We report on a rapid high-frequency somatic embryogenesis and plant regeneration protocol for Zea mays. Maize plants were regenerated from complete shoot meristem (3-4 mm) explants via organogenesis and somatic embryogenesis. In organogenesis, the shoot meristems were directly cultured on a high-cytokinin medium comprising 5-10 mg x L(-1) 6-benzylaminopurine (BAP). The number of multiple shoots produced per meristem varied from six to eight Plantlet regeneration through organogenesis resulted in just four weeks. Callus was induced in five days of incubation on an auxin-modified Murashige and Skoog (MS) medium. Prolific callus, with numerous somatic embryos, developed within 3-4 weeks when cultured on an auxin medium containing 5 mg 2,4-dichlorophenoxyacetic acid x L(-1). The number of multiple shoots varied from three to six per callus. Using R23 (Pioneer, Hi-Bred, Johnston, Iowa), the frequency of callus induction was consistently in excess of 80% and plant regeneration ranged between 47 and 64%. All regenerated plantlets survived in the greenhouse and produced normal plants. Each transgenic plant produced leaves, glumes, and anthers that uniformly expressed green fluorescent protein (GFP). The GFP gene segregated in the pollen. Based on this data it is concluded that the transgenics arose from single-cell somatic embryos. The rate of transfer DNA (T-DNA) transfer to complete shoot meristems of Zea mays was high on the auxin medium and was independent of using super-virulent strains of Agrobacterium.

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A protocol for plant regeneration from mesophyll/protoplasts of sorghum [Sorghum bicolor (L.) Moench] was developed. The yield of intact protoplasts, their subsequent divisions and regeneration were genotype-dependent. The genotype 296B was always more responsive than IS 32266. For 296B, the sixth leaf from 18-day-old plants kept in dark for 2 days before harvesting was found to be the most suitable source of viable protoplasts. The first division was observed 10-12 days after plating, and the second division after 12-14 days. The maximum plating efficiency was 4.8% in 296 B, followed by 2.48% in IS 32266. Microcolonies were visible after 25-30 days, and microcalli after 60-75 days. Whole plants were obtained after 6-8 weeks of culture of microcalli on MS medium containing 0.2 mg l-1 kinetin and 2 mg l-1 BAP. The frequency of regeneration in 296B and IS 32266 was 12.80% and 10.58%, respectively. Ten plants transferred to pots in the glasshouse established well. The seeds collected from glasshouse-grown plants were sown in the field where plants were grown to maturity. Key words Sorghum 7 (Mesophyll) protoplasts 7 Cereals 7 Tissue culture 7 Regeneration Abbreviations BAP 6-Benzylaminopurine 7 MS Murashige and Skoog's medium 7 KM8 Kao and Michyaluk's medium 7 NAA a-Naphthaleneacetic acid 7 2,4-D Dichlorophenoxyacetic acid 7 IAA Indole aceatic acid

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A system for rapid plant regeneration through somatic embryogenesis from shoot tip explants of sorghum [Sorghum bicolor (L.) Moench] is described. Somatic embryogenesis was observed after incubation of explants in dark for 6-7 weeks through a friable embryogenic callus phase. Linsmaier and Skoog medium supplemented with 2,4dichlorophenoxyacetic acid (2 mg l −1 ) and kinetin (0.1 mg l −1 ) was used for induction of friable embryogenic calli and somatic embryos. Germination of somatic embryos was achieved about 5 weeks after transfer onto Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (2 mg l −1 ) and indole-3-acetic acid (0.5 mg l −1 ) under light. Seeds from in vitro-regenerated plants produced a normal crop in a field trial, and were comparable to the crop grown with the seeds of the mother plant used to initiate tissue culture. The simplicity of the protocol and possible advantages of the system for transformation over other protocols using different explants are discussed.Abbreviations: 2,4-D -2,4 -dichlorophenoxy acetic acid; BA -6-benzylaminopurine; FEC -friable embryogenic callus; IAA -indoleacetic acid; MS -Murashige and Skoog; LS -Linsmaier and Skoog