Zea mays L., the queen of cereals, commonly called as maize, is the most important and major cereal crop occupied the third position next to rice and wheat because of its huge consumption in tropical and sub-tropical zones throughout the world. Its C4 photosynthetic mechanism makes this plant stood ahead among cereal crops in producing high grain yields and biomass through performing higher rates of photosynthetic activity. Not only food products, but it also has great value for producing industrial products (bio-ethanol), animal feed, and fodder. As it has diversified uses with higher productivity and adaptability, its demand was increased throughout the world, particularly in Asia. Hence, still several research programs are being done in all disciplines to improve its genome quality through adding, modifying or altering the traits of its genome not only through conventional breeding, but also using advanced biotechnological and molecular toolsviz., tissue culture, genetic transformation and molecular assisted selection (MAS). Among the methods adopted in crop improvement, integration and expression of the defined genes into the plants through genetic transformation is the most widely used technique, for which reliable and highly reproduciblein vitro protocol is pre-requisite. Callus induction and regeneration of maize were first reported by Green and Phillips (1975) using immature embryos. Since then, different studies have been carried out on regeneration of maize through organogenesis and somatic embryogenesis by using different explants namely, mature embryos (Al-Abed et al. 2006;Ali et al. 2014;Tiwari et al. 2015;Mushke et al. 2016), coleoptile nodes (Sreenu et al. 2016b;Pavan Kumar et al. 2018), nodal regions (Vladimir et al. 2006), leaf tissues (Conger et al. 1987;Ray and Ghosh 1990;Ahmadabadi et al. 2007), anthers (Barloy and Beckert 1993;Ismaili and Mohammadi 2016), tassel and ear meristems (Pareddy and Petolino 1990), protoplast (Morocz et al. 1990), and shoot meristems (Zhong et al. 1992;O’Connor et al. 2002;Sairam et al. 2003). Further studies revealed that immature embryos (IEs) are the most widely used explants than any other explants, because of their high efficiencies in giving embryogenic calli and regeneration. Though the model genotype of maize, A188, and its hybrid, Hi-II are known to yield high frequency of embryogenic callus and plantlet regeneration, they have poor agronomical values, which raise the necessity to search a wide range of genotypes for identifying the high regenerative lines. The genotype of the plant plays very crucial role onin vitro plant response. Hence it is necessary to screen the local lines that would be widely used in the breeding programmes. Inbred lines, which involved in the development of commercially successful hybrids, have to be screened for knowing theirin vitro competency to regenerate plantlets. Along with the genotype, explants and its size and hormonal concentrations and combinations influence thein vitro response. Hence, the present study has been undertaken to study the distinctivein vitro response of maize genotypes through studying the effect of different hormonal concentrations and combinations and explant sizes on fivein vitro charactersviz., frequency of callus induction, callus growth rate, frequency of embryogenic callus induction, its volume and number of plantlets regenerated by using immature embryos.

Plant material

Seeds of four inbred maize lines (NM74C, NM81A, NM5883, and NM5884) of Nuziveedu Seeds Limited were sown in the field. To ensure the supply of immature embryos (IEs) continuously, staggered planting was adapted, and seeds were sown in four lines in 3~4 days intervals. Plants were allowed to grow until flowering, and cobs were masked with butter paper bags before the emergence of silk, which were hand pollinated.

Isolation of explants

Ears were collected on different days after pollination (8~20 days); after removing sheaths, naked cobs were surface sterilized for 15 min using a 10% sodium hypochlorite (4% w/v) solution along with few drops of surfactant, Tween 20. Then the cobs were thoroughly washed three times with sterile distilled water (five min each). After kernel crowns were removed using a sterile scalpel, different sizes of embryos were carefully isolated and inoculated on callus induction medium by keeping the scutellar side facing up and flat surface down. These culture plates were incubated in the dark at 26±1°C.

Callus induction Medium

Linsmaier and Skoog’s (LS) medium (Linsmaier and Skoog, 1965) supplemented with different concentrations of 2, 4-dichloro phenoxyacetic acid (2,4-D)viz., 0.5, 1.5, 2.5, 4.0 and 5.0 mg/L along with 0.7 g/L L-proline, 0.5 g/L MES, 20 g/L sucrose and 3 g/L gelrite were used for callus induction. The pH of the medium was adjusted to 5.8 before adding gelrite and then autoclaved at 121°C temperature with 15 lbs pressure for 15 minutes. Filter sterilized silver nitrate (AgNO₃) solution at a concentration of 1.7 mg/L was added to the medium after autoclaving.

