Transient gene expression in electroporated Solanum protoplasts

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts –250 V/cm; Désirée mesophyll protoplasts –225 V/cm; Désirée suspension culture protoplasts –225 V/cm; and Désirée tuber protoplasts –150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36–48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the -glucuronidase (gus) gene, showed expression (at DNA concentrations between 0–10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20–30 pmol/ml) the patatin promoter directed 4–5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.     

Transformed calli obtained by direct gene transfer into sunflower protoplasts

Helianthus annuus protoplasts were transformed with the plasmid pCaMVNEO (Frommet al. 1986) conferring kanamycin resistance to plant. Transformed calli were selected with a frequency of 4 calli for 10⁶ treated protoplasts. DNA was extracted from kanamycin resistant calli. Analysis of this DNA shows the presence of the NPTII gene.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA 1-naphtalenoacetic acid - NPT Neomycin phosphotransferase - PEG Polyethyleneglycol     

Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression. W. Craig and D. Gargano contributed equally to this work     

Differentiation of potato (Solanum tuberosum L.) plants from cultured leaf protoplasts

Through induction of totipotent stage in cultured cells plants were regenerated from protoplast derived callus colonies of potato. Growing of the plants in vitro with optimal aeration and pretreatment of the leaves in dark and cold prior to protoplast isolation has improved the protoplast yield and frequency of cell division. Protoplasts of four potato genotypes have started to divide within 2-5 days after plating them into culture medium containing 2,4-D (0.2 mg/l); ZEA (0.5 mg/l); and NAA (1 mg/l) as growth regulators. Embedding of the cells into agarose proved to be favourable to avoid cell browning and to increase colony formation. The series of hormone treatments based on complex action of NAA and BAP promoting colony growth and greening, ZEA and IAA inducing shoot redifferentiation, and GA3 plus NAA supporting shoot elongation and rooting, finally resulted in high frequency of plant regeneration from microcolonies.     

Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L.

Protoplasts from potato mesophyll of two strains (Solannum tuberosum L. cv. Xiao Yie Zi x Duo Zi Bia and Solanum tuberosum L. cv. Wu Meng 601) were induced to callus in culture medium of protoplasts. The callus derived from mesophyll protoplasts were transferred to MS medium with 2 mg/l ZT+0.1 mg/L IAA. Shoots regenerated from the callus were detected after 70 days of culture.The shoots which had grown to a height of 2–3 cm were transferred to MS medium with 0.05 mg/L NAA. Roots were coming out in a few days.Complete plantlets were achieved. Stern segments with 1–2 leaves were then transferred to a mixture of sterilized soil and grown, and produced tuber.     

One-step transformation of two Andean potato cultivars (Solanum tuberosum L. subsp. andigena)

Most Agrobacterium-mediated transformation procedures described in the literature have proven to be inefficient for transformation of Andean potato cultivars. We describe here a more successful method for transformation of two of these cultivars, Diacol Capiro (DC) and Parda Pastusa (PP). Leaf explants were transformed using vector pBI121 carrying the #-glucuronidase and kanamycin resistance genes. Calli and shoot formation occurred after 5-8 weeks on a selection medium containing 3 mg/l zeatin and 1 mg/l indole acetic acid. The shoot regeneration frequency was 28% and 34% for DC and PP, respectively. Of the kanamycin-resistant plantlets obtained, 51% of those of DC and 13% of those of PP were confirmed to be transformants by histochemical assays, polymerase chain reaction analyses and Southern blotting techniques.     

Paclobutrazol stimulates bud regeneration in Solanum tuberosum L. primary explant cultures

The growth retardant paclobutrazol (PBZ) inhibited stem internode growth of in vitro cloned potato plants. The extent of growth inhibition caused by10-9 M PBZ in Murashige and Skoog medium was genotype-specific, varying between 10 - 60% of the stem growth of untreated controls in ten cultivars examined. An increase in percentage of de novo bud regenerating stem internode segments (SIS) as well as in the total number of buds per explant was observed in SIS taken from PBZ pretreated plants. PBZ applied directly into the regenerative media had no stimulatory effect on there generation process. We assume that the enhancing effect of PBZ on regeneration may be attributed to its interaction with cytokinin metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Salt Tolerance of Solanum tuberosum L. Overexpressing an Heterologous Osmotin-like Protein

Potato (Solanum tuberosum L. cv. Bintje) was transformed with a cDNA clone encoding an osmotin-like protein. Transgenic and non-transgenic in vitro plants were subjected to NaCl for 3 weeks. The shoot and root development was slightly affected by salinity indicating that the salt condition used was a mild stress. The endogenous proline content of the osmotin-like transformed clone only raised slightly as compared to the non-transformed genotype, where a marked increase in proline content could be observed as a result to salt stress. These data provide evidence for the involvement of osmotin-like proteins in the mechanisms of salt tolerance in potato plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Factors affecting PEG-mediated stable transformation of maize protoplasts

Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 11 ratio to 65% at a 1001 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.Abbreviations PEG polyethylene glycol - BMS Black Mexican Sweetcorn - MS salts of Murashige and Skoog (1962) culture medium - MaCa 0.2M mannitol, 80mM calcium chloride - MaMg 0.4M mannitol, 15mM magnesium chloride, 0.1% MES, pH 5.6 - CM conditioned medium - SE standard error of the mean     

Growth and photosynthesis of photoautotrophic callus derived from protoplasts of Solanum tuberosum L.

