International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 3(6), 66-72, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 66 Callus Induction and Plant Regeneration in Solanum tuberosum L. cultivars (Kufri Chipsona 3 and MP-97/644) via Leaf ExplantsVijay Kumar, Deep Rashmi and Madhuparna Banerjee* Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi-835215, INDIA College of Biotechnology, Birsa Agricultural University, Kanke, Ranchi- 834006, INDIA Available online at: www.isca.in, www.isca.me Received 24th January 2014, revised 6th March 2014, accepted 3rd April 2014 Abstract The aim of this study was to establish a protocol for rapid callus induction and plant regeneration of potato. The leaf explants of two potato (Solanum tuberosum L.) cultivars viz. (Kufri Chipsona 3 and MP-97/644) were cultured for callus induction and plant regeneration. Best callus growth from both the cultivars was observed on Murashige and Skoog (MS) (1962) media containing 3.0 mg/l of 2,4-D (2,4-dichlorophenoxy acetic acid) and1.0 mg/l of kinetin. MS medium supplemented with different concentrations and combinations of BA, Kinetin and AdSO were employed for shoot regeneration. Best shoot regeneration from callus was observed on MS media containing 1.5 mg/l BA (6-benzyladenine) and 25.0 mg/l AdSO (Adenine sulphate). The mean number of shoots/callus clump was 21.00 (Kufri Chipsona 3) and 18.67 (MP-97/644) after 60 days of inoculation. When the plants were transferred to green house, primary hardening was achieved within 10 days and about 99% plants survived. Keywords: leaf explants, 2,4-dichlorophenoxyacetic acid (2,4-D), MS medium, Solanum tuberosum L. Introduction Potato (Solanum tuberosum L.) is the most important non-cereal food crop of the world. In monetary terms it ranks fourth in the world after wheat, rice and maize. It produces the largest quantity of carbohydrates per day per unit area among the food crops. Potato recommended as food security crop, is consists of 80% water, 2-3% protein and 18% carbohydrate. Central Potato Research Institute, Shimla, India over the past 55 years has resulted in the development of 35 high yielding potato varieties for diverse agro climatic conditions and innovation of seed plot technique for augmenting the seed production. ICAR (Indian Council of Agricultural Research) has identified a new hybrid variety ‘Kufri Chipsona-3’of potato for release. Kufri Chipsona-3 can be used both for processing (chips and flakes) as well as table purposes. Tissue culture techniques are used worldwide to produce pre-basic, virus-free seed potatoes known as microtubers. Several tissue culture techniques have been used for huge number of potato cultivars. Recently in vitro conservation protocol for potato is also confirmed. Disease free and genetically uniform plantlets may be produced by callus culture. The callus induction in potato was first observed by Steward and Caplin. However, the past studies have shown that in vitro callus induction in S. tuberosum cultivars has been reported by few authors from different explants including nodal, intermodal and leaf explants, internodal and leaf explants, nodal explant, internodal explants9, 10, stem segment11, 12, and leaf discs13, 14. It is important to establish the protocol for plant regeneration through callus culture of two important indian potato cultivars (Kufri Chipsona 3 and MP-97/644). Regenerated plantlets through callus are genetically identical, disease free and produce a large number of plantlets in a very short period of time. Therefore, our objective is to introduce an efficient protocol for callus induction and regeneration from the leaf explants of potato cultivars (Kufri Chipsona 3 and MP-97/644). Material and Methods Plant material and callus induction: Two potato cultivars (Kufri Chipsona 3 and MP/97-644) were obtained from Central Potato Research Institute, Shimla. In our study leaf explants