Maintenance of cultures and parameters measured

After twelve days of inoculation, embryo axes were removed, and primary calli were sub-cultured onto the same media, which is repeated periodically until they were transferred onto the regeneration medium. These cultures were maintained in the dark at the same temperature (26±1°C).In vitro parameters,viz., frequency of callus induction, the growth rate of total callus, frequency of embryogenic callus induction, and its volume were studied in all genotypes. Explants and/or calli cultured on basal LS medium with no phytohormones were served as controls throughout this study. Callus induction was represented as frequency and calculated as the number of explants induced the callus; growth rate of total callus was represented as percentage of increase in callus growth during the I, II and III sub-cultures, in terms of increase in callus volume; Immature embryo explants, after removing the developed radicals, were allowed to grow in same conditions. After 12 days, proliferated callus was measured for the volume and it is considered as initial volume of the callus. Then calli were transferred onto the same medium. All the calli derived from different genotypes, different sizes of IEs and concentrations of 2,4-D were maintained separately and data were collected for measuring the growth rate of callus individually to evaluate the genotypic / hormonal / explant size effect on callus growth rate. The measurementsi.e., volume of the calli were taken at an interval of two weeks. The volume at zero weeks, while transferring onto fresh medium, was designated as initial volume and then transferred onto the respective media; after two weeks, at the end of I sub-culture, again the volumes of calli were measured and calculated the growth rate and represented as per cent of increase in volume. After taking the data, again calli were sub-cultured onto the fresh medium having the same constituents. These were repeated for two more times at the end of four and six weeksi.e., at the end of II and III sub-cultures. Embryogenic callus induction has also been represented as frequency and calculated as the number of calli induced embryogenic callus; embryogenic callus volume was represented as its volume in cubic millimeters before going to transfer onto the regeneration medium.

Regeneration, Rooting, and Acclimatization of plants

Embryogenic calli were transferred onto regeneration medium i.e., LS medium supplemented with 0.5 g/L MES, 10 µM CuSO₄, 20 g/L sucrose, 3 g/L gelrite and growth regulatorsviz., 6-benzylaminopurine (BAP) and kinetin (Kn) each at a concentration of 0.5 mg/L. Cultures were maintained at 26±1°C with 16 h light/ 8 h dark cycle. After two weeks, shoots were counted as number of plantlets grown from each embryogenic callus mass developed from a single explant. Regenerated shoots of 3~4 cm height were transferred onto rooting mediumi.e., MS basal (Murashige and Skoog 1962) having 0.8% agar. Profusely rooted plantlets were transferred to plastic cups filled with autoclaved cocopeat and covered with polythene bags, which were maintained at an ambient temperature of 28±1°C for primary hardening. After acclimatization, plants were moved to poly house and kept for 2 days in the same conditions. Then, the polythene covers were removed and transferred onto the soil for further growth.

Statistical analysis

Statistical analyses were done to evaluate the effect of IEs size and hormonal concentrations among different genotypes on differentin vitro parameters. Raw data were collected from three replications and subjected to two-way ANOVA (Snedecor and Cochran 1968).

In vitro regeneration of maize by using immature embryo explants

Cobs of different agesi.e., days after pollination, were harvested and sterilized; then, IEs of six different sizesviz., 1, 1.5, 2, 2.5, 3 and 3.5 mm were isolated (Fig. 1A, B and C) and inoculated onto the media supplemented with different concentrations of 2,4-D (0.5, 1.5, 2.5, 4.0 and 5.0 mg/L) for evaluating the effect of size or age of IEs and phytohormonal concentrations on fivein vitro characters among four maize genotypes. The last one (number of plantlets regenerated) was studied by supplementing BAP and Kn each at a concentration of 0.5 mg/L.

Figure 1. Explant (immature embryos) source (A), dissection and isolation of different sizes of explants (B & C), and their initial response on media supplemented with different concentrations of 2,4-D (D-F) in maize (Zea mays L.); immature embryos of 2 mm in size inoculated on the medium lacking hormones develop profuse roots and shoots without callus induction (D), immature embryos of <1 and >3 mm in size inoculated on the medium supplemented with 2,4-D seldom initiate callus formation (E & F)

