Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic cry1Ab gene

Abstract

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8-12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F 1 plants obtained through crossing of T 0 plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.

Transgenic sugar beet tolerant to imidazolinone obtained by Agrobacterium-mediated transformation

“…Transgenic cells had not any unam biguous selective advantages over the non trans genic ones in direct regeneration from the meris tem zones of explants. Selection on kanamycin resulted in production of escapes amounted to 50-95 % among the regenerated kanamycin tol erant shoots [33,34,38,41,44,45]. Similar results were obtained when 15 mg/l phosphinothricin was used for selection after transformation of embryo genic callus of cotyledonary origin [44] or 10 mg/l hygromycin after transformation of bud clumps from immature inflorescence [43].…”

“…Whereas transformation frequen cy depends on efficient and reliable tissue culture regeneration system the early experiments on genetic transformation of sugar beet were mainly focused on the search for the genotypes with high regenerative potential and the explants whose cells were the most competent for regeneration. Such high efficiency regeneration systems for sugar beet are (1) direct regeneration from petioles [32][33][34], cotyledonary node explants [35,36], shoot bases [37,38], bud clumps from immature inflorescence and (2) indirect regeneration via callus from cotyledons and hypocotyls [39,40]. Sugar beet transformation frequency of different explants was reported mainly within 1-5 % [28,35,[39][40][41][42] and arrived at 19.2 % in case of shoot base transforma tion [37,38].…”

A protocol for sonication-assisted Agrobacterium rhizogenes-mediated transformation of haploid and diploid sugar beet (Beta vulgaris L.) explants.

“…So far, haploid explants have never been used in sugar beet genetic transformation in contrast to the use of diploid tissues (Kishchenko et al, 2005, Jafari et al, 2009). Haploids may be valuable material for genetic transformation as diploidization induced after a gene transfer would lead to the development of a homozygous tissue, including a transgene locus.…”

Optimization of agrobacterium‐mediated transformation of sugar beet: Glyphosate and insect pests resistance associated genes

“…Transformation of sugar beet with several genes collected in a single vector accelerates improvement of this species to cope with adverse effects of pests and weeds in the field. Most studies used a single transgene for transformation of sugar beet and little information is available on multiple-gene transformation events (Jafari et al, 2009;Kishchenko et al, 2005;Krens et al, 1988;Lytvyn et al, 2014;Mannerlöf, Tuvesson, Steen Tenning, 1997;Mohammadzadeh et al, 2015). In the protocol used for transformation of sugar beet, promoters used in the binary vector helped expression of the genes in target tissues.…”