of both cultivars were chosen for callus induction. Leaf explants were washed thoroughly under tap water for 15 minutes, and then all the explants were treated with 0.2% bavistin for 10 minutes. Then all the explants were washed thoroughly with distilled water (4-5 times). The explants were further treated with 2-3 drops of teepol for 3-5 minutes followed by thorough washing with distilled water. The explants were then surface sterilized with 0.1 % (W/V) HgCl for 5 minutes under laminar airflow. After that, explants were washed 4-5 times with sterile double distilled water to remove traces of mercuric chloride. The leaf explants, approximately 1.0 cm size, were excised and inoculated on MS15 media containing different plant growth regulators (PGRs) (table 1). All culture bottles contained 25 ml of agarified medium. All media contained 30 g l-1 sucrose and pH was adjusted to 5.8 with 1.0 N HCl or 1.0 N NaOH before adding 0.8 % agar and autoclaving at 121 ºC for 20 min. Culture bottles were placed in a culture room at 25 ± 2 ºC and exposed to 40 µmol m-2 s-1 fluorescent light in a 16h photoperiod. Callus initiation was observed within 7-10 days of inoculation. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 66-72, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 67 Shoot regeneration from Callus: Callus induced on MS media containing different combinations of 2,4-D and kinetin were subcultured on MS media incorporated with different concentrations of BA and kinetin as well as BA and AdSO for shoot regeneration and their effects on the number of shoots regenerated were recorded at regular time intervals (table 2, 3, 4 and 5). Sprouting from Microtubers: The shoot regeneration potential of microtubers formed on media supplemented with different hormones was verified in vitro. The media supplemented with different concentrations and combinations of BA and AdSOshowed varied response after 60 days of culture (table 6 and 7). Root Induction: The plantlets of S. tuberosum cultivars (Kufri Chipsona 3 and MP-97/644) grown in vitro showed self rooting in the multiplication media if kept for a longer period. No different hormonal regime was provided to induce rooting. Hardening of in vitro plantlets: The rooted plantlets were first transferred to protray containing 50% moist coco peat and 50% vermicompost as potting mix and kept in poly tunnel for 10 days in the green house to provide humidity. After 10 days, they were taken away from poly tunnel and found healthy. The plantlets were transferred to polybags containing soil: sand: FYM in1:1:1 proportion and kept in net house. About 99% plants survived in the green house and the plants were in healthy condition when transferred to the field also. Statistical analysis: The callus induction and shoot regeneration experiments were conducted with a minimum of three replicates and all experiments were repeated three times. Data obtained from all experiments were presented as the mean ± SE of three replications. Statistically significant differences were determined by analysis of variance (ANOVA) and the Duncan multiple range test (DMRT) at a 0.05 level of significance. Results and Discussion In the present study complete regeneration was successfully achieved from in vivo leaf explants of S. tuberosum cultivars (Kufri Chipsona 3 and MP-97/644) through callus culture (figure 1 and 2). The sterilizedleaves were inoculated on MS media containing different concentrations of 2,4-D (1.0-3.0 mg/l) and kinetin (1.0-3.0 mg/l). Callus induction from leaves of S. tuberosum on callusing media were observed and recorded periodically at 10 days interval (10 days, 20 days, 30 days and 40 days). Leaf explants induced callus on MS medium supplemented with different concentrations of 2,4-D and kinetin and their response was observed at different time period (Table 1). It is well known that various combination and concentration of auxins and