Plant Cell Tiss Organ Cult (2010) 100:3137 DOI 10.

1007/s11240-009-9613-z

ORIGINAL PAPER

Callus induction and whole plant regeneration in elite Indian maize (Zea mays L.) inbreds
Sujay Rakshit Zerka Rashid J. C. Sekhar T. Fatma Sain Dass

Received: 3 December 2008 / Accepted: 10 September 2009 / Published online: 7 October 2009 Springer Science+Business Media B.V. 2009

Abstract Callus induction and regeneration ability of ve elite maize inbred lines, CM 111, CM 117, CM 124, CM 125 and CM 300 were investigated using 14-day-old immature embryos as explants. Genotype, medium, source of auxin and their concentrations inuenced induction of callus. Explants grown on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid at 1 mg l-1 showed the highest frequency of callusing. Among all the media tested, explants grown on N6 medium gave the highest frequency of organogenic callus. Moreover, N6 supplemented with Dicamba promoted higher callus response in terms of both frequency of induction as well as quality, compared to N6 medium with 2,4-D. N6 supplemented with 2 mg l-1 Dicamba induced the highest frequency of organogenic callus. Among the ve genotypes tested, CM 124, CM 125, and CM 300 gave the best callus. Explants of both CM 124 and CM 300 incubated on MS medium supplemented with 1 mg l-1 benzyladenine and 0.5 mg l-1 indole acetic acid promoted the highest
Sujay Rakshit and Zerka Rashid have equal contribution. S. Rakshit Z. Rashid S. Dass Directorate of Maize Research, Pusa Campus, New Delhi 110 012, India J. C. Sekhar Winter Nursery, Directorate of Maize Research, ARI Campus, Rajendranagar, Hyderabad 500 030, India T. Fatma Department of Biosciences, Jamia Milia Islamia, Jamia Nagar, New Delhi 110 025, India S. Rakshit (&) Directorate of Sorghum Research, Rajendranagar, Hyderabad 500 030, India e-mail: rakshit@nrcsorghum.res.in; srakshit@rediffmail.com

frequency of shoot induction. Though CM 124 induced higher percentage of shoot formation than CM 300, the mean number of developed shoots per explant was higher for CM 300. The highest frequency of root formation was observed when shoots were grown on MS medium supplemented with 2 mg l-1 naphathalene acetic acid. Percentage of regenerated plants ranged from 54 to 66. Keywords Callusing Immature embryo Maize Regeneration Shoot development Zea mays Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid BA 6-Benzyladenine IAA Indole-3-acetic acid NAA Naphthaleneacetic acid AgNO3 Silver nitrate PGR Plant growth regulator

Introduction Maize, Zea mays L., is the most widely grown cereal crop across the world. Globally it is top ranking cereal in terms of productivity and has worldwide signicance as human food, animal feed and fodder as well as source of large number of industrial products. It is used as a raw material for manufacture of large number of industrial products like corn starch and starch-based products, and in fermentation and distillation industries. Due to uses of maize and maizebased products, demand for maize is increasing across the world, and more predominantly in Asia (Wada et al. 2008). Maize production is affected by both biotic and abiotic stresses. Genetic transformation of maize with genes

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conferring resistance or tolerance against biotic and abiotic stresses is expected to address many of these issues synergistically with conventional breeding strategies. Green and Philips (1975) have rst reported regeneration from immature embryos of maize. Since then, maize regeneration has been reported from immature embryos (Duncan et al. 1985; Bohorova et al. 1995; Ishida et al. 1996; Aguado-Santacruz et al. 2007), mature embryos (Huang and Wei 2004; Al-Abed et al. 2006), nodal regions (Vladimir et al. 2006), leaf tissues (Conger et al. 1987; Ahmadabadi et al. 2007), anthers (Ting et al. 1981; Barloy and Beckert 1993), tassel and ear meristems (Pareddy and Petolino 1990), protoplast (Morocz et al. 1990), and shoot meristems (Sairam et al. 2003). However, immature zygotic embryos are predominantly used for establishing regeneration-competent cells or callus cultures for genetic transformation (Ahmadabadi et al. 2007). Gordon Kamm et al. (1990) rst established transgenic maize with bialophos resistance. Koziel et al. (1993) developed insect-resistant transgenic maize with cry 1Ab for the rst time. However, maize genotypes adapted to temperate regions have been used in these studies on regeneration and transformation (Prioli and Silva 1989; Bohorova et al. 1995). To harness the benets of genetic transformation in breeding progrmme under Indian condition it is important to develop protocols of regeneration and transformation with maize inbreds adapted to tropical and sub-tropicl climatic conditions of India. Therefore, the objectives in the present investigation were to establish a reproducible regeneration protocol for well-adapted Indian maize inbred lines and to compare the efciency of different sources of auxins on callus induction and regeneration in the Indian inbred lines.