Summary We describe a photoautotrophic culture procedure of potato (cvs Kennebec, Haig, DTO-33) callus derived from mesophyll protoplasts. The protoplast culture was initiated at very low concentration of glucose (down to 0.25 g l^–1). Callus was subcultured under CO₂ enriched air and glucose suppressed by the successive dilutions with glucose free media. Regeneration was successfully obtained under photoautotrophic conditions. The characterization of oxygen exchange and of some enzymes and metabolites of carbon assimilation indicated that chlorophyllous callus, grown on carbohydrate free medium, developed the photosynthetic pathway typical of C3 plants. By comparing the fresh weight of callus cultivated in the light or in non-photosynthetic conditions (in darkness or in the light +3-(3,4-Dichlorophenyl)-1,1-dimethylurea) we concluded that growth depended to about 70 to 88 % on photosynthesis.Abbreviations BAP 6-Benzylaminopurine - cv Cultivar - DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea - 2,4-D (2,4-Dichlorophenoxy) acetic acid - GA3 Gibberellin A₃ - IAA Indole-3-acetic acid - NAA 1-Naphthaleneacetic acid - PePcase Phosphoenolpyruvate carboxylase - RuBPcase Ribulo1,5-bisphosphate carboxylase - ZEA Zeatin     

光质对马铃薯试管薯形成的影响

以两个试管薯形成能力不同的马铃薯脱毒试管苗为材料,研究不同光质(白光、红光、蓝光)对马铃薯试管苗生长和试管薯形成的影响.结果表明：红光下试管苗叶片的净光合速率、可溶性糖含量和生物量最高,试管苗叶片数多.蓝光对试管苗干物质含量和试管苗发育后期的结薯数量以及结薯期提前有明显促进作用,但对试管苗株高有明显抑制作用.白光下试管苗净光合速率和干物质含量最低.不同品种试管薯的形成对光质的要求有一定差异.总之,壮苗培养阶段采用红光,试管薯诱导阶段采用蓝光处理利于提高试管薯产量.     

In vitro microtuberization in potato (Solanum tuberosum L.) cultivars

Mechanism of microtuberization in three elite cultivars kufri badhsha (KB), kufri chandramukhi (KCM) and kufri jawahar (KJ) of potato was studied. Sprouts of all the three cultivars were used to obtain in vitro shoot cultures. MS medium supplemented with chlorocholine chloride was found to be most suitable for all the cultivars. Maximum tuberization was obtained under incubation conditions of continuous darkness at 20 degrees +/- 1 degrees C. The highest number of micro-tubers per plant basis was produced under continuous darkness and KCM recorded the highest yield of micro-tubers and was found significantly superior to KJ and KB.     

Expression and promoter activity of the desiccation-specific Solanum tuberosum gene, StDS2

Environmental stresses induce the expression of several plant genes via multiple and cross‐talking signalling pathways. Previously it was shown that ScDS2, a gene of the wild potato species, Solanum chacoense, is highly inducible by dehydration but not by abscisic acid (ABA), the mediator of many plant stress responses. Herein it is shown that ScDS2‐related genes are present in the cultivated potato, Solanum tuberosum (StDS2) and also in the non‐tuberizing Solanum species, Solanum brevidens (SbDS2). We show that expression of StDS2 is dehydration‐specific, is not inducible by cold, heat, salt, hypoxia or oxidative stresses, and is independent of ABA. Signalling of StDS2 induction, however, is dependent on the synthesis of novel proteins because cycloheximide can block StDS2 expression. To analyse the promoter region of StDS2 a genomic library of Solanum tuberosum was established and 1140 and 498 bp regions of the StDS2 promoter were isolated. The promoter fragments were fused to the β‐glucuronidase (GUS) reporter gene and tested in transgenic potato plants. Both promoter fragments were able to induce GUS activity in response to dehydration. This result suggests that drought‐specific cis‐elements are located within 498 bp upstream to the StDS2 coding sequence.     

Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens

Summary Leaves of the in vitro grown potato cultivars Bintje, Berolina, Desiree, and Russet Burbank were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For Russet Burbank, it was necessary to include AgNO₃ in the medium to obtain efficient shoot regeneration. The transformed plants have one to a few copies of the T-DNA, show NPT-II and PAT activities, and are resistant to high doses of the commercial preparation of phospinotricin (glufosinate). Almost no somaclonal variation was detected in trans-genic plants.     

