Genetic transformation ofRhododendron byAgrobacterium tumefaciens

Summary TransgenicRhododendron plants were obtained byAgrobacterium tumefaciens-mediated gene transfer.A. tumefaciens harboring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and (3-glucuronidase (GUS) genes was co-cultivated with stem and leaf segments fromRhododendron tissues culturedin vitro. Adventitious buds were fonned and shoots were regenerated on kanamycin selection medium 3-4 months after inoculation. Integration of the NPTII and the GUS genes was confirmed by polymerase chain reaction (PCR) and by Southern hybridization analyses. Histochemical GUS assay showed that the inserted gene was expressed in all tissues with the cauliflower mosaic virus (CaMV) 35S promoter. This transformation procedure has the potential to expand the range of genetic variation inRhododendron.     

High-frequency transformation ofArabidopsis thaliana byAgrobacterium tumefaciens

Incorporation of 5 mg/L silver thiosulphate into media for seed germination and callus induction, as used in the transformation protocol originally described by Valvekens et al. (1988), was found to increase the frequency of regeneration of transformants ofArabidopsis thaliana ecotypes C24 and Landsbergerecta by at least 10- to 100-fold. Other factors, such as density of the bacterial inoculation culture, density of the root explants and duration of bacteria-plant cocultivation period, were also found to influence the efficiency of recovery of transformants.     

Genetic transformation ofEustoma grandiflorum byAgrobacterium tumefaciens

Callus cultures were initiated from micropropagated Artemisia absinthium plantlets on MS basal medium supplemented with different concentrations of BA, Kn, NAA, IAA and 2,4-d in combination or singly. Supplementing the medium with low doses of both BA in combination with NAA, and Kn in combination with NAA enhanced the growth rate of callus cultures. However, cultures grew slowly following the second subculture and the majority turned brown and died within the next month. Initiation of root and shoot primordia occured directly from leaf explants cultured on 1.81 M 2,4-d, while adventitious shoot formation from callus was observed occasionally when BA was added to the medium in combination with IAA. Furthermore, medium containing 2.22 M BA and 2.69 M NAA stimulated both callus growth and organogenesis on some callus cultures derived from leaves and stems of young stock material. The best results were obtained with leaf explants. Cytological analysis of root meristems revealed that all regenerants were diploid (2n=18), as expected.Abbreviations MS Murashige & Skoog's salts and vitamins (1962) - BA 6-benzyladenine - NAA alphanaphthaleneacetic acid - Kn Kinetin (6-furfurylaminopurine) - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - FW fresh weight - Bi biomass increase     

Genetic transformation of willows (Salix spp.) byAgrobacterium tumefaciens

Anin vitro transformation method has been developed for stem explants of fast-growing willow clones (Salix spp.) usingAgrobacterium tumefaciens as a vector. Transformants obtained with the strains C58 and GV3101 (pGV3851::pLD1) were selected on hormone-free medium and on medium containing kanamycin, respectively. Transformation was confirmed by Southern blot analysis and nopaline assay. Inoculation of green-house grown plants with nopaline and octopine wildtype strains and shoot or root inducing mutant strains caused undifferentiated tumors at a frequency of 0 to 80%, depending on theSalix genotype and the bacterial strain used.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Km kanamycin - NPT neomycin phosphotransferase     

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and -mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of -mercaptoethanol did not enhance transformation frequency.     

Efficient transformation of Korean rice cultivars (Oryza sativa L) mediated byAgrobacterium tumefaciens

The purpose of this study was to improve transformation efficiency for three Korean rice cultivars, Ilpum, Dasan, and Namyang. Using two different media with or without light, efficiencies of callus induction, regeneration, and transformation of the Korean cultivars were compared to Japanese cultivar, Nipponbare, as a control. Immature cv. Nipponbare seeds produced 35.5% and 16.1% regeneration efficiency on CIM and N6D media, respectively. Among the Korean cultivars, only cv. Ilpum induced on CIM in the dark was actively regenerated with efficiency of 8.2%. With LBA4404 (pTOK233), no difference for the efficiency of transformation was found between mature and immature seeds of cv. Ilpum. This result reveals that mature seeds can be substituted for this study with no difference. The anther-derived calli of cv. Namyang inoculated with either LBA4404 (pTOK233) or EHA101 (pSMABuba) showed regeneration efficiencies of 14.5% and 20.9%, respectively, even though efficiency of transformation did not differ with these two vectors. We suggest that the anther-derived calli are better-materials for transformation experiment due to their genotype-independent regeneration. In the assay of GUS, all of the calli that survived on the second selection medium were strongly stained. PCR-Southern blot analyses confirmed that T-DNA was stably transformed into all tissues selected. Cvs. Nipponbare and Namyang transformed by LBA4404 (pTOK233) showed positive color in the NPTII ELISA.     

