Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature.

Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.

Mature embryo axis-based high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (Hordeum vulgare L.).

A highly reproducible regeneration system through somatic embryogenesis from the excised mature embryos (MEs) of dry seeds of a range of European barley cultivars was developed. By minimizing the germination of plated MEs, primary callus could be obtained with high frequency which permitted efficient embryogenesis and regeneration of a large number of green plants. Different approaches were tested to reduce or prevent normal germination: (i) the use of a well defined balance of maltose and 2,4-D in the induction medium, (ii) soaking of seeds in water containing 2,4-D solution, (iii) direct culture of excised embryonic axes, (iv) longitudinally bisected MEs giving two halves, and (v) complete removal of the elongated main shoot including any roots within a week of culture initiation. Culturing of bisected MEs and whole embryonic axes gave the best responses with respect to large amounts of callus combined with minimal germination. The incorporation of BAP at low levels in the medium was found to be most effective for embryogenesis and the maintenance of long-term morphogenic capacity (more than 11 months up to now). This procedure allows the complete regeneration of plants in 16-20 weeks, from the initial isolation of MEs through all the steps to the development of plants ready to be transferred to the soil. The protocol was first developed for cv. Golden Promise and successfully applied to commercial cultivars. All cultivars tested formed embryogenic callus, with overall rates ranging from 22-55% and an average number of green plants per embryogenic callus from 1.5 to 7.5 across the genotypes.

A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos.

A fast and highly efficient short-term in vitro regeneration system was developed for barley (Hordeum vulgare L.) based on readily available explants. Clumps of multiple shoots and buds suitable for transformation were obtained 9-10 weeks after culture initiation from model and current commercial cultivars. Meristematic shoot segments (MSSs) excised from mature embryo-derived seedlings and subsequently cultured on MS-based medium containing 2 mg/l Picloram and 3 mg/l thidiazuron (TDZ) differentiated up to ten multiple shoots after 3-4 weeks with no or very little callus formation. Sectors of the already multiplied shoot clumps were further multiplied on proliferation-maintenance medium containing 2 mg/l Picloram and 2.5 mg/l TDZ. Biweekly subcultures resulted in a continuous process of multiplication of these highly differentiating green sectors without any loss of morphogenic potential. The differentiated small shoots and shoot buds gave rise to normal shoots on medium with 0.1 mg/l Picloram and 1 mg/l TDZ. After rooting on basal medium with 0.5 mg/l or 1 mg/l IBA the plants were transferred to soil and showed normal growth and fertility compared to the seed-grown plants. All of the genotypes tested formed multiple shoots. The percentage of relative MSS multiplication was 63-83%, and the average number of multiplied shoots per MSS ranged from 16 to 34 among the genotypes after 9-11 weeks.