Production of human lactoferrin in transgenic cell suspension cultures of sweet potato

Shoot apical meristem-derived calli were transformed with a hLF cDNA in an attempt to produce human lactoferrin (hLF) in transgenic cell suspension cultures of sweet potato [Ipomoea batatas (L.) Lam.]. Calli were bombarded with tungsten particles coated with the binary vector pLSM1 containing a hLF cDNA under the control of the 35S promoter and the neomycin phosphotransferase gene as a selection marker. Calli were then transferred to Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 100 mg dm⁻³ kanamycin. Kanamycin-resistant calli were selected at four-week intervals and subcultured. Cell suspension cultures were established in liquid MS medium with 4.52 μM 2,4-D. Southern and Northern blot analyses confirmed that hLF cDNA was incorporated into the plant genome and was properly expressed in the cells. ELISA analysis showed that transgenic cells produced hLF up to 3.2 μg mg⁻¹ (total protein).     

Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura

A sweet potato (Ipomoea batatas cv. Tainong 57) trypsin inhibitor gene was introduced into tobacco plants (Nicotiana tabaccum cv. W38) by Agrobacterium tumefaciens– mediated transformation. From 30 independent transformants, three lines with high level of expression were further analyzed. The trypsin inhibitor gene, under control of the 35S CaMV promoter, led to the production of the trypsin inhibitor proteins up to 0.2% of the total protein. In insecticidal bioassays of transgenic tobacco plants, larval, growth of Spodoptera litura (F.), the tobacco cutworm, was severely retarded as compared to their growth on control plants. This observation implied that expression of sweet potato trypsin inhibitor can provide an efficient method for crop protection. Received: 29 July 1996 / Revision received: 15 November 1996 / Accepted: 8 December 1996     

Establishment and characteristics of an anthocyanin-producing cell line from sweet potato storage root

 Anthocyanin pigments accumulated in a cell line derived from storage-root explants of sweet potato (Ipomoea batatas L.) cv 'Ayamurasaki'. Somatic pro-embryos were induced on the explants cultured on Murashige and Skoog medium supplemented with 1 mg/l 2,4-D. The pro-embryo structures produced callus when transferred to MS medium with 0.5 mg/l 2,4-D. A cell line was isolated from this callus which accumulated anthocyanin pigment. The color value of the pigment extracted after 27 days of culture in MS medium with 2 mg/l 2,4-D was 8.2, which was very close to that of a pigment extracted from roots, which was 8.9. Most of the pigments from the cell extract were hydrophilic and appeared on the ODS-column HPLC with a lower retention time than the main anthocyanins of the root tissues. The majority of the pigments were identical with the root anthocyanins. Cell line-specific anthocyanins were detected. Received: 8 January 1999 / Revision received: 2 March 1999 / Accepted: 30 June 1999     

Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts

A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0–5.0×10⁶ were obtained following 4–6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 10⁵ protoplasts/ml was optimal for subsequent division. An initial division frequency of 12–15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μm), and zeatin (2.3 μm). Colonies consisting of 100–200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1–2 mm in diameter) were formed after an additional 4–6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μm) and benzylaminopurine (2.2 μm). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μm gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation. Received: 10 October 1997 / Revision received: 10 February 1998 / Accepted: 2 March 1998     

Computer vision analysis of somatic embryos of sweet potato [Ipomoea batatas (L.) Lam.] for assessing their ability to convert to plants

Apical and axial shoot tips of sweet potato were cultured to produce somatic embryos that mature and develop into plants in basal nutrient medium. However, the lack of high regeneration efficiency is an impediment to the use of somatic embryos to produce synthetic seeds. Conversion experiments with mature embryos over a 20-day period revealed that 80–90% of the embryos formed roots but only 40–50% formed shoots. Using computer vision and canonical or Fisher discriminant function (CDA) analysis along with conversion results, it was possible to correctly classify competent embryos 40–50% of the time based on size features, 50–60% of the time based on shape features, and 55–60% of the time based on color features. Non-competent embryos were correctly classified 65–75%, 55–60%, and 70–75% of the time based on size, shape, and color, respectively. These results can be used effectively to identify and select competent embryos for improved regeneration efficiency. Received: 2 January 1997 / Revision received: 21 January 1998 / Accepted: 12 February 1998     

Starch granule size and the domestication of manioc (Manihot esculenta) and sweet potato (Ipomoea batatas)

Archaeological studies of plant remains have indicated that an increase in seed size is frequently correlated with both intensive cultivation and domestication of seed crop plants. To test if starch granules of domesticated root crops are significantly larger than those of wild or less intensively cultivated plants, archaeological and modern specimens of manioc and sweet potato were sampled for starch granules, and granule size was compared across a temporal sequence. The results indicate that a gross generalization can be made that modern specimens of both manioc and sweet potato yield larger starch granules than some archaeological specimens. It does appear, however, that modern domesticated manioc roots produce significantly larger-sized starch granules than those of its purported wild ancestor. Additionally, there exist two lines of evidence that the coastal Peruvian and lowland Neotropical regional types of manioc differ from one another and have been separate for several millennia. These findings indicate that manioc may have been domesticated more than once.     