Immature embryos of 1.02.0 mm size were aseptically excised from surface sterilized kernels under laminar ow and placed with scutellar side up and at surface down on the callus induction medium solidied with 0.8% agar. Callus induction Callus induction media used included N6 medium (Chu et al. 1975) and Murashige and Skoog (MS) (1962) supplemented with various levels of either 2,4-dichlorophenoxyacetic acid (2,4-D) or Dicamba. MS medium was supplemented with 1, 2, 3, or 4 mg l-1 2,4-D. N6 medium containing 3% sucrose, 2.303 g l-1 L-proline, 200 mg l-1 casein hydrolysate, 15 mg l-1 silver nitrate (AgNO3) was supplemented with 1, 2, 3, or 4 mg l-1 of either 2,4-D or Dicamba. pH of the different media was adjusted to 5.8 prior to autoclaving at 121C (108 kPa) for 20 min. Ten explants per treatment/Petri plate were used. Four replications per treatment was used and arranged in a completely randomized block design. Explants were incubated in the dark for 24 h at 28C. Then, these were transferred to 16 h photoperiod, 5070 lE m-2 s-1 light intensity, and 28C. After 2 weeks, number of explants (in percentage) producing primary callus was recorded. Calli were subcultured to fresh medium of the same composition for 1520 days. Regeneration The organogenic calli of the inbred lines CM 124 and CM 300, showing the highest callusing response on MS containing 2,4-D 1 mg l-1 and N6 with either 2 mg l-1 Dicamba or 2 mg l-1 2,4-D, were transferred to regeneration medium. Two different regeneration media were tried in the investigation. According to Rooz (2002) in the rst regeneration medium, referred to as regeneration medium 1 (RM1), calli were rst incubated on MS medium supplemented with 0.1 mg l-1 2,4-D, 0.1 mM abscisic acid (ABA), 0.25 mg l-1 Ca-pantothenate, 1.0 mM asparigine, and sucrose 2%. After 10 days, these were transferred to N6 medium with 6% sucrose and without any plant growth regulator (PGR). Developing shoots were subsequently placed for rooting on MS medium supplemented with 0.25 mg l-1 Ca-pantothenate and 1.0 mM asparigine. A total of 6 calli per plate were used, and these were replicated 8 times. In another set of regeneration media, referred to as regeneration media 2 (RM2), calli were transferred to MS medium supplemented with different combinations of 0, 1, and 2 mg l-1 6-benzyladenine (BA) with 0, 0.5, and 1 mg l-1 indoleacetic acid (IAA). This experimental design was a four factorial randomized block design with induction medium and genotype at two levels and three levels each of BA and IAA. Shoot percentage and number

Materials and methods Plant material Five well-adapted tropical Indian maize inbred lines, viz., CM 111, CM 117, CM 124, CM 125 and CM 300 were used in the study. These lines are from diverse genetic background and are parental lines of many promising Indian maize hybrids. Except CM 300 all the inbred lines were yellow maize, while CM 300 was white seeded. Seeds of these lines were planted in the greenhouse at the National Phytotron Facility, Indian Agricultural Research Institute, New Delhi. Plants were self-pollinated and the whole ears were collected 1314 days following pollination. Kernels were aseptically extracted and washed with Tween-20 (23 drops) followed by surface-sterilization with sodium hypochloride (0.6%) for 20 min. Subsequently, immature kernels were washed with 70% ethanol for 30 s and rinsed ve times with sterilized distilled water.

