Agrobacterium-mediated transformation and regeneration of kiwi fruit

Abstract

Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the β-glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25μg/ml kanamycin and 500μg/ml Claforan. After one month in culture, shoots had regenerated from the cuttings. Green shoots were analyzed for NPTII activity and GUS activity. Eighty-five percent of the green shoots examined expressed the nptII and GUS genes. GUS histochemical assays revealed strong GUS expression in guard cells, mesophyll cells, and trichomes.

Δ12-Oleate Desaturase-related Enzymes Associated with Formation of Conjugated trans-Δ11, cis-Δ13 Double Bonds

“…pGEM-T Easy/TkFac was digested with BamHI and SacI and ligated at the BamHI/SacI site of the pGEM-3Z plasmid vector (Promega). To generate plant expression vectors with the CaMV 35S promoter and nos terminator (pKS-TkFac and pKS-PgFac), the cDNA sequences were released from pGEM-3Z/TkFac and from pGEM-T Easy/PgFac with XbaHI/SacI digestion and then cloned into the corresponding sites of a binary vector pLAN421 (14) in which the GUS gene has been eliminated with XbaI/SacI digestion. To generate plant expression vectors with a strong seed-specific promoter of the Brassica napus napin gene (15) (pKN-TkFac and pKN-PgFac), the XbaI-SacI fragments were cloned into binary vector pNGKM (16) in which the GUS gene had been eliminated with XbaI/SacI digestion.…”

Expression of a rice homeobox gene causes altered morphology of transgenic plants.

“…The promoter-GUS cassette was cut out with Hindlll and EcoRl and ligated into the same site of the binary vector, pLAN4Z1, which mntains the right border sequence of the T-DNA and the gene for the selectable marker neomycin phosphotransferase II (Uematsu et al, 1991). Histochemical assays for GUS activity were performed by a previously reported method .…”

Applications of Biotechnology in Kiwifruit (Actinidia)

“…Initially, the development of Actinidia transformation focused on the integration into the plant genome of reporter and selectable marker genes Janssen & Gardner, 1993;Uematsu et al, 1991), but transformation of various heterologous genes has followed. These include: A. rhizogenes rol genes (Rugini et al, 1991); a soybean -1,3 endoglucanase cDNA (Nakamura et al, 1999); a rice OSH1 homeobox gene (Kusaba et al, 1999), and an Arabidopsis Na + /H + antiporter gene (Tian et al, 2011), in attempts to improve kiwifruit disease resistance or drought tolerance; a synthetic gene encoding human epidermal growth factor (Kobayashi et al, 1996); and a grape stilbene synthase (Kobayashi et al, 2000), in attempts to accumulate bioactive compounds; citrus geranylgeranyl diphosphate synthase, phytoene desaturase, -carotene desaturase, -carotene hydroxylase and phytoene synthase, to modify the lutein or -carotene content of kiwifruit (MiSun Kim et al, 2010) and the A. tumefaciens isopentyl transferase (ipt) gene, to alter vine architecture (Honda et al, 2011).…”

“…Compared with other woody species, e.g. apple (James et al, 1989), relatively high A. deliciosa transformation and regeneration rates have been achieved (Uematsu et al, 1991), and A. chinensis transformation efficiencies of up to 27.8% have been reported (T. Wang et al, 2007). However, A. arguta transformation was less successful when applying the transformation protocols developed for A. chinensis or A. eriantha, with co-cultivated explants suffering considerable browning and necrosis during callus induction and shoot regeneration stages.…”

“…Wang et al (2006) made similar observations with A. eriantha where the highest shoot regeneration rates were obtained using medium containing a combination of 2 mg/l zeatin and 3 mg/l BAP. Uematsu et al (1991) reported that the regeneration frequency varied with the basal medium used, and B5 basal medium containing zeatin was most suitable for obtaining transformed A. deliciosa shoots. Using A. deliciosa MCS explants for transformation, Kim et al (2010) used half-strength MS medium containing 0.001 mg/l 2,4-D and 0.1 gm/l zeatin, for callus induction and shoot regeneration.…”