Transgenic sorghum plants via microprojectile bombardment.

Abstract

Transgenic sorghum plants have been obtained after microprojectile bombardment of immature zygotic embryos of a drought-resistant sorghum cultivar, P898012. DNA delivery parameters were optimized based on transient expression of R and Cl maize anthocyanin regulatory elements in scuteflar cells. The protocol for obtaining transgenic plants consists of the delivery of the bar gene to immature zygotic embryos and the imposition of bialaphos selection pressure at various stages during culture, from induction of somatic embryogenesis to rooting of regenerated plantlets. One in about every 350 embryos produced embryogenic tissues that survived bialaphos treatment; six transformed callus lines were obtained from three of the eight sorghum cultivars used in this research.Transgenic (To) plants were obtained from cultivar P898012(two independent transformation events). (1). It is a primary staple in the semiarid tropics of Africa and Asia for over 300 million people. These are evidentiary statistics of the significance of this crop in these regions, which are predominated by subsistence agriculture. In the western hemisphere sorghum is used primarily as livestock feed (2). The development of hybrid varieties of sorghum in the 1950s contributed substantially to the increase in production in the United States. Presently, sorghum is third amongst cereals in U.S. production and is the preferred crop in areas oflow water availability because of its yield stability under drought conditions. Advances in biotechnology are now beginning to be used to augment traditional approaches for crop improvement. Restriction fragment length polymorphism (RFLP) linkage maps are being constructed that should greatly facilitate plant breeding efforts for marker-assisted backcross programs (3,4) and, in the near future, may be used to clone agriculturally important genes through the use of map-based cloning strategies (5). To this date, however, there are no programs in sorghum to access the pool of genes that are available as the result of genetic engineering research, because of the lack of a transformation system. Transformation of protoplasts byThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. electroporation (6) or cell suspensions by microprojectile bombardment (7) has resulted in stable expression of transferred genes; however, transgenic plants were not obtained.Microprojectile bombardment as a method to introduce DNA into cells circumvents two major constraints of cereal transformation. These are the lack of an available natural vector such as Agrobacterium tumefaciens and the difficulty to regenerate plants when protoplasts are used for transformation. Particle bombardment can target cells within tissues or organs that have high morphogenic potential. Immature or mature zygotic embryos (8-10), immature inflorescences (11,12), and shoot tips (13) of sorghum exhibit emb...

Genetic transformation of Sorghum bicolor

“…During the first decade of sorghum transformation studies, there are only a few reports of successful recovery and analysis of transgenic sorghum plants (Casas et al, 1993;Casas et al, 1997;Zhu et al, 1998). So far, PDS-1000/He gene delivery device of Biorad laboratories, Richmond, California has been the most common and successful device used for direct gene transfer.…”

“…The disadvantage of the protoplast or cell suspensions is that the method is laborious, needs special skills, tends to be cultivar specific and have very low regeneration frequency. Casas et al (1993) were first to report successful transformation of sorghum immature embryos via biolistic method of gene transfer. They reported that among the surviving calli, only a few exhibited shoot development indicating regeneration of plants at low frequency.…”

“…However, use of R and C1 maize anthocyanin regulatory elements (Casas et al, 1993) and luc, a fire fly luciferase (Kononowicz et al, 1995) as reporter genes in sorghum transformation is also reported. Apart from these individual reports, these genes were not used in sorghum transformation studies.…”

“…The key to establish a successful transformation strategy lies in the adoption of an effective and foolproof selection strategy. The usefulness of a selectable marker gene to optimize sorghum transformation system that will eventually allow the introduction of agronomically important traits to sorghum by genetic transformation was demonstrated (Casas et al, 1993;Harshavardhan et al, 2003).…”

“…The herbicide phosphinothricin is used as selection agent at a concentration of 5.0 mg L -1 (Zhao et al, 2000) while others used low concentrations of the same i.e. 2-3 mg L -1 (Rathus et al, 1996;Casas et al, 1993). Bialophos (a tripeptide) and Basta (shows nonsystemic and localized effect) are the derivatives of PPT.…”