In vitro Regeneration of Citrullus lanatus cv. Round Dragon

MATERIALS AND METHODS

Plant tissue culture media: Germination medium used was Murashige and Skoog (MS) salt supplied with 20 g L^-1 sucrose, 100 mg L^-1 myo-inositol and 3.2 g L^-1 phytagel. Shoot-regeneration medium (MS medium) supplied with 2.3 mg L^-1 BAP, 30 g L^-1 sucrose, 100 mg L^-1 myo-inositol and 3.2 g L^-1 Phytagel. Root-inducing medium (MS medium) contained 0.1 mg L^-1 IBA, 30 g L^-1 sucrose, 100 mg L^-1 myo-inositol and 3.2 g L^-1 Phytagel.

Plant material and shoot regeneration: Citrullus lanatus cv. Round Dragon was used as experimental material in this study. Citrullus lanatus seeds imported from Taiwan were purchased from local supplier ACE Seed Trading (Malaysia) Sdn Bhd. The seeds were rinsed thoroughly for 30 min under running tap water. The seed coat was removed and the embryos were surface sterilized for 15 min by immersing in 5% (v/v) sodium hypochlorite (commercial bleach) followed by successive five times rinses with sterile distilled water to remove the trace of sterilent. The seeds were then germinated on germination medium.

Cotyledons were excised from 5-day-old seedlings and cut crosswise into distal and proximal halves with 2 mm of hypocotyls segments using scalpel blade. The distal half was discarded and the proximal tissue was cultured horizontally in 90x15 mm plastic Petri dishes that contained 25 mL of shoot regeneration medium. All cultures were incubated for 3 weeks at 25±1°C with 16 h photoperiod under 12.16 μmol/m/sec from cool white fluorescent lamps in a tissue culture chamber. After 3 weeks, explants with adventitious shoots were subcultured to fresh shoot-regeneration medium in Magenta jar for another 3 weeks. Explants with shoots were transferred to Magenta jar that contained 50 mL of shoot regeneration medium without hormone for 4 weeks for shoot elongation.

After shoot regeneration for a total of 10 weeks, shoots longer than 2 cm were excised and transferred to 50 mL of root induction medium in Magenta jar for 3 weeks. After 3 weeks on rooting medium, the plantlets were transplanted under ex vitro condition into plastic pots containing autoclaved soil and sand in the proportion of 1:1. All the pots were covered with clear plastic covers for 7 days to maintain high humidity and incubated at 25±1°C under 16 h photoperiod in a tissue culture chamber. Plantlets in the pot were watered every 2 days with nutrient solution. After 7 days, the humidity was reduced by progressively removing the lids over 3 days. During this time, plants are watered 2 to 3 times alternatively with nutrient solution and water.

Optimizing shoot regeneration: To optimize shoot regeneration experiments, the effect of ages of in vitro germinated seedlings were tested for regeneration potential. The influence of cotyledonary explants age was evaluated using cotyledon excised from 5 days, 7 days and 9 days old seedlings after placement of the seeds onto germination medium. Cotyledons were excised 1-2 mm beyond the point of attachment to the stem and the distal halves were discarded. The proximal tissue was cultured abaxial side down in 90x15 mm plastic Petri dishes (Brandon) that contained 25 mL of regeneration medium for 3 weeks.

The explants were subcultured to fresh medium in sterile wide mouth conical flask (GQ) containing 100 mL of regeneration medium after 3 weeks and remained in conical flask for a total of 6 weeks. Shoot induction efficiency based on cotyledonary explant age was assessed using two criteria: the percentage of explants with shoots and the number of shoots per responding explant were counted by stereomicroscope after 6 weeks on shoot induction medium.

