Heng Zhong

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.

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.

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.

Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.)

We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4-dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and embryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 μM dicamba, addition of 2.25 to 9 μM BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 μM dicamba and 2.25 μM BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse.

In-vitro morphogenesis of corn (Zea mays L.) : II. Differentiation of ear and tassel clusters from cultured shoot apices and immature inflorescences.

The objective of this research was to study the in-vitro morphogenetic pattern of corn (Zea mays L.) shoot tips excised from aseptically-grown seedlings, and of expiants of axillary shoot buds, immature tassels and ears (staminate and pistillate inflorescences) obtained from greenhouse-grown corn plants. The seedling shoot tips and immature ears first regenerated clumps of multiple shoots within four weeks of culture on Murashige and Skoog (MS) basal medium supplemented with 500 mg/L casein hydrolysate (CH) and 9.0 μM N6-benzyladenine (BA). Multiple shoot clumps were also differentiated from spikelets of immature tassels cultured on MS medium containing 500 mg/L CH, 4.5 μM BA and 0.45 μM 2,4-dichlorophenoxy acetic acid (2,4-D). All these multiple shoot clumps in turn differentiated clusters of ears after further four subcultures at four-week intervals under light on MS medium supplemented with 500 mg/L CH and 2.25, 4.5, 9.0 or 18 μM BA. Axillary shoot buds readily differentiated clusters of ears within four weeks of the initial culture on these media. Secondary and tertiary ear clusters were initiated following subculture of primary ears on MS medium containing 500 mg/L CH and 4.5 or 9.0 μM BA. Most of the ear primordia developed into ears with well-developed ovaries and styles on subculture on MS medium containing 500 mg/L CH and 1.0 μM BA. Corn kernels were obtained after pollination of in-vitro-formed ears with pollens collected from greenhouse-grown corn. These kernels germinated in vitro and developed into mature corn plants in the greenhouse. Clusters of tassels were also differentiated in darkness from the multiple shoot clumps after six months successive subcultures but the spikelet primordia of tassels failed to develop fully under the in-vitro conditions tested. Somatic embryos arose directly from spikelet primordia of young tassels or ears on MS medium containing 500 mg/L CH and 4.5 μM 2,4-D, or indirectly from calli derived from spikelets of young tassels and ears on MS medium containing 500 mg/L CH and 9.0 μM 2.4-D.

Analysis of the functional activity of the 1.4-kb 5′-region of the rice actin 1 gene in stable transgenic plants of maize (Zea mays L.)

Abstract The activity of the 5′-region of the rice actin 1 gene (Act1), covering a region 1.4 kb upstream of the Act1 translation initiation codon, was extensively analyzed in transgenic maize plants. The 5′-region of Act1 fused to the β-glucuronidase (GUS) gene (gus) coding region was co-transformed to maize with the phosphinothricin acetyltransferase gene (bar) and the potato proteinase inhibitor II gene (pin2). One and 29 independent transformation events with expression of both bar and gus were recovered from bombardment of immature embryo-derived embryogenic callus of Hi-II derivative and bombardment of shoot tips of Honey N Pearl and Illinois Golden Extra Sweet, respectively. Expression of gus in tissues of transgenic plants was examined by histochemical assay, immunoblot analysis, and fluorometric GUS specific activity assay. A constitutive expression of the introduced gus was observed throughout the evelopmental stages of the vegetative and reproductive organs in transgenic maize plants. Quantitative analysis of GUS in transgenic plants showed that GUS, as percent of total soluble protein, was as much as 3.1% in leaves and 2.8% in roots. The functional activity of the 5′-region of Act1 was inherited to transgenic progeny. The results indicate that the 1.4-kb 5′-region of Act1 is an efficient and strong promoter for gene expression in stable transgenic maize plants.

Transformation of Oats and Its Application to Improving Osmotic Stress Tolerance

Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic stress due to drought and/or salinity. To genetically transform the available commercial oat cultivars, a genotype-independent and efficient regeneration system from shoot apical meristems was developed using four oat cultivars: Prairie, Porter, Ogle, and Pacer. All these oat cultivars generated a genotype-independent in vitro differentiated multiple shoots from shoot apical meristems at a high frequency. Using this system, three oat cultivars were genetically co-transformed with pBY520 (containing hva1 and bar) and pAct1-D (containing gus) using biolistic trade mark bombardment. Transgenic plants were selected and regenerated using herbicide resistance and GUS as a marker. Molecular and biochemical analyses of putative transgenic plants confirmed the co-integration of hva1 and bar genes with a frequency of 100%, and 61.6% of the transgenic plants carried all three genes (hva1, bar and gus). Further analyses of R0, R1, and R2 progenies confirmed stable integration, expression, and Mendalian inheritance for all transgenes. Histochemical analysis of GUS protein in transgenic plants showed a high level of GUS expression in vascular tissues and in the pollen grains of mature flowers. Immunochemical analysis of transgenic plants indicated a constitutive expression of hva1 at all developmental stages. However, the level of HVA1 was higher during the early seedling stages. The characteristic of HVA1 expression for osmotic tolerance in transgenic oat progeny was analyzed in vitro as well as in vivo. Transgenic plants exhibited significantly (P<0.05) increased tolerance to stress conditions than non-transgenic control plants. The symptoms of wilting or death of leaves as observed in 80% of non-transgenic plants due to osmotic stress was delayed and detected only in less than 10% of trans-genic plants. These observations confirmed the characteristic of HVA1 protein as providing or enhancing the osmotic tolerance in transgenic plants against salinity and possible water-deficiency stress conditions.