Pak. J. Biotechnol. Vol. 14 (4) (2017) ISSN Print: ISSN Online:

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1 Pak. J. Biotechnol. Vol. 4 (4) (207) ISSN Print: ISSN Online: MICROPROPAGATION OF BANANA CULTIVAR BASRAI UNDER DIFFERENT CONCENTRATIONS OF N 6-BENZYLAMINOPURIN FOR SHOOT AND INDOLE-3-BUTYRIC ACID FOR ROOT INDUCTION Parvez Hussain Dahari, 2 Ghulam Shah Nizamani, Muharam Ali, 2 Abdullah Khatri, 3 Muhammad Rashid Nizamani, 2 Shafquat Yasmeen and Shah Nawaz Mari Sindh Agriculture University and 2 Nuclear Institute of Agriculture, Tando Jam, Pakistan 3 College of Forestry, Northwest A&F University, Yangling, China. 2 : nizamanigs@gmail.com Article received , Revised , Accepted ABSTRACT The present study was carried out to check different concentrations of N6-benzylaminopurin (BAP) on multiplication of shoots and along with different concentrations of indole-3-butyric acid (IBA) for root induction in banana variety Basrai. Data recorded for various parameters were subjected to completely randomized design. The suckers were taken as explants source and cultured on (Murashige and Skoog, 962) medium with various concentrations (0.0,.0, 2.0, 3.0 and 4.0 mg l - ) of BAP for shoot induction and ½MS medium under the concentrations (0.0, 0.5,.0,.5 and 2.0 mg l - ) of IBA were used for root induction. After four weeks of culturing, the external leaf primordia of explants turned green which were initially creamy white and from these balls approximating structure, adventitious plantlets were developed. The results indicated that highest shoot length 4.5, 5.23 and 8.59 and more multiplication of shoots was achieved 3.25, 4.39 and 6.29 explants - under the concentrations of 2.0 mg l - BAP at 20, 40 and 60 days after inoculation. The highest number of leaves 2.49, 3.6 and 4.0 explants - observed under the concentrations of 3.0 mg l - BAP at 20, 40 and 60 DAI, respectively. For root induction ½MS media under various concentrations of IBA indicated that the more numbers of roots were recorded (.25 and 2.6) under the concentration of.50 mg l - IBA at 20 and 40 days, while 3.67 numbers of roots was achieved at 60 days with concentration of.0 mg l - IBA. The maximum root length (.38,.92 and 2.72 cm) was observed at 20, 40 and 60 DAI under the concentration of.50 mg l - IBA at 20, 40 and 60 DAI, respectively. Key words Banana, tissue culture, MS medium, growth hormones, concentrations INTRODUCTION Banana (Musa spp.) is one of the most vital fruit crops of the world and grown in the tropical and subtropical world locations (Darvari et al., 200; Rahman et al., 203). Banana is a major fruit crop of Pakistan, it is cultivated near about 34,800 hectares with production of 54,800 tons and mostly grown in province of Sindh, where environment of soil and climate are suitable for its successful production and the total share of province alone in its cultivation is 87 percent (Memon et al., 206). Ninety five percent of area is cultivated under Basrai variety (Cavendish dwarf), and the remaining under William Hybrid (Iqra, 204). Plant growth regulators play a vital role to determine the developmental pathway of the plant cells. To gain disease free healthy plant materials for the enhancement of a protocol for meristem culture of banana Basrai variety (Kabir et al., 2008). Nowadays, the technique of plant tissue culture has become a powerful tool for studying, solving basic and applied problems in plant biotechnology (Yadav, et al., 202). This technique provides high rates of multiplying genetically homogeneous, pest and disease free planting materials through tissue culture techniques (Madhulatha et al., 2004, Strosse et al., 2006) and long-standing alternate for increasing banana yield which could be used for resistant genotypes. MATERIALS AND METHODS The six months old suckers of Basrai variety of bananas were taken from experimental farm, Nuclear Institute of Agriculture (NIA) and brought in the laboratory. Outer tissues, roots of suckers were removed with the help of knife. The experiments were conducted on MS medium with different concentration of N6-benzylaminopurine for shoot induction and IBA were used for root induction. Surface sterilization of explants: The suckers were treated with 70% alcohol for one minute followed by 0% sodium hypochlorite for twenty minutes and washed three times after sterilization in sterilized distilled water for five minutes. Media and culture condition: Murashige and Skoog (962) medium supplemented were used for shoot and root inductions with different growth hormones concentrations added according to the requirement of the experiment. The medium was prepared from the stock solutions (macro, micro nutrients and growth regulators) in the laboratory through mixing of all nutrients on hot plate and medium was solidified with gelrite 3.0 g l -. The ph of the medium was adjusted to before putting in autoclave at.06 kg per cm 2 pressure for 20 minutes at 2 C (Pinaki and Jahan, 202, Ali et al., 20). After culturing, all jars were transferred to growth room at 25 ± 2 C under 6/

