CALLUS INDUCTION AND SOMATIC EMBRYOGENESIS FROM MAIZE MATURE EMBRYOS (ZEA MAYS L.)

Transcription

1 Journal of Cell and Tissue Research Vol. 13(1) (2013) (Available online at ISSN: ; E-ISSN: Original Article CALLUS INDUCTION AND SOMATIC EMBRYOGENESIS FROM MAIZE MATURE EMBRYOS (ZEA MAYS L.)? DHILLON, N. K. 1 AND GOSAL, S. S. 2 1 Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana ; 2 Director of Research, Punjab Agricultural University, Ludhiana E. mail: dhillon.navjot@gmail.com Received: January 15, 2013; Accepted: February 10, 2013 Abstract: Ten maize lines prevalently used by breeders in the maize program were screened for their ability to form type II callus. Mature seeds were used as explant for in vitro culturing. Inbred lines showed high embryogenic response and formed friable, type II calli. And also exhibited successful plant regeneration. Various culture media compositions based on Murashige and Skoog (1962) salts thus investigated differed with respect to the combinations of concentrations of auxins (picloram, 2, 4-D) and cytokinins (kinetin). Highest percent callus induction and subsequent somatic embryogenesis were observed on MS medium supplemented with picloram. The embryogenic calli were maintained and multiplied on medium containing reduced levels of auxins. Embryogenesis was characteristed, using callus morphology such as colour/nodulation and scanning electron microscopy. This inbred is considered suitable for further evaluation using a transformation system Key words: Maize, Mature seed, Somatic embryogenesis INTRODUCTION With the expanding population and limitation of land and water resources, and environmental stresses, there is great demand for rapid genetic improvement of maize (Zea Mays L). Maize is the most important fodder crop among cereals in industrialized countries and many developing countries. Genetically transformed maize plants have been obtained by various approaches, such as particle bombardment [1] and Agrobacterium infection [2]. However, an efficient plant tissue culture procedure with high regeneration frequency is prerequisite for most of the approaches. A method for the induction of embryogenic and organogenic calli from maize mature embryos is reported in this investigation. Previously, Plant regeneration has been achieved from callus cultures from mature embryos in cereals [3-6]. The use of mature embryos from dry seeds has several advantages as mature embryos are available year round in bulk quantities and easy to handle. Callus induction from mature embryo of maize was first reported by Green [7] but no plantlets were regenerated. Wang [8] successfully regenerated plants from mature embryos of two maize inbreds B73 and Mo17, but the frequency was only 4 to 5%. The objective of this research was to develop an efficient system from mature embryo of maize for the ultimate utility in genetic improvement of maize crop. MATERIALS AND METHODS The mature grains of locally developed maize inbred line being used as parent for developing commercial maize hybrids were obtained from Maize Section, Department of Plant Breeding and Genetics, Punjab Agricultural University Ludhiana. The seeds were 3565

