A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.

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Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.

Friable, embryogenic maize (Zea mays L.), inbred line A188, callus was established and maintained for more than one year without apparent loss of friability or embryogenic potential. Embryoid development was abundant in these cultures and plants were easily regenerated. Frequencies of friable-callus initiation and somatic-embryoid formation increased linearly with addition to N6 medium (C.C. Chu et al. 1975, Sci. Sin. [Peking] 18, 659–668) of up to 25 mM L-proline. Proline additions up to 9 mM to MS medium (inorganic elements of T. Murashige and F. Skoog 1962, Physiol. Plant. 15, 473–497, plus 0.5 mg 1-1 thiamine hydrochloride and 150 mg 1-1
L-asparagine monohydrate) did not stimulate embryoid formation. A major part of the difference between MS and N6 media could be attributed to their respective inorganic nitrogen components. L-Glutamine was not a satisfactory substitute for L-proline. Of 111 regenerated plants grown to maturity from three independent friable, embryogenic cell lines ranging in age from three to seven months, only four plants were abnormal based on morphology and pollen sterility. Seed was produced by 77% of the regenerated plants.

Establishment and maintenance of friable, embryogenic maize callus and the involvement of L-proline.

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K+ uptake by roots. When F1 hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes.

To improve the iron content of rice, we have transferred the entire coding sequence of the soybean ferritin gene into Oryza sativa (L. cv. Kita-ake) by Agrobacterium-mediated transformation. The rice seed-storage protein glutelin promoter, GluB-1, was used to drive expression of the soybean gene specifically in developing, self-pollinated seeds (T1 seeds) of transgenic plants, as confirmed by reverse transcription PCR analysis. Stable accumulation of the ferritin subunit in the rice seed was demonstrated by western blot analysis, and its specific accumulation in the endosperm by immunologic tissue printing. The iron content of T1 seeds was as much as threefold greater than that of their untransformed counterparts.

Iron fortification of rice seed by the soybean ferritin gene.

This invention relates to a reproducible system for the production of stable, genetically transformed maize cells, and to methods of selecting cells that have been transformed. One method of selection disclosed employs the Streptomyces bar gene introduced by microprojectile bombardment into embryogenic maize cells which were grown in suspension cultures, followed by exposure to the herbicide bialaphos. The methods of achieving stable transformation disclosed herein include tissue culture methods and media, methods for the bombardment of recipient cells with the desired transforming DNA, and methods of growing fertile plants from the transformed cells. This invention also relates to the transformed cells and seeds and to the fertile plants grown from the transformed cells and to their pollen.

Methods and compositions for the production of stably transformed fertile monocot plants and cells thereof

The experiments have revealed that (NH4)2SO4 combined with KNO3 at low concentration is of advantage to the formation, growth and differentiation of pollen callus in rice, whereas the high concentration of (NH4)2SO4, whether used separately or in combination with KNO3, obviously inhibits the pollen callus formation. The optimum NH4+ concen- tration is about 7.0 mM (equal to 3.5 mM (NH4)2SO4). A basic medium containing 3.5 mM (NH4)2SO4 and 28 mM KNO3 as nitrogen sources has been established. On such medium the frequency of the pollen callus formation is higher than that on Millers me- dium, and the differentiation of shoot from pollen callus is satisfactory.

Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources

The components of soluble protein and ribosomal RNA in green and albino pollen plantlets of rice were studied by means of polyacrylamide gel electrophoresis. The results were as follows: (1) Soluble protein: the soluble proteins in green pollen plantlets may be fractionated into 15 bands of varying intensities in which the highest content and the most prominent one is band 3 (fraction I protein). Band 3, however, is nearly absent in albino pollen plantlets. (2) Ribosomal RNA (rRNA): rRNA of high molecular weight in green pollen plantlets may be fractionated into 4 bands, i.e. 25S RNA and 18S RNA in cytoplasmic ribosomes, and 23S RNA and 16S RNA in plastid ribosomes. Little or no 23S RNA and 16S RNA, however, is found in albino pollen plantlets. Together with the evidence obtained by other workers, it is suggested that albino forms of pollen plantlets is caused by the impairment of DNA.

The Deficiency of Soluble Proteins and Plastid Ribosomal RNA in the Albino Pollen Plantlets of Rice

The course of androgenesis in the anthers of wheat (Triticum aestivum) cultured in vitro has been studied. The first mitosis of some uninucleate pollens was equational and resulted in two cells, which gave rise to multicellular mass by repeated divisions. Further development of the multicellular mass produced morphologically irregular callus. The differentiation of young shoots from callus was found to depend not only on the medium employed but also on the feature of the callus cells. Formation of cmbryoids which eventually developed into shoots occurred in the anther cultured on certain medium. Chromosome counts made on the root tips of the pollen plants showed the haploid number n =21.

The androgenesis in wheat (triticum aestivum) anthers cultured in vitro

Embryogenic callus cultures were obtained by culturing young inflorescence tissues of Hordeum vulgare cv. ‘PF51811’ (2x)XTriticum aestivum cv. ‘Chinese Spring’ (6x) hybrids on 2,4-D-containing N6 medium. After subculture for about 10 months the calli retained a high potentiality for somatic embryogenesis and plant regeneration. Of about 300 regenerated plants, approximately 100 were transplanted to potting soil. Eight embryoids and three regenerated plants examined had 28 chromosomes identical to the original hybrid plants, while one regenerated plant was found to be a mixploid composed of cells with 28 and 56 chromosomes. The possibility for obtaining amphiploid hybrids through tissue culture is discussed.

Ching-San Sun

Papers

Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources

The Deficiency of Soluble Proteins and Plastid Ribosomal RNA in the Albino Pollen Plantlets of Rice

The androgenesis in wheat (triticum aestivum) anthers cultured in vitro

Somatic embryogenesis and plant regeneration in callus from inflorescences of Hordeum vulgare X Triticum aestivum hybrids