Abstract: In this study, we have investigated the effect of polyamines (PA) and silver nitrate (AgNO3) on the high frequency regeneration from cotyledon explants of bottle gourd containing Murashige and Skoog (MS) media supplemented with different kind of Cytokinin alone or in the combination. Synergistic effect of kinetin (1 mg L-1) and benzyl adenine (BA) (2 mg L-1) itself showed highest shoot regeneration (80.6%) efficiency than BA or Kinetin alone in cotyledon explants of bottle gourd without adding AgNO3 or PAs. We have also observed that PAs and AgNO3 show their sensitivity on the regeneration, which is hormonal dependent. Regenerated shoots were rooted in half strength MS media containing 0.1 mg L-1 IAA.
INTRODUCTION
The genus Lagenaria belonging to the family of cucurbitaceae is an important vegetable in the tropics, subtropics and many other regions around the world. Morphological analysis and archeological evidence suggest the oceanic dispersals of wild bottle gourd fruit from Africa to Asia and America by 10000-15000 BP followed by independent domestication on all three Continents (Deena et al., 2001). The young fruits and tender tips of fast growing vines are eaten as a green vegetable. This vegetable contains high moisture carbohydrates, proteins, fats, fibers, minerals and vitamins, especially vitamin C and B complex (Heiser, 1979). With maturation, the rind becomes very hard and durable; the fruit is used to make utensils or fabricated into works of art (Yamaguchi, 1980). Bottle gourd have commercially important, especially in Japan, Korea, the Mediterranean area and Southern Europe, as they are being used as root stocks for grafting of watermelon and other cucurbits crops (Lee and Oda, 2003).
Cytokinins are known as ethylene inducing plant hormones and play an essential and crucial role in various aspects of plant growth and development of morphogenesis. In recent years there has been increasing evidence that the occurrence of morphogenesis in cultured plant cell may be associated with ethylene. Ethylene stimulated shoot morphogenesis in rice callus (Adkins et al., 1990) and Pinus radiata cotyledons (Kumar et al., 1987), embryogenesis from anther culture of Barley (Cho and Kasha, 1989) and flower bud formation in tobacco explants (Smulders et al., 1990). On the contrary, inhabitor ethylene action (AgNO3) in Nicotiana (Huxter et al., 1981), Triticum (Purnhauser et al., 1987), Zea mays (Songstad et al., 1988) and Brassica (Chi et al., 1991). The inhibitory effects of ethylene can be prevented by inhibition of ethylene action and bio synthesis (Pua, 1993). However the role of ethylene in plant cells and tissues grown in vitro is not well understood. Cytokinins are also known to promote ethylene production several folds in many plants, at least partially through the increase in ACC synthase activities (Abeles et al., 1992). N6-benzyladenine (BA), a synthetic cytokinin, synergistically enhances ethylene production in the presence of IAA in Mungbean hypocotyls (Yoshii and Imaseki, 1982). Kinetin alone slightly stimulates ethylene production by etiolate seedling of several species, but a remarkable synergistic effect of Kinetin on IAA induced ethylene production has been observed (Fuches and Lieberman, 1968). It has not yet been proven that a plant hormone might operate a specific function by itself. On the contrary, there are several potential mutual iterating points between hormones (Coenen and Lomax, 1997), depending upon the plant species and tissue type (Scumulling et al., 1997). The possibility of different hormonal receptors controlling growth and development is another new question, as well as whether different hormone types may compete for a common receptor or at least operate in separate signaling pathways (Timpte et al., 1995). In addition, Polyamines (PA) have been implicated in somatic embryogenesis and organogenesis in vitro in several plant species (Pua, 1999). In view of the fact that PAs and ethylene compete for the same precursor, S-adenosylmethionine for their synthesis (Evans and Malmberg, 1989), it was thought that their possible interactions and/or mutual regulation might play an important role in plant morphogenesis in vitro.
To address these questions, in this study, we investigated the capacity of shoot regeneration in different cytokinins in response to ethylene inhibitor (AgNO3) and PAs from cotyledon explants of bottle gourd.
MATERIALS AND METHODS
Explants preparation and phytohormones: Decoated seeds of Barsa-F were sterilized by soaking in 70% ethanol for 1 min followed by 45 min in 20% Sodium hypochlorite (1% a.i) containing 0.2% Tween-20, rinsed 4 times with sterile distilled water and finally soaking in autoclave sterile water for 2 h. Sterilized seeds were placed on germination medium containing MS basal salts and vitamins, 2% (w/v) sucrose. The pH of the medium was adjusted to 5.8 before the addition of the 0.8% agar (INA AGAR BA-30). Cotyledon explants of seedling at various ages, ranging from 2 to 14 days were tested for their regeneration capacity on MS medium containing 2 mg L-1 BA and 1 mg L-1 kinetin combination. Four days proximal part of cotyledon were isolated from seedlings and cultured on MS basal medium supplemented with different level of BA and kinetin alone or both combinations to optimize the regeneration %, bud proliferation, number of shoot per explants and shoot elongation. Seed germination and all the cultures including shoot initiation and shoot growth cultures were maintained 27±1°C with 16 h photoperiod.