cytokinins are effective for callus induction8, 9. Theoretically, equal amount of auxin and cytokinin promotes callusing, but in practice it differs to a good extend may be due to the variation in endogenous level of phytohormones. In the present study, curling as well as swelling was observed initially from the periphery and later on all the surface of leaf on MS medium containing 3.0 mg/l 2,4-D and 1.0 mg/l kinetin after 10 days of inoculation. The most effective media for Kufri Chipsona 3 for callus induction was found with high concentration of auxin, 2,4-D (3.0 mg/l) in combination with low concentration of cytokinin, kinetin (1.0 mg/l). Similarly, cultivar MP-97/644 showed better response in the same media. The calli were creamy and light yellowish in color. Similarly, best callus response were found in different potato cultivars viz. Diamant, Multa, Atlus and Lalpakri on MS medium supplemented with (3.0 mg/l) 2, 4-D. The present findings were also similar to Gavinlertvatanaand Li13 who induced creamy white friable callus from S. tuberosum on media containing 3.0 mg/l 2,4-D and 0.3 mg/l kinetin. In several other plant species also like Swertia angustifolia and Swertia mussotti higher concentration of 2,4-D (3.0 mg/l ) and Kinetin (2.5 mg/l) exhibited best callusing in leaf explants16, 17. Onamu et al18obtained highest callus induction from different explants in three potato cultivars (Alfa, Cambray Rosa Morelos and Atlantic) on MS medium supplemented with 4.0 mg/l BA and 1.0 mg/l NAA. Afrasiab and Iqbal reported best callogenesis on MS medium supplemented with 1.0 mg/l NAA and 0.5mg/l BA in cv. Diamant. In contrast, Haque et al19 reported 2, 4-D (1.0 mg/l) + Kinetin (0.25 mg/l) to be best for callus length and weight for cv. Diamant. The present results are also in accordance with the results of Omidi and Shahpiri20 who obtained callus from leaf and internodes on MS media containing 5.0 mg/l 2, 4-D and 0.25 mg/l kinetin that is high concentration of 2,4-D and low concentration of 0.25 mg/l kinetin. The interaction of both hormones had significant result on callus induction but low concentration of 2, 4-D with high concentration of kinetin was found ineffective. In the present investigation BA in combination of AdSO was essential for the regeneration of the calli. Callus induced from both cultivars (Kufri Chipsona 3 and MP-97/644) were subcultured on MS media supplemented with different concentrations of BA and Kinetin as well as BA and AdSO for shoot regeneration and their effects on the number of shoots regenerated were recorded at regular time intervals (table 2, 3, 4 and 5). The different stages of shoot regeneration and multiplication from the callus clump of both cultivars are shown in (Figure-1 and 2). In the present study, best shoot regeneration per callus clump was observed on MS media containing BA (1.5 mg/l) and AdSO (25.0 mg/l) where mean number was 21.00 (Kufri Chipsona 3) and 18.67 (MP-97/644) shoots/callus clump after 60 days of inoculation (Table 4 and 5). AdSO (adenine sulphate) play a vital role for the mass multiplication as PGR. It is well known that cytokinins stimulate plant cell division and participate in the release of lateral bud dormancy, in the induction of adventitious bud formation, in the growth of lateral buds and in the cell cycle control21, 22. The benefits of adenine sulphate are often only noticed when it is associated together with cytokinins such as BAP or kinetin23. The simulative role of AdSO in shoot multiplication has been emphasized from time International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 66-72, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 68 to time in various plants24, 25. Among all PGRs used, BA (1.5 mg/l) in combination with AdSO (25.0mg/l) was the most effective for multiple shoot induction. A similar effect of BA was also observed by Bhuiyan26. Our results suggest that BA, in combination