Evidence for regional diversity and host adaptation in Iranian populations of the Russian wheat aphid

Effective pest management is greatly facilitated by knowledge of the genetic structure and host adaptation of the pest species in question. The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko) (Homoptera: Aphididae: Macrosiphini), is an important economic pest in many cereal‐growing areas of the world, and in this study we investigated these aspects of its populations, using microsatellite markers and host plant response assays. Diuraphis noxia was sampled from 38 locations in Iran and genotyped at four polymorphic microsatellite loci that had been isolated from various Sitobion species. We identified 50 multilocus genotypes in 376 individuals. The overall observed heterozygosity was 0.134. F‐statistics showed a regional partitioning in D. noxia populations with overall F_ST = 0.231. In addition, there was a significant correlation between genetic and geographic distances. In order to test for the ecological consequences of genetic variability in D. noxia, biotypic variation amongst the isolates collected from wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) was evaluated on a number of resistant and susceptible wheat varieties. The plant variables we measured were damage rating (based on leaf chlorosis, leaf rolling, wilting, and death of the host plant), host plant dry weight, and root length. Damage rating was the best criterion for detecting biotypic variation in D. noxia. Discriminant analysis correctly classified the isolates in respective groups in 80–91.8% of the cases. The barley isolate showed no differences in performance on resistant and susceptible wheat, indicating a lack of gene‐by‐gene relationship with wheat plants. In contrast, wheat isolates differentially damaged the resistant and susceptible plants and showed moderate to severe virulence.     

Direct shoot regeneration from mature embryo as a rapid and genotype-independent pathway in tissue culture of heterogeneous diverse sets of cumin (Cuminum cyminum L.) genotypes

Summary A rapid and one-step protocol for direct regeneration of shoots from cumin embryo explants has been developed. Embryo explants with shoot meristems were cultured on shoot regeneration medium for 15–22 d. After embryo culture, shoots were regenerated from the area adjacent to the region between the cotyledons and embryo axis within 2 wk, without any intermediate callus phase. Shoot proliferation and elongation were achieved on shoot regeneration medium without subculture. Among the different combinations of 6-benzylaminopurine, α-naphthaleneacetic acid (NAA), and indole-3-acetic acid (IAA) tested, 0.8 mgl^−1 (4.3 μM) NAA in combination with 0.3 mgl^−1 (1.71 μM) IAA in the B5 medium resulted in the most efficient direct shoot regeneration. No significant difference was detected for the number of regenerated explants when different heterogeneous endemic varieties were compared. This plant regeneration procedure was applicable to different cumin genotypes and regenerated plants were phenotypically normal.     

Proteomic analysis of rice leaves during drought stress and recovery

Three-week old plants of rice (Oryza sativa L. cv CT9993 and cv IR62266) developed gradual water stress over 23 days of transpiration without watering, during which period the mid-day leaf water potential declined to approximately -2.4 MPa, compared with approximately -1.0 MPa in well-watered controls. More than 1000 protein spots that were detected in leaf extracts by proteomic analysis showed reproducible abundance within replications. Of these proteins, 42 spots showed a significant change in abundance under stress, with 27 of them exhibiting a different response pattern in the two cultivars. However, only one protein (chloroplast Cu-Zn superoxide dismutase) changed significantly in opposite directions in the two cultivars in response to drought. The most common difference was for proteins to be up-regulated by drought in CT9993 and unaffected in IR62266; or down-regulated by drought in IR62266 and unaffected in CT9993. By 10 days after rewatering, all proteins had returned completely or largely to the abundance of the well-watered control. Mass spectrometry helped to identify 16 of the drought-responsive proteins, including an actin depolymerizing factor, which was one of three proteins detectable under stress in both cultivars but undetectable in well-watered plants or in plants 10 days after rewatering. The most abundant protein up-regulated by drought in CT9993 and IR62266 was identified only after cloning of the corresponding cDNA. It was found to be an S-like RNase homologue but it lacked the two active site histidines required for RNase activity. Four novel drought-responsive mechanisms were revealed by this work: up-regulation of S-like RNase homologue, actin depolymerizing factor and rubisco activase, and down-regulation of isoflavone reductase-like protein.     

Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) gene

Rice cultivars of isozyme group V include high-quality, aromatic rices that are difficult to improve by traditional methods because of the loss of quality characters upon sexual hybridization. Their low-tillering plant type predisposes them to economic loss from attack by stem borers, a group of insects to which they are susceptible. We report here the enhancement of stem borer resistance in cv. Tarom Molaii through transformation by microprojectile bombardment. Embryogenic calli derived from mature seeds were bombarded with gold particles coated with plasmid pCIB4421, carrying a synthetic truncated toxin gene based on the cryIA(b) gene from Bacillus thuringiensis, and plasmid pHygII, carrying the hygromycin phosphotransferase (hpt) selectable marker gene. Inclusion of 50 mg/l hygromycin B in culture media from bombardment through to rooting of plantlets eliminated escapes. The procedure generated three independent hpt transformants of which two also contained the cryIA(b) gene. One such line (No. 827) produced truncated (67 kDa) CryIA(b) protein equivalent to about 0.1% of total soluble protein. The cryIA(b) gene was controlled by the promoter of the maize C₄ PEP carboxylase gene and was expressed in leaf blades but was not expressed to a detectable level in dehulled mature grain. Line 827 contained about 3 copies of the cryIA(b) gene which segregated as a single dominant Mendelian locus in the second (T₁) and third (T₂) generations and co-segregated with enhanced resistance to first-instar larvae of striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas). T₂ line 827-6 homozygous for the cryIA(b) gene showed no dead hearts or whiteheads after infestation with stem borers, whereas T₂ line 827-25 lacking the gene averaged 7 dead hearts per plant and 2.25 whiteheads per plant. These results establish that transformation of high-quality rices of group V is a feasible alternative to sexual hybridization.     

Expression of recombinant human IFN-γ protein in soybean (Glycine max L.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Globular androgenic haploid embryos of TV21 and TV19 cultivars of Camellia ssp., obtained on embryo induction medium (EIM), Murashige and Skoog medium...     

A rapid and efficient method for regeneration of plantlets from embryo explants of cumin (Cuminum cyminum)

A new, simple and efficient method was developed for multiple shoot regeneration of cumin from imbibed embryo cultures. This method yielded a large number of shoots within short period of time (30–50 days) without any subculturing. The effects of different media, different embryo explants and various combinations of plant growth regulators (PGRs) on callus formation and shoot regeneration in cumin were investigated. Simultaneous callus formation and shoot regeneration was obtained. The best response for multiple shoot regeneration was observed on B5 medium containing 1.0 mg l^–1 BAP, 0.2 mg l^–1 NAA and 0.4 mg l^–1 IAA, with an average of 140 shoots per explant.     

Enhanced salt stress tolerance in transgenic potato plants (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) expressing a bacterial <Emphasis Type="Italic">mtl</Emphasis>D gene

Bacterial mannitol 1-phosphate dehydrogenase (mtlD) gene was introduced into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were selected on a medium containing 100 mg l⁻¹ kanamycin and confirmed by polymerase chain reaction (PCR), Southern blotting, and RT-PCR analyses. All of the selected transformants accumulated mannitol, a sugar alcohol that is not found in wildtype potato. Experiments designed for testing salt tolerance revealed that there was enhanced NaCl tolerance of the transgenic lines both in vitro and in hydroponic culture. Compared to 0 mM NaCl, the shoot fresh weight of wildtype plants was reduced by 76.5% at 100 mM NaCl under hydroponic conditions. However, under the same condition, the shoot fresh weight of transgenic plants was reduced only by 17.3%, compared to 0 mM NaCl treatment. The improved tolerance of this transgenic line may be attributed to the induction and progressive accumulation of mannitol in the roots and shoots of the plants. In contrast to in vitro experiments, the mannitol content in the transgenic roots and shoots increased at 50 mM NaCl and decreased slightly at 75 and 100 mM NaCl, respectively. Overall, the amount of accumulated mannitol in the transgenic lines was too small to act as an osmolyte; thus, it might act as an osmoprotectant. However, the results demonstrated that mannitol had more contribution to osmotic adjustment in the roots (but not in shoots). Finally, we concluded that mtlD expression in transgenic potato plants can significantly increase the mannitol accumulation that contributes to the enhanced tolerance to NaCl stress. Furthermore, although this enhanced tolerance resulted mainly from an osmoprotectant action, an osmoregulatory effect could not be ruled out.     