Primary callus induction

IEs isolated from any of these four genotypes inoculated onto the medium lacking 2,4-D (control) seldom initiated callus from any of their sizes, but resulted in profuse roots and shoots (Fig. 1D). IEs of sizes <1 and >=3.5 mm did not respond in giving callus in any of the genotypes (Fig. 1E and F), hence data has not been presented. Callus initiation was observed from the explants after 7~8 day of inoculation. Among the explants, very small and large IEs measuring 1 and 3 mm were not suitable and showed no callus induction in NM81A and NM5883 in any of the 2,4-D concentrations, whereas very low induction percentages were observed in NM74C and NM5884 at medium 2,4-D concentrations (1.5 and 2.5 mg/L) (Fig. 2). Only 1.5 and 2 mm size IEs showed callus induction in NM81A and NM5883 in all 2,4-D concentrations except at 5.0 mg/L in 1.5 mm size explants. On the other hand, three sizes of IEsviz., 1.5, 2 and 2.5 mm resulted in callus induction in NM74C as well as NM5884 with varying degree of variation in frequency at all 2,4-D concentrations (Fig. 2). When the genotypes were considered, NM74C and NM5884 resulted in callus induction from all sizes of explants with higher frequencies than other two genotypesviz., NM81A and NM5883 by having variation among 2,4-D concentrations (Fig. 2). Frequency of callus induction was widely ranged from 6.66 to 100% in different sizes of explants, 2,4-D concentrations and genotypes used. Frequency of callus induction was increased with increasing 2,4-D concentration up to 1.5 mg/L, then decreased in further concentrations not only in any of the genotypes but also in sizes of the explants (Fig. 2), and also increased with increasing size of the explants up to 2 mm and then decreased in further explant sizes. Though the maximum frequency of callus induction (100%) was observed in both the genotypes of NM74C and NM5884 in 2 mm size IEs at 1.5 mg/L 2,4-D, frequencies at other concentrations of 2,4-D and explant sizes were on the higher side in NM5884 than NM74C. NM5884 resulted in higher frequencies of callus induction than other genotypes in all concentrations and explant sizes. Even in the NM5884 genotype, distinctive variation was observed in the callus induction among different sizes of explants. This wide range of variation observed in the frequencies of callus induction among the genotypes, 2,4-D concentrations and sizes of explants was subjected to two-way analysis of variance (ANOVA), which revealed that all genotypes showed significant variation among the 2,4-D concentrations as well as sizes of explants except in NM81A and NM5883 between 2,4-D concentrations (Table 1), which indicated that, not only the hormonal concentrations but also size of the explants showed significant effect on frequency of callus induction (Table 1). It also revealed that, at all 2,4-D concentrations and sizes of IEs, inbred lines showed significant effect on frequency of callus induction. Size of the explants showed significant effect on frequencies of callus induction at all 2,4-D concentrations, likewise, 2,4-D concentrations have also showed significant effect on callus induction except in the explants measuring 1 and 3 mm (Table 1).

Figure 2. Effect of 2,4-D concentrations and different sizes of immature embryos on frequency of callus induction in different genotypes (inbred lines) of maize

Table 1 . Analysis of variance of the effect of phytohormonal (2,4-D) concentrations, size of the immature embryos, and inbred lines on frequency of callus induction.

		Source of variation	MS	F - value
In Different Inbred Lines
	NM74C	Between 2,4-D concentrations	1003.65	5.08*
		Between IE sizes	4664.93	23.61*
	NM81A	Between 2,4-D concentrations	78.95	1.69
		Between IE sizes	572.41	12.32*
	NM5883	Between 2,4-D concentrations	241.26	1.54
		Between IE sizes	3127.94	20.05*
	NM5884	Between 2,4-D concentrations	1296.72	5.28*
		Between IE sizes	5871.68	23.93*
In Different Concentrations of 2,4-D
	0.50 mg/L	Between Inbred Lines	1126.31	4.19*
		Between IE sizes	2222.77	8.29*
	1.50 mg/L	Between Inbred Lines	1437.47	8.03*
		Between IE sizes	3699.11	20.66*
	2.50 mg/L	Between Inbred Lines	1017.12	7.14*
		Between IE sizes	3206.09	22.53*
	4.00 mg/L	Between Inbred Lines	802.01	5.76*
		Between IE sizes	2836.35	20.39*
	5.00 mg/L	Between Inbred Lines	738.77	4.47*
		Between IE sizes	2817.72	17.05*
In Different Sizes of Immature Embryos
	1.00 mm	Between Inbred Lines	16.63	3.46*
		Between 2,4-D concentrations	10.72	2.23
	1.50 mm	Between Inbred Lines	857.42	20.48*
		Between 2,4-D concentrations	481.01	11.49*
	2.00 mm	Between Inbred Lines	4019.13	20.51*
		Between 2,4-D concentrations	3317.81	16.94*
	2.50 mm	Between Inbred Lines	1966.33	19.59*
		Between 2,4-D concentrations	295.16	2.94*
	3.00 mm	Between Inbred Lines	63.36	5.31*
		Between 2,4-D concentrations	32.66	2.74

^*significant at p=0.05.

Total callus growth rate

Explants of 1 and 3 mm sizes did not show callus growth in any of four genotypes and three sub-cultures at 0.5 and 5.0 mg/L 2,4-D concentrations. When the growth rate among genotypes was taken into consideration, the genotypesviz., NM81A and NM5883 showed significantly lower rate in their growth when compared with other two genotypes. 1 and 3 mm explants of these two genotypes did not show any growth in any of 2,4-D concentrations and also in any of sub-cultures (Fig. 3). Almost both of these genotypes showed similar growth rates and pattern at first sub-culture, whereas some discrepancies were observed among these two in II and III sub-cultures respectively in 2 mm explants size on 1.5 and 2.5 mg/L 2,4-D concentrations. On the other hand, two other genotypesviz., NM74C and NM5884 resulted in better growth rates at each sub-culture and 2,4-D concentrations with all sizes of explants than NM81A and NM5883. Though the difference in growth rate between NM74C and NM5884was not significant enough in first sub-culture, they showed significant differences in II and III sub-cultures particularly in 2 mm size IEs, where the genotype NM5884 resulted in maximum rate of its growth in all concentrations of 2,4-D used. Among the genotypes used in present study, maximum growth rates of calli were observed with NM5884 followed by NM74C, NM81A and NM5883 genotypes in this given order at all 2,4-D concentrations and explants sizes respectively during II and III sub-cultures (Fig. 3).

