Research Article
Regeneration and Establishment of Potato Plantlets Through Callus Formation with BAP and NAA
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K.M. Nasiruddin
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R. Begum
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S.K. Talukder
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Potato is a staple food in some countries and important vegetable in Bangladesh. In Bangladesh, potato represents about 53% of the total edible vegetables. The potential value of tissue culture in crop improvement has been widely recognized. The regeneration of plants from cell and tissue culture is an important and essential component of biotechnology that is required for the genetic manipulation of plants. High frequency regeneration of plants from in vitro cultured tissues and cells is a pre-requisite for successful application of tissue culture and genetic engineering technologies for crop improvement. Many attempts have been made to enhance the frequency of plant regeneration from potato callus and a lot of research has been devoted to investigate the factors affecting plant regeneration. Great progress has been made in potatoes for plant regeneration in recent years (Ehsanpour and Jones, 2000; Fiegert et al., 2000; Ahn et al., 2001). Both callus induction and plant regeneration from explants require the presence of appropriate combinations and concentrations of plant growth regulators in the culture media. Callus induction, subsequent plant regeneration and microtuberization are somehow dependent on the type of explants, media components, growth conditions and varieties. Remarkable work has been done on plant regeneration with different explant and growth regulators. Many author work to standardize the optimum concentrations of BAP and NAA for regeneration of potato (Dobranszki et al., 1999; Hansen et al., 1999; Zel et al., 1999; Fomenko et al., 2000; Asthma et al., 2001). It is important to standardize the protocol for regeneration of potatoes grown in Bangladesh. The present study was undertaken to investigate the in vitro callus induction ability and regenerability of potato with optimum concentrations of BAP and NAA.
The present investigation was carried out in the USDA Biotechnology Laboratory, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh in 2002. Potato tubers cv. Cardinal were collected from Bangladesh Agricultural Research Institute, Gazipur. Disease-free potato tubers were sprouted in the dark condition at room temperature.
The present experiment consisted of three factors eg. explant, NAA and BAP. There were 5 levels of both of NAA (0, 1.25, 2.5, 5 and 10 mg l-1) and BAP (0, 0.5, 1, 2 and 4 mg l-1) and two types of explants eg. leaf and internodal segment The experiment was laid out in Completely Randomized Design (CRD) with 5 replications. For surface sterilization, etiolated sprouts of potato were first sterilized with 70% (v/v) ethanol for few seconds. The sprouts were then rinsed twice with sterile distilled water. Afterwards the sprouts were surface sterilized by immersing in 0.1% HgCl2 solution for 2 min and then washed several times with sterile distilled water. The pH medium was adjusted to 5.8. MS media was prepared and then autoclaved at 1.16 kg cm-2 pressure at 121°C for 20 min. The sprouts were allowed to grow on MS solidified medium with 3% sucrose. At 25±2°C under 16 h photoperiod. One month old in vitro grown micro plants were used as the source of explants. Four explants were directly inoculated to each glass petridish (70 mm dia.) containing 10 ml of MS medium with different hormone concentrations as per treatment. The petridishes were covered and sealed with parafilm and were kept at 25±2°C illuminated 16 h daily (light intensity of 1500 lux) with 1.83 m florescent tubes (C84 TDFL/Phillips). Callus was initiated after 7-18 days of explant inoculation. Plantlets were developed from calli in this media after 10-25 days. Plantlets of the age of 30 days with well developed roots were removed from petridishes. Plantlets were then individually transplanted in pots containing sand, soil and cowdung (1:1:1) mixtures. Immediately after transplantation, the plantlets were irrigated with a fine spray of water and the plantlets along with pots were covered with transparent polythene bags to prevent desiccation. The pots were kept in the laboratory and were irrigated regularly. The plantlets were established within 5 to 7 days and then the polythene bags were removed. Data were recorded on different parameters of callus and plantlets. The analysis of variance for different characters were performed and means were compared by the Duncans Multiple Range Test (DMRT).
Results and Discussion
Auxin and cytokinin showed significant variation for plant regeneration via induced calli from leaf and internodal stem explants.
Callus formation: The main effect of explants and different supplements of BAP and NAA on callus induction have been presented in Table 1. Twenty explants were inoculated in each treatments. Leaf was more responsive for induction of callus and showed better callusing (11.38) than internodal segment (9.14) out of 20 cultured explants. The highest callusing was obtained in 5 mg l-1 NAA (13.00) followed by 2.5 mg l-1 NAA (12.90). On the other hand, 2 mg l-1 BAP showed highest callusing (12.60) followed by 1 mg l-1 BAP (12.50) from 20 explants.