Rooting of micropropagated shoots: Prior to rooting studies, an adequate number of microshoots for rooting experiment were established from 5-day-old seedlings germinated in vitro. Regenerated adventitious shoots (with length >2 cm) were excised aseptically from the cotyledon explants for rooting after 3 weeks. The shoots were then cultured individually in Magenta jar (MAGENTA) with 50 mL of rooting medium having different strength and combination of auxins. Rooting in vitro were tested on half-strength MS salts (1/2 MS) and full strength MS salts supplemented with 30 g L^-1sucrose, 100 mg L^-1 myo-inositol, 3.2 g L^-1 Phytagel and different concentrations of IBA (0, 0.1, 0.5, 1.0 and 10.0 mg L^-1) , IAA (0, 0.1, 0.5, 1.0 and 3.0 mg L^-1) and NAA (0, 0.1, 0.5, 1.0 and 10.0 mg L^-1). Auxins were filter sterilized prior adding into the autoclaved medium. Cultures were incubated under a 16 h photoperiod of 12.16 μmol/m/sec from cool white fluorescent lamps at 25±1°C in a tissue culture chamber for 3 weeks. At week 3, the frequency of shoots that developed roots and the number of roots per shoot were counted.

Statistical analysis: All statistical analysis was performed using SPSS for Windows software (SPSS Windows Version 15). The experimental design were completely randomized with each experiment were performed with three replicates per treatment and each set within the replicate consisted of 10-20 explants. Data were analyzed using one-way ANOVA (analysis of variance) and Tukey’s Honestly Significant Difference Test (HSD value) and the significance were determined at the p<0.05 level. Data are presented as means and standard errors.

RESULTS

Effect of explant age on shoot regeneration: Cotyledon explants from 5, 7 and 9 days old seedlings were tested for the efficiency of shoot regeneration on MS medium supplemented with 2.3 mg L^-1 BAP alone for 6 weeks. Six weeks exposure of explants on MS medium containing 2.3 mg L^-1 BAP led to high frequency of shoot formation on 5-day-old seedlings. The shoots on 5-day-old seedlings were longer and appeared earlier and grew vigorously than those on 7 and 9-day-old seedlings (Fig. 1a-f). Apart from that, 5-day-old seedlings produced significantly higher number of shoots than 7 and 9-day-old seedlings (Table 1). On the other hand, explants from 7-day-old seedlings produced fewer adventitious shoots while shoots from 9-day-old seedlings remained short and smaller in number even after 6 weeks in culture. In this experiment, cotyledon explants from 5-day-old seedlings of Citrullus lanatus found to be more potential to regenerate rather than 7 and 9-day-old seedlings.

In vitro rooting experiment: For the initiation of roots, microshoots (>2 cm) derived from 5-day-old seedlings were individually transferred to half strength and full strength MS medium each supplemented with various concentrations of IBA, IAA and NAA for 3 weeks. Among different concentrations of auxins tested in half strength media, IBA was found to be comparatively better to response than IAA and NAA. It was observed that the microshoots induced high frequency of roots in half strength MS medium supplemented with 0.1 mg L^-1 IBA followed by 0.5 and 1.0 mg L^-1 IBA, however no significance differences were observed for these concentrations (Table 2). On the other hand, microshoots on half strength MS medium supplemented with IAA gave a slightly higher level of response compared to NAA. However, no significance differences on root formation were observed between IAA and NAA.

Fig. 1:	Adventitious shoot development (a) based on explant ages. Shoot initiation on 5-day-old seedlings after 3 weeks and after (b) 6 weeks. Shoot growth on (c) 7-day-old seedlings after 3 weeks and (d) 6 weeks. Shoot development on (e) 9-day-old seedlings after 3 weeks and (f) 6 weeks

Table 1:	Shoot regeneration from different explant ages after six weeks in culture on MS medium supplemented with 2.3 mg L-1 BAP
Values within a column followed by different superscripted letters are significantly different at the p<0.05 level

Root initiation on full-strength MS media showed that IBA as an auxin source was significantly better than IAA and NAA (Table 3). Optimal root development was observed on full strength MS medium supplemented with 0.1 mg L^-1 IBA yielded the best regeneration rate of 100% followed by 0.5 and 1.0 mg L^-1 IBA. The IBA promoted root development more efficiently than IAA and NAA. The IBA induced several long primary roots which grew more rapidly and branched more frequently. However, IAA induced normal lateral shoots and abundant callus at the higher concentration nevertheless NAA led to thicker and much shorter roots with a few lateral roots.