Figure 3. Effect of a range of concentrations of 2,4-D on growth rate (percent increase in volume) of total callus developed from different sizes of immature embryos isolated from different inbred lines of maize at three growth stages

Data on callus growth rate obtained from different concentrations of 2,4-D, sizes of explants of four genotypes at three sub-cultures were subjected to two-way ANOVA, it revealed that 2,4-D concentrations and explants size influence significantly in all genotypes except in NM81A in first sub-culture (Table 2); significant variation was observed between explant sizes in all 2,4-D concentrations except at 2.5 mg/L in II sub-culture, whereas, between inbred lines, at medium 2,4-D concentrations (1.5, 2.5 and 4.0 mg/L) significant variation was observed in all sub-cultures. In contrary, at 0.5 mg/L (low) 2,4-D concentration, significant variation was observed in II and III sub-cultures; and in 5.0 mg/L (high) concentration significant variation was observed only in III sub-culture (Table 2). On the other hand, in 1 mm size explants, no significant effect was observed between 2,4-D concentrations in any of three sub-cultures. In other sizes of the explants (1.5 to 3 mm) variation was significant in all three sub-cultures. Significant variation was observed between genotypes in all sizes of explants and three sub-cultures, except in 2.5 mm at I sub-culture (Table 2). Callus growth rate showed significant variation at all growth stages between genotypes and in all explants sizes.

Table 2 . Analysis of variance of the effect of phytohormonal concentrations, size of the immature embryos, and inbred lines on callus growth rate.

Source of variation		I Sub-Culture		II Sub-Culture		III Sub-Culture
		MS	F - value	MS	F - value	MS	F - value
In Different Inbred Lines
NM74C	Between 2,4-D concs.	88.84	9.89*	13832.24	8.71*	53716.25	8.68*
	Between IE sizes	62.26	6.93*	5974.96	3.76*	57835.61	9.35*
NM81A	Between 2,4-D concs.	27.78	2.61	4046.31	3.24*	13456.88	3.26*
	Between IE sizes	96.18	9.01*	12322.93	9.87*	38976.71	9.45*
NM5883	Between 2,4-D concs.	31.89	3.89*	3275.01	3.93*	9982.01	3.81*
	Between IE sizes	122.88	14.99*	12036.81	14.45*	35512.19	13.54*
NM5884	Between 2,4-D concs.	127.16	12.58*	20363.33	13.87*	91983.57	12.67*
	Between IE sizes	69.46	6.87*	4386.21	2.98*	90634.63	12.49*
In Different Concentrations of 2,4-D (mg/L)
0.50	Between Inbred Lines	11.41	1.75	4617.82	3.98*	23399.36	3.55*
	Between IE sizes	122.61	18.84*	11400.92	9.85*	79091.67	12.02*
1.50	Between Inbred Lines	59.98	8.13*	14476.09	11.21*	75488.93	29.74*
	Between IE sizes	61.09	8.27*	5421.56	4.21*	44462.41	17.52*
2.50	Between Inbred Lines	61.04	8.30*	8744.93	5.61*	57183.19	19.14*
	Between IE sizes	54.11	7.36*	4594.89	2.95	31623.35	10.58*
4.00	Between Inbred Lines	42.72	5.09*	6141.45	3.98*	44914.79	11.16*
	Between IE sizes	63.61	7.58*	5557.51	3.61*	39219.28	9.75*
5.00	Between Inbred Lines	15.54	1.91	1501.14	3.01	20135.68	4.13*
	Between IE sizes	125.77	15.42*	14773.01	29.64*	66441.57	13.63*
In Different Sizes of Immature Embryos (in mm)
1.00	Between Inbred Lines	34.79	3.49*	7914.86	3.99*	24908.58	3.74*
	Between 2,4-D concs.	21.91	2.19	4515.84	2.27	15501.94	2.32
1.50	Between Inbred Lines	22.08	3.45*	3272.76	8.68*	25626.92	12.07*
	Between 2,4-D concs.	77.87	12.17*	9829.21	26.08*	44996.32	21.18*
2.00	Between Inbred Lines	32.93	15.51*	1741.74	9.43*	95501.73	22.74*
	Between 2,4-D concs.	104.23	49.09*	12164.51	65.91*	74795.53	17.81*
2.50	Between Inbred Lines	7.15	1.39	3429.41	3.71*	21507.19	7.08*
	Between 2,4-D concs.	76.94	15.02*	11050.36	11.97*	31900.82	10.51*
3.00	Between Inbred Lines	63.34	4.99*	11601.35	4.94*	28606.65	4.95*
	Between 2,4-D concs.	37.61	2.96*	7036.37	2.99*	17292.13	2.99*

^*significant at p=0.05.

