Mohsin Abbas Zaidi

Inheritance and field performance of transgenic Korean Bt rice lines resistant to rice yellow stem borer

Transgenic Korean rice plants containing the cry1Ab gene were developed for resistance against yellow stem borer (Scirpophaga incertulas, YSB). More than 100 independent transgenic lines from three Korean varieties (P-I, P-II and P-III) were generated. The amount of Cry1Ab in transgenic T0 plants was as high as 2.88% of total soluble proteins. These levels were sufficient to cause 100% mortality of YSB larvae. The majority of T1 transgenic lines originated from the varieties P-I and P-II followed a Mendelian fashion of segregation. Deviation from the expected segregation ratio was observed in a small number of the transgenic lines of P-I and P-II origins. However, this deviation was primarily observed in the P-III originated lines. Segregation analysis of the T1 generation indicated that 1–3 copies of the cry1Ab gene were integrated into the genome of the majority of the transgenic lines originating from varieties P-I and P-II. Stunted and semi-fertile mutants were observed in some transgenic lines. These aberrations were either independent or closely linked to the introduced cry1Ab gene loci in different transgenic lines. Reduction in GUS expression levels and loss of toxicity against YSB larvae were found in some transgenic lines. The transgenic T3 and T4 lines causing 100% mortality of third instar YSB larvae in the lab were completely protected in the field. Analysis of important yield components on nine selected transgenic lines indicated that stem length, panicle length, grain number per panicle, and seed setting rates were reduced in transgenic plants compared to those in non-transgenic parental rice lines. Number of panicles per cluster, however, was significantly higher in transgenic plants. The numerical value of the average yield was in general greater in the controls than in all the transgenic lines, indicating some ‘yield drag’. Since some selected lines were highly resistant to the YSB with good yielding potential, they offer effective potential for use in insect resistance management programs.

Resistance to Tecia solanivora (Lepidoptera: Gelechiidae) in Three Transgenic Andean Varieties of Potato Expressing Bacillus thuringiensis Cry1Ac Protein

Abstract Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazúcar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 &mgr;g/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7–100% mortality, whereas the mortality levels on nontransgenic lines were 0–2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of Cry1Ac potatoes in the insect pest management strategies.

Resistance to Anticarsia gemmatalis Hübner (Lepidoptera, Noctuidae) in transgenic soybean (Glycine max (L.) Merrill Fabales, Fabaceae) cultivar IAS5 expressing a modified Cry1Ac endotoxin

Somatic embryos of the commercial soybean (Glycine max) cultivar IAS5 were co-transformed using particle bombardment with a synthetic form of the Bacillus thuringiensis delta-endotoxin crystal protein gene cry1Ac, the β-glucuronidase reporter gene gusA and the hygromycin resistance gene hpt. Hygromycin-resistant tissues were proliferated individually to give rise to nine sets of clones corresponding to independent transformation events. The co-bombardment resulted in a co-transformation efficiency of 44%. Many histodifferentiated embryos and 30 well-developed plants were obtained. Twenty of these plants flowered and fourteen set seeds. The integration and expression of the cry1Ac, gusA and hpt transgenes into the genomes of a sample of transformed embryos and all T0, T1 ,T 2 and T3 plants were confirmed by Gus activity, PCR, Southern and western blot, and ELISA techniques. Two T0 plants out of the seven co-transformed plants produced seeds and were analyzed for patterns of integration and inheritance until the T3 generation. Bioassays indicated that the transgenic plants were highly toxic to the velvetbean caterpillar Anticarsia gemmatalis, thus offering a potential for effective insect resistance in soybean.

The Bt gene cry2Aa2 driven by a tissue specific ST-LS1 promoter from potato effectively controls Heliothis virescens.

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100 mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21 of the total soluble proteins. Bioassays with R1 transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.

Molecular Characterization of the 16S rRNA Gene of Phytoplasmas Detected in Two Leafhopper Species Associated with Alfalfa Plants Infected with Witches' Broom in Oman

Two leafhopper species, Austroagallia avicula and Empoasca sp., were consistently found in alfalfa fields infected with witches’ broom phytoplasma (OmanAlfWB) in the Al-Batinah, Dakhliya, North and South Sharqiya, Muscat, and Al-Bureimi regions of the Sultanate of Oman. Phytoplasmas from both leafhoppers were detected by specific polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the spacer region in direct PCR using P1/P7 primer pairs. Comparative RFLP profiles of the amplified rRNA gene and the spacer region from leafhopper phytoplasmas and from 20 phytoplasma controls yielded patterns referable to phytoplasmas belonging to the peanut witches’ broom group (16SrII group). In particular, extensive RFLP analyses with the endonucleases HpaII, Tru9I, Tsp509I, and RsaI indicated that the phytoplasmas in A. avicula and Empoasca sp. were identical but showed some differences from OmanAlfWB; however, RFLP patterns obtained with TaqI showed the OmanAlfWB and the phytoplasmas from the two leafhoppers to be identical. Direct PCR products amplified from phytoplasma leafhopper DNA using the P1/P7 primer pair were cloned and sequenced yielding 1758 bp and 1755 bp products from A. avicula and Empoasca sp. respectively; the homology of these sequences with OmanAlfWB and papaya yellow crinkle phytoplasmas was more than 98%. A phylogenetic tree based on the 16S rRNA gene and spacer region sequences from 44 phytoplasmas revealed that the phytoplasmas from the leafhoppers clustered with OmanAlfWB, papaya yellow crinkle, and gerbera phyllody phytoplasmas, all belonging to 16SrII group, but were distinct from lime witches’ broom phytoplasma, the most commonly found phytoplasma in the Sultanate of Oman.