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of shoots per calli were calculated 3 weeks of culturing. Developing shoots were subsequently separated and transferred to rooting medium consisting of MS with NAA 0, 1, or 2 mg l-1. A total of 10 shoots were used, with four replications per treatment. Cultures of RM1 and RM2 were maintained under 16 h photoperiod, 5070 lE m-2 s-1 light intensity, and 28C. Plantlets with well developed roots were transferred overnight to liquid 1/2 strength MS medium (pH 5.8) without sucrose and then transferred to Jiffy pots containing sterilized Soilrite mix (Karnataka Explosives, Bangalore, India) for acclimatization under 16 h photoperiod for a period of 7 days. Following acclimatization, plants were moved to the greenhouse for further growth. Data analysis All data were subjected to ANOVA. Effects of various treatment combinations on callus induction were compared using contrast analysis using SPSS ver. 13.0. All percent data were subjected to arcsin transformation and shoot numbers were square root transformed before analysis of variance. Mean comparisons were conducted using respective critical differences.

supplemented with 2,4-D. Thus, we made an effort to induce regeneration in Indian maize using both MS and N6 media supplemented with various doses of 2,4-D and Dicamba. Callus induction Based on analysis of variance, all the four parameters, including genotype, induction medium, type of auxin, and level of auxin inuenced the frequency of callus induction. Frequency of callus induction in MS supplemented with 2,4-D ranged from 26.7 to 84.1%. Callus initiation from cultured embryos was observed within 1 week following culture, with swelling of the scutellum and formation of mass on the surface of scutellum. Within 15 days of incubation, the swollen scutellum developed into irregular callus, which turned into organogenic callus after an additional 15 days (Fig. 1a). The highest frequency of callus induction was observed on the MS medium supplemented with 1 mg l-1 2,4-D for all the ve genotypes but the response for callusing reduced as the level of 2,4-D increased in the medium (Table 1). These ndings were similar to those reported by Al-Abed et al. (2006) whereby elevated levels of 2,4-D decreased maize callus induction and resulted in browning of calli at levels C4 mg l-1 2,4-D. Among the ve maize genotypes investigated in this study, inbred line CM 125 yielded the highest frequency (84.1%) of callus induction on MS medium. Contrast analysis for callus induction showed signicant differences (P = 0.023) between N6 and MS media. Bohorova et al. (1995) suggested that N6 medium, which contained lower level of nitrogen than that of MS, showed better callus induction and maintenance. Frequency of callus induction for explants incubated in N6 medium in the presence of Dicamba, ranged between 0 and 100%; while, in the presence of 2,4-D, this ranged between 40 and 85% (Table 1). Quality of calli derived from N6 medium was better than the calli obtained upon incubation under MS medium. Among the 2,4-D levels on N6 medium, callus response was highest with medium containing 2 mg l-1 and this medium was designated as ND2. In ND2, CM 111 and CM 124 showed maximum callusing percentage (84.9%). Dicamba beyond 3 mg l-1 had drastic effect in majority of cases, where the calli turned watery. In genotypes like CM 124, CM 125 and CM 300 no callus could be obtained at this concentration of Dicamba. N6 supplemented with Dicamba gave a better callusing response than 2,4-D supplemented N6, both in terms of callusing percentage and quality of the calli (P = 0.011). This supported the observation of Furini and Jewell (1994) who reported dicamba as superior to 2,4-D in promoting the callus induction.

