Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes




Abstract. Carsono N, Juwendah E, Liberty, Sari S, Damayanti F, Rachmadi M. 2021. Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes. Biodiversitas 22: 2555-2560. The development of callus in the course of transgenic rice avoids the somaclonal variants. To obtain a high number of normal phenotypes and a low number of somaclonal variants requires an appropriate 2,4-D concentration. In this study, we obtained the best callus induction time and a high number of green plant regeneration for three responsive rice genotypes on different 2,4-D concentrations in NB5 medium. The mature seeds of rice embryos were used as explants. A completely randomized factorial design was applied with four levels of 2,4-D concentrations (0, 1, 3, and 5 ppm), two levels of induction time (one and two weeks), and three rice genotypes (cv. Fatmawati, Nipponbare, and Kitaake). The study revealed that there was no interaction effect among genotype, 2,4-D concentration, and callus induction time. Three rice genotypes performed best in callus proliferation and regeneration. One-week callus induction time showed higher callus growth capacity (CGC) as compared to two-week callus induction time. Shoot regeneration capacity (SRC) was independently affected by genotype as well as by callus induction time. The interaction effect between 2,4-D concentration and callus induction time was observed on plant regeneration capacity (PRC). Without the addition of 2,4-D and 1 ppm of 2,4-D, the green plant regeneration capacity (GRC) was comparatively higher. Addition of 2,4-D showed a significant effect, especially at the plant regeneration stage. We found that one-week callus induction was the best treatment for callus proliferation and plant regeneration. We recommend the use of one-week callus induction and 1 ppm of 2,4-D for rice callus proliferation (sub-culture) and subsequent plant regeneration.


Abe T, Futsuhara Y. 1986. Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L). Theoretical and Applied Genetic 72: 3-10. doi:10.1007/bf00261446.
Abiri R, Maziah M, Shaharuddin NA, Yusof ZN, Atabaki N, Hanafi MM, Sahebi M, Azizi P, Kalhori N, Valdiani A. 2016. Enhancing somatic embryogenesis of Malaysian rice cultivar MR219 using adjuvant materials in a high-efficiency protocol. Int. J. Environ. Sci. Technol. (2017) 14:1091–1108. DOI 10.1007/s13762-016-1221-y.
Akins PO, Vasil IK. 1982. Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L. (Wheat): evidence for somatic embryogenesis. Protoplasma 110: 95-105. doi:10.1007/bf01281535.
Anai T, Koga M, Tanaka H, Kinoshita T, Rahman SM, Takagi Y. 2003. Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene. Plant Cell Reports 21:988-327. doi: 10.1007/s00299-003-0609-6.
Carsono N, Yoshida T. 2006. Plant regeneration capacity of calluses derived from mature seed of five Indonesian rice genotypes. Plant Production Science 9: 71-77. DOI:10.1626/pps.9.71.
Carsono N, Yoshida T. 2008. Transient expression of green fluorescent protein in rice calluses: optimization of parameters for Helios gene gun device. Plant Prod Science 11: 88-95. DOI: 10.1626/pps.11.88.
Cha-um S, Boriboonkaset T, Pichakum A, Kirdmanee C. 2009. Multivariate physiological indices for salt tolerance classification in indica rice (Oryza sativa L. spp. indica). General and Applied Plant Physiology 35: 75-87.
Datta K, Baisakh N, Oliva N, Torizzo L, Tan J, Rai M, Abrigo E, Rehana S, Al-Babili S, Beyer P, Potrykus I, Datta SK. 2003. Bioengineered ‘golden’ indica rice cultivars with ?-carotene metabolism in the endosperm with hygromycin and mannose selection systems. Plant Biotechnology Journal 1: 81-90. doi: 10.1046/j.1467-7652.2003.00015.x
Din AJM, Ahmad FI, Wagiran A, Rahmat AAZ, Sarmidi MR. 2016. Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas). Saudi J Biol Sci. 23(1): S69–S77. doi: 10.1016/j.sjbs.2015.10.022.
