Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying mutant forms of a native acetohydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the A19 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones only (642 substitution). The herbicide-resistant phenotype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were transformed with the mutated forms of the A19 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the A19 gene. In these transformation experiments the mutated A19 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A19 transgene in one of the biolistically transformed R₀ plants, and a single copy in one of the R₀ plants transformed with Agrobacterium. As expected. progeny seedlings derived from outcrosses involving the R₀ plant transformed with Agrobacterium segregated in a 1:1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 g/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.Abbreviations AHAS acetohydroxyacid synthase - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - FW fresh weight - GUS -glucuronidase - IC₅₀ herbicide concentration that produces a 50% reduction in the fresh weight growth of cells - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962)     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility

The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.     

I-superfamily conotoxins: sequence and structure analysis

I-superfamily conotoxins have four-disulfide bonds with cysteine arrangement C-C-CC-CC-C-C, and they inhibit or modify ion channels of nerve cells. They have been characterized only recently and are relatively less well studied compared to other superfamily conotoxins. We have detected selective and sensitive sequence pattern for I-superfamily conotoxins. The availability of sequence pattern should be useful in protein family classification and functional annotation. We have built by homology modeling, a theoretical structural 3D model of ViTx from Conus virgo, a typical member of I-superfamily conotoxins. The modeling was based on the available 3D structure of Janus-atracotoxin-Hv1c of Janus-atracotoxin family whose members have been suggested as possible biopesticides. A study comparing the theoretically modeled structure of ViTx, with experimentally determined structures of other toxins, which share functional similarity with ViTx, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels.     

Production of 2-hydroxy-4-methoxybenzaldehyde in roots of tissue culture raised and acclimatized plants of Decalepis hamiltonii Wight & Arn., an endangered shrub endemic to Southern India and evaluation of its performance vis-a-vis plants from natural habitat

Axillary buds obtained from field grown plants of D. hamiltonii were used to initiate multiple shoots on Murashige and Skoog's medium (MS) supplemented with 2 mg L(-1) 6-benzyl aminopurine (BA) and 0.5 mg L(-1) indole-3-acetic acid (IAA). Profuse rooting was achieved when the actively growing shoots were cultured on MS medium supplemented with 1.0 mg l(-1) indole-3-butyric acid (IBA). Regenerated plants were grown successfully in the plains, in contrast to wild growth in high altitudes and rocky crevices of hilly regions. Roots of different sizes from one-year-old tissue culture raised field grown plants had the same profile of 2-hydroxy-4-methoxybenzaldehyde as that of wild plants. A maximum of 0.14% and 0.12% 2-hydroxy-4-methoxybenzaldehyde was produced in roots of one year old tissue culture derived plants and greenhouse grown plants respectively.     

Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.)

Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0°C/min). A cooling rate of 0.5°C/min provided a satisfactory recovery rate (30%) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of 1°C/min. Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soil.Abbreviations DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) - MMS modified MS - NAA -naphthaleneacetic acid     

Production of clonal plantlets of Grevillea robusta in in vitro culture via axillary bud activation

A micropropagation method for Grevillea robusta A. Cunn. was developed using explants from mature trees cultured on Woody Plant Medium plus 4.4 M benzyladenine and 0.27 M naphthaleneactic acid (NAA) for shoot proliferation. Each nodal explant produced three to five shoots within 10 to 12 weeks in three successive transfers. Rooting was obtained by dipping the shoots in 0.54 mM NAA solution. Rooted shoots were established well in soil.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indolebutyric acid - NAA naphthaleneactic acid