In vitro PROPAGATION OF SELECTED MALAYSIA CASSAVA (Manihot esculenta Crantz) VARIETIES BY USING NODAL EXPLANTS

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1 Malays. Appl. Biol. (2018) 47(1): 1 5 In vitro PROPAGATION OF SELECTED MALAYSIA CASSAVA (Manihot esculenta Crantz) VARIETIES BY USING NODAL EXPLANTS HUZAIMAH MAHDI 1* and REBICCA EDWARD 1 1 Department of Plant Science, Faculty of Resource Science and Technology, University Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia * huzaimah.mahdi@gmail.com Accepted 20 January 2018, Published online 31 March 2018 ABSTRACT Cassava (Manihot esculenta Crantz) is one of the perennial shrub belongs to Euphorbiaceae family and is widely found in Africa and Asia. In Malaysia, Cassava is an important industrial crop for starch processing and other common industrial products such as animal pellets and for food industries. Hence, an in vitro propagation technique by using rapid shoot multiplication is needed to produce these highly demand industrial crop. This study aims to compare the shoot multiplication of three local Malaysia Cassava varieties which were Putih, Sawah and Baloi by using Murashige and Skoog (MS) media supplemented with plant growth regulators of 6-Benzylaminopurine (BAP) at 0.5, 1, 2, and 3 mg/l with or without 1- Naphthalaneacetic acid (NAA) at 0.01 mg/l. The results showed that the best media for the induction of shoot multiplication and leaves was MS media supplemented with 1.0 mg/l BAP mg/l NAA for all varieties. The best media for induction of shoot height was MS media free of BAP and NAA. The results also revealed that the shoot multiplication, induction of shoot height and induction of leaves were inhibited by high concentration of BAP with or without combination of NAA for each Cassava varieties. Key words: In vitro, Manihot esculenta, shoot multiplication, nodal explant INTRODUCTION Manihot esculenta or also known as Cassava, Tapioca, Manioc, Yuca (Spanish), and Ubi Kayu (Malay) is a perennial woody shrub from Euphorbiaceae family which is native to Central and South America (FAO, 2000). Cassava is categorized as an important source of carbohydrate after rice and corn which provides important component of diet to more than 800 million of people around the world (Richardson, 2013). In Asia, the main countries which produced highest Cassava tuber in fresh form are Thailand which are (29.19 million mt in 1.18 ha land) followed by Indonesia (20 million mt in 1.05 ha land) (MyAgri, 2015). Cassava contains high level of cyanogenic glucosides in all parts of plant with the highest distribution is on the leaf part which can determine the sweetness or bitterness of the Cassava tuber roots (O Hair, 1995). Cassava cultivation in Malaysia has * To whom correspondence should be addressed. faced a lot of problems including its susceptibility to diseases such as leaf spots, bacterial blight and white root disease (Sahadevan, 1987). In vitro propagation techniques is used to overcome the problems in cultivation of Cassava by selecting disease-free mother plants, produce large amount of plantlets from disease-free mother plants, and to shorten the growth cycle of Cassava (Dugassa & Feyissa, 2011). In Malaysia, Cassava is used for industrial purpose which is mainly for starch processing and other common industrial products such as animal pellets and for food industries such as monosodium glutamate and manufacturers of glucose (Lian & Idris, 2000). According to Department of Agriculture (DOA) Sarawak (2015), the Cassava industry for production of chip and snack has been increasingly in demand and been a source of income generation for small scale farmers which are really promising (DOA Sarawak, 2015). Hence, tissue culture propagation technique is needed for the rapid production of plantlets which have uniform genetic characteristics and free from