Ezhova TA. 2007. Genetic control of totipotency of plant cells in an in vitro culture. Russian Journal of Developmental Biology 34 (4): 197-204. DOI: 10.1023/A:1024940130511.
Filho JCB, Adison KK, Luiz FPP, Zuleide H, Luiz GEV. 2001. In vitro adventitious shoot regeneration from sweet orange using thin epicotyls section. Crop Breeding and Applied Biotechnology 1(1): 27-34. DOI:10.13082/1984-7033.v01n01a03.
Gao T, Wu Y, Zhang Y, Liu L, Ning Y, Wang D, Tong H, Chen S, Chu C, Xie Q. 2011. OsSDIR1 overexpression greatly improves drought tolerance in transgenic rice. Plant Molecular Biology 76: 145-156. doi: 10.1007/s11103-011-9775-z.
He GY, Lazzer PA. 2001. Improvement of somatic embryogenesis and plant regeneration from durum wheat (Triticum turgidum var durum Derf) scutellum and inflorescence culture. Euphytica 119(3): 369-376.
Henke RR, Mansur M, Constantine MJ. 1978. Organogenesis and plantlet formation from organ and seedling-derived calli of rice (Oryza sativa L). Physiologia Plantarum 44: 11-14. DOI:10.1111/J.1399-3054.1978.TB01605.X.
Hiei Y, Ohta S, Komari T, Kumashiro T. 1994. Efficient transformation of rice (Oryza Sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 6: 271-282. doi: 10.1046/j.1365-313x.1994.6020271.x.
Hoque ME, Mansfield JW. 2004. Effect of genotype and explants age on callus induction and subsequent pant regeneration from root-derived callus of Indica rice genotypes. Plant Cells, Tissue and Organ Culture 78: 217-223.
Jiang SE, Xie L, Ramachandran S. 2016. Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants. Sci. Rep. 6, 34427; doi: 10.1038/srep34427 (2016).
Jha S, Chattoo BB. 2010. Expression of a plant defensin in rice confers resistance to fungal phytopathogens. Transgenic Research 19: 373-384. DOI 10.1007/s11248-009-9315-7.
Jiménez VM. 2001. Regulation of in vitro somatic embryogenesis with emphasis on to the role of endogenous hormones. Revista Brasileira de Fisiologia Vegetal 13(2): 196-223. doi:10.1590/s0103-31312001000200008.
Karp A. 1995. Somaclonal variations as a tool for crop improvement. Euphytica 85: 295-302. DOI: 10.1007/978-94-011-0357-2_35.
Khaleda L, AlForkan M. 2006. Genotypic variability in callus induction and plant regeneration through somatic embryogenesis of five deepwater rice (Oryza sativa L.) cultivars of Bangladesh. African Journal of Biotechnology 5: 1435-14440
Khanna HK, Raina SK. 1998. Genotype x media culture interaction effect on regeneration respons three indica rice cultivars. Plant Cell, Tissue and Organ Culture, 52(3), 145–153. doi:10.1023/a:1006032303195.
Kumar V, Shriram V, Kishor PBK, Jawali N, Shitole MG. 2010. Enhanced proline accumulation and salt stress tolerance of transgenic indica rice by over-expressing P5CSF129A gene. Plant Biotechnology Reports 4: 37-48. doi:10.1007/s11816-009-0118-3.
Lapitan VC, Haruhiko W, Yoshifumi T, Fumio T. 2005. Transfer of soybean glycinin gene into rice via agrobacterium tumefaciens-mediated transformation. Philippine Journal of Crop Science 30(2): 39-45.
Larkin PJ., Scowcroft WR. 1981. Somaclonal variation a novel source of variability from cell cultures for plant improvement. Theoretical and Applied Genetic 60: 197-214. doi:10.1007/bf02342540.