with Adenine sulphate, improves the process of organogenesis. The shoot regeneration gradually increased with time period. Although the media enriched with 2,4-D and kinetin also showed good response. Khatun et al obtained 70 percent regeneration from callus on medium containing 5.0 mg/l BA, 1.0 mg/l IBA. But in our study reduced concentration of BA (1.5 mg/l) was found to be best for shoot multiplication. MS medium containing 4.0 mg/l Kinetin + 0.5 mg/l NAA was the best for maximum shoot regeneration from four potato cultivars. Hamdi et al27 reported regeneration from callus on MS media containing other hormones, 0.02 mg/l NAA, 2.0 mg/l Zeatin riboside and 0.02 mg/l gibberllic acid. Table-1 Callus induction from leaves of S. tuberosum cvs. (Kufri Chipsona 3 and MP-97/ 644), In table columns with different letters are significantly different at 0.05 according to Duncan’s multiple range test 2,4-D + Kn (mg/l) Induction of callus (%) 10 days 20 days 30 days 40 days K.chip 3 MP-97/644 K.chip 3 MP-97/644 K.chip 3 MP-97/644 K.chip 3 MP-97/644 1.0+1.0 - - - - - - - - 1.0+2.0 - - 7.9c 2.9c 16.1c 11.6c 14.3c 9.8c 2.0+1.0 24.5b 22.9ab 38.3b 29.8b 54.9b 43.9b 54.6b 39.8b 2.0+2.0 31.7ab 27.3a 43.6b 39.5b 60.2b 48.6b 58.9b 41.1b 3.0+1.0 41.3a 36.1a 67.5a 58.6a 91.0a 80.6a 87.9a 78.1a 1.0+3.0 - - 8.9c - 12.9c - 13.5c - - No callus (c)(b)(a)(e)(d) Figure-1 Different stages of callusing from the in vitro grown leaves of S. tuberosum (Cv. Kufri chipsona 3). (a) Curling and callus initiation from the periphery of leaves; (b) Callus induction after 30 days of inoculation (c) Shoot regeneration from callus after 20 days of inoculation; (d) Shoot regeneration from callus after 60 days of inoculation; (e) In vitro sprouting from microtubers of S.tuberosum L. (Kufri Chipsona 3) (Bars = 1.0 cm) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 66-72, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 69 (c)(b)(a)(e)(d) Figure-2 Different stages of callusing from the in vitro grown leaves of S.tuberosum (Cv. MP-97/644). (a) Callus induction after 30 days of inoculation; (b) Shoot regeneration from callus clump after 10 days of inoculation; (c) Shoot regeneration from callus after 20 days; (d) Shoot regeneration and multiplication; (e) In vitro sprouting from microtubers of S.tuberosum L. (MP-97/644) (Bars = 1.0 cm) Table-2 Shoot regeneration from callus of Kufri Chipsona 3 in vitro induced by different concentrations of 2, 4-D and Kinetin. In table columns with different letters are significantly different at 0.05 according to Duncan’s multiple range test 2,4-D + Kn (mg/l) Mean no. of shoots/ callus clump 20 Days 40 Days 60 Days 0.5 + 0.5 1.33 ± 0.58b 4.00 ± 0.33b 5.00 ± 0.88c 0.5 + 1.0 4.33 ± 0.67a 6.00 ± 0.58a 8.33 ± 0.33a 1.0 + 0.5 2.00 ± 0.88b 4.33 ± 0.67b 6.67 ± 1.00b Table-3 Shoot regeneration from callus of MP-97/644 in vitro induced by different concentrations of 2, 4-D and Kinetin. In table columns with different letters are significantly different at 0.05 according to Duncan’s multiple range test 2,4-D + Kn (mg/l) Mean no. of shoots/callus clump 20 Days 40 Days 60 Days 0.5 + 0.5 1.67 ± 0.67bc 2.33 ± 0.58c 3.67 ± 0.33c 0.5 + 1.0 3.00 ± 0.88a 4.33 ± 0.67a 7.33 ± 0.58a 1.0 + 0.5 2.00 ± 0.33ab 3.67 ± 0.33ab 5.00 ± 0.67b International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 66-72, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 70 Table-4 Effect of different concentrations of BAP and AdSO4 on shoot regeneration from callus of Kufri Chipsona 3. In table columns with different letters are significantly different at 0.05 according to Duncan’s multiple range test BA + AdSO(mg/l) Mean no. of shoots/callus clump 20 Days 40 Days 60 Days 1.0 + 15.0 2.67 ± 0.67bc 4.33 ± 0.88d 8.00 ± 0.58d 1.0 + 25.0 3.33 ± 0.58b 7.00 ± 1.00c 14.33 ± 0.88c 1.5 + 15.0 4.00 ± 0.33b 9.67 ± 0.67b 16.67 ± 0.33b 1.5 + 25.0 6.67 ± 0.67a 11.33 ± 0.88a 21.00 ± 0.58a Table-5 Effect of different