菠萝蜜低聚肽对db/db小鼠炎症反应、血糖及血脂的影响

目的：研究菠萝蜜低聚肽（JOPs）干预对db/db糖尿病模型小鼠炎症反应、血糖及血脂的影响作用。方法：选择db/db糖尿病小鼠模型,将其随机分为3个JOPs组（0.2、0.4、0.8g/kg·BW）以及糖尿病模型对照组、二甲双胍对照组、乳清蛋白对照组,并选用db/m小鼠作为非糖尿病小鼠空白对照。经过为期6个月的干预,检测小鼠空腹血糖（FPG）、血清胰岛素（INS）、白细胞介素6（IL 6）、白细胞介素8（IL 8）、白细胞介素10（IL 10）和肿瘤坏死因子α（TNF α）、C反应蛋白（CRP）以及脂代谢指标。结果：JOPs可显著降低db/db糖尿病小鼠空腹血糖水平及胰岛素抵抗指数;可使血清IL 6、TNF α、总胆固醇（TC）和甘油三酯（TG）显著降低,并使高密度脂蛋白胆固醇（HDL C）显著升高。结论：JOPs干预可有效降低糖尿病小鼠的血糖水平,改善胰岛素抵抗,同时有效调节炎症反应及血脂代谢。     

转BADH基因马铃薯无性繁殖二代耐盐性检测

在0%、0.3%、0.6%NaCl胁迫下,检测转BADH基因马铃薯及其受体亲本‘甘农薯2号’的盆栽植株耐盐性的结果表明:BADH基因的遗传性稳定且转基因植株的耐盐性比非转基因的强。     

Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs

Summary The techniques of microspore and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A₁ plant lines and of several hundred protoplast derived plantlets has become possible. Further, from the dihaploid Solanum species S. phureja the regeneration of microspores to plants, and from the species S. infundibuliforme, S. sparsipilum and S. tarijense the regeneration of protoplasts to calluses, has been achieved. The plants descending from the two single cell culture systems are compared with reference to phenotypic markers and economic qualities. Some principles characteristic for either microspore or protoplast derived plants are examined and their significance is discussed. The results are compiled into an extended analytical synthetic breeding scheme based on a stepwise reduction of the autotetraploid to the monohaploid level and a subsequent controlled combination to a new synthetic completely heterozygous tetraploid potato.     

Metabolite profiling of potato (Solanum tuberosum L.) tubers during wound-induced suberization

Suberin is a specific cell wall-associated biopolymer characterized by the deposition of both a poly(phenolic) domain (SPPD) associated with the cell wall, and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. In planta, suberin functions to prevent plants from desiccation and pathogen attack. Although the chemical identity of the monomeric components of the SPPD and SPAD are well known, their concerted biosynthesis and assembly into the suberin macromolecule is poorly understood. To expand our knowledge of suberin biosynthesis, a GC/MS-based metabolite profiling study was conducted, using wound healing potato (Solanum tuberosum L.) tubers as a model system. A time series of both non-polar and polar metabolite profiles were created, yielding a broad-based, dynamic picture of wound-induced metabolism, including suberization. Principal component analysis revealed a separation of metabolite profiles according to different suberization stages, with clear temporal differences emerging in the non-polar and polar profiles. In the non-polar profiles, suberin-associated aliphatics contributed the most to cluster formation, while a broader range of metabolites (including organic acids, sugars, amino acids and phenylpropanoids) influenced cluster formation amongst polar profiles. Pair-wise correlation analysis revealed strong correlations between known suberin-associated compounds, as well as between suberin-associated compounds and several un-identified metabolites in the profiles. These data may help to identify additional, as yet unknown metabolites associated with suberization process.     

Culture of Cotyledon and Hypocotyl Protoplasts from True Potato Seedlings in Solanum tuberosum L.

Culture of protoplast using cotyledon and hypocotyl as the donor tissue from true potato seedlings (TPSs) of 3 breeding lines (DTO-33, ND 860-2 and BN 9815-3) of Solanum tuberosum L. was studied. The cotyledons and hypocotyls of TPSs just extended were excised and digested in an enzyme solution containing 1 % cellulase and 0. 5 % macerozyme for 17—20 h after vacuum infiltration of the tissue in the solution. The protoplasts were cultured in an improved liquid medium and transferred onto solid media for callus culture and shoot regeneration. Some factors affecting the efficiency of cotyledon and hypocotyl protoplast culture were studied. The results showed that using the cotyledons and hypocotyls as donor tissues for protoplast isolation and culture in potato, the division frequency of protoplast derived cells was significantly higher than that using the leaves and shoot-tips of the test-tube plantlets: the yield and quality of the protoplast from TPSs cultured under continuous high light intensity (3000 Ix) were much higher than the TPSs cultured under low light intensity (1000 Ix), and no intact protoplast was ever obtained from the TPSs cultured in continuous dark condition. Vacuum infiltration of the cotyledon and hypocotyl segments in enzyme solution before digestion increased protoplast yield. The yield of protoplasts from hypocotyl tissue was significantly higher than from the cotyledon, but there was no significant difference in quality between the protoplast derived from the two tissues. The significance, advantages and shortcomings of using the cotyledons and hypocotyls as the donor tissues for isolation and culture of potato protoplasts are dicussed.