Mariam Sticklen

Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol

The catalytic domain of Acidothermus cellulolyticus thermostable endoglucanase gene (encoding for endo-1,4-β-glucanase enzyme or E1) was constitutively expressed in rice. Molecular analyses of T1 plants confirmed presence and expression of the transgene. The amount of E1 enzyme accounted for up to 4.9% of the plant total soluble proteins, and its accumulation had no apparent deleterious effects on plant growth and development. Approximately 22 and 30% of the cellulose of the Ammonia Fiber Explosion (AFEX)-pretreated rice and maize biomass respectively was converted into glucose using rice E1 heterologous enzyme. As rice is the major food crop of the world with minimal use for its straw, our results suggest a successful strategy for producing biologically active hydrolysis enzymes in rice to help generate alcohol fuel, by substituting the wasteful and polluting practice of rice straw burning with an environmentally friendly technology.

In-vitro morphogenesis of corn (Zea mays L.) : I. Differentiation of multiple shoot clumps and somatic embryos from shoot tips.

In-vitro methods have been developed to regenerate clumps of multiple shoots and somatic embryos at high frequency from shoot tips of aseptically-grown seedlings as well as from shoot apices of precociously-germinated immature zygotic embryos of corn (Zea mays L.). About 500 shoots were produced from a shoot tip after eight weeks of culture (primary culture and one subculture of four weeks) in darkness on Murashige and Skoog basal medium (MS) supplemented with 500 mg/L casein hydrolysate (CH) and 9 μM N6-benzyladenine (BA). In this medium, shoots formed in shoot tips as tightly packed “multiple shoot clumps” (MSC), which were composed of some axillary shoots and many adventitious shoots. When the shoot tips were cultured on MS medium containing 500 mg/L CH, 9 μM BA and 2.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D), most of the shoots in the clumps were adventitious in origin. Similar shoot tips cultured on MS medium containing 500 mg/L CH, 4.5 μM BA and 2.25 μM 2,4-D regenerated many somatic embryos within eight weeks of culture. Somatic embryos were produced either directly from the shoot apical meristems or from calli derived from the shoots apices. Both the MSC and the embryos produced normal shoots on MS medium containing 2.25 μM BA and 1.8 μM indole-3-butyric acid (IBA). These shoots were rooted on MS medium containing 3.6 μM IBA, and fertile corn plants were grown in the greenhouse. The sweet-corn genotype, Honey N Pearl, was used for the experiments described above, but shoot-tip cultures from all of 19 other corn genotypes tested also formed MSC on MS medium containing 500 mg/L CH and 9 μM BA.

Dutch elm disease research : cellular and molecular approaches

Dutch elm disease is a significant problem in forestry and horticulture which has proven remarkably difficult to ameliorate. Since the introduction of the Dutch elm disease pathogen to North America, the disease has devastated the elm population of this continent and has been the subject of intensive research. This book summarizes the range of approaches that have been taken to address the disease, and emphasizes the significant progress over the past decade in applying methods from cell and molecular biology.

The Competence of Maize Shoot Meristems for Integrative Transformation and Inherited Expression of Transgenes

We have developed a novel and reproducible system for recovery of fertile transgenic maize (Zea mays L.) plants. The transformation was performed using microprojectile bombardment of cultured shoot apices of maize with a plasmid carrying two linked genes, the Streptomyces hygroscopicus phosphinothricin acetyltransferase gene (bar) and the potato proteinase inhibitor II gene, either alone or in combination with another plasmid containing the 5[prime] region of the rice actin 1 gene fused to the Escherichia coli [beta]-glucuronidase gene (gus). Bombarded shoot apices were subsequently multiplied and selected under 3 to 5 mg/L glufosinate ammonium. Co-transformation frequency was 100% (146/146) for linked genes and 80% (41/51) for unlinked genes. Co-expression frequency of the bar and gus genes was 57% (29/51). The co-integration, co-inheritance, and co-expression of bar, the potato proteinase inhibitor II gene, and gus in transgenic R0, R1, and R2 plants were confirmed. Localized expression of the actin 1-GUS protein in the R0 and R1 plants was extensively analyzed by histochemical and fluorometric assays.

Heterologous Acidothermus cellulolyticus 1,4-β-Endoglucanase E1 Produced Within the Corn Biomass Converts Corn Stover Into Glucose

Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-β glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic® (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active form.

SHOOT APICAL MERISTEM: A SUSTAINABLE EXPLANT FOR GENETIC TRANSFORMATION OF CEREAL CROPS

SummaryImmature zygotic embryo has been the widely used explant source to develop embryogenic callus lines, cell suspensions and protoplasts for transformation of cereal crops including maize, wheat, rice, oat, barley, sorghum, and millet. However, the lack of competence of immature embryos in certain elite lines is still a barrier to rontine production of transgenic cereal crops in certain commercial cultivars. In addition, a great deal of effort is required to produce immature embryos, manipulate cultures, of immature embryos or their cell suspensions, and cryoperserve cultures for further use. In addition, undifferentiated cells may have reduced regenerability after a few months, of in vitro culture. Alternative explants and regeneration systems for efficient transformation of cereal crops are needed to avoid or reduce the above limitations. During the past decade, scientists have successfully manipulated the shoot apical meristerms from seedlings of maize oat, sorghum, millet, wheat, and barley in an effort to develop a less genetype-dependent and efficient cereal regneration system that can be maintained in vitro for long pertiods of time without the need for cryopreservation. Furthermore, apical mesistem regeneration systems were used to stably transform maize, wheat, rice, oat, barley, sorghum, and millet.