2 2 In vitro PROPAGATION OF SELECTED MALAYSIA CASSAVA (Manihot esculenta Crantz) diseases. Besides, there is no research on the in vitro propagation of local Malaysia Cassava varieties has been reported. MATERIALS AND METHODS The local Cassava (Manihot esculenta Crantz) varieties in Sarawak which were Putih, Sawah and Baloi varieties vegetative germplasms were obtained from Agriculture Research Centre (ARC) Semongok. The nodal cuttings were obtained from three months old plant stem which consists of node number 2 to node number 4 from the shoot tips. The nodal explants of each varieties were cut into cm length before surface sterilized by immersing in 70% ethanol for 1 minute followed by agitation in 25% Clorox (active ingredients: 5.25% Sodium Hypochlorite (NaOCl)) for 10 minutes with two drops of Tween-20 before rinsed with sterile distilled water for five minutes. The sterilized nodal explants were cultured onto full strength Murashige and Skoog (MS) media supplemented with 30 g/l sucrose, 7 g/l Phytagel with 6-Benzylaminopurine (BAP) at 0.5, 1, 2, and 3 mg/l with or without combination of 1-Naphthalaneacetic acid (NAA) at 0.01 mg/l. Each plantlets were subcultured every four weeks on the same MS media according to the previous treatments until week 12. The cultures were kept at culture room at temperature of 25±1ºC under 16 hours photoperiod condition under fluorescent lamp. There were thirty replicates of each treatment and one explant in each replicate. The observation on growth parameters such as number of shoots, plant height, and number of leaves were observed on each four weeks interval until week 12. One way analysis of variance (ANOVA) was used for data analysis and comparison of means by using Tukey test (p<0.05). RESULTS AND DISCUSSION The effect of BAP and NAA on the number of shoots From the treatments of BAP at concentrations of 0, 0.5, 1.0, 2.0, and 3.0 mg/l with or without combination of NAA at concentration of 0.01 mg/l, there was a statistically significant difference on the mean number of shoots of cassava Putih variety where the highest mean number of shoots produced from nodal explants was obtained from T7 (1.0 mg/l BAP mg/l NAA) which was 3.90±0.31. For Sawah variety, there was a statistically significant difference on the mean number of shoots where the highest mean number of shoots produced from nodal explants was obtained from T7 (1.0 mg/l BAP mg/l NAA) which was 7.87±0.35. For Baloi variety, there was a statistically significant difference on the mean number of shoots where the highest mean number of shoots produced from nodal explants was obtained from T7 (1.0 mg/l BAP mg/l NAA) which was 5.73±0.45. The similar findings of shoot multiplication of Cassava was also reported by Beyene et al. (2010) on Quelle and Kello Cassava varieties which produced the highest shoot multiplication on MS supplemented with 0.5 mg/l BAP mg/l NAA + 1 mg/l GA3. In contrast, the result from this experiment showed that MS media supplemented with 1.0 mg/l BAP only produced lower mean number of shoot compared to the MS media supplemented with BAP combined with NAA. This finding was supported by Venkatachalam and Jayabalan (1997) on their study on Groundnut (Arachis hypogaea L.) where the maximum number of shoot of cotyledonary-node explants of Groundnut (Arachis hypogaea L.) was obtained from the combination of BAP with NAA at 5 mg/l and 1 mg/l respectively. Treatment with the higher concentration of BAP from T5 (3.0 mg/l BAP mg/l NAA) and T9 (3.0 mg/l BAP mg/l NAA) did not produced shoot at all. According to Arab et al. (2014), BAP is only required at low concentrations from 0.5 mg/l to 2.5 mg/l and higher concentrations can inhibit shoot proliferation rate. Table 1 shows the mean number of shoots of