Embryogenic callus induction

Among the four genotypes used, NM74C and NM5884 were chosen to study other twoin vitro charactersviz., frequency of E. callus induction and volume of E. callus obtained at different sizes of explants maintained on different concentrations of 2,4-D. Induction of embryogenic callus was observed at the end of II sub-culture from the calli maintaining on different 2,4-D concentrations. E. callus induction was observed in all calli, except the calli derived from 1 mm explants maintained on 0.5, 4.0 and 5.0 mg/L 2,4-D concentrations and also in 3 mm explants maintained on 0.5 and 5.0 mg/L 2,4-D concentrations. In NM74C, E. callus induction was comparatively less than in NM5884, in all 2,4-D concentrations and explant sizes. Frequency of E. callus induction was ranged from 23.52 to 90.37 per cent in NM74C, whereas it was ranged from 30.40 to 100 per cent in NM5884. Highest frequency (100 per cent) of E. callus induction was observed from the calli derived from 2 mm size explants of NM5884 on three of five 2,4-D concentrations used for total callus maintenance (viz., 0.5, 1.5 and 2.5 mg/L) (Fig. 4A and B). In both the genotypes, frequency of E. callus induction was increased with increasing size of explants up to 2 mm and then decreased with further increase in size except in calli derived from 1.5 mg/L of 2,4-D in NM5884. The same trend has also been observed with 2,4-D concentrations, where increase in frequency of E. callus induction was observed up to 1.5 mg/L and then decreased in further concentrations except in 2 mm size explants of NM5884 (Fig. 4A and B). When the data was subjected to two-way ANOVA, in both the genotypes, significant variation was observed not only between 2,4-D concentrations but also in explant sizes. Variation observed in frequency of E. callus induction was significant between explant sizes at all 2,4-D concentrations, whereas, significant variation was observed between genotypes only at 1.5, 2.5 and 4.0 mg/L 2,4-D concentrations, and no significant variation was observed at low and high (0.5 and 5.0 mg/L respectively) 2,4-D concentrations. On the other hand, variation was significant between the genotypes in all explant sizes except in 2 mm, whereas significant variation between 2,4-D concentrations were observed only at 1.5, 2.5 and 4.0 mg/L concentrations, but not at low and high (1 and 3 mm respectively) sizes of explants (Table 3).

Figure 4. Frequency of E. callus induction (A & B), volume (C & D), and average number of plantlets (E & F) regenerated from different sizes of immature embryos from different inbred lines cultured on media supplemented with a range of concentrations of 2,4-D.

Table 3 . Analysis of variance of the effect of phytohormonal (2,4-D) concentrations, size of the immature embryos, and inbred lines on frequency of embryogenic callus induction.

	Source of variation	MS	F - value
In Different Inbred Lines
NM74C	Between 2,4-D concs.	822.35	20.81*
	Between IE sizes	4291.1	108.58*
NM5884	Between 2,4-D concs.	963.17	8.13*
	Between IE sizes	6899.61	58.27*
In Different Concentrations of 2,4-D
0.50 mg/L	Between Inbred Lines	508.55	5.56
	Between IE sizes	3193.33	34.96*
1.50 mg/L	Between Inbred Lines	896.04	22.89*
	Between IE sizes	1622.95	41.46*
2.50 mg/L	Between Inbred Lines	617.91	15.98*
	Between IE sizes	1422.83	36.81*
4.00 mg/L	Between Inbred Lines	711.04	7.75*
	Between IE sizes	2423.57	26.42*
5.00 mg/L	Between Inbred Lines	658.27	4.99
	Between IE sizes	2767.16	21.01*
In Different Sizes of Immature Embryos
1.00 mm	Between Inbred Lines	850.39	13.22*
	Between 2,4-D concs.	171.32	2.66
1.50 mm	Between Inbred Lines	263.75	47.47*
	Between 2,4-D concs.	2295.38	413.17*
2.00 mm	Between Inbred Lines	42.13	2.77
	Between 2,4-D concs.	695.45	45.79*
2.50 mm	Between Inbred Lines	435.38	13.61*
	Between 2,4-D concs.	1241.13	38.79*
3.00 mm	Between Inbred Lines	698.36	68.67*
	Between 2,4-D concs.	49.91	4.91

^*significant at p=0.05.