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens

Summary Leaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 g/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 g/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense. Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - LB Luria Broth - EDTA ethylenediamine-tetraacetic acid     

Genetic transformation of Medicago species by Agrobacterium tumefaciens and electroporation of protoplasts

Shoot and leaf segments of a non-regenerable Medicago sativa L. genotype were cocultivated with the shooty mutant of Agrobacterium tumefaciens carrying the pGV 2206 plasmid. Transformed callus lines were selected and regenerated on the hormone free B5 medium. Southern blot analysis demonstrated integration of T-DNA in to the genome of the regenerated plants.Transgenic plants resistant to kanamycin were obtained by electroporation of Medicago borealis protoplasts with the pGA 472 plasmid DNA.Abbreviations 2.4 D 2.4 dichlorophenoxyacetic acid - BAP 6-benzyladenine - T-DNA transferred DNA into plants from Ti-plasmid of A. tumefaciens     

Role of iron in the enhancement byAgrobacterium tumefaciens infection in mice

Microorganisms require iron for their growth and usually compete with their host for available iron from the system. Iron supplementation to host causes an increase of available iron both to host and to potential microbial invaders and favours the latter more than the former as the bacteria release siderophores which are responsible for iron transport mechanism. In view of this observation a study was done to deal with the distribution of storage and injected iron given as an overload within a physiological pool, taking mice as the host, with a correlation to its utilization by Agrobacterium tumefaciens and with bacterial growth and multiplication. The results obtained help in understanding the host--parasite relationships, regarding bacterial virulence and infection and the growth-promoting effect of iron, as iron promoted the development and progression of serum-exposed A. tumefaciens in mice.     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside     

Reversion of protoplasts and spheroplasts in the yeast Nadsonia elongata

Summary The time rate of regeneration of the cell wall and reversion of protoplasts of the yeast Nadsonia elongata to cells of normal shape and size has been compared with the capability for regeneration of spheroplasts of this yeast. Nearly all protoplasts in a given culture were able to regenerate new walls and had usually reverted to cells of normal appearance by the 30th h of cultivation. Spheroplasts required only half this time to do this. These results can be interpreted as evidence that regeneration of a wall by protoplasts does not depend upon the presence of a cell wall primer, because the proportion of reverting protoplasts (which lack wall remnants) was the same as that of reverting spheroplasts (which possess them). The presence of wall remnants in spheroplasts appears to have merely an accelerating effect on the formation of a new wall and on subsequent reversion of the spheroplasts to complete cells of normal shape and size.     

Cultivar dependence of transformation rates in moth bean after co-cultivation of protoplasts with Agrobacterium tumefaciens

Summary A simplified protoplast regeneration system for Vigna aconitifolia was developed. A plating efficiency of 60% was obtained using mesophyll protoplasts from 10-day-old seedlings. By co-cultivation of protoplasts with Agrobacterium tumefaciens containing the Ti plasmid derivative pGV 38501103 neo kanamycin-resistant colonies were obtained; 23% of the transformed lines showed expression of the nonselected co-transferred nopaline synthase gene. Transformation was confirmed by Southern blot analysis using a nonradioactive detection system. The plant cultivar used was an important factor in determining transformation frequencies since one of the cultivars had an 85 fold higher transformation rate than the other.On deputation from: Bhabha Atomic Research Centre, Bombay, India, under the Indo-FRG Bilateral Programme     

Low temperature protocol for efficient transformation ofMycobacterium smegmatis spheroplasts