Cassava Putih, Sawah and Baloi varieties by using different concentrations of BAP with or without combination of NAA after 12 weeks of cultures. The effect of BAP and NAA on the shoot height For the shoot height, there was a statistically significant difference on the mean shoot height for Putih variety on the treatments of different concentrations of BAP with or without combination of NAA in which T1 (0.0 mg/l BAP mg/l NAA) gave the highest mean shoot height at 8.90±0.23. For Sawah variety, there was also a statistically significant difference on the mean shoot height produced where T1 (0.0 mg/l BAP mg/l NAA) showed the statistically highest mean shoot height compared to other treatments which was 8.25±0.21. For Baloi variety, there was a statistically significant difference on the mean shoot height produced where T1 (0.0 mg/l BAP mg/l NAA) produce the highest mean shoot height which was 8.74±0.21. Taha et al. (2013) reported the results of in vitro shoot length of Cassava in which the treatment of MS free plant growth regulator produced the second highest shoot length after MS supplemented with 0.5 mg L -1 Kin and 0.05 mg L -1 NAA. Sukmadjaja and Widhiastuti (2011) also

3 In vitro PROPAGATION OF SELECTED MALAYSIA CASSAVA (Manihot esculenta Crantz) 3 Table 1. Mean number of shoots of Cassava Putih, Sawah and Baloi varieties by using different concentrations of BAP with or without combination of NAA after 12 weeks of cultures Treatment BAP NAA Mean no. of shoots Cassava Putih Cassava Sawah Cassava Baloi variety variety variety ±0.00 b 1.00±0.00 b 1.00±0.00 b ±0.00 b 2.83±0.38 c 1.73±0.45 c ±0.38 c 3.63±0.49 d 2.63±0.49 e ±0.41 c 3.37±0.49 d 2.27±0.45 d ±0.00 a 0.00±0.00 a 0.00±0.00 a ±0.51 d 4.67±0.48 e 3.27±0.52 f ±0.31 e 7.87±0.35 g 5.73±0.45 h ±0.47 e 7.13±0.35 f 4.83±0.38 g ±0.00 a 0.00±0.00 a 0.00±0.00 a *Mean followed by the same letter is not statistically significant (p> 0.05). *Data represents mean of thirty replicates, each replicate consists of one explant. Table 2. Mean shoot height of Cassava Putih, Sawah and Baloi varieties by using different concentrations of BAP with or without combination of NAA after 12 weeks of cultures Treatment BAP NAA Mean shoot height Cassava Putih Cassava Sawah Cassava Baloi variety variety variety ±0.23 e 8.25±0.21 g 8.74±0.21 g ±0.21 c 6.99±0.15 e 6.51±0.27 d ±0.29 b 5.64±0.30 c 5.95±0.35 c ±0.51 b 4.28±0.39 b 5.04±0.32 b ±0.00 a 0.00±0.00 a 0.00±0.00 a ±0.35d e 7.71±0.40 f 7.92±0.22 f ±0.16 d 6.91±0.24 e 7.28±0.41 e ±0.28 cd 5.96±0.20 d 6.67±0.20 d ±0.00 a 0.00±0.00 a 0.00±0.00 a *Mean followed by the same letter is not statistically significant (p> 0.05). *Data represents mean of thirty replicates, each replicate consists of one explant. reported that the highest length of shoot for Cassava Malang-6 variety was reported on MS media without plant growth regulator. In contrast, lower shoot height was recorded on the treatment with higher concentration of BAP which was 2.0 mg/l and 3.0 mg/l BAP on Cassava Putih, Sawah and Baloi varieties. Ashraf et al. (2014) has stated that BAP at higher concentration has an enhancing role on bud proliferation followed by higher shoot multiplication but less effective on shoot elongation. Table 2 shows the mean shoot height of Cassava Putih, Sawah and Baloi varieties by using different concentrations of BAP with or without combination of NAA after 12 weeks of cultures. The effect of BAP and NAA on the number of leaves For Putih variety, there was a significant difference on the mean number of leaves in which the highest mean number of leaves was obtained from T7 (1.0 mg/l BAP mg/l NAA) at 19.68±0.48. For Sawah variety, there was a significant difference on the mean number of leaves where the highest mean number of leaves was obtained from T7 (1.0 mg/l BAP mg/l NAA) at 23.29±0.54. For Baloi variety, T7 (1.0 mg/l BAP mg/l NAA) also produced the statistically highest mean number of leaves at 19.46±0.51. Taha et al. (2013) supported the results of this experiment in which the highest mean number of leaves was