Embryogenic callus volume

E. calli induced in II sub-culture were allowed to grow for two weeks and at the end of III sub-culture, its volumes were measured and data was collected (Fig. 4C and D). At this stage E. callus was fully grown. Based on the texture and compactness of the calli, it has been identified as two typesviz., Type I and Type II. Type I callus was compact and tightly packed with hard appearance which shows slow growth and will not spread much through sub-cultures. On the other hand, Type II callus was soft, nodular, loose, friable, grow quickly and more regenerative than Type I and multiplies fastly through sub-cultures (Fig. 5B and F). E. calli volumes were measured from each of 2,4-D concentrations as well as explant sizes (Fig. 4Cx and D). Data obtained on E. callus volume has exhibited the same trends as observed in frequency of E. callus induction,i.e., volume of E. callus obtained from different 2,4-D concentrations showed increase in volume up to 1.5 mg/L and then decreased in further concentrations; likewise, the volume of E. callus showed increasing trend up to 2 mm explant size and then decreased further in larger sizes of explantsi.e., in 2.5 and 3.0 mm. In NM5884, volumes of E. calli derived from any of the 2,4-D concentrations and explant sizes were more when compared with NM74C. Maximum volume of E. callus (114 cu. mm) was observed in NM5884 from 2 mm IE cultured on media augmented with 1.5 mg/L 2,4-D. For understanding the discrepancies showed in the data and to interpret the effect of hormonal concentrations and explant sizes among these two genotypes, the data was subjected to two-way ANOVA and represented inTable 4. In both genotypes, variation in E. callus volume was significant not only between hormonal concentrations but also in explant sizes. On the other hand, differences observed in different hormonal concentrations revealed that variation between the explant sizes was significant in all 2,4-D concentrations, whereas, between the genotypes variation was significant only in 2.5 and 4.0 mg/L. When the data obtained from different explant sizes were analysed, significant variation in E. callus volume was observed neither in genotypes nor between hormonal concentrations in 1 mm explants. Only in 2.5 and 3.0 mm explants, significant variation was observed between hormonal concentrations; likewise, between genotypes, variation was significant in 1.5, 2.0 and 2.5 mm explants (Table 4), which indicated that in smaller explants up to 2.0 mm size, hormonal concentrations have no significant effect in giving E. callus, whereas in explants measuring 2.5 and 3.0 mm size, 2,4-D concentration has an effect in giving E. callus rather than explants sizes.

Figure 5. Immature embryos of different maize genotypes develop two types of calliviz., non-embryogenic (A) and embryogenic calli (B-F). Embryogenic calli further differentiated into Type I, which is compact and less regenerative (B), and Type II embryogenic calli that are friable, loose, and more regenerative (C-F). Genotypes NM81A and NM5883 resulted in non-embryogenic calli (A). At some hormonal concentrations, few calli develop into an E. callus, which is a Type I callus along with a non-embryogenic callus (B); NM5884 has the most suitable genotype, which results in more Type II calli and more plantlet regeneration (C); genotype NM74C showed mixed results based on hormonal concentration and has comparatively less Type II callus

Table 4 . Analysis of variance of the effect of phytohormonal (2,4-D) concentrations, size of the immature embryos, and inbred lines on embryogenic callus volume.

	Source of variation	MS	F - value
In Different Inbred Lines
NM74C	Between 2,4-D concentrations	122.15	11.43*
	Between IE sizes	1432.01	134.01*
NM5884	Between 2,4-D concentrations	461.62	10.71*
	Between IE sizes	4474.08	103.75*
In Different Concentrations of 2,4-D
0.50 mg/L	Between Inbred Lines	325.93	3.74
	Between IE sizes	1324.81	15.21*
1.50 mg/L	Between Inbred Lines	1133.19	5.26
	Between IE sizes	1671.25	7.75*
2.50 mg/L	Between Inbred Lines	536.56	7.83*
	Between IE sizes	971.84	14.19*
4.00 mg/L	Between Inbred Lines	537.12	10.92*
	Between IE sizes	766.78	15.58*
5.00 mg/L	Between Inbred Lines	172.49	3.13
	Between IE sizes	911.27	16.54*
In Different Sizes of Immature Embryos
1.00 mm	Between Inbred Lines	32.64	2.93
	Between 2,4-D concentrations	67.29	6.04
1.50 mm	Between Inbred Lines	585.83	44.01*
	Between 2,4-D concentrations	61.87	4.65
2.00 mm	Between Inbred Lines	3157.02	44.98*
	Between 2,4-D concentrations	332.56	4.74
2.50 mm	Between Inbred Lines	366.56	62.01*
	Between 2,4-D concentrations	52.84	8.94*
3.00 mm	Between Inbred Lines	35.28	5.17
	Between 2,4-D concentrations	177.06	25.98*

^*significant at p=0.05.