2 762 P.H. Dahari et al., Pak. J. Biotechnol. 8 hours in light and dark period under white florescent light with intensity 2000 lux for the growth and development. Laminar airflow beach was sterilized by 70 % alcohol and switching on the UV light of the cabinet for ten minutes before starting the culturing and sub-culturing work. Statistical analysis: The experimental data were recorded and subjected to analysis of variance under linear models of statistics to observe statistical difference among various traits using computer software Statistix 8. version. Furthermore, least significant difference (LSD) test was applied to test the level of significance between mean performances of traits (Gomez and Gomez, 984). RESULTS AND DISCUSSIONS Days to shoot initiation: The results indicated that the days to shoot initiation varied significantly between the different concentrations of N6-benzylaminopurine (BAP) and data are presented in Table. Results indicated that the lower amount of BAP showed early days to shoot initiation 5.4 and 7.45 under the concentration of 0.0 and.0 mg l -, while as compared to higher concentrations of 2.0 and 3.0mg l - showed late days to shoot initiation and respectively. The results are fully supported by Ali (996) and Al-Amin et al., (2009). Table-: Effect of different concentrations of BAP on days to initiation of banana variety Basrai at different days after inoculation Concentrations MS + BAP (mg l - ) No. of cultures Mean days to initiation e d a b c Days, S.E. (0.8623), LSD (5%) (.7663), BAP, S.E. (0.6679), LSD (5%) (.3682), Days x BAP, S.E. (.4935), LSD (5%) (3.0594) Shoot length (cm): Effect of different treatments of N6-benzylaminopurine for shoot length was statistically highly significant and data are depicted in Table 2. Mean of the different concentrations showed highest shoot length were recorded 5.99cm, followed by 5.52cm under the concentrations of 2.0 and 3.0mg per litre, while minimum shoot length 3.8cm under control. The maximum shoot length 4.5, 5.23 and 8.59cm were achieved under the concentration of 2.0 mg l - BAP, followed by 3.80, 4.72 and 8.03 cm under concentration of 3.0 mg l - BAP and minimum shoot length 2.7, 3.8 and 4.9 cm were obtained under control at 20, 40 and 60 days to inoculation, respectively. Rahman et al., (2004), Mangrio et al., (204) concluded that highest shoot length found with the treatment of.5 to 2.0 mg l - BAP. Table-2: Effect of different concentrations of BAP on shoot length (cm) of banana variety Basrai at different days after inoculation Concentrations No. of Shoot length (cm) Mean MS + BAP (mgl - ) cultures 20 DAI 40 DAI 60 DAI j 3.8 h 4.9 f 3.8 e i 3.95 g 5.23 d 4.03 d f 5.23 d 8.59 a 5.99 a g 4.72 e 8.03 b 5.52 b hi 4.24 f 7.54 c 4.96 c Mean 3.23 c 4.26 b 6.72 a Days, S.E. (0.097), LSD (5%) (0.0403), BAP, S.E. (0.052), LSD (5%) (0.032), Days x BAP, S.E. (0.034), LSD (5%) (0.0698) Multiplication of shoots: The effect of different concentrations of N6-benzylaminopurine for multiplication of shoot was statistically highly significant and data are shown in Table 3. The mean of the different concentrations indicated that highest multiplication of shoots was recorded 4.64, followed by 3.97 under the concentrations 2.0 and 3.0 mg l - of N6-benzylaminopurine, while mini-mum 2.26 under control. The maximum multiplication of shoots was recorded 3.25, 4.39 and 6.29 under the concentration of 2.0mg l - N6-benzylaminopurine, followed by 2.8, 3.90 and 5.9 under the concentration of N6-benzylaminopurine 3.0 mg l -, and minimum multiplication of shoots were observed.24, 2.8 and 3.36 under control (0.0mg l - N6-benzylaminopurine) at 20, 40 and 60 days to inoculation, respectively. The same concentration of medium was used for two times to maintain multiplication of shoots in the medium. For multiplication of shoots and BAP stimulates shoot proliferation in bananas and have mutagenic effect when supplemented from low to high concentrations (Rahman et al., 2004).

