation of tetraploid C. lanceolata, low formation (0.8) was observed in 2 MS medium at highest mineral salt concentration, and there was no big difference in other culture media presenting the range of 1.7 ~ 1.8. Shoot growth showed a decrease at higher mineral salt concentration and active growth (2.9 cm) was observed in 1/4MS medium at low mineral salt concentration. Adventitious root formation also exhibited the decrease at higher mineral salt concentration. Particularly, in the case of 2 MS medium, there was almost no adventitious root formation presenting 0.3. Adventitious root growth also showed similar result with that of adventitious root formation. Active growth (3.2 cm) was observed in 1/ 4 MS medium. In the case of Wasabia japonica, it has been reported earlier that the shoot propagation is proportionate to concentration of mineral salt (Park et al., 2007). In the case of Prunella vulgaris albiflora, low concentration of mineral salt is reported to be effective in adventitious root formation (Kwon et al., 2012). Furthermore, it is also known to be effective in adventitious root formation and growth in Hypericum patulum (Hwang et al., 2007). As a result of conducting this experiment, it was revealed that shoot growth and adventitious root formation and growth of diploid and tetraploid C. lanceolata are different based on the concentration of inorganic matters. Therefore, it was determined that demand for mineral salt concentration within MS medium which has influence on differentiation and growth differs based on polyploidy even for same plant.

Sucrose Concentration

The results obtained from in-vitro cultured diploid and tetraploid C. lanceolata with various concentration of sucrose are presented in Fig. 3. On the contrary, shoot formation of diploid C. lanceolata exhibited favorable result at higher concentration, shoot growth did not present consistent trend based on high or low concentration and the highest growth (2.3 cm) was presented at 3% concentration. Adventitious root formation was suppressed to 0.8 at low concentration (1%), and there were no significant differences for other concentrations presenting the range of 2.0 ~ 2.6. Although there was no consistent inhibition based on concentration for adventitious root growth, a little higher growth (2.0 cm) was presented in 7% concentration. In the case of tetraploid C. lanceolata, there was no difference in shoot growth for all concentrations regardless of high or low concentration presenting the range of 1.7 ~ 1.8. However, there was little decrease of shoot growth at higher concentration. Adventitious root formation was inhibited at higher concentration. Particularly, in the case of 7% concentration, no adventitious root formation was observed, thus it showed opposite result compared to adventitious root formation of diploid C. lanceolata. Adventitious root growth did not present potential difference with the range of 0.4 ~ 0.6 cm in all concentrations except 7% concentration which did not present adventitious root formation. It was reported that there was favorable shoot formation at 3% concentration for Rhodiola sachalinensis (Bae et al., 2009) and 5% for Veronica rotunda var. subintegra (Cha et al., 2007). However, as a result of conducting the experiment, favorable organogenesis and growth were presented by low concentration sucrose for diploid balloon flower, but it did not have an effect on adventitious root induction and growth in the case of tetraploid P. grandiflorum.

Agar Concentration

The results observed from the diploid and tetraploid C. lanceolata by differing the agar concentration are presented in Fig. 4. Although shoot formation of diploid C. lanceolata did not present difference in all concentration with the range of 1.6 ~ 1.9, shoot growth did not present consistent trend based on high or low concentration. Active growth of 4.4 cm and 4.2 cm was exhibited in 0.6% and 0.8% concentration respectively. Adventitious root formation also showed similar result with shoot growth at high formation in 0.6% and 0.8% concentration. Adventitious root growth was the highest (4.1 cm) in 0.8% concentration, and there was no difference in other concentrations with the range of 2.8 ~ 3.3 cm. Shoot formation of tetraploid C. lanceolata showed little inhibition at higher concentration. Shoot growth also exhibited similar result and favorable shoot growth (2.0 cm) was observed in 0.6% concentration. Adventitious root formation showed the inclination of suppress at higher concentration and the highest formation (3.7) was observed in 0.4% concentration. Adventitious root growth in 0.6% concentration was found to be little higher (1.9 cm) than other concentrations. It was reported that adventitious root formation and growth exhibited favorable trend at lower agar concentration in the case of P. grandiflorum A. DC. with yellow green petals (Kwon et al., 2014) and organogenesis was most favorable in 0.4% concentration in the case of Lysimachia vulgaris var. davurica (Kang et al., 2007). However, in the present study, tetraploid C. lanceolata exhibited similar result with that of P. grandiflorum A. DC., but it was little different from that of diploid C. lanceolata. This revealed the fact that there is a difference in solidity of culture medium with influence on adventitious root formation and growth based on polyploidy even in the same specifies.

Effect of pH

The results of the effects of pH range (3.8 ~ 7.8) observed from the diploid and tetraploid C. lanceolata to culture medium are presented in Fig. 5. There was a little higher shoot formation of diploid C. lanceolata at lower pH. Although there was no consistent inhibition for shoot growth, favorable result (2.3 cm) was presented at pH 3.8. Adventitious root formation also presented similar trend with shoot formation which is higher formation with lower pH. Adventitious root growth showed a little higher result (2.4 cm) at pH 3.8, but there was no difference in other treatment group with the range of 1.9 ~ 2.1 cm. Although there was no difference in shoot formation of tetraploid C. lanceolata with the range of 1.7 ~ 1.8 regardless of high or low pH, its growth showed the inclination of suppress at higher pH. Adventitious formation did not present consistent trend based on high or low pH, and higher formation was observed at pH 3.8 and pH 5.8 with 2.0 and 2.2 respectively. Adventitious growth observed a little higher result (2.0 cm) at pH 5.8. Normal pH range of culture medium used for plant tissue culture is 5.5 ~ 5.8. It was reported the range of pH 5.3 ~ 5.8 is appropriate for P. grandiflorum which belongs the same family as C. lanceolata (Chung & Cho, 2002; Choi et al., 2005), and pH 6.8 is adequate for adventitious root and shoot formation of Hypericum Perforatum (Hwang et al., 2009). However, different result was showed as culture medium of adequate pH for organogenesis of C. lanceolata is culture medium with high acidity with pH of 5.8 or lower.

Taken together, the overall results obtained from the present study revealed that the potential result was observed for organogenesis from in-vitro cultured C. lanceolata when culture was performed with stem segment rather than leaf explant regardless of type and concentration of culture medium compositions and polyploidy of C. lanceolata. Moreover, favorable organogenesis and growth were observed at the low concentration sucrose for diploid plant, but it did not have an effect on adventitious root induction and growth towards tetraploid P. grandiflorum. However, the shoot formation and adventitious root formation of diploid C. lanceolata was increased at lower pH and shoot growth was the highest at pH.

적 요