Silver nitrate and exogenous polyamines: The effect of ethylene action inhibitor, AgNO3 of different concentrations viz. 5, 10 and 15 μM on BA or kinetin alone or their synergistic combination was examined on the regeneration potential of bottle gourd. The role of polyamines was also investigated by culturing explants on BA and kinetin alone or their synergistic combination supplemented with putrescine 5, 10, 15, 20 mM, respectively or spermidine 1, 2, 3, 4 μM, respectively on regeneration. Silver nitrate, putrescine and spermidine were sterilized by filtration and added to the medium after autoclaving.
Experimental design and data analysis: Each treatment was consisted of 4 explants with 8 replicates and each experiment was repeated three times. Data on the regeneration percentage, number of shoots per explants and shoot length were statistically tested by analysis of variance (ANOVA). The differences among the means were analyzed by Duncan’s Multiple Range Test (DMRT) at 5% level of significance.
Root induction: Elongated young shoot were isolated from the cotyledon explants and cultured on a half strength MS medium containing 0.1 mg L-1 IAA for root induction.
RESULTS
Explants age: Age of plantlets greatly influenced the regeneration of Cotyledon explants of bottle gourd is shown in Fig. 1 and results are shown in Table 1. Cotyledon explants of various ages were tested for their organogenic potential for multiple shoot induction as well as regeneration. Shoot differentiation frequency varied remarkably when cotyledon explants from seedling of different ages were cultured on the MS medium supplemented with 2 mg L-1 BA and 1 mg L-1 kinetin. Proximal part of explants from 4 days seedling, gave highest (80.6%) regeneration compared to that obtained from 2, 7, 10 and 14 days (Table 1). Proximal part of cotyledon showed higher regeneration than distal part for all the ages tested.
Effect of cytokinins on regeneration: Different kinds of cytokinin and
concentration were tested for their organogenic potential on regeneration from
four days proximal part of cotyledon explants of bottle gourd (Table
2).
| Fig. 1: | The cotyledon explants of different ages of bottle gourd seedling of genotype Barsa-F; 2, 4, 7, 10 and 14 days after sowing of the seeds in vitro |
| Table 1: | Effect of explants age and explants types (proximal part % or distal part %) on adventitious shoot regeneration |
| Values in column followed by different letters differ significantly at the 5% levels (Duncan’s multiple range test) | |
| Table 2: | Effect of different plant growth regulators on shoot initiation, shoot elongation and shoot regeneration from cotyledon explants |
| Values in column followed by different letters differ significantly at the 5% levels (Duncan’s multiple range test) | |
| Fig. 2: | Effect of different concentrations of AgNO3 on shoot regeneration from proximal part of cotyledon explants of bottle gourd genotype as compared with BA, Kinetin alone or both. Bar indicates the standard error |
Highest number of regeneration (80.6%) and shoot/explants (4.06) was produced
in MS medium supplemented with 2 mg L-1 BA and 1 mg L-1
kinetin combination (Fig. 4) among all the combination tested.
Among the different concentrations of BA or Kinetin, 2 mg L-1 BA
(25.33%) or 2 mg L-1 kinetin (24%) performed higher regeneration,
respectively. The combination of 2 mg L-1 BA with higher concentrations
of kinetin (2 and 3 mg L-1) studied, bud proliferation state performed
good but restricted to initiate the shoot, resulted in poor effect in terms
of regeneration. In kinetin containing media, a positive effect on shoot elongation
was found.
| Fig. 3a: | Effect of different concentrations of putrescine on shoot regeneration from proximal part of cotyledon explants of bottle gourd genotype as compared with BA, Kinetin alone or both. Bar indicates the standard error |
| Fig. 3b: | Effect of different concentrations of spermidine on shoot regeneration from proximal part of cotyledon explants of bottle gourd genotype as compared with BA, Kinetin alone or both. Bar indicates the standard error |
Two milligram per liter kinetin proved to be best (3.7 cm) among all of the treatments (Table 2 and Fig. 4c).
Effect of AgNO3 and polyamines on regeneration: The effect
of AgNO3 (Fig. 2) and Polyamines (Fig.
3a and b) was tested for their organogenic potential
on regeneration in MS media containing BA and kinetin alone or both. There were
no significant effect found on regeneration improvement in MS media containing
2 mg L-1 kinetin or synergistic combination of 2 mg L-1
BA and 1 mg L-1 Kinetin in the presence of AgNO3 (Fig.