Results and discussion An efcient regeneration protocol is prerequisite to a successful genetic transformation work in any crop plant. Many published reports are available in maize suggesting successful regeneration from mature embryos (Huang and Wei 2004), nodal culture (Vladimir et al. 2006), split seeds (Al-Abed et al. 2006) other than use of immature embryos as explant (Duncan et al. 1985; Furini and Jewell 1994; Bohorova et al. 1995). Efforts were made to obtain regeneration through either of these protocols using popular Indian inbred lines. However, we met with little success in this direction (data not shown). Therefore, we made an effort to obtain regeneration using immature embryos as this has been most commonly reported as best explant in maize (Ahmadabadi et al. 2007). Towards this endeavour we principally followed the protocols suggested by Bohorova et al. (1995), who reported that N6 medium containing dicamba and AgNO3 give efcient callus induction and plant regeneration in tropical and sub-tropical maize germplasm. Furini and Jewell (1994) also suggested that callus obtained from immature embryos in presence of dicamba developed into somatic embryos than the callus obtained with 2,4-D. Huang and Wei (2004) mentioned the role of 2,4-D with MS media in inducing highly regenerable calli from mature embryos. Rooz (2002) also reported successful regeneration from immature embryos using MS

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Fig. 1 Callusing and regeneration of plantlets from immature embryos of CM 300. a Organogenic calli in NDA2 medium 30 days after inoculation. b Initiation of shoots in shooting medium (MS with BAP 1 mg l-1 and IAA 0.5 mg l-1). c Green shoots from

organogenic calli in shooting medium. d Plant growth in shooting medium. e Plantlets with healthy roots in rooting medium (MS with NAA 1 mg l-1). f Normal plant regenerated from immature embryos in green house

Signicant genotypic differences were recorded among the inbred lines. CM 125 was the most responsive, while CM 117 was the least responsive on MS media (Table 1). However, on N6 medium with 2,4-D CM 125 was the least responsive. At the concentration of 2 mg l-1 of 2,4-D all the genotypes gave nearly similar callusing response. In case of Dicamba, CM 124, CM 125 and CM 300 produced nearly similar response, while CM 111 and CM 117 showed poor response. Comparison of means suggested that CM 125 in N6 with 2 mg l-1 Dicamba as well as CM 300 in N6 with either 1 or 2 mg l-1 dicamba gave best callusing response. In terms of calli quality CM 124 and CM 300 gave the best response in N6 supplemented with 2 mg l-1 Dicamba. Genotypes are reported to play an important role in callusing response in various crop plants including maize (Bohorova et al. 1995; Furini and Jewell 1994; Aguado-Santacruz et al. 2007). We have found that N6 supplemented with 2 mg l-1 Dicamba (subsequently referred to as NDA2) was the most optimum in terms of quality and quantity of organogenic calli (Fig. 1a).

Regeneration response Rooz (2002) reported good regeneration in calli derived from MS with 2,4-D and regeneration in RM1 (refer materials and methods). However, when the organogenic calli developed on MS with 1 mg l-1 2,4-D were transferred to regeneration medium, RM1, for plantlet formation, inbred line, CM 300 did not give any shoot development on this medium, while up to 12.1% shoot formation was recorded in case of CM 124 with an average number of 1.25 shoots per callus. Due to low efciency of the regeneration, RM1 medium was not tried subsequently. Organogenic calli obtained from NDA2 (Fig. 1a) and ND2, when induced for shoot development in MS supplemented with various combinations of IAA and BAP, gave rise to shoot initiation (Fig. 2). Analysis of variance suggested highly signicant effect of induction media, BAP and IAA on both shoot percentage and shoot number per calus, while genotype had a signicant effect only on shoot percentage. Genotype had no interaction with either of the

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Plant Cell Tiss Organ Cult (2010) 100:3137 30.5 0.5 (33.5) 21.7 1.4 (27.6)