2 J. Cell Tissue Research surface sterilized by using mercuric chloride (0.1%) for 10 minutes followed by three washings with sterile distilled water. Sterilized seeds were then cultured on MS (Murashige and Skoog) medium [9] supplemented with 2,4-D (3.0 mg/l), (6.0 mg/l), (10.0 mg/l), Picloram (2.5 mg/l), (5.0 mg/l), (10.0 mg/l) along with NAA (0.75 mg/l), BAP (0.5 mg/l) and sucrose (30 g/l) in test tubes. All these callus induction media were solidified with agar (8.0 g/l). Different media combinations evaluated are given in table 1 and figure1. The cultures were incubated in total dark in a growth room maintained at 25±2 C. Subculturing of primary calli was done onto medium with low concentrations of auxins. The concentrations, especially of 2, 4-D and picloram were reduced to half in each of the callus maintenance media and solidified with 8.0 g/l agar (Table 2; Fig. 2) to promote somatic embryogenesis. Each experiment was repeated three times in Completely Randomized Block Design with hundred explants per replication. Statistical analysis was done using CPCS-1 software package developed at Punjab Agricultural University [10]. Scanning electron microscopy (SEM) of maize calli was performed for deciphering the gross morphology and surface topographical differences in somatic and embryonic tissues. Tissue samples were fixed in 2.5% glutaraldehyde washed with buffer and post fixed in osmium tetraoxide followed by dehydration by treatment with graded ascending ethanol series (from 30 to 100%). The dehydrated samples were dried overnight in vacuum dessicator, and sputter coated with nm gold layer in Hitachi E-1010 sputter coater before imaging in SEM (Hitachi 15.0 KV (Fig. 5) [11]. RESULTS AND DISCUSSSION The mature seed explant was used with a thought of keeping the seed contents intact at the time of culturing, in view of the fact that natural nutrients and growth hormones are stored within the endosperm. Besides, maize seed can be handled as a dicotyledonous seed (Fig. 2). Mature embryos have been used to induce callus and regenerate plants [7,8,12]. Plant regulators play the most significant role in callus culture, and the effect of growth regulators on maize callus cultures have been investigated [13]. Earlier reports have confirmed the use of 2,4-D to induce callus from maize immature embryos was a critical factor [1,14]. The percent callus induction and percent somatic embryogenesis was studied using 10 media compositions based on MS salts. The callus induction frequency ranged from 42.06% to 88.06% on MS + NAA (5.0) + and MS + Pic (10.0) + BAP(0.5) (Table 1, Fig. 1). However, there was significant difference at 5% level of significance in callus induction frequency between any of the ten media thus tried. The use of picloram alone or in combination with 2,4-D resulted in higher frequency of callus induction. The combination of BAP and 2,4- D also promoted callusing but callusing was higher on picloram containing medium. Bhaskaran and Smith [13] have also shown that the use of cytokinins in combination with auxins induces somatic embryogenesis from callus cultures in cereals. It is thus evident that maize is difficult system and it requires the use of strong auxin to initiate callusing especially from the mature seeds. Picloram has also been used to induce callus from mature seeds earlier [12,15]. The primary calli were whitish, smooth and watery in colour (Fig. 3), which were maintained on same medium that was used for callus induction and multiplied on medium by reducing auxins to its half strength. Generally, the higher levels of 2,4-D and picloram used for callus induction prevented embryogenesis. Frequent embryogenesis was exhibited when the obtained calli were transferred to medium supplemented with reduced (half levels) of