Summary
A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons Calli induced from scutella were very good starting materials A strain of A tumefaciens that carried a so-called ‘super-binary’ vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1994.6020271.x

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

1 The Basic Features of Self-Incompatibility.- 2 The Genetics of Self-Incompatibility.- 3 Cellular and Molecular Biology of Self-Incompatibility.- 4 Breakdown of the Self-Incompatibility Character, S Mutations and the Evolution of Self-Incompatible Systems.- 5 Incompatibility and Incongruity Barriers Between Different Species.- 6 Conclusions.- References.

Incompatibility and Incongruity in Wild and Cultivated Plants

In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor.

https://science.sciencemag.org/content/sci/286/5445/1697.full.pdf

The male determinant of self-incompatibility in Brassica.

Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). We describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that resides at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus glycoprotein (SLG) gene and is connected via a single pass transmembrane domain to a protein kinase catalytic center. SRK alleles derived from different S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

https://www.pnas.org/content/pnas/88/19/8816.full.pdf

Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

We have recovered transgenic rice plants from a number of commercially important cultivars, including until now recalcitrant Indica varieties, using electric discharge particle acceleration Immature embryos from greenhouse–grown plants were bombarded with gold particles carrying DNA, and transgenic plants were recovered following a simple culture protocol Mendelian segregation of foreign genes was observed in R1 progeny and stable integration was demonstrated by Southern blot analysis of genomic DNA isolated from progeny plants Alternative transformation protocols that are dependent on the development of protoplast and suspension culture systems are no longer necessary as we have shown that a wide variety of diverse cultivars can be transformed Transgenic plants expressing agronomically useful traits such as herbicide resistance have been obtained and are currently undergoing further evaluation This report also demonstrates that it is possible to produce transgenic monocoty–ledonous plants by transforming scutellar tissue of immature embryos

Production of Transgenic Rice ( Oryza Sativa L.) Plants from Agronomically Important Indica and Japonica Varieties via Electric Discharge Particle Acceleration of Exogenous DNA into Immature Zygotic Embryos

We have regenerated whole plants of rice (Oryza sativa L.) derived from protoplasts, which had been electroporated with plasmid DNA possessing a chimeric gene encoding aminoglycoside phosphotransferase II (APH(3′)II). Transformed calli were selected on the basis of tolerance to the antibiotic, G418. Several plants were regenerated possessing functional APH(3′)II activity due to the integration of intact foreign DNA into their genome.

/pdf/transgenic-rice-plants-after-direct-gene-transfer-into-1tcmligwd6.pdf

Transgenic Rice Plants After Direct Gene Transfer into Protoplasts

Cell suspension and protoplast culture in rice

The regeneration of haploid and diploid plants was demonstrated from protoplasts that were isolated from cell suspensions of anther callus in rice. The cell suspension in the AA medium that contained 4 amino acids as the sole nitrogen source was friable, finely dispersed, and readily released a large number of protoplasts. These protoplasts, subsequently cultured in NO3 medium that contained nitrate as the sole nitrogen source, formed compact calli. The compact calli produced green plants with a frequency of 24%. Out of 15 flowering plants, 4 were haploids, the others were diploids which showed a uniform morphology but varied in seed fertility from 95 to 0%.

Haploid and diploid plant regeneration from protoplasts of anther callus in rice

An SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.

Kinya Toriyama

Papers

Transgenic Rice Plants After Direct Gene Transfer into Protoplasts

Cell suspension and protoplast culture in rice

Haploid and diploid plant regeneration from protoplasts of anther callus in rice

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype.

Production of Somatic Hybrid Plants, ‘Brassicomoricandia’, Through Protoplast Fusion between Moricandia Arvensis and Brassica Oleracea