Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants

Wu, Emily; Lenderts, Brian; Glassman, Kimberly; Berezowska-Kaniewska, Maya; Christensen, Heather M.; Asmus, Tracy; Zhen, Shifu; Chu, Uyen Cao; Cho, Myeong‐Je; Zhao, Zuo‐Yu

doi:10.1007/s11627-013-9583-z

Cited by 114 publications

(146 citation statements)

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“…One reason was the recalcitrance of sorghum to genetic modification via transformation. Recent improvements in sorghum transformation have largely overcome this barrier and offer an alternative approach to genetic improvements in sorghum (25).…”

mentioning

confidence: 99%

Elevated vitamin E content improves all- trans β-carotene accumulation and stability in biofortified sorghum

Che

Zhao

Glassman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Micronutrient deficiencies are common in locales where people must rely upon sorghum as their staple diet. Sorghum grain is seriously deficient in provitamin A (β-carotene) and in the bioavailability of iron and zinc. Biofortification is a process to improve crops for one or more micronutrient deficiencies. We have developed sorghum with increased β-carotene accumulation that will alleviate vitamin A deficiency among people who rely on sorghum as their dietary staple. However, subsequent β-carotene instability during storage negatively affects the full utilization of this essential micronutrient. We determined that oxidation is the main factor causing β-carotene degradation under ambient conditions. We further demonstrated that coexpression of homogentisate geranylgeranyl transferase (HGGT), stacked with carotenoid biosynthesis genes, can mitigate β-carotene oxidative degradation, resulting in increased β-carotene accumulation and stability. A kinetic study of β-carotene degradation showed that the half-life of β-carotene is extended from less than 4 wk to 10 wk on average with HGGT coexpression.β-carotene accumulation | β-carotene stability | vitamin E | HGGT | biofortified sorghum T he importance of vitamin A for human health has been widely addressed (1-6). A 2009 Global Report (7) summarized vitamin A as being "vital for survival and sight; to boost the immune system, vitamin A is a critical micronutrient for survival and physical health of children exposed to disease." In Africa, malnutrition is a serious challenge, but micronutrient deficiency also plays a dominant role in the overall food security of that continent. Based on this global report, the five countries having the highest proportions of preschool age children with vitamin A deficiency were all located in Africa: 95.6% in Sao Tome and Principe, 84.4% in Kenya, 75.8% in Ghana, 74.8% in Sierra Leone, and 68.8% in Mozambique. Sorghum (Sorghum bicolor L.) is one of the most important staple foods for an estimated 500 million people, primarily those living in arid and semiarid areas. In Africa, it is the second most important cereal; about 300 million people rely on it as their daily staple food. Although sorghum is gluten-free and could be an attractive replacement for wheat-allergy sufferers, it is considered a nutrient-poor crop (8, 9) with very low amounts of β-carotene (10). The improvement of micronutrients in food crops has attracted considerable attention, and significant advances have been made in a range of major crops (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Nutritional improvement in sorghum was undertaken a decade ago (23, 24); however, progress has lagged behind the progress in other crops. One reason was the recalcitrance of sorghum to genetic modification via transformation. Recent improvements in sorghum transformation have largely overcome this barrier and offer an alternative approach to genetic improvements in sorghum (25).One of our objectives is to develop sorghum lines with enhanced and stabilized provitamin A (β-car...

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mentioning

confidence: 99%

Elevated vitamin E content improves all- trans β-carotene accumulation and stability in biofortified sorghum

Che

Zhao

Glassman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Using immature embryos harvested from the same plant and transformed on the same day, the transformation frequency in sorghum cultivar Tx430 was increased from a control level of 1.9 to 18.3% when the morphogenic genes were present in the T-DNA (Table 1). The sorghum transformation frequencies reported here are low compared with those published by Wu et al (2014), as this work was performed much earlier than that of Wu et al (2014). These transgenic sorghum lines (produced using our desiccationinduced maize rab17 pro :CRE excision method as described earlier) were characterized by PCR and verified to contain the marker genes and no longer to contain Bbm, Wus2, and the CRE recombinase gene.…”

Section: Transformation Of Other Monocot Cropsmentioning

confidence: 85%

“…Transformation of sorghum immature embryos was performed in a similar fashion to maize immature embryos, following the detailed methods and medium formulations described by Wu et al (2014).…”