auxins (Fig. 4). The statistical comparison of ten different media compositions used for callus induction from immature embryos of five inbreds (LM5, LM6, LM13, LM15 and LM16) revealed that media composition, i.e., MS + 2,4-D (3 mgl -1 ) + Picloram (10.0 mgl -1 ) was significantly better than rest of the nine media compositions for all inbreds. Picloram has been used to induce callusing even in recalcitrant genotypes [16]. There were genotypic differences with respect to callus induction. For LM5, the callus induction on two media, i.e., MS + 2,4-D (3 mgl -1 ) + Picloram (10.0 mgl -1 ) and MS + 2,4-D (3 mgl -1 ) + Picloram (5.0 mgl -1 ) concentrations were statistically at par. For inbred LM6, media, MS + 2,4-D (3 mgl -1 ) + Picloram (10.0 mgl -1 ) was found to be the best and callus induction was significantly better than other nine media. Among different inbreds, LM13 showed highest number of calli that was significantly better than the number of calli induced in the other inbreds on the same medium. Therefore, MS + 2,4-D (3 mgl

3 Table 1: The effect of different growth hormones on percent callus induction in mature grains. *Values represent means ± SD of three replications with twenty explants per replication **Values in the parentheses are arc sine transformed values MS media + Growth hormones mg/l) Dhillon and Gosal Callus induction (%) Mature Grain LM 5 LM 6 LM 13 LM 15 LM 16 2,4-D (3.0) ±0.8 ( 44.2) 42.3±0.3 (40.6 ) 55.6±1.4 (48.2 ) 53.3±1.4 (46.9 ) 51.6± 0.8 (46.0) 2,4D (6.0) ±1.2 ( 42.5) 50.6±1.2 ( 45.4) 53.3±1.4 ( 46.9) 52.3±1.4 ( 46.3) 43.3±0.8 ( 41.1) 2,4-D (10.0)+ 45.6±1.4(42.5 ) 45.6±1.4(42.5 ) 59.6±0.8( 50.6) 49.3±0.8 ( 44.6) 46.6±1.4 ( 43.0) NAA (5.0) ±1.4 ( 46.3) 44.3±0.8 ( 41.7) 56.6±1.4 ( 48.8) 42.6±0.8 ( 40.8) 43.3±0.8 ( 41.1) NAA (10.0)+ 49.3±0.8 ( 44.6) 54.6±1.2 ( 47.7) 57.3±0.8 ( 49.2) 48.3±0.8 ( 44.0) 45.3±0.8 ( 42.3) 2,4-D (3.0)+ Pic (5.0) 62.3±1.2 ( 52.1) 62.3±0.8 ( 52.2) 73.3±1.4 ( 59.0) 63.3±0.8 ( 52.7) 66.6±0.8 ( 54.7) 2,4-D(3.0) + Pic (10.0) 61.3±0.8 ( 51.5) 68.3±0.8 ( 55.7) 72.3±1.2 ( 58.3) 66.3±0.8 ( 54.5) 70.3±0.8 ( 57.0) Pic (2.5) +BAP (0.5) 60.3±0.8 ( 51.0) 68.3±1.2 ( 55.7) 72.6±0.8 ( 58.5) 65.3±0.8 ( 54.0) 66.3±1.2 ( 54.5) Pic (5.0) + BAP(0.5) 72.3±0.8 ( 58.2) 76.6±0.8 ( 61.1) 78.3±0.8 ( 62.3) 73.6±0.8 ( 59.1) 74.6±0.8 ( 59.7) Pic (10.0) + BAP(0.5) 72.6±1.2 ( 65.9) 80.3±1.7 ( 70.0) 88.6±1.4 ( 70.3) 80.6±1.4 ( 70.4) 78.3±0.8 ( 70.3) Table 2: The effect of different growth hormones on somatic embryogenesis induction in mature grains. *Values represent means ± SD of three replications with twenty explants per replication. **Values in the parentheses are arc sine transformed values MS media Somatic embryogenesis (%) + Growth hormones (mg/l) Mature Grain LM 5 LM 6 LM 13 LM 15 LM 16 2,4-D (1.5) + Kn 0.75) 26.3±3.1 (30.8) 21.3±2.9 (27.4) 28.6±3.2 (32.3) 24.3±2.7(29.5) 19.3±2.3 (26.0) 2,4-D (3.0)+ 22.6±3.1 (28.3) 33.3±3.1 (35.2) 27.3±2.4(31.5) 26.6±3.3 (31.0) 24.6±2.9 (29.7) 2,4-D (5.0)+ 31.6±3.5 (34.2) 39.6±3.4 (39.0) 46.6±2.6 (43.1) 34.6±2.4 (36.0) 32.6±2.9 (34.8) NAA (2.5) ±3.5 (27.4) 23.3±2.3 (28.8) 27.3±1.7 (31.5) 17.3±2.8 (24.5) 20.3±2.3 (26.7) NAA (5.0)+ 18.3±1.8 (25.3) 19.6±1.4 (26.3) 21.6±2.3 (27.7) 14.6±2.9 (22.3) 13.0±2.6 (20.9) 2,4-D (1.5)+ Pic (1.0) 24.3±2.9 (29.5) 29.6±3.4 (32.9) 32.3±3.1 (34.6) 20.6±2.7 (26.9) 19.6±3.1 (26.1) 2,4-D(1.5) + Pic (2.5) 19.6±3.2 (26.2) 25.6±2.4 (30.4) 34.3±2.9 (35.8) 18.3±2.9 (25.2) 25.6±1.8 (30.4) Pic (1.0) +BAP (3.0) 23.3±3.4 (28.7) 31.3±2.6 (34.0) 29.3±2.3 (32.7) 