Lee KS, Jeon HS, Kim MY. 2002. Optimization of a mature embryo based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars. Plant Cell, Tissue and Organ Culture 71: 9–13. doi:10.1023/a:1020305432315.
Lin YZ, Zhang Q. 2005. Optimizing the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Reports 23: 540-547. doi: 10.1007/s00299-004-0843-6.
Lutts S, Kinet JM, Bouharmont J. 2004. Somaclonal variation in rice after two successive cycles of mature embryo derived callus culture in the presence of NaCl. Biologia Plantarum 44: 489-495. doi:10.1023/a:1013761814407.
Ma J, Song Y, Wu B, Jiang M, Li K, Zhu C, Wen F. 2011. Production of transgenic rice new germplasm with strong resistance against two isolations of Rice stripe virus by RNA interference. Transgenic Research 20: 1367-1377. doi: 10.1007/s11248-011-9502-1.
Maeda E, Radi SH, Nakamura T, Yamada S. 1988. Cellular differentiation and morphogenesis in plant tissue culture. In Cell and tissue culture in field crop improvement. FFTC Book series no 38. p. 13- 23.
Meneses A, Flores D, Munoz M, Arrieta G, Espinosa AM. 2005. Effect of 2.4-D Hydric stress and light on indica rice (Oryza sativa) somatic embryogenesis. Revista de Biologia Tropical 53: 361-368. DOI: 10.15517/rbt.v53i3-4.14598.
Michalczuk L, Cooke TJ, Cohen JD. 1992a. Auxin levels at different stages of carrot somatic embryogenesis. Phytochemistry 31: 1097–1103. doi:10.1016/0031-9422(92)80241-6.
Michalczuk L, Ribnicky DM, Cooke TJ, Cohen JD. 1992b. Regulations of indole-3-acetic acid biosynthetic pathways in carrot cell cultures. Plant Physiology 100: 1346–1353. doi: 10.1104/pp.100.3.1346.
Miroshnichenko D, Chernobrovkina M, Dolgov S. 2016. Somatic embryogenesis and plant regeneration from immatureembryos of Triticum timopheevii Zhuk. and Triticum kiharaeDorof. et Migusch, wheat species with G genome. Plant Cell Tiss Organ Cult 125:495–508. DOI: 10.1007/s11240-016-0965-x.
Mitsuoka K, Honda H, Xing XH and Unno H. 1994. Effect of intracellular 2,4-D concentration on plantlet regeneration of rice (Oryza sativa L.) callus. Appl Microbiol Biotech 42: 364-366. https://link.springer.com/article/10.1007/BF00902743
Mostafiz SB, Wagiran A. 2018. Efficient callus induction and regeneration in selected indica rice. MDPI Agronomy 8 (5): 77. doi.org/10.3390/agronomy8050077.
Oh SJ, Song SI, Kim YC, Jang HJ, Kim SY, Kim M, Kim YK, Nahm BH, Kim JK. 2005. Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth. Plant Physiology 138: 341-351. DOI: 10.1104/pp.104.059147.
Ozawa K, Kawahigashi H, Kayano T, Ohkawa Y. 2003. Enhancement of regeneration of rice (Oryza sativa L.) calli by integration of the gene involved in the regeneration ability of the callus. Plant Science 165: 395-402. doi:10.1016/S0168-9452(03)00200-0.
Patkar RN. Chatto BB. 2006. Transgenic indica rice expressing ns-LTP-like protein shows enhanced resistance to both fungal and bacterial pathogens. Molecular Breeding 17: 159-171. doi:10.1007/s11032-005-4736-3.
Puhan P, Siddiq EA. 2013. Protocol optimization and evaluation of rice varieties response to in vitro regeneration. Advances in Bioscience and Biotechnology 4: 647-653. DOI: 10.4236/abb.2013.45085.
Rashid H, Yokoi S. Toriyama K, Hinata K. 1996. Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Reports 15: 727-730. doi: 10.1007/BF00232216.