4 4 In vitro PROPAGATION OF SELECTED MALAYSIA CASSAVA (Manihot esculenta Crantz) Table 3. Mean number of leaves of Cassava Putih, Sawah and Baloi varieties by using different concentrations of BAP with or without combination of NAA after 12 weeks of cultures Treatment BAP NAA Mean no. of leaves Cassava Putih Cassava Sawah Cassava Baloi variety variety variety ±0.82 bx 5.20±0.41 bz 4.37±0.49 by ±0.57 bx 10.56±0.51 cz 5.68±0.48 cy ±0.40 cx 14.68±0.48 ez 9.57±0.50 ey ±0.67 cx 11.35±0.49 dz 8.19±0.40 dy ±0.00 ax 0.00±0.00 ax 0.00±0.00 ax ±0.49 dx 20.56±0.51 fz 10.46±0.51 ey ±0.48 fx 23.29±0.54 gy 19.46±0.51 gx ±0.41 ex 20.21±0.42 fz 17.79±0.42 fy ±0.00 ax 0.00±0.00 ax *Mean followed by the same letter is not statistically significant (p> 0.05). *Data represents mean of thirty replicates, each replicate consists of one explants. reported from MS supplemented with 0.3 mg L -1 BA in combination with 0.05 mg L -1 NAA. This is due to the fact that according to Yuniastuti et al. (2016), the appropriate balance ratio of auxin and cytokinin are effective in triggering organogenesis especially for leaf formation, in which auxin plays a role in cell elongation whereas cytokinin plays a role in cell division, so that combination of both of them able to affect leaf length. Table 3 shows the mean number of leaves of Cassava Putih, Sawah and Baloi varieties by using different concentrations of BAP with or without combination of NAA after 12 weeks of cultures. CONCLUSION The best Murashige and Skoog (MS) media for the shoot multiplication and induction of leaves for Cassava Putih, Sawah and Baloi varieties was MS media supplemented with 1.0 mg/l BAP mg/l NAA. For induction of shoot height, the best media was MS media free of plant growth regulator for cassava Putih, Sawah and Baloi varieties. ACKNOWLEDGEMENTS The authors would like to thank the Plant Tissue Culture Laboratory, Universiti Malaysia Sarawak (UNIMAS) for providing the laboratory facilities through conducting the research. The authors would also like to thank En Haji Yakup Pendak, Senior Agronomist and Senior Research Officer at Agriculture Research Centre (ARC) Semonggok and the ARC staff for providing the germplasms of cassava varieties for this research. REFERENCES Arab, M.M., Yadollahi, A., Shojaeiyan, A., Shokri, S. & Ghojah, S.M Effects of nutrient media, different cytokinin types and their concentrations on in vitro multiplication of G N15 (hybrid of almond peach) vegetative rootstock. Journal of Genetic Engineering and Biotechnology, 12(2): Ashraf, M.F., Aziz, M.A., Kemat, N. & Ismail, I Effect of cytokinin types, concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. & Fernandez. Electronic Journal of Biotechnology, 7(6): Beyene, D., Feyissa, T. & Bedada, G Micropropagation of selected cassava (Manihot esculenta Crantz) varieties through meristem culture. Ethiopian Journal of Biological Science, 9(2): Department of Agriculture (DOA) Sarawak Pakej teknologi penanaman ubi kayu. Jabatan Pertanian Sarawak, Sarawak, Malaysia pp. Dugassa, G. & Feyissa, T In vitro production of virus-free sweet potato (Ipomoea batatas (L.) Lam) by meristem culture and thermotherapy. Ethiopia Journal of Science, 34(1): FAO Cassava. Retrieved on [October 22, 2015], Lian, T.S. & Idris, K Present situation and future potential of Cassava in Malaysia. Proceedings of the 6th Regional Workshop on Cassava s Potential in Asia in the 21st Century, Ho CHi Minh, Vietnam. pp MyAgri Penanaman ubi kayu. Retrieved on [November 25, 2015], /11/penanaman-ubi-kayu/.

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