Culture conditions were developed that induce Arabidopsis thaliana (L.) Heynh. root cuttings to regenerate shoots rapidly and at 100% efficiency. The shoots produce viable seeds in vitro or after rooting in soil. A transformation procedure for Arabidopsis root explants based on kanamycin selection was established. By using this regeneration procedure and an Agrobacterium tumor-inducing Ti plasmid carrying a chimeric neomycin phosphotransferase II gene (neo), transformed seed-producing plants were obtained with an efficiency between 20% and 80% within 3 months after gene transfer. F(1) seedlings of these transformants showed Mendelian segregation of the kanamycin-resistance trait. The transformation method could be applied to three different Arabidopsis ecotypes. In addition to the neo gene, a chimeric bar gene conferring resistance to the herbicide Basta was introduced into Arabidopsis. The expression of the bar gene was shown by enzymatic assay.

https://www.pnas.org/content/pnas/85/15/5536.full.pdf

Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection.

When Vicia hajastana Grossh. cells or protoplasts were cultured at a high population density (ca. 5000 cells or protoplasts/ml), they were able to grow in a mineral-salt solution supplemented with sucrose (or glucose), a few vitamins, and 2,4-dichlorophenoxyacetic acid. They were not able to survive when cultured at a low population density unless the medium was supplemented with zeatin, naphthalene-1-acetic acid, nucleic-acid bases, amino acids, other sugars, sugar alcohols, and organic acids. Vicia cells were able to grow at an initial population density of 25-50 cells/ml in this defined medium. The population density could be lowered to 1-2 cells/ml with good growth when the mineral-salt medium was enriched with organic acids, sugars, sugar alcohols, coconut water, and casamino acids. The protoplasts also grew best in a medium enriched with these supplements. Three individual protoplasts were isolated and each one was cultured in a separate dish containing 4 ml of this medium. Within 30-40 days, each one had grown indefinitely and formed a mass of cells (ca. 10(7)).

Nutritional Requirements for Growth of Vicia hajastana Cells and Protoplasts at a Very Low Population Density in Liquid Media

Phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase in plants and is used as a non-selective herbicide. The bar gene which confers resistance in Streptomyces hygroscopicus to bialaphos, a tripeptide containing PPT, encodes a phosphinothricin acetyltransferase (PAT) (see accompanying paper). The bar gene was placed under control of the 35S promoter of the cauliflower mosaic virus and transferred to plant cells using Agrobacterium-mediated transformation. PAT was used as a selectable marker in protoplast co-cultivation. The chimeric bar gene was expressed in tobacco, potato and tomato plants. Transgenic plants showed complete resistance towards high doses of the commercial formulations of phosphinothricin and bialaphos. These data present a successful approach to obtain herbicide-resistant plants by detoxification of the herbicide.

Engineering herbicide resistance in plants by expression of a detoxifying enzyme.

We introduced a synthetic gene encoding a truncated version of the CryIA(b) protein derived from Bacillus thuringiensis into immature embryos of an elite line of maize using microprojectile bombardment. This gene was expressed using either the CaMV 35S promoter or a combination of two tissue specific promoters derived from maize. High levels of CryIA(b) protein were obtained using both promoter configurations. Hybrid maize plants resulting from crosses of transgenic elite inbred plants with commercial inbred lines were evaluated for resistance to European corn borer under field conditions. Plants expressing high levels of the insecticidal protein exhibited excellent resistance to repeated heavy infestations of this pest.

Field Performance of Elite Transgenic Maize Plants Expressing an Insecticidal Protein Derived from Bacillus thuringiensis

Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins

Nutrient requirements of suspension cultures of soybean root cells.

Cell division in newly formed cells from protoplasts of soybean.

THERE have been several reports of plant cell wall regeneration1–4 and two reports of division following protoplast formation3, 4 but only one of sustained cell division3.

Cell Divisions in Cells regenerated from Protoplasts of Soybean and Haplopappus gracilis

: Somatic cell hybridization is a potentially useful technique for the introduction of genetic variability into plant species. One proposed method involves fusion of plant protoplasts (which is a cell lacking its wall--defined here on the basis of light microscope examination) to form a hybrid cell which can be induced to form a plant. Plant protoplasts produced by the use of crude commercial lytic enzymes are not always healthy (as defined by consideration of factors such as cytoplasmic streaming, presence of cytoplasmic strands and position of the nucleus within the cell, in relation to survival time). Peroxidase, basic proteins and ribonuclease have been implicated as possible deleterious agents. The use of purified enzymes offers a more rigorously defined system for obtaining healthy protoplasts. Purified preparations of cellulase (EC 3.2.1.4), pectate lyase (EC 4.2.99.3) and endopolygalacturonase (EC 3.2.1.15) have been used to prepare protoplasts from a range of plant cell suspension cultures. Inducible streptomycete enzymes lytic towards plant cell walls have also been prepared. (Author)

Plant protoplasts for use in somatic cell hybridization.

Aging manifests as progressive deterioration in cellular and systemic homeostasis, requiring systems-level perspectives to understand the gradual molecular dysregulation of underlying biological processes. Here, we report systems-level changes in the molecular regulation of biological processes under multiple lifespan-extending interventions in mice and across age in humans. In mouse cohorts, Differential Rank Conservation (DIRAC) analyses of liver proteomics and transcriptomics show that mechanistically distinct prolongevity interventions tighten the regulation of aging-related biological modules, including fatty acid metabolism and inflammation processes. An integrated analysis of liver transcriptomics with mouse genome-scale metabolic model supports the shifts in fatty acid metabolism. Additionally, the difference in DIRAC patterns between proteins and transcripts suggests biological modules which may be tightly regulated via cap-independent translation. In a human cohort spanning the majority of the adult lifespan, DIRAC analyses of blood proteomics and metabolomics demonstrate that regulation of biological modules does not monotonically loosen with age; instead, the regulatory patterns shift according to both chronological and biological ages. Our findings highlight the power of systems-level approaches to identifying and characterizing the biological processes involved in aging and longevity.

R. A. Miller

Papers

Nutrient requirements of suspension cultures of soybean root cells.

Cell division in newly formed cells from protoplasts of soybean.

Cell Divisions in Cells regenerated from Protoplasts of Soybean and Haplopappus gracilis

Plant protoplasts for use in somatic cell hybridization.

Systems-level patterns in biological processes are changed under prolongevity interventions and across biological age