Sah SK, Kaur A, Sandhu JS. 2014. High frequency embryogenic callus induction and whole plant regeneration in japonica rice cv. Kitaake. Journal Rice Res 2:2. DOI: 10.4172/jrr.1000125.
Sahoo KK, Tripathi AK, Pareek A, Sopory SK, Pareek SLS. 2011. An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7: 49. doi:10.1186/1746-4811-7-49.
Sato Y, Masuta Y, Saito K, Murayama S, Ozawa K. 2011. Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene, OsAPXa. Plant Cell Reports 30: 399-406. doi: 10.1007/s00299-010-0985-7.
Sawahel W. 2003. Improved performance of transgenic glycinebetain-accumulating rice plants under drought stress. Biologia Plantarum 47: 39-44. doi:10.1023/a:1027372629612.
Sivamani E, Shen P, Ong CA, de Kochka A, Fauquet C, Beachy R. 1999. Rice Plant Containing Rice Tungro Spherical Virus (RTSV) coat protein transgenes to virus infection. Molecular Breeding, 5(2): 177–185. doi:10.1023/a:1009633816713.
Takesawa T, Ito M, Kanzaki H, Kameya N, Nakimura I. 2002. Over-expression of ? glutathione S-transferase in transgenic rice enhances germination and growth at low temperature. Molecular Breeding 9(2): 93-101. doi:10.1023/a:1026718308155.
Tang Y, Bao X, Zhi Y, Wu Q, Guo Y, Yin X, Zeng L, Li J, Zhang J, He W, Liu W, Wang Q, Jia C, Li Z, Liu K. 2019. Overexpression of a MYB family gene, OSMYB6, increases drought and salinity stress tolerance in transgenic rice. Front. Plant Sci. 10: 168. https://doi.org/10.3389/fpls.2019.00168
Tsukahara M, Hirosawa T. 1992. Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. Journal of Plant Research 105(2): 227-233. doi:10.1007/BF02489417.
Upadhyaya G, Sen M, Roy A. 2015. In vitro callus induction and plant regeneration of rice (Oryza sativa L.) var. ‘Sita’, ‘Rupali’ and ‘Swarna Masuri’. Asian Journal of Plant Science and Research 5(5): 24-27. https://www.imedpub.com/articles/in-vitro-callus-induction-and-plant-regeneration-of-rice-oryza-sativa-l-var-sita-rupali-and-swarna-masuri.pdf
Wada Y, Carsono N, Anas, Tong L, Yoshida T. 2009. Genetic transformation of high molecular weight glutenin (Glu-1Dx5) to rice cv. Fatmawati. Plant Production Science 12(3): 341-344. DOI: 10.1626/pps.12.341.
Wagiran A, Ismanizan I, Che RCMZ, Ruslan A. 2008. Improvement of plant regeneration from embryogenic suspension cell culture of Japonica rice. Journal of Biological Sciences 8: 570-576. doi:10.3923/jbs.2008.570.576.
Wójcik A, Wójcikowska B, Gaj MD. 2020. Current Perspectives on the auxin-mediated genetic network that controls the induction of somatic embryogenesis in plants. Journal Mol. Sci. 21(4) 1333. doi:10.3390/ijms21041333.
Yang Z, Chen H, Tang W, Hua H, Lin Y. 2011. Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes. Pest Management Science 67: 414-422. doi: 10.1002/ps.2079.
Zhou YL, Xu MR, Zhao MF, Xie XW, Zhu LH, Fu BY, Li ZK. 2010. Genome-wide gene responses in a transgenic rice line carrying the maize resistance gene Rxo1 to the rice bacterial streak pathogen, Xanthomonas oryzae pv. Oryzicola. BMC Genomics 11:78. doi: 10.1186/1471-2164-11-78.
Zimmerman JL. 1993. Somatic embryogenesis: a model for early development in higher plants. The Plant Cell 5: 1411–1423. doi:10.1105/tpc.5.10.1411.