Competence of oat (Avena sativa L.) shoot apical meristems for integrative transformation, inherited expression, and osmotic tolerance of transgenic lines containing hva1

Abstract.Three oat (Avena sativa L.) cultivars have been successfully transformed using an efficient and reproducible in vitro culture system for differentiation of multiple shoots from shoot apical meristems. The transformation was performed using microprojectile bombardment with two plasmids (pBY520 and pAct1-D) containing linked (hva1-bar) and non-linked (gus) genes. The hva1 and bar genes cointegrated with a frequency of 100% as expected, and 61.6% of the transgenic plants carried all three genes. Molecular and biochemical analyses in R0, R1 and R2 progenies confirmed stable integration and expression of all transgenes. Localization of the GUS protein in R0 and R1 plants revealed that high-expression of gus occurred in vascular tissues and in the pollen grains of mature flowers. The constitutive expression of HVA1 protein was observed at all developmental stages of transgenic plants, and was particularly stronger during the early seedling stages. R2 progeny of five independent transgenic lines was tested in vitro for tolerance to osmotic (salt and mannitol) stresses. As compared to non-transgenic control plants, transgenic plants maintained a higher growth and showed significantly (P < 0.05) increased tolerance to stress conditions. Less than 10% of transgenic plants showed symptoms of wilting or death of leaves and, when these symptoms present were delayed in transgenic plants as compared to 80% of non-transgenic plants, either wilted or died. These symptoms confirmed the increased in vitro tolerance in hva1-expressing transgenic plants to non-transgenic plants, providing strong evidence that the HVA1 protein may play an important role in the protection of oats against salinity and possible water-deficiency stress conditions.

In vitro morphogenesis of Sorghum bicolor (L.) moench: Efficient plant regeneration from shoot apices

Summary An efficient and reproducible plant regeneration system has been developed from shoot apices of aseptically germinated seedlings of sorghum. After four passages of culture at two-week intervals on MS basal medium containing 2 or 4 mg/L BA, the shoot apices produced multiple shoot clumps, each capable of regenerating more than 100 shoots, via an intensive differentiation of both axillary and adventitious buds. When the shoot apices were cultured on MS medium supplemented with 0.5 mg/L 2,4-D and 2 or 4 mg/ L BA, a highly efficient differentiation of adventitious buds was initiated from enlarged apical domes and thickened leaf bases of the shoot apices six weeks after inoculation. Over 200 shoots were induced from a shoot apex eight weeks after culture. Somatic embryos were produced direcdy from the enlarged and organized apical domes of primary and secondary shoots without apparent callus formation after subsequent subcultures of the multiple shoot clumps on MS medium with 0.5 mg/L 2,4-D and 2 or 4 mg/L BA. All 18 sorghum genotypes tested followed a similar differentiation and regeneration pattern at a high frequency. Most of the multiple shoots and somatic embryos developed into fertile plants after rooting on IBA-containing MS medium and transfer to greenhouse.

Shoot apical meristem: In vitro regeneration and morphogenesis in wheat (Triticum aestivum L.)

SummaryWe report a less genotype-dependent in vitro regeneration system capable of producing multiple shoot clumps and whole plants in four different wheat genotypes. Shool apical meristems from 7-d-old-seedlings produced axillary and adventitious shoots and somatic embryos on media containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). All four genotypes responded positively to shoot multiplication depending upon media composition. Scanning electron microscopies of cultures showed a proliferating budding state that gave rise to adventitious shoots and somatic embryos on further multiplication. The percentage of relative shoot apical meristem multiplication was 80–90%, and the average number of shoot meristems per multiplied shoot was 40–50 in all genotypes. Among different concentrations of phytohormones, 2 and 4 mgl−1 BA (8.8 and 17.7 μM) in combination with 0.5 mg l−1 2,4-D (2.26 μM) gave the best results. Actively multiplying shoot clumps were recovered with high frequency among 3-mo.-old cultures. These shoot clumps regenerated normally and produced fertile plants containing viable seeds. This in vitro system might prove useful for the production of transgenic plants of wheat in a relatively genotype-independent manner.

In vitro morphogenesis of pearl millet [Pennisetum glaucum (L.) R.Br.]: efficient production of multiple shoots and inflorescences from shoot apices

Abstract This report presents a procedure for high-frequency multiple shoot production from cultured shoot apical meristems of pearl millet [Pennisetum glaucum (L.) R. Br.]. Shoot apices from 1-week-old aseptically germinated seedlings were cultured in vitro on MS medium containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) with biweekly subculture. A low concentration of 2,4-D coupled with four different concentrations of BA induced the production of adventitious shoots from the enlarged shoot apical meristems. Somatic embryogenesis was also observed at higher concentrations of BA. The use of higher levels of 2,4-D resulted in callusing of shoot apical meristems, while the shoot tips produced many leaves and in vitro flowering in 2,4-D-free media containing BA. All four pearl millet genotypes produced similar results. Fertile pearl millet plants were produced from in vitro-produced multiple shoots.