Motoyasu Otani

Physicochemical properties of amylose-free starch from transgenic sweet potato

A transgenic amylose-free sweet potato has been obtained by introduction of granule-bound starch synthase I (GBSSI) cDNA of sweet potato in sense orientation (Plant Cell Reports (2001)). In this study, starches from 6 transgenic sweet potatoes produced by introduction of GBSSI cDNA, including the amylose-free transformant, were analyzed for their physicochemical properties, granule-size distribution, enzymatic digestibility, amylopectin structure, gelatinization properties and pasting properties. We observed little difference in granule size distribution between amylose-free starch and control starch. Amylose-free starch was more susceptible to glucoamylase digestion than control starch. The amylopectin of the amylose-free transformant was found to have a slightly lower content of short chains. Amylose-free starch showed higher gelatinization temperature and gelatinization enthalpy and lower setback in comparison with control starch. Thus, it was found that the starch from the amylose-free transgenic sweet potato possessed unique physicochemical properties.

Transformation of sweet potato (Ipomoea batatas (L.) Lam.) plants by Agrobacterium rhizogenes

Abstract Transgenic sweet potato plants were obtained after Agrobacterium rhizogenes -mediated transformation. Leaf disks of in vitro plants were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on leaf disks by both agropine-type and mikimopine-type strains. Whole plants transformed with Ri-T-DNA were regenerated from the hairy roots in five cultivars. These plants had wrinkled leaves, altered shape of flowers, reduced apical dominance, shortened internodes, small storage roots and abundant, frequently branching roots that showed reduced geotropism. Transgenic sweet potato plants possessing both NPT II gene and GUS gene were also obtained from the hairy roots by infection with Agrobacterium rhizogenes containing the binary vector pBI121 in addition to the wild-type Ri-plasmid.

Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants

Granule-bound starch synthase I (GBSSI) is one of the key enzymes catalyzing the formation of amylose, a linear α(1,4)D-glucan polymer, from ADP-glucose. Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato GBSSI gene expression through RNA interference. The gene construct consisting of an inverted repeat of the first exon separated by intron 1 of GBSSI driven by the CaMV 35S promoter was integrated into the sweet potato genome by Agrobacterium tumefaciens-mediated transformation. In over 70% of the regenerated transgenic plants, the expression of GBSSI was inactivated giving rise to storage roots containing amylopectin but not amylose. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene had occurred. These results clearly demonstrate that amylose synthesis is completely inhibited in storage roots of sweet potato plants by the constitutive production of the double-stranded RNA of GBSSI fragments. We conclude that RNA interference is an effective method for inhibiting gene expression in the starch metabolic pathway.

Effect of six promoter-intron combinations on transient reporter gene expression in einkorn, emmer and common wheat cells by particle bombardment

Abstract The effect of six promoter-intron combinations on transient expression of β-glucuronidase (GUS) as the reporter gene was estimated in cultured einkorn ( Triticum monococcum ), emmer ( Triticum durum ) and common ( Triticum aestivum ) wheat cell lines. Four promoters; cauliflower mosaic virus (CaMV) 35S, tandem CaMV35S, maize alcohol dehydrogenase gene ( Adh1 ) and rice actin gene ( Act1 ) promoters, and two introns; Adh1 intron1 and castor bean catalase intron, were tested. Six vectors having different promoter-intron combinations were introduced into wheat cells by using particle bombardment. Different levels of GUS gene activity in three wheat calli were detected by an in situ enzyme assay. The CaMV35S promoter gave the lowest level of transient expression in wheat cells. On the other hand, the rice Act1 promoter showed the highest level of transient expression in all three wheat cells and was also the most efficient promoter of all, suggesting that the rice Act1 promoter is efficient for use in wheat transformation.

Transgenic sweet potato expressing thionin from barley gives resistance to black rot disease caused by Ceratocystis fimbriata in leaves and storage roots

Black rot of sweet potato caused by pathogenic fungus Ceratocystis fimbriata severely deteriorates both growth of plants and post-harvest storage. Antimicrobial peptides from various organisms have broad range activities of killing bacteria, mycobacteria, and fungi. Plant thionin peptide exhibited anti-fungal activity against C. fimbriata. A gene for barley α-hordothionin (αHT) was placed downstream of a strong constitutive promoter of E12Ω or the promoter of a sweet potato gene for β-amylase of storage roots, and introduced into sweet potato commercial cultivar Kokei No. 14. Transgenic E12Ω:αHT plants showed high-level expression of αHT mRNA in both leaves and storage roots. Transgenic β-Amy:αHT plants showed sucrose-inducible expression of αHT mRNA in leaves, in addition to expression in storage roots. Leaves of E12Ω:αHT plants exhibited reduced yellowing upon infection by C. fimbriata compared to leaves of non-transgenic Kokei No. 14, although the level of resistance was weaker than resistance cultivar Tamayutaka. Storage roots of both E12Ω:αHT and β-Amy:αHT plants exhibited reduced lesion areas around the site inoculated with C. fimbriata spores compared to Kokei No. 14, and some of the transgenic lines showed resistance level similar to Tamayutaka. Growth of plants and production of storage roots of these transgenic plants were not significantly different from non-transgenic plants. These results highlight the usefulness of transgenic sweet potato expressing antimicrobial peptide to reduce damages of sweet potato from the black rot disease and to reduce the use of agricultural chemicals.