2).
| Fig. 4: | Regeneration of shoots from the cotyledon explants of bottlegourd. (a) bud proliferation within 2 weeks of culture at 2 mg L-1 BA and 1 mg L-1 kinetin medium, (b) Shoot initiation at 3 weeks of culture in MS medium containing 2 mg L-1 BA an kinetin combination, (c) shoot elongation in 2 mg L-1 kinetin media and (d) root induction |
But 2 mg L-1 BA (25.33%) was significantly increased the regeneration (54.67%) in the presence of AgNO3 (10 μM). The application of PAs to the regeneration medium did not produce any significant improvement of regeneration on 2 mg L-1 BA with 1 mg L-1 kinetin combination. The application of putrescine and spermidine at several concentrations was tested on the regeneration potential of cotyledon explants of bottle gourd on kinetin, BA alone or their synergistic combination. Putrescine and spermidine containing 2 mg L-1 kinetin or synergistic combination (2 mg L-1 BA and 1 mg L-1 kinetin) had no effect on regeneration improvement (Fig. 3a). BA containing media (25.33%) increased their regeneration in the presence of 1, 5, 10 and 15 mM putrescine i.e., 36, 41.33, 52 and 56%, respectively (Fig. 3a). One micro molar spermidine also increased the regeneration (34.67%) in the presence of BA (Fig. 3b). Putrescine and spermidine are more effective on BA but not in 2 mg L-1 kinetin or combination of 2 mg L-1 BA and 1 mg L-1 kinetin for regeneration. AgNO3, Putrescine and spermidine showed a cytokinin dependent sensitivity on regeneration.
DISCUSSION
Cotyledon explants of various ages were tested for their organogenic potential for multiple shoot induction as well as regeneration. The highest shoot regeneration was found in the 4 days old seedling explants compared with others. Proximal part of cotyledon showed a higher frequency of regeneration when compared with the distal part (Fig. 1). Although plant growth regulator modifications during the culture period are important for the successful regeneration, the most determining factor for regeneration competence of bottle gourd was their optimal explants age. These results agree well with the reports of bottle gourd (Han et al., 2004), cucumber (Mohiuddin et al., 1997) and squash (Lee et al., 2003). A possible explanation is that the 4 days proximal part of young cotyledons are very active in physiology and are easily affected by environmental factors, such as, exogenous hormones.
According to the previous study of Han et al. (2004), only BA may be considered a crucial factor for adventitious shoot regeneration of bottle gourd and AgNO3, a potent inhibitor improves the regeneration. The results obtained from our study are not only compatible with this finding, but also clearly indicates the use of AgNO3 is more potent on BA. Concurrently it has no significant effect on kinetin or 2 mg L-1 BA and 1 mg L-1 kinetin combination for higher regeneration (Fig. 2). Similar results obtained from nodal explants of Pistachio (Ozden-Tokatli et al., 2005). They suggested that the medium containing BA could possibly release higher amount of ethylene than kinetin containing media. As a result, the inhibitory effect of AgNO3 on ethylene action could be more significant on BA containing media for higher regeneration. So, our results strongly support it. But it is not clear yet, how the kinetin keeps a role on ethylene inhibition in the presence of BA in vitro. Shoot regeneration capacity of explants and stimulation of ethylene biosynthesis may vary; depending on the growth regulator used (Kumar et al., 1998).
The effect of polyamines on the regeneration was effective on BA medium. But kinetin and synergistic combination did not found any significant improvement on regeneration (2 mg L-1 BA and 1 mg L-1 kinetin) with polyamines (Fig. 3a and b). PAs play an important role in shoot regeneration from Passiflora leaves (Desai and Metha, 1985). However, in morphogenesis of different species or different explants, spermidine and putrescine have been observed to exhibit dissimilar effects (Zhu and Chen, 2004). Exogenously added PAs recover browning tissues into normal callus cultured by decreasing oxidative damage and improving plant regeneration by acting as plant growth substance in Pine as reported by Tang et al. (2004). The accumulative evidences of culture in vitro indicate that polyamines showed a cytokinin dependent sensitivity on regeneration. May be it is related with ethylene. The role of PAs has been reported in a wide range of biological and physiological processes, but the precise mode of PAs action is still unclear.
From the foregoing discussion it can be concluded that synergistic effect of 2 mg L-1 BA and 1 mg L-1 kinetin itself performed best on the regeneration from cotyledon explants of bottle gourd. We report such an opposite effect of AgNO3 and PAs within the same explants, which may be explained in terms of regeneration pattern induced by different exogenous growth regulators, likely resulting in changed sensitivity to the hormone dependent manner. Similar results were found in Japanese variety, Yu-Gao but regeneration % found very poor compared to Barsa-F. Relatively little is known about the molecular mechanisms of cross-talk and integration between hormone responses on regeneration. A few downstream genes are known to modulate or integrate different hormonal signals, deserve further investigations.
ACKNOWLEDGMENTS
The authors are grateful to East West Seed Bangladesh Ltd. and Peacock Seed Company, Japan for supplying seed. The authors are thankful to Hori Information Science of Promotion Foundation for providing research grant for this work. Special thanks are due to Makio Kato, Plant Genetics and Breeding Lab., Nagoya University, for his contribution to the computer lay out of figures.