Spheroplasts ofMycobacterium smegmatis LM15, strain 607, were prepared by a combined treatment with glycine and lysozyme. The spheroplasts were tested for ability to take up and express purified mycobacteriophage DNA. Exposure of 1.0×10⁸ to 1.0×10⁹ spheroplasts to saturing DNA (1 g) for 15 min at 5°C resulted in a transfection efficiency of approximately 0.009%. The transfer of the -lactamase marker with DNA purified from strain LM15 to spheroplasts of a -lactamase-negative mutant, strain LM144, was achieved. The DNA-treated cultures, after reversion to the bacillary form, contained 20-fold more penicillinresistant cells than the nontreated control culture. Approximately 80% of the penicillin-resistant colonies from the DNA-treated cells were positive for -lactamase. Cell-free extracts of penicillin-resistant transformants contained -lactamase activity that ranged from 0.046 to 0.134 mol of benzylpencillin hydrolyzed/min per mg protein. This low temperature procedure is recommended for high efficiency transformation ofM. smegmatis.     

Further evidence for a membrane potential-dependent shape transformation of the human erythrocyte membrane

The influence of various factors (pH, temperature, sodium gluconate) on the ionic strength-dependent stomatocyte-discocyte-echinocyte transformation of the human erythrocyte membrane was investigated. The results give further evidence for a correlation between shape of erythrocyte membrane and the transmembrane potential of the cells.     

Further evidence of a cybridization requirement for plant regeneration from citrus leaf protoplasts following somatic fusion

Summary Somatic hybridization experiments in Citrus that involve the fusion of protoplasts of one parent isolated from either nucellus-derived embryogenic callus or suspension cultures with leaf-derived protoplasts of a second parent, often result in the regeneration of diploid plants that phenotypically resemble the leaf parent. In this study, plants of this type regenerated following somatic fusions of the following three parental combinations were analyzed to determine their genetic origin (nuclear and organelle): (embryogenic parent listed first, leaf parent second) (1) calamondin (C. microcarpa Bunge) + Keen sour orange (C. aurantium L.), (2) Cleopatra mandarin (C. reticulata Blanco) + sour orange, and (3) Valencia sweet orange (C. sinensis (L.) Osbeck) + Femminello lemon (C. limon (L.) Burm. f.). Isozyme analyses of PGI, PGM, GOT, and IDH zymograms of putative cybrid plants, along with RFLP analyses using a nuclear genome-specific probe showed that these plants contained the nucleus of the leaf parent. RFLP analyses using mtDNA-specific probes showed that these plants contained the mitochondrial genome of the embryogenic callus donor, thereby confirming cybridization. RFLP analyses using cpDNA-specific probes revealed that the cybrid plants contained the chloroplast genome of either one or the other parent. These results support previous reports indicating that acquisition of the mitochondria of embryogenic protoplasts by leaf protoplasts is a prerequisite for recovering plants with the leaf parent phenotype via somatic embryogenesis following somatic fusion.Abbreviations cp chloroplast - GOT glutamateoxaloacetate transaminase - IDH isocitrate dehydrogenase - mt mitochondria - PEG polyethylene glycol - PGI phosphoglucose isomerase - PGM phosphoglucomutase - RFLP restriction fragment length polymorphism Florida Agricultural Experiment Station Journal Series No. R-04631.     

Optimum conditions for efficient transformation ofStreptomyces venezuelae protoplasts

Summary Optimum conditions for protoplast regeneration and transformation ofStreptomyces venezuelae ETH 14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10⁵ transformants/g DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30°–32°C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10⁵ regenerating protoplasts per plate. Higher protoplast densities considerably decreased the regeneration frequency of the transformants.     

Transformation of meristematic cells in the shoot apex of cultured pea shoots byAgrobacterium tumefaciens andA. rhizogenes

Summary Different tissues in cultured pea shoots were inoculated withAgrobacterium tumefaciens wild types C 58 and ACH 5 andA. rhizogenes wild type 9402. The C 58 and 9402 bacteria induced the formation of tumours and hairy roots respectively while the ACH 5 was inactive. The younger the tissue the more rapidly it responded to the active bacteria. The shoot apex was the most reactive organ and developed into a tumour, theA. rhizogenes tumours subsequently giving rise to transformed hairy roots. Histological examination showed that transformed cells (including those within the apical dome) initially became highly vacuolate before dividing rapidly to form a tumour. These changes were accompanied by cell division in surrounding tissues.