Callus induced from all these four genotypes were identified as embryogenic and non-embryogenic calli. The non-embryogenic callus was watery, not organized and loose. This callus does not give the regeneration and become brown through age (sub-cultures). On the other hand, embryogenic callus was loose to compact, friable and cream colored which is again sub-divided into two typesviz., Type I and II. Among the genotypes studied, NM81A and NM5883 resulted in non embryogenic callus (Figs. 5A and6B), which turned to brown after few weeks (sub-cultures). Hence these two genotypes were omitted and only NM74C and NM5884 which yielded E. callus were taken into consideration for further regeneration studies.

Figure 6. Regeneration,in vitro rooting, and primary hardening of plantlets in different maize (Zea mays L.) genotypes: Maximum E. callus induction and plantlet regeneration were observed in NM5884 followed by NM74C (A); in contrast NM5883 (B) and NM81A showed scanty E. callus formation without regeneration. Plantlets sub-cultured onto MS basal medium (C) and fully grown plantlets showing root development on rooting medium (D); primary hardened rooted plantlets in plastic cups filled with sterile cocopeat (E)

Plantlet regeneration and hardening of plantlets

E. calli obtained from different sizes of explants maintained on different 2,4-D concentrations were harvested individually at the end of III sub-culture and transferred and maintained separately on the regeneration medium having 0.5 mg/L BAP and 0.5 mg/L Kn. After 15 days of growth on regeneration medium, shoot bud induction and plantlet regeneration was observed from E. callus masses. Then these plantlets were transferred on to MS media lacking any hormones (basal medium). Shoot elongation (Fig. 6C) and rooting was induced, which were continued to maintain for two weeks in bottles (Fig. 6D). Then these plantlets were primary hardened and transferred onto soil in poly house (Fig. 6E).

The data revealed that no plantlet regeneration was observed from any of the E. calli masses derived from 1 and 3 mm explants at any of the 2,4-D concentrations in both the genotypes except in 1.5 and 2.5 mg/L 2,4-D from 1 mm explants in NM5884. All remaining three sizes of explants on all concentrations of 2,4-D, showed plantlet regeneration in both the genotypes except from 1.5 and 2.5 mm explants maintained at 0.5 mg/L 2,4-D in NM74C, where no plantlet regeneration was observed (Fig. 4E and F). In all these concentrations and sizes, number of plantlet regenerated from E. calli were higher in NM5884 than its counterpartviz., NM74C (Fig. 6A). Maximum number of plantlets were regenerated from 2 mm explants at 1.5 mg/L 2,4-D in both the genotypes. NM5884 resulted in highest number of plantlets of 18, whereas, in NM74C only 5 plantlets were observed (Fig. 4E and F). In almost all concentrations and sizes of explants, where the plantlet regeneration was observed, NM5884 resulted in double the number of plantlets than the number obtained from NM74C. Among these two genotypes, NM74C resulted in more of Type I E. callus on majority of the media with different explants sizes. In contrary, NM5884 yielded more of Type II E. callus and produced more number of plantlets. Hence NM5884 was considered as best genotype forin vitro studies and to improve the germplasm through genetic transformation.

In vitro regeneration with very high frequency of plantlet regeneration is highly essential pre-requisite for the genetic transformation of any plant system. In monocots, establishment of regeneration protocol is more critical than dicot plants due to structural variation and composition of the tissues. Many factors like explants and its age, plant genotype, media compositionviz., major and minor nutrients and vitamins, nitrogen content, hormonal concentration and their combinationsetc., play a major vital role in controlling regeneration and plantlet development.

Bohorova et al. (1995) suggested that N6 medium, with lower level of nitrogen than MS, showed better callus induction and maintenance.Rooz (2002) reported high frequency of regeneration in calli derived from MS with 2,4-D in IEs.Ishida et al. (2007) andSreenu et al. (2016a) used LS medium for maize IEs. In present study LS medium proposed byIshida et al. 2007 supplemented with only 2,4-D has been used for callus induction and E. callus induction, later which were transferred onto media supplemented with BAP and Kn for plant let regeneration. Our preliminary studies revealed that the LS medium was more suitable for culturing the IEs and MS media for coleoptile nodes (Sreenu et al. 2016a;Sreenu et al. 2016b;Pavan Kumar et al. 2018) with these genotypes; hence in present study LS medium has been used. Presence of AgNO₃ in medium enhances the E. callus induction by influencing the embryogenic potential of fresh callus (Giridhar et al. 2003) by acting in antagonistic way to the endogenous ethylene which damages the tissue. Both N6 and LS media used byBohorova et al. (1995) andIshida et al. (2007) respectively were supplemented with AgNO₃.