35

hormones, while other second order interactions were signicant or highly signicant. Three factor interaction was highly signicant only for induction media and both the hormones. MS medium with increasing concentration of BAP without IAA allowed the shoot induction in NDA2derived calli (Fig. 2ad). However, the efciency was low. BAP beyond 1 mg l-1 had a diminishing effect on shoot initiation as well as on the number of shoots induced. IAA alone (without BAP) did not give any response barring some sporadic shoot formation observed with CM 300. Interaction between IAA and BAP was highly signicant and was recognized as important for efcient shoot induction. BAP 1 mg l-1 and IAA 0.5 mg l-1 proved to be the best combination for shoot induction for both CM 124 and CM 300 inbreds (Fig. 1b, c). This observation is in concurrence with that reported by Bohorova et al. (1995) and Kennedy et al. (2001). In terms of shoot percentage CM 124 gave better response (81.8%) as compared to CM 300 (63.6%; Fig. 2a, c). However, in terms of number of shoots per explant, CM 300 gave better response (4.3) than CM 124 (2.3; Fig. 2b, d). Genotype plays an important role in tissue culture response across crop plants. Bohorova et al. (1995) reported genotype dependent regeneration response among tropical and sub-tropical maize lines. Genotype dependent regeneration response has also been reported by various authors (Wenbin et al. 2002; AguadoSantacruz et al. 2007). Genotypic differences in terms of regeneration response might be related to variations in endogenouse hormone levels (Norstong 1970; Bhaskaran and Smith 1990). Organogenic calli obtained from ND2 were also tried for shoot initiation with similar hormonal combinations and the response obtained was similar to that obtained with NDA2-derived calli. The only exception was that increased IAA alone without BAP also gave some limited shoot induction, particularly at 0.5 mg l-1 (Fig. 2eh). The best response was obtained when 0.5 mg l-1 IAA and 1 mg l-1 BAP were tried. However, the overall shoot percentage and number of induced shoots were much lower in this case as compared to NDA2 derived calli. Well developed multiple shoots (Fig. 1d) were separated and transferred to MS medium supplemented with 0, 1 or 2 mg l-1 NAA for root induction. MS medium without hormone also led to rooting but the rooting efciencies were better at both 1 and 2 mg l-1 NAA. Quality of rooting was the best in MS supplemented with 2 mg l-1 NAA (Fig. 1e). In terms of root initiation and proliferation genotype or induction media had no effect. Regenerated plants were successfully established into complete plants. Establishment percentage for CM 124 and CM 300 were 54 and 66, respectively. Among the established plants, 20% showed oral or structural abnormalities, which may be due to culture stresses induced during various stages of development. However, normal

0 (0.9)

0 (0.9) 91.2 2.4 (72.5) 96.9 1.7 (82.6) 91.7 2.2 (73.4) 48.5 2.9 (44.1)

52.5 5.2 (46.3)

33.3 1.4 (35.2)

57.4 1.1 (49.2)

N6 with Dicamba (mg l-1)

50.0 1.3 (45.2)

78.9 1.6 (62.7)

90.3 1.8 (72.0)

69.1 1.0 (56.3)

50.8 0.6 (45.2)

52.5 1.8 (46.6)

51.6 2.9 (46.2)

40.6 1.0 (39.6) Values in parenthesis are transformed values, CDs are 3.48 and 5.38 at 5 and 1% level of signicance, respectively CM 300 72.9 0.9 (58.6) 62.7 0.6 (52.3) 47.4 1.2 (43.4) 35.1 1.0 (36.2) 55.0 0.7 (47.9) 82.1 1.6 (64.7) 64.2 1.1 (53.1)

71.7 1.0 (57.7)

58.3 2.1 (49.8)

Table 1 Callus response of explants from ve inbred lines of maize incubated on different media

80.8 1.5 (64.1)

67.7 0.4 (55.3)

84.9 2.3 (67.2)

N6 with 2,4 D (mg l-1)

68.8 6.2 (55.9)

84.9 1.9 (67.1)

44.4 4.7 (41.8)

26.7 1.4 (30.8)

71.7 1.9 (57.6)

36.4 0.5 (37.1)

34.9 2.5 (36.2)

42.2 0.8 (40.5)

51.7 1.0 (45.9)

Frequency of callus development (%)

42.4 0.7 (40.6)

52.5 1.7 (46.4)

67.2 1.0 (54.9)

MS with 2,4 D (mg l-1)

82.3 1.4 (65.0)

50.0 1.8 (44.9)

65.4 1.3 (53.9)

Genotype

73.3 1.7 (58.7)

CM 117

CM 124

CM 111

CM 125

84.1 2.3 (66.4)