21.6±2.6 (27.6) 22.3±3.5 (28.1) Pic (2.5) + BAP(3.0) 18.6±3.7 (25.4) 33.6±2.7 (35.4) 37.3±3.5 (37.6) 28.6±2.6 (32.3) 26.6±2.4 (31.0) Pic (5.0) + BAP(3.0) 48.3±2.6 (44.0) 66.3±2.9 (54.5) 72.6±2.6 (58.5) 57.3±3.2 (49.2) 43.6±2.9 (41.3) LM5 LM6 LM13 LM15 LM16 Fig ,4-D (1.5) + 2,4-D (3.0)+ 2,4-D (5.0)+ NAA (2.5) + NAA (5.0)+ 2,4-D (1.5)+ Pic (1.0) 2,4-D(1.5) + Pic (2.5) Pic (1.0) +BAP (3.0) Pic (2.5) + BAP(3.0) Pic (5.0) + BAP(3.0) Fig. 1: The effect of different growth hormones on percent callus induction in mature grains Fig. 2: Callus induced from mature embryos. Fig. 3: Formation of whitish friable non-embryogenic callus (soft and watery). Fig. 4: Occurrence of compact and globular embryogenic callus in somatic embryo induction medium. Fig. 5: Scan. Electron Micrograph of embryogenic callus depicting granular surface with somatic embryos at 3500X using SE detector 3567

4 J. Cell Tissue Research Fig. 2 Fig. 3 Fig. 4 Fig

5 Dhillon and Gosal 1 ) + Picloram (10.0 mgl -1 ) was found to be the best medium for embryogenic callus induction in maize inbreds. Callus induction from mature seeds was highest in LM13 on callus induction medium MS + 2,4-D (3 mgl -1 ) + Picloram (10.0 mgl -1 ). The callus induction frequency was statistically at par for LM6 line as the results were non significant (Table 2; Fig. 1). Further the callus induction frequency was significantly less in LM5, LM15 and LM16 (Table 2). [12] Huang, X.Q. and Wei, Z.M.: Plant Cell Rep., 22: (2004). [13] Bhaskaran, S. and Smith, R.H.: Crop Sci 30: (1990). [14] Armstrong, C.L. and Green, C.E.: Planta 164: (1985). [15] Abebe, D.Z., Teffera, W. and Machuka, J.: African J. Biotech., 7: (2008). [16] Eisinger, W.R. and Morre, D.J.: Canadian J. Botany 49: (1971). Nevertheless, it should be point out that the inbred lines, each with different parental population, presented somatic embryogenesis. Therefore, for practical purposes, all genotypes tested could be cultured efficiently. Considering the year round availability and abundance, the use of mature embryos as an alternative explant source can be useful in maize tissue culture and transformation studies. Abbreviations: MS: Murashige and Skoog medium, BAP: 6-Benzylamino purine, Pic: 4- Amino-3,6,6- trichloropicolinic acid (picloram), 2, 4-D: 2, 4- Dichlorophenoxyacetic acid, CH: Casein hydrolysate, SEM: Scanning Electron Microscopy. REFERENCES [1] Bohorova, N.E., Zhang, W., Julstrum, P., McLean, S., Luna, B., Briton, R.M., Diaz, L., Ramos, M.E., Estanol, P., Pacheco, M., Salgado, M. and Hoistington, D.A.: Theoretical and Applied Genetics, 99: (1999). [2] Ishida, Y., Satto, H., Ohta, S., Hiei, Y., Komari, T. and Kumashiro, T.: Nature Biotec., 14: (1996). [3] Rueb, S., Leneman, M., Schilperoort, R. and Hensgens, L.: Plant Cell Tissue Organ Cult., 36: (1994). [4] Ozgen, M., Turet, M., Altmok, S. and Sancak, C.: Plant Cell Rep., 18: (1998). [5] Akula, C., Akula, A. and Henry, R.: Biol. Plant. 42: (1999). [6] Ward, K. and Jordan, M.: In Vitro Cell Dev. Biol. Plant 37: (2001). [7] Green, C. E., Phillips, R. L., Kleese and R. A.: Crop Science, 14: (1974). [8] Wang, A.S.: Plant Cell Reports, 6: (1987). [9] Murashige, T. and Skoog, F.: Physiol Plant 15: (1962). [10] Cheema, H.S. and Singh, B.: A user s manual to CPCS- 1. A Computer Programme Package for the Analysis of Commonly used Experimental Designs, PAU, Ludhiana (1990). [11] Bozzola J.J. and Russell L.D. Electron Microscopy. Jones and Bartlett Publishers, Sudbury, Massachusetts (1999). 3569