Gene technology in agriculture, environment and biopharming: beyond Bt-rice and building better breeding budgets for crops

Applications of gene technology in agriculture, the environment and human health fields are reviewed. This case study of the intricate historical details of the development of Bt crops like cotton and rice unveils essential elements of productive funding schemes and effective multinational collaborations. Gene technology applied to pest resistance traits in global cotton is analyzed using nation-specific data from India to demonstrate ‘ricochet’ results: Regulatory approval for one crop catalyzes an ‘Enhancer Effect’ for promoting more research funding and more competitive results for other crops-in-waiting, namely rice. Just as cotton commerce promoted philanthropy in unpredictable situations like the Kreenholm dynasty of Ludwig Knoop, research budgets for pesticide and biocide technology have yielded intended effects, but several surprising unintended effects as well. Finally, the case is made for greater control of gene flow and identity preserve issues in plant biotechnology research by invoking Appellation d’Origine Contrôlée for Bt genes.

A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase from Pseudomonas stutzeri in transgenic plants

Wan, S., Greenham, T., Goto, K., Mottiar, Y., Johnson, A. M., Staebler, J. M., Zaidi, M. A., Shu, Q. and Altosaar, I. 2014. A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase from Pseudomonas stutzeri in transgenic plants. Can. J. Plant Sci. 94: 1013-1025. As a stable greenhouse gas, nitrous oxide (N2O) plays a significant role in stratospheric ozone destruction. The primary anthropogenic N2O source is the use of nitrogen in agriculture. Currently, the annual N2O emissions from this soil-plant-microbial system is more than 2.6 Tg (1 Tg=1 million metric tonnes) of N2O-N globally. So it is important to explore some innovative and effective biology-based strategies for N2O mitigation. If shown to be effective in field trails as well as laboratory-scale experiments, such GMO plants could help guide international policies on adaptation to climate change. The bacterial enzyme nitrous oxide reductase (N2OR) is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N2O to N2. To “scrub” the N2O emissions, bacterial N2OR was heterologously expressed in plants. Structurally, the enzyme N2OR is encoded by nosZ, but its biosynthesis and assembly in prokaryotes require the products of several nos genes, including a putative ABC-type transporter encoded by nosDFY, and the copper chaperone NosL for biogenesis of the metal centre. We have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other nos genes were co-expressed under the control of a root-specific promoter (rolD) and a constitutive promoter (d35S). The nosZ gene from Pseudomonas stutzeri heterologously expressed in tobacco produced active recombinant N2OR. The positive results in the preliminary proof-of-principle experiments indicated that plants heterologously expressing N2OR could mitigate emissions at the source before N2O reaches the stratosphere or troposphere.

Bacterial nitrous oxide reductase expressed in transgenic plants: Evidence for sufficient anaerobicity to permit activity

Wan, S., Goto, K., Mottiar, Y., Staebler, J. M., Johnson, A. M., Voronova, A., Blais, D. R., Zaidi, M. A. and Altosaar, I. 2012. Bacterial nitrous oxide reductase expressed in transgenic plants: Evidence for sufficient anaerobicity to permit activity. Can. J. Plant Sci. 92: 1283-1294. Soil nitrogen enrichment practices enhance crop yields but greatly exacerbate the production and release of nitrous oxide (N2O), a potent greenhouse gas. The conversion of N2O to dinitrogen (N2) in soil denitrifiers is normally driven by the anaerobic enzyme nitrous oxide reductase (N2OR). Since this step is often limited in fertilised soils, we have transferred this unique microbial biocatalyst from the soil bacterium Pseudomonas stutzeri to transgenic Nicotiana tabacum plants. Our results confirm that engineered plants expressing the N2OR structural gene nosZ are capable of producing functional reductase enzyme without the involvement of the native accessory gene products. Since bacterial N2OR normally exists in the anaerobic environment of the soil bacteriums periplasmic space, this novel in planta activity suggests that plant cells can provide a similar hypoxic refuge for anaerobic enzyme activity. Genetically engineered crops containing N2OR could have considerable potential in the phytoremediation of atmospheric N2O.