75.8 1.3 (60.5)

64.2 1.1 (53.2)

44.8 1.1 (42.0)

54.8 4.4 (47.8)

66.2 1.0 (54.6)

59.7 1.1 (50.4)

100.0 2.1 (89.1)

26.6 10.6 (30.9)

85.5 14.7 (67.7)

96.8 1.9 (82.2)

66.7 1.2 (54.6)

0 (0.9)

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(a)
100 IAA 0 mg/l IAA 0.5 mg/l IAA 1 mg/l

(b)
5 IAA 0 mg/l IAA 0.5 mg/l IAA 1 mg/l

Shoot percentage

No. of shoots

80 60 40 20 0 0 1 2

4 3 2 1 0 0 1 2

(c)
Shoot percentage

100 80 60 40 20 0 0 1 2

(d) 8
No. of shoots
6 4 2 0 0 1 2

(e)100
Shoot percentage
80 60 40 20 0 0 1 2

(f)
No. of shoots

5 4 3 2 1 0 0 1 2

(g)
100

(h) 5
No. of shoots
4 3 2 1 0 0 1 2 0 1 2

Shoot percentage

80 60 40 20 0

BAP (mg/l)

BAP (mg/l)

Fig. 2 Regeneration response of orgnogenic calli derived from NDA2 (a, b, c, d) and ND2 (e, f, g, h) under various combinations of BAP and IAA in CM 124 (a, b, e, f) and CM 300 (c, d, g, h). CDs

for shooting percentage are 15.64 and 20.59, and for shoot number are 0.66 and 0.87 at 5 and 1% level of signicance, respectively

regenerated plants owered and cobs were harvested (Fig. 1f). Seeds derived from the regenerated plants were established into normal plants in the eld. In terms of establishment of complete plants from immature embryos, CM 300 was the best genotype followed by CM 124. Though shoot induction percentage in CM 124 was higher than CM 300, number of shoots derived from CM 300 and establishment percentage were higher than CM 124, thus allowing more established plantlets. For callus induction, NDA2 proved to be the best media, while for shoot induction, MS with 1 mg l-1 BAP and 0.5 mg l-1 IAA and 2 mg l-1 NAA for rooting were identied as the best. The regeneration method reported here is quick, efcient and highly reproducible and could be perhaps used in regular transformation studies. Till date, to the best of

our knowledge, no published literature has discussed about the regeneration response of established Indian maize inbred lines. CM 124 and CM 300, the two genotypes identied in this study as responsive to regeneration are well adapted Indian maize inbred lines and have already been used in breeding programme. CM 124 is parent of a popular hybrid, Sartaj, while CM 300 is a common parent of number of hybrids, viz., Ganga Safed-2, Ganga hybrid-4 and High Starch. These also posses tolerance against number of foliar diseases like rust and turcicum leaf blight and are very good pollen shedder. Thus, the established regeneration protocol for these lines might make it possible to use the protocol in transformation studies towards development of genetically modied maize hybrids readily adapted to Indian condition.

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Plant Cell Tiss Organ Cult (2010) 100:3137 Acknowledgments This work has been sponsored by ICAR Network Project on Development of Stem borer resistant transgenic maize. The authors thank Drs. V. Dinesh Kumar, A. Pattanayak and R. C. Bhattacharya for their kind input in improving the manuscript.

