https://www.cell.com/trends/plant-science/pdf/S1360-1385(02)02251-3.pdf

GATEWAY vectors for Agrobacterium-mediated plant transformation.

It is concluded from a review of the literature that plant cell culture itself generates genetic variability (somaclonal variation). Extensive examples are discussed of such variation in culture subclones and in regenerated plants (somaclones). A number of possible mechanisms for the origin of this phenomenon are considered. It is argued that this variation already is proving to be of significance for plant improvement. In particular the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species. It may also be used to generate variants of a commercial cultivar in high frequency without hybridizing to other genotypes.

Somaclonal variation — a novel source of variability from cell cultures for plant improvement

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.

https://science.sciencemag.org/content/sci/287/5451/303.full.pdf

Engineering the Provitamin A (β-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm

Acetylation of the amino group in position 3 of the 2-desoxystreptamine moiety [AAC(3)] of gentamycin is a potent and wide-spread resistance mechanism in bacteria. Several marker genes coding for AAC(3) enzymes with varying substrate ranges or differing molecular properties have been identified. The enzymes AAC(3)-I and AAC(3)-II have a narrow substrate range and confer resistance to gentamycin and its close structural derivatives only, whereas genes encoding AAC(3)-III and AAC(3)-IV enzymes have a broad substrate range, modifying kanamycins, tobramycin, neomycin and paromomycin [1, 2]. Genes encoding AAC(3)-III and AAC(3)-IV enzymes were expressed in plants and shown to confer resistance to gentamicin, kanamycin (one of the genes), and possibly to other antibiotics [3]. Resistance to more than one antibiotic limits the use of the chimeric marker gene. We report that the narrow substrate range AAC(3)-I enzyme from the transposable element Tn21 [2] also provides a useful gentamycin resistance marker in plants. Materials and methods

Gentamycin resistance in Nicotiana conferred by AAC(3)-I, a narrow substrate specificity acetyltransferase.

Chapter 11 – cell culture mutants and their uses

The plastid genome (ptDNA) of higher plants is highly polyploid, and the 1000-10 000 copies are compartmentalized with up to approximately 100 plastids per cell. The problem we address here is whether or not a newly arising genome can be established in a developing tobacco shoot, and be transmitted to the seed progeny. We tested this by generating two unequal ptDNA populations in a cultured tobacco cell. The parental tobacco plants in this study have an aurea (yellowish-golden) leaf color caused by the presence of a bar au gene in the ptDNA. In addition, the ptDNA carries an aadA gene flanked with the phiC31 phage site-specific recombinase (Int) attP/aftB target sites. The genetically distinct ptDNA copies were obtained by Int, which either excised only the aadA marker gene (i.e. did not affect the aurea phenotype) or triggered the deletion of both the aadA and bat au transgenes, and thereby restored the green color. The ptDNA determining green plastids represented only a small fraction of the population and was not seen in a transient excision assay, and yet three out of the 53 regenerated shoots carried green plastids in all developmental layers. The remaining 49 Int-expressing plants had either exclusively aurea (24) or variegated (25) leaves with aurea and green sectors. The formation of homoplastomic green shoots with the minor green ptDNA in all developmental layers suggests that the ptDNA population in a regenerating shoot apical meristem derives from a small number of copies selected through a stochastic process.

Plastid genomes in a regenerating tobacco shoot derive from a small number of copies selected through a stochastic process

In this chapter we provide an overview of cytosine-to-uridine (C-to-U) RNA editing in the plastids of higher plants Particular emphasis will be placed on the role plastid transformation played in understanding the editing process We discuss how plastid transformation enabled identification of mRNA cis elements for editing and gave the first insight into the role of editing trans factors The introduction will be followed by a protocol for plastid transformation, including vector design employed to identify editing cis elements We also discuss how to test RNA editing in vivo by cDNA sequencing At the end, we summarize the status of the field and outline future directions

Transformation of the Plastid Genome to Study RNA Editing

The SR180 cell line has been isolated in a callus culture derived from a haploid Nicotiana sylvestris (n = X = 12) plant by its ability to proliferate on a selective medium containing 2,000 μg/ml streptomycin sulphate. From the cell line diploid plants have been regenerated. The SR180 selfs are resistant to streptomycin. Streptomycin sensitivity in F1, and a 3∶1 (sensitive to resistant) segregation in F2 indicate that resistance in the SR180 mutant is the result of a recessive Mendelian mutation.

Pal Maliga

Papers

Gentamycin resistance in Nicotiana conferred by AAC(3)-I, a narrow substrate specificity acetyltransferase.

Chapter 11 – cell culture mutants and their uses

Plastid genomes in a regenerating tobacco shoot derive from a small number of copies selected through a stochastic process

Transformation of the Plastid Genome to Study RNA Editing

Streptomycin resistance is inherited as a recessive Mendelian trait in a Nicotiana sylvestris line