Additives Promote Adventitious Buds Induction from Stem Segments of Bitter Melon (Momordica charantia L.)

The effects of thidiazuron (TDZ), silver nitrate (AgNO3) and triacontanol on adventitious buds induction from stems of balsam pear (Momordica charantia L.) were investigated. It was found that TDZ was necessary for bud development and the higher concentration of it could induce adventitious buds efficiently, while 0.1 mg/L was the best concentration to induce adventitious buds considered effect and cost. Bud formation was significantly affected by AgNO3 and triacontanol. Best results were obtained at concentration of 2.0 mg/L separately.


Introduction
Bitter melon (Momordica charantia L.) is an annual tendril herbage plant of balsam pear genus of Cucurbitaceae family, which is a kind of important and valuable vegetable crop and medicinal plant (Tang et al., 2009).It is difficult and taking too much time to breed using normal method, while tissue culture is an efficient method for plant breeding and can shorten breeding period.However there were few reports on tissue culture in bitter melon and the results revealed that it was easy to induce callus and roots but very difficult to differentiate buds (Yang et al., 2002;Song et al., 2004).In the former study, TDZ was shown to provide sufficient stimulus for induction of shoot regeneration in a variety of plant species.(Murthy et al., 1998;Zahoor et al., 2009;Shagufta et al., 2009).Additionally, AgNO 3 and triacontanol have been considered as promoters of adventitious bud induction in many species (Tantos et al., 2001;Liu et al., 2008).
In the present investigation, factors that promote adventitious buds formation from stems of balsam pear were studied.The aim of the paper is to provide the theoretical and technical basis for rapid propagation and breeding.

Plant materials
Stems about 2mm in diameter of balsam pear cv.Bixiu were the experimental materials in the present investigations.The mother plants were grown in the experimental plots using standard agronomic practices.

Callus induction
Stems harvested from field were rinsed with a little detergent, and washed in running water for 1h.Then they were surface sterilized with 75% (v/v) alcohol for 1 minute, and immersed in 0.1% (w/v) mercuric chloride solution with periodic agitation for 6 minutes in a laminar air-flow cabinet under aseptic conditions.After surface-sterilized, stems were finally washed 5 times with sterile distilled water.Then stems divided in 5-mm-long were inoculated on MS medium (Murashige & Skoog, 1962), supplemented with 3% (w/v) sucrose, 0.6% (w/v) agar, IBA 1.0mg/ L and BA 2.0 mg/L.The pH was adjusted to 5.8.

Adventitious bud induction
After 20 days the newly formed callus were separated from the explants and transferred to subculture medium.Subculture medium consisted of MS mineral salts and vitamins, IBA 0.2 mg/ L and different concentrations of TDZ (Table 1).In order to evaluate the effects of additives on adventitious bud induction, different concentrations of AgNO 3 and triacontanol were supplemented in media separately.Subsequent subcultures were conducted every 20 days.Without special explanation, all the MS media contained 3% (w/v) sucrose, 0.6% (w/v) agar, IBA 0.2 mg/ L and TDZ 0.01mg/L, and the pH was adjusted to 5.8 before autoclaving.After 40 days of culture, the data of adventitious buds were recorded.
Average number of buds per callus = number of adventitious buds / number of callus induced adventitious buds

Culture conditions
Cultures were maintained in growth chambers at 28℃ in the dark for 7 days, and then at 28℃ under 16 h daily illumination with 1500 lx fluorescent light.

Effect of TDZ on adventitious bud induction
The effect of TDZ providing sufficient stimulus for induction of shoot regeneration has been reported for many plant species (Li et al., 2000).Statistical analysis showed that the diffierentiation rate of balsam pear was influenced significantly by TDZ (Table 1).Poor result was obtained at TDZ free medium with no adventitious bud formation.With the increasing of TDZ concentration from 0.01 mg/L up to 0.5 mg/L, callus exhibited the higher differentiation rate.The results showed that TDZ was necessary for bud development and the higher concentration of it could induce adventitious buds efficiently.It was notable that TDZ concentration up to 0.5 mg/L, the bud differentiation rate was the highest as the same as result obtained at 0.1 mg/L TDZ, however the average number of buds per callus was relative low, and the buds prolongated more than proliferated (Fig. 1).When concentration of TDZ was 0.1mg/L, the adventitious bud induction rate reached the highest and callus gave the most dense clumps of buds (Fig. 2).Moreover, TDZ was relatively costly, so 0.1 mg/L was the situable concentration concluded in the present study.

Effect of AgNO3 on adventitious bud induction
Analysis of variance of the adventitious bud induction rate indicated that bud formation was significantly affected by AgNO 3 and its concentration.According to the data in table 2, the highest inductivity of buds and average number of buds per callus were obtained when media added AgNO 3 2.0 mg/L or 4.0 mg/L, which was similar with the result of canola (Hazrat et al., 2007).In previous studies, it was widely supposed that too high concentration of AgNO 3 had negative effect on bud formation and resulted in decreased regenerative ability even death (Zhou & Zhuang, 2007;Liu et al., 2007).According to this experiment, with the concentration of AgNO 3 up to 8.0 mg/L, the callus was prone to necrosis (Fig. 3).It can be concluded that high concentration of AgNO 3 was toxic for cultures, while relatively low concentration was optimal.<Table 2>

Effect of triacontanol on adventitious bud induction
Effect of triacontanol on adventitious bud induction was statistically significant (Table 3).Apparently, in medium with low concentration of triacontanol few buds similar with triacontanol free medium were obtained.And it was advantageous to differentiate buds when medium added with triacontanol 2.0 mg/L or 4.0 mg/L, which was in agreement with the result of watermelon (Wang & Wei, 2007).The existence of triacontanol even at low concentration was efficacious for average number of buds per callus.When callus cultured in medium supplemented with triacontanol 4.0 mg/L, it occured not only adventitious buds but also few leaves (Fig. 4).It was speculated that formation of buds and leaves desired different contents of triacontanol respectively.<Table 3> <Figure 1-4>

Figure 1 .
Figure 1.Buds prolongated more than proliferated Figure 2. Callus gave the most dense clumps of buds

Figure 3 .
Figure 3. Callus prone to necrosis Figure 4. Callus formed adventitious buds and leaves

Table 1 .
Effect of TDZ on adventitious bud induction

TDZ concentration (mg/L) Average number of buds per callus Differentiation rate (%)
Means having the same letter in the columns were not significantly different according to Duncan's multiple range test at P=0.05 (The same was the following tables).

Table 2 .
Effect of AgNO 3 on adventitious bud induction

Table 3 .
Effect of triacontanol on adventitious bud induction