The main effect showed that the percentage of callus induction from leaf was higher (56.58%) than internodal segment (45.76%). Also the percentage of callus induction was found significantly higher in 5 mg l-1 NAA (65.00%) followed by 2.5 mg l-1 NAA (64.50%); and in 2 mg l-1 BAP (63.00%) followed by 1 mg l-1 BAP (62.50%). Lowest callus was found when the treatment contained no NAA (12.30%) and no BAP (27.50%).
Table 1: | Main effect of different combinations of NAA and BAP in MS medium on callus induction from leaf and internodal segment of potato cv. Cardinal. Data were recorded at 10 days of explantation. Twenty explants were placed in each treatment |
Leaf explants required minimum time (12.26 days) for callus induction and internodal explants required more time (14.48 days). Minimum time was required for 2.5 mg l-1 NAA (12.30 days) and 1 mg l-1 BAP (12.50 days) than that of other supplements.
Among the different combinations, 2 mg l-1 BAP + 2.5 mg l-1 NAA and 1 mg l-1 BAP + 1.25 mg l-1 NAA showed better callus induction i.e 19.00 and 18.00, respectively in a shortest period out of 20 explants (Table 2). The explants cultured without growth regulator did not produce any callus; these results are in support of the results obtained by Fiegert et al. (2000) and Jayasree et al. (2001).
The percentage of callus induction was the highest in MS media containing 2 mg l-1 BAP + 2.5 mg l-1 NAA (95%) followed by 1 mg l-1 BAP + 1.25 mg l-1 NAA (90%) from leaf explants (Table 2). NAA was essential for callus induction and the amount of callus induction increased with increasing concentrations of NAA and BAP which was similar to the results of Martel et al. (1992).
Regeneration through callus: The results on the main effect of explants and different supplements of BAP and NAA on regeneration of potato cv. Cardinal have been presented in Table 3. Leaf showed better performance in regeneration (5.40) through callus from 20 cultured explant. The percentage of regeneration was recorded higher in case of leaf (25.60%) than internodal segment (11.60%). Also leaf took minimum time (23.68 days) for regeneration compared to internodal segment (28.96 days). The highest regeneration potentiality was found in 2.5 mg l-1 NAA (6.60) followed by 1.25 mg l-1 NAA (6.10) out of 20 explants. But there was no regeneration without growth regulator. The percentage of regeneration was the highest in 2.5 mg l-1 NAA (33.00%) followed by 1.25 mg l-1 NAA (30.50%) and 2 mg l-1 BAP (30.50%).
Table 2: | Combined effect of different combinations of NAA and BAP in MS medium on callus induction from leaf and internodal stem of potato cv. Cardinal. Data were recorded at 10 days of explantation. Twenty explants were placed in each treatment |
Table 3: | Main effect of different combinations of NAA and BAP in MS medium on plant regeneration of potato cv. Cardinal. Data were recorded at 21 days of explantation. Twenty explants were placed in each treatment |
Table 4: | Combined effect of different combinations of NAA and BAP in MS medium on plant regeneration of potato cv. Cardinal. Data were recorded at 21 days of explantation. Twenty explants were placed in each treatment |
No regeneration was noticed in absence of either of NAA or BAP |
Various combinations of supplements showed significant variation in regeneration ability. Among the combinations used, 2 mg l-1 BAP +2.5 mg l-1 NAA showed the highest regeneration of plantlets from leaf (16.00) and internodal segment (9.00), respectively out of 20 cultured explants (Table 4).
The percentage of regeneration was recorded the highest (80%) in MS media containing 2 mg l-1 BAP + 2.5 mg l-1 NAA followed by 1 mg l-1 BAP + 1.25 mg l-1 NAA (70%) from leaf (Table 4). Also it was observed that the above combinations required shorter period of time for regeneration (25.80 and 26.10 days, respectively). It was also found that leaf was always more responsive explant than internodal segment. Yadav and Sticklen (1995), Alphonse et al. (1998), Hamdi et al. (1998) observed that leaf was the best explant for regeneration. The regenerants were successfully established ex vitro on sand, soil and cowdung mixture (1:1:1).
We acknowledge the financial support of USDA funded project on Salinity and drought resistant genes in potato: cloning, expression, transformation and variety development.