Table 2:	Effect of different concentrations of auxins in half strength MS medium on root formation from microshoots in citrullus lanatus
Values within a column followed by different superscripted letters are significantly different at the p<0.05 level

Table 3:	Effect of different concentrations of auxins in full strength MS medium on root formation from microshoots in Citrullus lanatus
Values within a column followed by different superscripted letters are significantly different at the p<0.05 level

In this experiment, both half strength MS medium supplemented with 0.1 mg L^-1 IBA (87%) and full strength MS medium supplemented with 0.1 mg L^-1 IBA (100%) improved the regeneration frequency of the roots. However, regeneration efficiency in full strength MS medium supplemented with 0.1 mg L^-1 IBA was slightly increased and better compared to half strength MS medium supplemented with 0.1 mg L^-1 IBA. Thus, the presence of 0.1 mg L^-1 IBA in full strength MS medium was essential to initiate and to induce in vitro root induction in Citrullus lanatus.

Plant regeneration: Cotyledon explants of 5-day-old seedlings were excised and the proximal region (Fig. 2a) was cultivated on MS regeneration medium supplemented with 2.3 mg L^-1 BAP. The BAP alone induced adventitious shoot bud differentiation from cotyledon explants in Citrullus lanatus. Cotyledonary explants on MS medium expanded considerably after 1 week and gradually turned to yellowish-green as little green callus appeared directly at the proximal end of cotyledon segments (Fig. 2b).

Fig. 2:

Adventitious shoot development and plant establishment in Citrullus lanatus. (a) Proximal region from 5-day-old seedling used as cotyledon explants. (b) Initial development of callus at proximal end after 1 week. (c) Adventitious shoots on explants at week 3. (d) Adventitious shoot induction on MS medium with 2.3 mg L-1 BAP at week 6. (e) Shoot elongation on MS medium without hormone. (f) Rooted plantlet on MS medium with 0.1 mg L-1 IBA. (g) Induction of root on MS medium supplemented with 0.1 mg L-1 IBA. (h) Regenerated plantlets 1 month after transferred to pot. (i) Ex vitro rooting of Citrullus lanatus

The cotyledon explants kept increasing in size and after 3 weeks on MS regeneration medium supplemented with 2.3 mg L^-1 BAP, cotyledon explants produced adventitious shoots from the proximal end without intervening callus (Fig. 2c).

Moreover, the cotyledon explants transferred to fresh MS regeneration medium containing 2.3 mg L^-1 BAP for another 3 weeks increase the size of the adventitious shoots and subsequently initiates a well-developed shoot tips (Fig. 2d). Further, MS medium lacking of hormone induced elongation of adventitious shoots with numerous shoot tips and leaves within 4 weeks (Fig. 2e). Independent fully developed shoots were transferred to MS medium containing 0.1 mg L^-1 IBA to developed roots at their base resulting in complete plantlets with expanded leaves (Fig. 2f, g). The plantlets with roots (3-5 cm with >1 roots) which were transferred and acclimatized in pot resume growth and their stems and leaves elongated and expanded in 1 month (Fig. 2h, i).

DISCUSSION

Cotyledon from in vitro germinated seedlings used as an explant in this study was the most efficient system for plant regeneration in Citrullus lanatus. These finding support previous research that cotyledon tissues has been successfully regenerate into adventitious shoots in diverse type of cucurbits species like Citrullus vulgaris (Srivastava et al., 1989), Citrullus colocynthis (Dabauza et al., 1997) and Cucumis melo L. (Gaba et al., 1999). Furthermore, cotyledon regions in cucurbits consist of high competent cells which easily response to exogenous plant growth regulator and enable to regenerate adventitious shoots (Choi et al., 1994; Compton, 2000; Ananthakrisnan et al., 2003). In current experiment, cotyledon explants were excised and the proximal region of the cotyledon segment was effectively used to induce adventitious shoot on MS medium supplemented with 2.3 mg L^-1 BAP. It has been reported that a higher percentage of explants forming adventitious buds was obtained from the cotyledon proximal region (Krug et al., 2005). Moreover, Compton and Gray (1993) have claimed that competent cells, which retained the capacity for a particular kind of cellular differentiation or regeneration located at the proximal region of the cotyledon segments in watermelon. In fact, proximal region of the cotyledon segments used in this study resulted in high adventitious shoot regeneration frequency.