Characteristics of Cd accumulation and distribution in two sweet potato cultivars

In this study, we compared the chemical forms and subcellular distribution of Cd in high-Cd (X16) and low-Cd (N88) sweet potato cultivars through hydroponic experiments and examined the Cd distribution in their roots by histochemical staining. The results showed that inorganic and pectate/protein-integrated Cd predominated in the leaves, and Cd concentrations were significantly higher in X16 than in N88. However, in the roots, Cd was mostly integrated with pectate and protein, and Cd concentration was higher in N88 than in X16. It was mainly stored through vacuolar sequestration and cell wall binding. In the leaves and stems, Cd concentrations in all subcellular fractions were higher in X16 than in N88; the opposite was observed in the roots. In X16, Cd was mostly accumulated in the root stele, and its Cd translocation factor was higher than that of N88. Overall, the subcellular fractions of X16 roots retained less Cd than N88 roots, and more Cd entered the root stele of X16 and subsequently moved to the shoots. The higher amounts of inorganic, water-soluble, and pectate/protein-integrated Cd with high mobility in the shoots of X16 than in N88 might facilitate Cd remobilization to other tissues, but this needs to be further studied.     

Combining chloroplast and nuclear microsatellites to investigate origin and dispersal of New World sweet potato landraces

We analysed a representative collection of New World sweet potato landraces (329 accessions from Mexico to Peru) with both chloroplast and nuclear microsatellite markers. Both kinds of markers supported the existence of two geographically restricted genepools, corresponding to accessions from the north-western part of South America and accessions from the Caribbean and Central America region. Our conservative cpSSRs markers revealed that the divergence between the two haplotype groups is associated with numerous mutation events concerning various markers, supporting the idea that this divergence may be ancient, predating domestication. For both kinds of markers, we found no significant difference in diversity between the two genepools and detected region-specific alleles in both groups. Previous studies have favoured the hypothesis of a single domestication of this crop. Our analysis suggests at least two independent domestications, in Central/Caribbean America and in the north-western part of South America. Sweet potato was then dispersed from these centres throughout tropical America. Comparison of nuclear and chloroplast data suggests that exchanges of clones and sexual reproduction were both important processes in landrace diversification in this clonally propagated crop. Our analysis provides useful tools for rationalizing the conservation and use of sweet potato germplasm collections.     

Growth stimulation produced by methylene blue treatment in sweet potato

Methylene blue as an alternative treatment to gamma rays to stimulate growth in sweet potato tissue cultures, was applied in two different ways: – pre-incubation of nodal explants with methylene blue for 1 h using urea as a permeabilizer; – methylene blue directly incorporated into the culture medium. Both treatments stimulated growth, but the better performance being obtained with the second treatment, which had no toxic effect. The activity and electrophoresis pattern of peroxidase after treatment ofIpomoea batatas plantlets with methylene blue or gamma rays did not show similar results for the two treatments. Peroxidase activity was greater in leaves of gamma ray treated plants compared to the non-treated control. The results obtained with the Methylene blue treatment did not significantly change the peroxidase activity relative to the control. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Reduced dispersal and survival in the sweet potato weevil (Euscepes postfasciatus) after irradiation

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Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification

 Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival of vitrified sweet potato shoot tips cooled to approximately –208  °C was increased by preculturing with 0.3 M sucrose for 24 h at 22  °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod. The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22  °C followed by dehydration with PVS2 for 16 min at 22  °C. Rapid cooling was used and achieved by the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise for cryopreserving sweet potato shoot tips. Received: 2 March 1999 / Revision received: 21 September 1999 / Accepted: 29 September 1999     

Production of dextran in transgenic potato plants

The production of dextran in potato tubers and its effect on starch biosynthesis were investigated. The mature dextransucrase (DsrS) gene from Leuconostoc mesenteroides was fused to the chloroplastic ferredoxin signal peptide (FD) enabling amyloplast entry, which was driven by the highly tuber-expressed patatin promoter. After transformation of two potato genotypes (cv. Kardal and the amylose-free (amf) mutant), dextrans were detected by enzyme-linked immunosorbent assay (ELISA) in tuber juices of Kardal and amf transformants. The dextran concentration appeared two times higher in the Kardal (about 1.7 mg/g FW) than in the amf transformants. No dextran was detected by ELISA inside the starch granule. Interestingly, starch granule morphology was affected, which might be explained by the accumulation of dextran in tuber juices. In spite of that, no significant changes of the physicochemical properties of the starches were detected. Furthermore, we have observed no clear changes in chain length distributions, despite the known high acceptor efficiency of DSRS.     

