Direct Embryogenesis from Mesophyll Cells of Orchardgrass

Conger, B. V.; Hanning, G. E.; Gray, D. J.; McDaniel, J. K.

doi:10.1126/science.221.4613.850

Cited by 150 publications

(54 citation statements)

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“…This is not the first report of direct leaf cell transformation in a monocot; transgenic events have been recovered after transformation of leaf cells in orchardgrass (Denchev et al, 1997). However, before performing these transformation experiments, these researchers selected accessions of orchardgrass that exhibited an atypically high level of somatic embryogenesis when leaf tissue was exposed to exogenous auxin (Conger et al, 1983). More recently, it has been reported that Agrobacterium-mediated transformation directly into young sugarcane leaf tissue has been accomplished (Eldessoky et al, 2011), but again in a species in which somatic embryogenesis from leaf cells in response to auxin has been well established (Lakshmanan et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

et al. 2016

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While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

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Section: Discussionmentioning

confidence: 99%

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

et al. 2016

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“…Genotypes ofDactylis glomerata L. used in this study included two clones (designated Ela and Elb) of one that exhibited a very high capacity for somatic embryogenesis (4,8) and two (designated NEi and NE2) which exhibited no capacity to produce somatic embryos. Tillers were collected from mature plants grown in an environmentally controlled chamber with a 12 h day/night cycle at 220/1 5C.…”

Section: Methodsmentioning

confidence: 99%

“…For the tissue culture experiments, basal sections of the two innermost leaves were split lengthwise along the midvein and surface sterilized as previously described (4 (14) and is expected since IAA is synthesized in highly meristematic regions, e.g., basal portions of grass leaves, and is transported basipetally. Although IAA concentration is positively correlated with the gradient response for somatic embryogenesis, it does not appear to be related to genotype differences in orchardgrass.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of Somatic Embryogenesis in Orchardgrass by Endogenous Cytokinins

Wenck¹,

Conger²,

Trigiano³

et al. 1988

Plant Physiol.

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ABSTRACIEndogenous indoleacetic acid (IAA) and cytokinin concentrations were measured by high performance liquid chromatography in leaf sections of an orchardgrass (Dactylis glomerata L.) genotype which exhibited a high capacity for somatic embryogenesis in vitro and in two genotypes that did not exhibit this capacity. The nonembryogenic genotypes contained 3-to 4-fold higher concentrations of zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, and total cytokinins than the embryogenic genotype. There were no significant differences in IAA concentrations between genotypes. Cytokinin concentrations between basal and distal sections of embryogenic genotype were not different, but the IAA concentration was significantly greater in basal sections. Somatic embryogenesis was inhibited in the embryogenic genotype by 0.001 micromolar exogenously added zeatin.Despite recent advances in regeneration of cereals and grasses from protoplasts (1,5,15,22), the development ofhighly efficient and repeatable regeneration systems remains a major hurdle in the advancement of biotechnological applications, including gene transfer, in these species. Somatic embryogenesis systems developed for Dactylis galomerata L. (orchardgrass or cocksfoot) are among the most advanced currently existing for the Poaceae. These include the formation of embryos directly from mesophyll cells in cultured leaf segments (4, 8) and the full development of somatic embryos in a single liquid medium (6,7,19).Somatic embryogenic capacity in orchardgrass is highly dependent on genotype (9, 13). Genotypic differences for in vitro response, including somatic embryogenesis, have also been reported for other cereal and grass species (2,11,18), but these differences have not been related to either endogenous or exogenous PGRs.2 The requirement for an exogenously added auxin to gramineous cell and tissue cultures has been long established (21); however, the influence of cytokinins is less clear. The objectives of this study were to measure and relate endogenous levels of IAA and cytokinins (Z, DHZ, ZR, DHZR) to the genotypic response for somatic embryogenesis in orchardgrass and to investigate the effect of exogenously added Z to cultured leaf segments of the embyogenic genotype. MATERIALS AND METHODSGenotypes ofDactylis glomerata L. used in this study included two clones (designated Ela and Elb) of one that exhibited a very high capacity for somatic embryogenesis (4, 8) and two (designated NEi and NE2) which exhibited no capacity to produce somatic embryos. Tillers were collected from mature plants grown in an environmentally controlled chamber with a 12 h day/night cycle at 220/1 5C. Individual leaves were separated and basal sections (0-3 cm) of the two innermost (youngest) were lyophilized. The next distal (3-6 cm) sections (nonembryogenic portion) of Ela and Elb were also collected. Plant growth regulators were extracted and purified according to a modification of existing procedures (10, 12) that is outlined in Figure 1.Cytokinins were analyzed ...

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“…Other strong auxins such as dicamba, picloram and or combination of these auxins have been used as alternatives (Conger et al 1983, Lührs and Lörz 1987, Przetakiewicz et al 2003). Most studies develop induction and regeneration systems for the model genotype Golden Promise which has high regeneration efficiency, but is agronomically obsolete (Wan andLemaux 1994, Tingay et al 1997) or for genotypes grown in Canada, U.S., Spain (Baillie et al 1993, Castillo et al 1998, Chant et al 2003.…”

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confidence: 99%

Callus induction and regeneration efficiency of spring barley cultivars registered in the Czech Republic

Šerhantová¹,

Ehrenbergerová²,

Ohnoutková³

2004

PLANT SOIL ENVIRON

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Immature zygotic embryos are frequently used for induction of cell cultures in vitro and for genetic transformation. The effect of three synthetic auxins: 2,4-dichlorophenoxyacetic acid (2,4-D), 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 4-amino-3,5,6-trichloropicolinic acid (picloram) on induction and subsequent regeneration capacity of barley. In vitro culture was studied in 12 spring barley cultivars registered in the Czech Republic and a variety of Golden Promise, with known high regeneration efficiency. The callus formation frequency and number of green regenerants were influenced significantly both by genotype and auxin. Most cultivars formed statistically significantly a higher mean number of regenerants (1.95) a�er the callus induction on the medium with 2.4-D as compared to the media with picloram and dicamba. Only two cultivars (Nordus and SG-S-261) did not respond to the used auxins differently. The highest average number of regenerants (from all three auxins) was obtained with Golden Promise (2.7 plants per one cultivated scutellum). From the set of genotypes used in our study, the cultivars Atribut, Forum, and Scarle� with the mean number of regenerants (2.09-1.57) would be the most suitable cultivars for further transformation use. They differ statistically significantly from the cultivars Nordus, Amulet, Akcent, SG-S-252, Orbit, and Granát (0.42-0.92) which had the lowest mean number of regenerants.

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Direct Embryogenesis from Mesophyll Cells of Orchardgrass

Cited by 150 publications

References 7 publications

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

Inhibition of Somatic Embryogenesis in Orchardgrass by Endogenous Cytokinins

Callus induction and regeneration efficiency of spring barley cultivars registered in the Czech Republic

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