Together with the explant type, seedling age is also important for adventitious shoot development. In this study, cotyledon explants were collected from 5-day-old seedlings was highly effective for the regeneration of shoots from the cotyledon explants. This phenomenon suggested that 5-day-old seedling is significantly different from 7 and 9-day-old seedlings in its efficiency level for shoot initiation. A possible explanation is that the young cotyledons are physiologically very active and response easily and efficiently to exogenous hormones (Dong and Jia, 1991). Another possible explanation for this is that young explants with many meristematic and undifferentiated cells are susceptible to tissue culture manipulation, thus regenerating shoots from their normal developmental pathway (Shin et al., 2000). Comparing the available information for Citrullus vulgaris with results obtained in Citrullus lanatus, it seems that 5-day-old cotyledons were the most sensitive to shoot induction compared to seedlings older than 7 days. Immature cotyledon of 5-day-old seedlings used as an explant showed to be an excellent source for plant regeneration on MS medium containing 2.3 mg L^-1 BAP in this study.

The type and the concentration of phytohormone in the induction media were found to be another crucial factor in regeneration of Citrullus lanatus. The presence of BAP alone (without auxin) in the MS medium promoted the highest frequency of shoot regeneration from cotyledon explants. This is accordance with earlier studies of the same Citrullus species (Chaturvedi and Bhatnagar, 2001; Pirinc et al., 2003). In addition, shoots originated at proximal region of cotyledon explants on MS medium supplemented with 2.3 mg L^-1BAP have well-developed shoots tips and highly elongated with expanded leaves. These result are consistent with those of Pirinc et al. (2003), who reported that the shoot regeneration for watermelon cotyledons was best on MS medium with BAP alone (1 or 2 mg L^-1). Compton and Gray (1993) and Srivastava et al. (1989) detected an inhibition of shoot organogenesis when both cytokinin and auxin was added to shoot induction medium indicate that auxin counteracts the effect of cytokinin on shoot regeneration. However, the presence of auxin is absolute necessary for root regeneration to occur. Microshoots cultured in full strength MS medium supplemented with auxin comparatively better and improve root initiation than the microshoots cultured in half-strength MS medium. Furthermore, these results are consistent with those of the other studies which found full strength MS medium supplemented with auxin induced high frequency in root formation (Thakur et al., 2005).

Full strength MS medium supplemented with 0.1 mg L^-1 IBA achieved high frequency of root initiation in Citrullus lanatus. It was significantly difference compared to various concentrations of NAA and IAA that has been tested for rooting efficiency. These findings agrees with Dabauza et al. (1997) and Ahn et al. (2007) that high frequency of shoots rooted and grew normally on MS medium supplemented with IBA. Moreover, IBA produced longer primary roots with numerous lateral roots on MS medium compared to IAA and NAA which promoted poor root development. For this reason, full strength MS medium supplemented with 0.1 mg L^-1 IBA was chosen as the optimum condition for root initiation in Citrullus lanatus cv. Round Dragon.

CONCLUSIONS

In current study, we have established a reproducible in vitro plant regeneration protocol using cotyledonary explant for Citrullus lanatus cv. Round Dragon. The adventitious shoot regeneration was successfully induced from the proximal region of 5-day-old seedlings on Murashige and Skoog (MS) media supplemented with 2.3 mg L^-1 BAP. For rooting, MS media was added with 0.1 mg L^-1 IBA and further incubated for another 3 weeks for the plantlets to grow under ex-vitro conditions. Establishments of some conditions would facilitate for future study in genetic transformation and seed production of transgenic Citrullus lanatus cv. Round Dragon.

ACKNOWLEDGMENTS

We wish to thank Malaysian Government (MOSTI) Votes 79073/78307 (e-science fund/MOHE) for financial support.