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Key Message: Major QTL on LG 1 and 3 control seed filling and seed coat development, thereby affecting seed shape, size, color, composition and weight, key determinants of crop yield and quality. A chickpea (Cicer arietinum L.) population consisting of 189 recombinant inbred lines (RILs) derived from a cross between medium-protein ICC 995 and high-protein ICC 5912 genotypes of the desi market class was analyzed for seed properties. Seed from the parental lines and RILs was produced in four different environments for determination of seed shape (SS), 100-seed weight (100-SW), protein (PRO) and starch (STA) concentration. Polymorphic genetic markers for the population were identified by Genotyping by Sequencing and assembled into a 522.5 cM genetic map. Phenotype data from the different growth environments were analyzed by QTL mapping done by single and multi-environment analyses and in addition, single marker association mapping. The analyses identified in total 11 QTL, of which the most significant (P < 0.05) loci were located on LG 1 (q-1.1), LG 2 (q-2.1), LG 3 (q-3.2, q-3.3), LG 4 (q-4.2), and LG 5 (q-5.1). STA was mostly affected by q-1.1, which explained 19.0% of the phenotypic variance for the trait. The largest QTL effects were demonstrated by q-3.2 that explained 52.5% of the phenotypic variances for 100-SW, 44.3% for PRO, and 14.6% for SS. This locus was also highly associated with flower color (COL; 95.2% explained) and showed q-3.2 alleles from the ICC 5912 parent conferred the blue flower color and production of small, round seeds with relatively high protein concentration. Genes affecting seed filling at q-1.1 and seed coat development at q-3.2, respectively, were considered to underlie differences in seed composition and morphology in the RIL population.

Low-temperature (LT) tolerance is an important economic trait in winter wheat (Triticum aestivum L.) that determines the plants' ability to cope with below freezing temperatures. Essential elements of the LT tolerance mechanism are associated with the winter growth habit controlled by the vernalization loci (Vrn-1) on the group 5 chromosomes. To identify genomic regions, which in addition to vrn-1 determine the level of LT tolerance in hexaploid wheat, two doubled haploid (DH) mapping populations were produced using parents with winter growth habit (vrn-A1, vrn-B1, and vrn-D1) but showing different LT tolerance levels. A total of 107 DH lines were analyzed by genetic mapping to produce a consensus map of 2,873 cM. The LT tolerance levels for the Norstar (LT(50)=-20.7 degrees C) x Winter Manitou (LT(50)=-14.3 degrees C) mapping population ranged from -12.0 to -22.0 degrees C. Single marker analysis and interval mapping of phenotyped lines revealed a major quantitative trait locus (QTL) on chromosome 5A and a weaker QTL on chromosome 1D. The 5A QTL located 46 cM proximal to the vrn-A1 locus explained 40% of the LT tolerance variance. Two C-repeat Binding Factor (CBF) genes expressed during cold acclimation in Norstar were located at the peak of the 5A QTL.

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.

The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli β-glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 μg/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 μg/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 μg/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 μg/ml kanamycin, but exhibited reduced rooting ability.

A comparative study on a pure cationic and a pure anionic protein from peanut cells and petunia stem tissue respectively, both with peroxidative activity, was made. The cationic protein weighs 44 Kd and the anionic 36 Kd. No immunological cross reactivity could be detected between the two proteins. In assays for peroxidative activity using the substrates 4-aminoantipyrine, guaiacol and eugenol it was noted that the anionic protein had 1.9, 12.7, and 27.7 fold greater enzymatic activity, respectively. For overall peroxidative measurements it is suggested that aminoantipyrine is probably the superior substrate. With regard to IAA oxidase activity of the two protein fractions it was noted that the cationic enzyme possessed optimal activity at pH 3.6 and the anionic protein at pH 7.0. The latter value could only be obtained by the addition of H2O2 and dichlorophenol (DCP). Since no additives were needed for the assay of IAA oxidation by the cationic protein it is suggested that this is a true IAA oxidase while the anionic fraction is a peroxidase involved in other reactions such as lignin biosynthesis.

Transient expression of chloramphenicol acetyl transferase gene has been detected in cultured barley (Hordeum vulgare L. cv. Heartland) cells and freshly isolated immature zygotic embryos (cv. Ellice) following the introduction of the gene by microprojectile bombardment. The DNA expression vector used to introduce the CAT gene, pCaMVI1CN, is a pUC8 derivative and consisted of a CaMV35S promoter, a fragment of alcohol dehydrogenase intron1, a CAT coding region and NOS polyadenylation region. The inclusion of the Adh1 intron1 was essential for the expression of CAT activity in cultured cells as well as immature zygotic embryos. Expression of CAT activity, which was dependent upon the DNA concentration used, could be detected as early as 20 h after bombardment. The results also suggested that the recipient cells have to be in an active state of cell division in order for the introduced gene to be expressed since mature zygotic as well as somatic embryos failed to reveal any gene expression. The effect of other parameters which influence the expression of the introduced gene as well as the potential of this novel technology for cereal transformation are also discussed.