3 Vol. 4 (4) 207 Micropropagation of banana 763 Table-3: Effect of different concentrations of BAP on multiplication of shoots of banana variety Basrai at different days after inoculation Concentrations No. of Multiplication of shoots Mean MS + BAP (mgl - ) cultures 20 DAI 40 DAI 60 DAI n 2.8 l 3.36 g 2.26 e m 3.00 i 3.99 e 2.98 d h 4.39 c 6.29 a 4.64 a j 3.90 f 5.9 b 3.97 b k 3.27 h 4.25 d 3.27 c Mean 2.3 c 3.35 b 4.6 a Days, S.E. (0.005), LSD (5%) (0.026), BAP, S.E. (0.060), LSD (5%) (0.0320), Days x BAP, S.E. (0.084), LSD (5%) (0.0374) Number of leaves: Effect of different treatments of N6-benzylaminopurine on number of leaves was statistically highly significant and data are appeared in Table 4. The mean of the different concentrations indicated highest numbers of leaves were recorded 3.25, followed by 3.06 under the concentrations 3.0 and 4.0 mg l - of N6-benzylaminopurine, while lowest number of leaves was recorded 2.0 under control N6-benzylaminopurine. The maximum number of leaves were obtained 2.49, 3.6 and 4.0 with 3.0 mg l - treatment of BAP, followed by 2.33, 2.99 and 3.86 under the concentration of 4.0 mg l - N6-benzylaminopurine and minimum number of leaves.28,.96 and 2.80 were achieved under control at 20, 40 and 60 days to inoculation. The results agreed with Rabbani et al., (996) and Rahman et al., (2004). Table -4: Effect of different concentrations of BAP on number of leaves of banana variety Basrai at different days after inoculation Concentrations No. of Number of leaves Mean MS + BAP (mg l - ) cultures 20 DAI 40 DAI 60 DAI l.96 k 2.80 g 2.0 e k 2.32 i 3.08 e 2.44 d j 2.8 g 3.72 c 2.86 c h 3.6 d 4.0 a 3.25 a i 2.99 f 3.86 b 3.06 b Mean 2.0 c 2.65 b 3.5 a Days, S.E. (0.053), LSD (5%) (0.033), BAP, S.E. (0.09), LSD (5%) (0.0243), Days x BAP, S.E. (0.0265), LSD (5%) (0.0543) Number of roots: Effect of different concentratons of indole-3-butyric acid for number of roots was statistically highly significant with 5% probability level (Table 5). The mean of different concentrations indicated that maximum number of roots were recorded 2.48 under.50mg l -, followed by 2.28 and 2.25 at par under the concentrations.0 and 2.0mg l - IBA. The highest number of roots were observed.25, 2.6 under the concentration of.5 mg l - indole-3-butyric acid, while 3.67 under.0mg l - indole-3-butyric acid at 20, 40 and 60 DAI, respectively. The results confirmed by Moshiur, et al., (2002) that the 2.0 mg l - concentration of indole-3-butyric acid proved the best concentration for rooting. Table -5: Effect of different concentrations of BAP on number of roots of banana variety Basrai at different days after inoculation Concentrations ½MS+IBA No. of Number of roots Mean concentrations (mg l - ) cultures 20 DAI 40 DAI 60 DAI o.64 j 2.93 e.79 e n.87 i 3.8 d 2.0 d m 2.03 h 3.67 a 2.28 b k 2.6 f 3.5 b 2.46 a l 2.26 g 3.3 c 2.25 c Mean.07 c 2.08 b 3.32 a Days, S.E. (0.005), LSD (5%) (0.026), BAP, S.E. (0.060), LSD (5%) (0.0320), Days x BAP, S.E. (0.082), LSD (5%) (0.0374) Root length (cm): The outcome of diverse amounts of indole-3-butyric acid on root length was statistically highly significant with 5% probability level and data are present in Table 6. The mean of