Effect of plant age, larval age, and fertilizer treatment on resistance of a cry1Ab-transformed aromatic rice to lepidopterous stem borers and foliage feeders

The resistance of vegetative, booting, and flowering stage plants of a variety of an aromatic rice, Oryza sativa L., transformed with a Bacillus thuringiensis Berliner cry1Ab gene under control of the maize phosphoenolpyruvate carboxylase (PEPC) promoter was evaluated against four lepidopterous rice pests--the stem borers Chilo suppressalis (Walker) (Lepidoptera: Crambidae) and Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae), and the foliage feeders Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae) and Naranga aenescens Moore (Lepidoptera: Noctuidae). Plants of the cry1Ab-transformed line (no. 827) were more resistant to young larvae of S. incertulas, C. suppressalis, and C. medinalis than control plants at the vegetative stage but not at the flowering stage. Survival of 10-d-old stem borer larvae did not differ on cry1Ab plants and control plants at either the vegetative or flowering stage, but the development of 10-d-old C. suppressalis larvae was retarded on the vegetative stage cry1Ab plants. Immunological analysis also showed an apparent decline in Cry1Ab titer in leaf blades and leaf sheaths at the reproductive stage. In experiments comparing three fertilizer treatments (NPK, PK, and none), there was a significant interaction between fertilizer treatment and variety on larval survival only in whole-plant assays at booting stage with C. suppressalis. On cry1Ab plants, larval survival did not differ significantly among the three fertilizer levels, whereas on control plants survival was highest with the NPK treatment. cry1Ab plants tested at the sixth and seventh generations after transformation were more resistant than control plants to N. aenescens and C. suppressalis, respectively, suggesting that gene silencing will not occur in line 827. The results of the experiments are discussed in terms of resistance management for B. thuringiensis toxins in rice.     

Globular embryo-like structures and highly efficient thidiazuron-induced multiple shoot formation in saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L.)

Saffron (Crocus sativus L.) is a monocotyledonous plant propagated via corms, but recently several alternative methods have been reported. To find the conditions suitable for saffron shoot formation from corms, the effect of different concentrations of the plant growth regulatory cytokinins N⁶-benzyladenine (BA) and N-phenyl-1, 2,3-thidiazol-5-ylurea, commonly known as thidiazuron (TDZ), were compared. In all corm explants, an average of 39.5 ± 5.1 shoots per corm were induced by 4.54 μM TDZ, whereas only 3.6-11.4% by BA. The outstanding result in the shoot formation stage is the generation of globular, translucent structures that are morphologically similar to globular embryos. To optimize the plant regeneration from the induced adventitious shoots obtained from the TDZ treatment, the shoots were transferred to MS and B5 media supplemented with different concentrations and combinations of NAA and BA. The highest rate of plant regeneration from developing shoots was observed in the B5 medium containing 2.22 μM NAA and 2.68 μM BA. With optimized hormonal conditions, an average of 19.55 ± 5.75 shoots and 3.18 ± 1.5 roots per explants were obtained. Based on this experiment, a simple, new and efficient protocol is presented to produce numerous plants from induced corm explants of saffron.