A comparison of shoot-forming and non-shoot-forming somatic embryos of sweet potato [Ipomoea batatas (L.) Lam.] using computer vision and histological analyses

Diagnostic structural features for competence to form shoots were tested among sweet potato embryos by combining morphological image capture (using a computer vision system) with anatomical analyses (using light microscopy). Five major morphological variants (`perfect', `near perfect', `limited/no meristematic activity', `disrupted internal anatomy', and `proliferating') were identified among torpedo- and cotyledonary-stage embryos. Among these, only the first two were found to be competent for conversion into plantlets. Lack of organized shoot development in somatic embryos of sweet potato was associated with the following abnormalities: lack of an organized apical meristem, sparcity of dividing cells in the apical region, flattened apical meristem, and multiple meristemoids and/or diffuse meristematic activity throughout the embryo. Diagnostic separation of most shoot-forming and non-shoot-forming torpedo and cotyledonary embryo variants was achieved. Received: 27 January 1997 / Revision received: 28 January 1998 / Accepted: 12 February 1998     

Production of HIV-1 p24 protein in transgenic tobacco plants

The production of antigens for vaccines in plants has the potential as a safe and cost-effective alternative to traditional production systems. Toward the development of a plant-based expression system for the production of human immunodeficiency virus type I (HIV-1) p24 capsid protein, the p24 gene was introduced into the genome of tobacco plants using Agrobacterium tumefaciens-mediated gene transfer. Southern blot analyses confirmed the presence of the p24 coding sequence within the genome of transgenic lines. Western blot analysis of protein extracts from transgenic plants identified plant-expressed p24 protein that cross-reacted with a p24-specific monoclonal antibody, thus confirming the maintenance of antigenicity. Quantification of the p24 protein using enzyme-linked immunosorbent assay (ELISA) estimated yields of approx 3.5 mg per g of soluble leaf protein. Similar accumulation levels of p24 were also detected in T1 plants, confirming that the p24 gene is transmitted stably. Our results indicate that plant-based transgenic expression represents a viable means of producing p24 for the development of HIV vaccine and for use in HIV diagnostic procedures.     

Antisense inhibition of cytosolic NADP-dependent isocitrate dehydrogenase in transgenic potato plants

Cytosolic NADP-dependent isocitrate dehydrogenase (cyt-NADP-ICDH; EC 1.1.1.42) has been suggested to play a major role in the production of 2-oxoglutarate, an important precursor for amino acid synthesis. Using an antisense RNA approach under the control of the cauliflower mosaic virus 35S promoter, transgenic potato plants were created in which NADP-ICDH activity was reduced to 8% of the wild-type level in leaves. Residual activity was almost completely due to mitochondrial and chloroplastic NADP-ICDH isoforms. Activity staining after non-denaturing polyacrylamide gel electrophoresis revealed the complete absence of a major activity band in leaves of antisense plants. No differences in growth or development, including flower formation and tuber yield, were observed between transgenic and wild-type plants. Photosynthesis and respiration were also unchanged. Levels of amino acids were the same in wild-type and cyt-NADP-ICDH antisense plants, even when accumulation of amino acids was induced by incubation of detached leaves in tap water in the dark (`induced senescence'). Consistent with a reduction in NADP-ICDH activity, however, were slight increases in the levels of isocitrate (up to 2.5-fold) and citrate (up to 2-fold). 2-Oxoglutarate was not reduced. Our data indicate that potato plants can cope with a severe reduction in cyt-NADP-ICDH activity without major shifts in growth and metabolism. Received: 28 July 1997 / Accepted: 3 November 1997     

转基因植物对农业生物多样性的影响

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Apparent absence of viruses in most symptomless field-grown sweet potato in Uganda

Heat tolerance of groundnut (Arachis hypogaea L.) genotypes was evaluated by solute leakage and chlorophyll fluorescence techniques in heat-hardened and non-hardened plants. To determine the appropriate hardening treatment, 1-month-old plants of two groundnut genotypes, ICGV 86707 and Chico were conditioned at five combinations of hardening (37°C) and non-hardening (30°C) air temperatures over a 5-day period. Heat injury, was assessed through measurements of electrolyte leakage after stressing leaf discs to 55°C for 15 min. The relative injury was significantly influenced by the conditioning temperatures and by the temperature during 24 h prior to measurement if those involved non-hardening conditions. Relative injury and chlorophyll fluorescence were measured after stressing leaves of six genotypes at a range of temperatures between 49°C and 55°C. Significant genotype × hardening treatment interactions were observed in relative injury and chlorophyll fluorescence. Chico was susceptible to heat stress, the relative injury test identified ICGV 86707 as tolerant, and the chlorophyll fluorescence test identified ICGV 86707 as tolerant under hardened conditions and ICGV 87358 as tolerant when non-hardened. When expressed as percentage of control values, the relative injury and chlorophyll fluorescence measurements over the 49–53°C stress temperature range were strongly correlated. Chlorophyll concentrations were increased by hardening in all genotypes except Chico. In Chico, chl_b concentration was decreased and the chl_a/b ratio increased by hardening, and chlorophyll concentrations were correlated with chlorophyll fluorescence parameters. Chlorophyll concentration may therefore provide an alternative means of screening for heat tolerance.