concentrations of BAP and AdSO4 on shoot regeneration from callus of MP-97/644, In table columns with different letters are significantly different at 0.05 according to Duncan’s multiple range test BA + AdSO(mg/l) Mean no. of shoots/callus clump 20 Days 40 Days 60 Days 1.0 + 15.0 2.00 ± 0.58cd 3.67 ± 0.88d 6.33 ± 1.00d 1.0 + 25.0 3.33 ± 0.67c 7.33 ± 1.00c 10.00 ± 0.33c 1.5 + 15.0 4.67 ± 0.88b 10.00 ± 0.33b 14.33 ± 0.67b 1.5 + 25.0 7.00 ± 0.33a 11.67 ± 0.88a 18.67 ± 0.58a Table-6 Role of different concentrations of BA and AdSO4 on shoot regeneration potential of microtubers of Kufri Chipsona 3, In table columns with different letters are significantly different at 0.05 according to Duncan’s multiple range test BA + AdSO(mg/l) Mean no. of shoots/microtuber 20 Days 40 Days 60 Days 1.0 + 20.0 1.33 ± 0.33bc 2.67 ± 0.67c 4.33 ± 0.88c 1.0 + 25.0 4.00 ± 0.58a 7.33 ± 0.88a 9.67 ± 0.58a 1.5 + 20.0 2.33 ± 0.88b 4.33 ± 0.33b 6.00 ± 1.00b 1.5 + 25.0 1.67 ± 0.58bc 4.00 ± 1.00b 5.00 ± 0.67bc Table-7 Role of different concentrations of BAP and AdSO4 on shoot regeneration potential of microtubers of MP-97/644. In table columns with different letters are significantly different at 0.05 according to Duncan’s multiple range test BA + AdSO(mg/l) Mean no. of shoots/microtuber 20 Days 40 Days 60 Days 1.0 + 20.0 1.00 ± 0.33bc 1.67 ± 0.58bc 2.67 ± 0.67cd 1.0 + 25.0 2.33 ± 0.67a 4.00 ± 0.88a 8.33 ± 0.67a 1.5 + 20.0 1.67 ± 0.88ab 2.33 ± 0.33b 5.00 ± 0.58b 1.5 + 25.0 1.33 ± 0.33bc 2.00 ± 0.88bc 3.67 ± 0.33c The shoot regeneration potential of microtubers formed on media supplemented with different PGRs was verified in vitro. The MS medium supplemented with different concentrations and combinations of BA and AdSO showed varied response after 60 days (table 6 and 7). The best response was on media with 1.0 mg/l BAP and 25.0 mg/l AdSO with mean number of shoots 9.67 and 8.33 of Kufri Chipsona 3 and MP-97/644 respectively (figure-1e, 2e). The media supplemented with 1.5 mg/l BA and 25.0 mg/l AdSO also showed good response. The plantlets of S. tuberosum cultivars (Kufri Chipsona 3 and MP-97/644) grown in vitro showed self rooting in the multiplication media if kept for a maximum two months of period (figure-3a). The rooted plantlets were first transferred to portray containing 50% moist coco peat and 50% vermicompost as potting mix and kept in poly tunnel for 10 days in the green house to provide humidity (figure-3b). After 10 days, they were taken away from poly tunnel and found healthy. The plantlets were transferred to soil: sand: FYM in1:1:1 proportion and kept in net house. About 99% plants survived in the green house and the plants were in healthy condition when transferred to the field also (figure-3c). After 120 days tubers formed in Kufri Chipsona 3 and in MP-97/644 (figure 3d, 3e). International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 66-72, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 71 (a) (b)(c)(d)(e) Conclusion The present study was undertaken with a view to optimizing in vitro propagation through callus culture in two potato varieties of India with disease free and could be propagated for large-scale production and also can be conserved as virus free germplasm. . In addition, S. tuberosum can be supplied year round for commercial cultivation without any geographical and seasonal constraints. AcknowledgementsAuthors are thankful to the Central Potato Research Institute, Shimla, India for providing the S. tuberosum (Cultivar. Kufri Chipsona 3 and MP-97/644) plants. Authors are also highly thankful to respected reviewers for their important comments which help to improve this manuscript. References1.Wang B., Ma Y.L., Zhang Z.B., Wu Z.M., Wu Y.F., Wang Q.C., Li M.F., Potato viruses in China, Crop Prot.,30, 1117–1123 (2011)2.Hashem A., Hussain M.M., Monnikhof G., Seed Potato production at the private sector in Bangladesh. 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