Inhibition of the expression of the starch synthase II gene leads to lower pasting temperature in sweetpotato starch

The sweetpotato cultivar Quick Sweet (QS) with a lower pasting temperature of starch is a unique breeding material, but the biochemical background of this property has been unknown. To assess the physiological impact of the reduced isoform II activity of starch synthase (SSII) on the starch properties in sweetpotato storage root, transgenic sweetpotato plants with reduced expressions of the SSII gene were generated and evaluated. All of the starches from transgenic plants showed lower pasting temperatures and breakdown measured by a Rapid Visco Analyzer. The pasting temperatures in transgenic plants were approximately 10–15°C lower than in wild-type plants. Distribution of the amylopectin chain length of the transgenic lines showed marked differences compared to that in wild-type plants: more chains with degree of polymerization (DP) 6–11 and fewer chains with DP 13–25. The starch granules from the storage root of transgenic plants showed cracking on the hilum, while those from wild-type plants appeared to be typical sweetpotato starch. In accordance with these observations, the expression of SSII in the storage roots of the sweetpotato cultivar with low pasting temperature starch (QS) was notably lower than in cultivars with normal starch. Moreover, nucleotide sequence analysis suggested that most of the SSII transcripts in the cultivar with low pasting temperature starch were inactive alleles. These results clearly indicate that the activity of SSII in sweetpotato storage roots, like those in other plants, affects the pasting properties of starch through alteration of the amylopectin structure.

Fertile transgenic plants of Ipomoea trichocarpa Ell. induced by different strains of Agrobacterium rhizogenes

Cotyledon explants of Ipomoea trichocarpa Ell. were inoculated with ten strains of Agrobacterium rhizogenes. Hairy roots were produced from the cut surface of explants by inoculation with all bacterial strains. No clear differences in rhizogenicity were observed among the bacterial strains of A. rhizogenes tested. Whole plants were regenerated from the hairy roots transformed by all of the bacterial strains. These hairy root-derived plants exhibited the expected transformed phenotype, which was sexually transmitted to the progenies in Mendelian fashion as a single dominant locus. Transgenic I. trichocarpa plants possessing both the npt II and gus genes were also obtained from the hairy roots by infection with Agrobacterium rhizogenes containing the binary vector pBI121 in addition to the wild-type Ri-plasmid.

Chromosomal regions controlling anther culturability in rice (Oryza sativa L.)

Diallel analysis has revealed that anther culturability in rice (Oryza sativa L.) is a quantitative trait controlled by the nuclear genome. Mapping of anther culturability is important to increase the efficiency for green plant regeneration from microspores. In the previous study, we detected distorted segregation of RFLP markers in rice populations derived from the anther culture of an F1 hybrid between a japonica cultivar ‘Nipponbare’ and an indica cultivar ‘Milyang 23’. To clarify the association between chromosomal regions showing distorted segregation and anther culturability, the anther culturability of doubled haploid lines derived from the same cross combination was examined, and the association between alleles of the RFLP markers, which exhibiting the most distorted segregation on chromosomes 1, 3, 7, 10 and 11, and the anther culturability was evaluated. One region on chromosome 1 was found to control callus formation from microspores, and one region on chromosome 10 appeared to control the ratio of green to albino regenerated plants. In both regions, the Nipponbare allele had positive effects. Three regions on chromosomes 3, 7 and 11, however, showed no significant effect on anther culturability.

Mesophyll protoplast culture of sweet potato (Ipomoea batatas L.)

Abstract Mesophyll protoplasts of sweet potato ( Ipomoea batatas L.) were readilyisolated by soaking chopped leaf tissue in distilled water for 16 h prior to enzymatic digestion. Isolated mesophyll protoplasts began to divide three days after start of culture in liquid modified N 6 medium and and formed colonies after 30 days of culture. The colonies transferred to solid medium grew rapidly and differentiated into calli. Some of the calli transplanted onto regeneration medium produced roots.

Improved Method for Anther Culture of an Indica Rice Cultivar of Thailand

At present, anther culture is a powerful tool for plant breeding. Anther culture offers the possibility to rapidly obtain the genetically diverse haploids or homozygous doubled haploids from Fl hybrids (Gosal et al., 1997). However, the use of anther culture as a routine technique for breeding of indica rice is extremely limited by the poor induction of androgenic calli and subsequent plant regeneration. The average frequency of green plant regeneration from cultured anthers of indica rice was only 1% in comparison with the 10% of japonica rice (Hu, 1985). Extensive works have been made on various factors influencing the anther culture efficiency of indica rice (Reddy et al., 1985) . Chu et al. ( 1997) developed the medium for the anther culture of javanica and tropical japonica rice varieties. In this report, we describe that the Chu medium is effective for the green plant regeneration from microspore-derived calli of an indica rice cultivar, Chinat 1, in Thailand.