Section: Particle Gun Transient Phenotype Observationsmentioning

confidence: 99%

“…qPCR (Wu et al, 2014) was used to estimate the copy number of the transgenes to determine if T-DNA integrations were intact or truncated, if recombinase-mediated excision had occurred, and to screen for the presence of Agrobacterium binary vector backbone integration. Genomic DNA was extracted from a single piece (200 ng) of fresh leaf tissue from each plant (Truett et al, 2000).…”

Section: Molecular Analysismentioning

confidence: 99%

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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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“…The R19 genotype was kindly donated by Dr. Leopoldo Mendoza Onofre from Montecillo campus of Colegio de Posgraduados, who developed this genotype from selective crosses between different sorghum lines with cold tolerance (Cisneros López et al, 2007;J Osuna-Ortega, 2003;Mendoza-Onofre et al, 2017). On the other hand, the variety TX430 is susceptible to cold and is also a genotype that has been successfully transformed (Wu et al, 2014;Wu & Zhao, 2017). The culture of both genotypes was done during the early spring of 20016 in a substrate composed of peat moss, perlite, vermiculite (3,1,1), the development of the plants was monitored until they reached the phenological phases V1, V3, V5 and HB (Vanderlip and Reeves, 1972) under semi-controlled conditions in greenhouse.…”

Section: Organisms and Exposure To Stressmentioning

confidence: 99%

Proline as a probable biomarker of cold stress tolerance in Sorghum (Sorghum bicolor)

et al. 2018

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Plants have developed physiological and molecular mechanisms to support and adapt to adverse environments. One response to abiotic stress is the accumulation of free proline (PRO). PRO can induce the expression of many genes, which have the proline-responsive element (PRE) in their promoters, nevertheless due to the complexity of interactions between stress factors and various molecular, biochemical and physiological phenomena it is still unclear whether a more efficient PRO accumulation can be considered a biomarker of tolerance in plants. In the present work, we evaluated the accumulation of PRO in two genotypes of sorghum with contrasting tolerance to cold stress. To explore the cause behind the accumulation of proline under cold stress conditions, we identified the Transcription Factors Binding Sites (TFBS) present in the promoter regions in the genes involved in the biosynthesis and degradation of proline in sorghum and other important crops, finding that the untranslated 3 'region P5CS gene contains different TFBS. We found TFBS that could allow the activation of genes involved in proline biosynthesis through the ornithine pathway under cold stress conditions, suggesting that ornithine route can be activated under cold stress conditions. Keywords: Biomarker, Cold, Proline, Sorghum, TFBS. Mexican Journal of Biotechnology 2018, 3(3):77-86 RESUMENLas plantas han desarrollado mecanismos fisiológicos y moleculares para soportar y adaptarse a entornos adversos. Una de las respuestas a condiciones de estrés abiótico es la acumulación de prolina (PRO). PRO puede inducir la expresión de distintos genes, que tienen el elemento de respuesta a prolina (PRE) en sus promotores, sin embargo, debido a la complejidad de las interacciones entre los factores de estrés y varios fenómenos moleculares, bioquímicos y fisiológicos aún no está claro si la acumulación de PRO puede considerarse un biomarcador de tolerancia en las plantas. En el presente trabajo evaluamos la acumulación de PRO en dos genotipos de sorgo con tolerancia contrastante a estrés por frío. Para explorar la causa detrás de la acumulación de prolina ante condiciones de estrés por frío, identificamos los TFBS presentes en las regiones promotoras en los genes involucrados en la biosíntesis y degradación de prolina en sorgo y otros cultivos importantes encontrando que la región 3' no transcrita del gen P5CS contiene diferentes TFBS. Encontramos TFBS que pudieran permitir la activación de los genes involucrados en la biosíntesis de prolina a través de la vía de ornitina ante condiciones de estrés por frío lo que sugiere que dicha vía puede activarse ante condiciones de estrés por frío.

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Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants

Cited by 114 publications

References 37 publications

Elevated vitamin E content improves all- trans β-carotene accumulation and stability in biofortified sorghum

Elevated vitamin E content improves all- trans β-carotene accumulation and stability in biofortified sorghum

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

Proline as a probable biomarker of cold stress tolerance in Sorghum (Sorghum bicolor)

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