In cereal tissue culture, influence of growth hormones is very crucial (Bhaskaran and Smith 1990) in giving the primary callus from the explants and differentiation into embryogenic callus (Rueb et al. 1994;Vikrant and Rashid 2003;Jogeswar et al. 2007).Armstrong and Green 1985,Rao et al. 1995, reported that in maize and sorghum, proline promoted the embryogenic callus production, on the other hand,Zhao et al. 2010, reported proline effect on callus induction was not significant in low concentrations. In the present study, only 2,4-D has been used for the callus induction,Zhao et al. 2010 also reported the same in sorghum that 2,4-D alone was sufficient to induce callus formation and the additional Kn in high concentrations (0.6 mg/L) decreased the callus induction frequency; whereas in our previous study with maize coleoptilar nodal explants, contrastingly callus induction was enhanced in presence of Kn (0.5 mg/L) along with 2,4-D (Pavan Kumar et al. 2018). For the regeneration, low concentration of Kn (0.5 mg/L) has been used for enhancing the frequency,Zhao et al. 2010, reported, low Kn concentration (0.2 mg/L) was beneficial for keeping the calli embryogenic. High concentrations of 2,4-D results in decreased regeneration frequency (Mendoza and Kaeppler 2002). At higher concentrations of 2,4-D callus growth was drastically effected not only in giving the regeneration but also the callus became brown and watery.

Plant genotype plays an important role in giving high frequency of regenerative embryogenic callus in different cultures, including in maize (Gorji et al. 2011,Grando et al. 2013;Bohorova et al. 1995;Furini and Jewell 1994;Aguado-Santacruz et al. 2007). The maize genotypes growing in temperate areas are extensively screened and others which are growing in tropical regions are yet to be screened for theirin vitro ability to give high frequencies of regenerations. Intense variation existing in maize genotypes makes it more difficult to respondin vitro for regeneration. This makes to explore and optimize the specificin vitro growth conditions for specific genotypes (Malini et al. 2015). Several reports have explained the genotypic differences in maize regarding the formation of embryogenic cultures from different explants (Frame et al. 2006;Ishida et al. 2007;Lee et al. 2007;Tomes and Smith 1985).Malini et al. 2015, focused on a standard protocol for regeneration of tropical Indian maize inbreds using IEs by adopting the protocol suggested by Bohorova et al., 1995.

Successful regeneration has been reported from different explantsviz., mature embryos (Tiwari et al. 2015), nodal culture (Vladimir et al. 2006), split seeds (Al-Abed et al. 2006) and immature embryos (Duncan et al. 1985;Furini and Jewell 1994 andBohorova et al. 1995). Among the different explants used and reported in different studies, IEs were found to be more suitable in yielding highest frequencies of callus induction and regenerative embryogenic callus. IEs also differ in giving regenerative E. callus basing on their age (days after pollination) size and season in a window of 8-20 days. Right developmental stages of IEs are obtained in a window of 8~15 days after pollination. Obtaining right stage of immature embryo is critical and easily identified with its size which varies from 1~3 mm in this window.Ishida et al. 2007 reported that this size varied among the genotypes in giving high frequencies of E. callus and regeneration. In present study, 2 mm IEs obtained in 8-14 days after pollination resulted in maximum regeneration. The same genotype yielded this size during 10~16 days in rabi season. In this window of 8~14 days, embryos attain 1.0~3.0 mm size which again shows variation in giving E. callus. Hence, both age and size of the explants have to be considered while selecting the IE explants. In tropical genotypes (in present study) the window extended up to 8~16 days to get the approximate size for giving maximum frequencies of E. callus. In present study, explants during that age varied from 1 to 3.5 mm, among which 2.0 mm size of IEs yielded maximum frequencies of calli. The size of explants varies among genotypes in that window of 8~16 days period, in turn, within the genotype also during kharif the optimum size of 2.0 mm size could be obtained during 12~14 days, whereas in rabi season it extends up to 14~16 days. Hence explants size is crucial along with the age and genotype. Genotype NM81A which did not yield embryogenic calli in this study, resulted in good frequency of E. callus induction and regeneration with coleoptilar nodal explants (Pavan Kumar et al. 2018). Hence the genotype shows significant effect on yielding high frequencies of E. callus induction and regeneration.

Effect of culture media and phytohormones on callus and E. callus induction shows much variation among different explants and different genotypes, the four genotypes studies in present study have also been used in our previous study (Pavan Kumar et al. 2018), by using coleoptilar nodal explants, where the callus induction and E. callus induction were recorded better on MS media supplemented with 2,4-D along with Kn. In contrary, in present study, Kn along with 2,4-D did not support in giving higher frequencies. Not only the culture media and hormones differed but also the genotypes showed contrasting results in giving regeneration, where NM5884 was found to be least responding genotype with coleoptilar nodal explant. Hence it can be concluded that the genotypes and explants play crucial role in giving high frequencies of regenerations. Next to these, media and hormones have their own effect in yielding the E. callus.

This work was financially supported by IGSTC (Indo-German Science and Technology Centre, New Delhi, India), a Binational Centre Supported by Govt. of India and Govt. of Germany. Project was sanctioned under IGSTC (2+2 scheme) (IGSTC/First Call/ICGEB-001/2011/157). The authors are thankful for the support.