4 764 P.H. Dahari et al., Pak. J. Biotechnol. different amounts indicated that maximum root length was recorded 2.0, followed by.9 under the concentrations of.5 and 2.0 mg l - indole-3 acetic acid. The mean of different concentrations indicated that maximum root length were observed.38,.92 and 2.72 cm, followed by.30,.84 and 2.60 cm under the concentrations of.50 and 2.0 mg l - IBA respectively after 20, 40 and 60 days to inoculation. The result of recent study is similar with the conclusion of Molla et al., (2004), Moshiur, et al., (2002), Bhosale et al., (203) Ahmed et al., (204) and Mehnaz et al., (205) for root length under concentrations of.5-3.0mg l - indole -3-butyric acid. Table -6: Effect of different concentrations of BAP on root length of banana variety Basrai at different days after inoculation Concentrations ½MS + IBA No. of Root length (cm) Mean concentrations (mg l - ) cultures 20 DAI 40 DAI 60 DAI n.34 jk.96 e.39 e m.5 i 2.5 d.57 d l.75 h 2.4 c.79 c j.92 f 2.72 a 2.00 a k.84 g 2.60 b.9 b Mean.6 c.67 b 2.37 a Days, S.E. (0.03), LSD (5%) (0.0232), BAP, S.E. (0.080), LSD (5%) (0.0360), Days x BAP, S.E. (0.096), LSD (5%) (0.0402) CONCLUSION It was concluded that MS media hold 2.0 mg l - N6- benzylaminopurine observed best for shoot length, shoots multiplication and number of leaves, while.50 mg l - indole-3-butyric acid (IBA) performed very well for highest number of roots and root length after 20, 40 and 60 days after inoculation of banana variety Basrai for micropropagation. The plants with well developed roots were transplanted inside pots for hardening and reach about 25 to 30 cm height were transferred in the field for further screening. REFERENCES: Ahmed, S., A. Sharma, B. Bhushan, A.K. Singh and V.K. Wali, Effect of carbohydrates sources, ph and supporting media on in vitro rooting of banana (Musa spp.) cv. Grand naine. Afri. J. Agri. Res. 9(4): (204). AL-Amin, M.D, M.R. Karim, M.R. Amin, S. Rahman and A.N.M. Mamun, In vitro micropropagation of banana (Musa spp.). Bangladesh J. Agril. Res. 34(4): (2009). Ali, H., Effect of BAP and IBA on micropropagation of some banana cultivars. M.Sc. thesis, Department of Horticulture, Bangladesh Agricultural University, Mymensingh. Pp. 73 (996). Ali, A., A. Sajid, N.H. Naveed, A. Majid, A. Saleem, U.A. Khan, F.I. Jafery and S. Naz, Initiation, proliferation and development of micropropagation system for mass scale production of banana through meristem culture. Afri. J. Biotech. 0(70): (20). Analytical Software, Statistix 8. User Manual, Tallahassee (2005). Bhosale, U.P., S.V. Dubhashi and N.S. Mali, In vitro rooting of different species of banana. Advances in Applied Sci. Res. 4(3): 5-8 (203). Darvari, F.M., M. Sariah, M.P. Puad and M. Maziah, Micropropagation of some Malaysian banana and plantain (Musa sp.) cultivars using male flowers. Afric. J. Biotech. 9(6): (200). Gomez, K.A. and A.A. Gomez, Statistical Procedure for Agricultural Research, (2 nd eds.), Wiley, New York, USA, Pp. 680 (984). Iqra Junejo, A Technical Book of Banana, Pp. -66 (204). Kabir Shiragi, M.H., M.A. Baque and K.M. Nasiruddin, Eradication of banana bunchy top virus (BBTV) and banana mosaic virus (BMV) from infected plant of banana cv. Amritasagar through meristem culture. South Pacific Studies 29(): 7-4 (2008). Larkin, P.J., and W.R. Scowcroft, Somaclonal variation: a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60:97-24 (98). Madhulatha, P., M. Anbalagan, S. Jayachandranand and N. Sakthivel, Influence of liquid pulse treatment with growth regulators on in vitro propagation of banana (Musa spp. AAA). Plant Cell Tissue Organ Cult. 76: (2004). Mangrio, G.S., A.S. Altaf, M.R. Rind, S.M. Mangrio, S.N.R Balouch and M.U. Dahot, In vitro regenerability of different sugarcane (Saccharum officinarum L.) varieties through shoot tip culture. Pak. J. Biotechnol. (): 3-23 (204). Mehnaz Q., S.T. Qureshi, I.A. Khan and S.

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