37 Ishida Y, Satto H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efciency transformation of maize (Zea mays L.) mediated by Agrobacterium tumrfaciens. Nat Biotech 14:745750 Kennedy MM, Burger JT, Berger DK (2001) Transformation of elite white maize using the particle inow gun and detailed analysis of a low copy integration event. Plant Cell Rep 20:721730 Koziel M, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, KMc Pherson, Meghji M, Merlin E, Rhodes R, Warren GW, Wright M, Evolas S (1993) Field performance of elite transgenic maize plant expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/ tech 11:194200 Morocz C, Donn G, Nemeth J, Dudits D (1990) An improved system to obtain fertile regenerants via maize protoplast isolated from highly embryogenic suspension culture. Theor Appl Genet 80:721726 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473497 Norstong K (1970) Induction of embryo like structures by kinetin in cultured barley embryos. Dev Biol 23:665670 Pareddy DR, Petolino JF (1990) Somatic embryogenesis and plant regeneration from immature inorescences of several elite inbreds of maize. Plant Sci 46:225232 Prioli LM, Silva WJ (1989) Somatic embryogenesis and plant regeneration capacity in tropical maize inbreds. Rev Brazil Genet 12:553566 Rooz BBK (2002) Plastid transformation in maize: construction and testing of vector. Dissertation, Post Graduate School, Indian Agricultural Research Institute, New Delhi, India Sairam RV, Paran M, Franklin G, Lifeng Z, Smith B, MacDougall J, Wilber C, Sheikhi H, Kashikar N, Meeker K, Al-Abed D, Berry K, Vierling R, Goldman SL (2003) Shoot meristem an ideal explant for Zea Mays L. transformation. Genome 46:323329 Ting YC, Yu M, Zheng WZ (1981) Improved anther culture of maize. Plant Sci Lett 23:139145 Vladimir S, Gilbertson L, Adae P, Duncan D (2006) Agrobacteriummediated transformation of seedling-derived maize callus. Plant Cell Rep 25:320328 Wada N, Feng C, Gulati A (2008) Introduction and over view. In: Gulati A, Dixon J (eds) Maize in Asia changing markets and incentives. Academic Foundation, New Delhi, pp 2775 Wenbin LI, Masilamany P, Kasha KJ P, Pauls K (2002) Developmental, tissue culture and genotypic factors affecting plant regeneration from shoot apical meristems of germinated Zea mays L. seedlings. In Vitro Cell Dev Biol Plant 38:285292

References
Aguado-Santacruz GA, Garcia-Moya E, Aguilar-Acuna JL, MorenoGomez B, Preciado-Ortiz ER, Jimenez-Bremont JF, Rascon-Cruz Q (2007) In vitro plant regeneration from quality protein maize. In Vitro Cell Dev Biol Plant 43:215224 Ahmadabadi M, Ruf S, Bock R (2007) A leaf based regeneration and transformation system for maize (Zea mays L.). Transgenic Res 16:437448 Al-Abed D, Rudrabhatla S, Talla R, Goldman S (2006) Split seed: a new tool for maize researchers. Planta 223:13551360 Barloy D, Beckert M (1993) Improvement of regeneration ability of androgenetic embryos by early anther transfer in maize plant. Plant Cell Tissue Organ Cult 33:4550 Bhaskaran S, Smith RH (1990) Regeneration in cereal tissue culture. Crop Sci 30:13281336 Bohorova NE, Luna B, Brito RM, Huerta LD, Hoisington DA (1995) Regeneration potential of tropical, sub tropical, mid altitude and highland maize inbreds. Maydica 40:275281 Chu CC, Wang CC, Sun CS, Hus C, Yin KC, Chu CY, Bi FY (1975) Establishment of an efcient medium for another culture of rice through comparative experiments on nitrogen source. Sci Sin 18:659668 Conger BV, Novak FJ, Afza R, Erdelsky KE (1987) Somatic embryogenesis from cultured leaf segments of Zea mays. Plant Cell Rep 6:345347 Duncan DR, Wiliams ME, Zehr BE, Widholm JM (1985) The production of callus capable of plant regeneration from immature embryos of numerous Zea mays (L.) genotypes. Planta 165: 322332 Furini A, Jewell DC (1994) Somatic embryogenesis and plant regeneration from immature and mature embryos of tropical and subtropical Zea Mays L. genotypes. Maydica 39:155164 Gordon Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O Brein JV, Chambers SA, Adams WR, Willetts JNG, ThB Rice, Backy CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603618 Green CE, Philips RL (1975) Plant regeneration from tissue culture of maize. Crop Sci 15:417421 Huang XQ, Wei ZM (2004) High frequency plant regeneration through callus initiation from mature embryos of maize. Plant Cell Rep 22:793800

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