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Isolation and characterization of streptomycin resistant Hyoscyamus muticus plants
Abstract
A distinct streptomycin resistant dark green colony of Hyoscyamus muticus was isolated after six weeks culture of leaf discs on modified MS medium with 2.0 mg/l NAA, 0.5 mg/l BAP with 1.0 mg/ml streptomycin sulphate for selection of streptomycin resistant lines. The rest of the leaf discs of H. muticus were bleached with slight callusing on the margins. This streptomycin resistant colony (HMS) was isolated and sub-cultured on the callusing medium with streptomycin sulphate (1.0 mg/ml). The callus derived from leaves of wild type H.muticus and that of the streptomycin resistant line were placed on the regeneration medium (MS+0.1 mg/l NAA+0.5 mg/l BAP) with 1.0 mg/ml streptomycin sulphate. The wild type callus showed bleaching and did not evince a regeneration response, however, the HMS line grew normally as a green callus evincing profuse shoot regeneration. The regenerated shoots developed roots on a half strength MS medium containing 2.0% sucrose with streptomycin sulphate (1 mg/ml). The plants were transferred to a glass-house and grown to maturity. The HMS plants were highly vigorous and diploid with bigger flowers and seeds. On reciprocal crossing with wild H. muticus, it was observed that the streptomycin resistance is maternally inherited. The crude tropane alkaloid content of these plants was also found to be higher than that of the parent plants.
... It is known that sucrose concentration in the medium has an important effect on the growth of root cultures and tropane alkaloid production (Rothe and Drager 2002;Rothe et al. 2001). However, scientific data to support the use of a certain concentration of sucrose in the medium to root H. muticus or H. niger shoots are not available and sucrose concentrations from 0 gL −1 to 50 gL −1 are used indistinctively (Rahman and Ahuja 1998;Tu et al. 1996Tu et al. , 2005Zeef et al. 2000). Therefore, to determine the most suitable sucrose concentrations in the rooting medium (RM) for H. muticus, a simple experiment was performed. ...
Hyoscyamus muticus is cultivated in Egypt, India, Pakistan and USA for the production of the medicinally important tropane alkaloids (TA) hyoscyamine and scopolamine. The TA pathway provides an excellent model to test the feasibility of transgenic strategies to modify secondary metabolic flux. However, H. muticus transformation and regeneration has been proven difficult. We report a successful experience obtaining several independent fertile plant lines, genetically engineered to redirect the TA metabolic flux into alternative directions. cDNA from H. niger encoding tropinone reductase enzymes (TR1 or TR2), which catalyse the reduction of tropinone into scopolamine and hyoscyamine or calystegines, respectively, were incorporated in a silencing vector, followed by Agrobacterium tumefaciens transformation. Most explants produced large amounts of calli. Shoot formation occurred in the majority of the calli, being ready for rooting after an average of 9 months. On average, 45% of the shoots formed roots after 4 months. However, most rooted shoots died during ex vitro acclimatization, regardless of substrate type and cultivation conditions. This problem was overcome by the development of a specialized protocol (cultivation on soil like substrate under sterile conditions) that allowed the recovery of a reasonable percentage (44%) of fertile plants per rooted shoot. In this study were obtained 11 tr2 lines (25 plants), three tr1 lines (3 plants), four empty vector lines (13 plants) and three untransformed-regenerated lines (3 plants); a significant result compared to previous attempts to stable transform H. muticus.
We report an efficient whole plant transformation system for Hyoscyamus muticus, an important medicinal plant of the Solanaceous family. We developed a system using a plasmid carrying the nptII and gusA genes, which was delivered into leaf explants by particle bombardment. Ten percent of bombarded leaf explants formed kanamycin-resistant callus, from which putative transgenic plants were recovered. The nptII gene conferring kanamycin resistance was found to be incorporated into the genome of all transgenic plants screened. Over 50% of the kanamycin resistant plants showed strong expression of the non-selected gusA gene. The majority of transgenic plants reached maturity, could be self pollinated, and produced fertile seed. A simple and efficient whole plant transformation system for this medicinal plant is an important step in furthering our understanding of tropane alkaloid production in plants.
Approximately 106 protoplast-derived cell colonies of sainfoin were stressed with streptomycin and two resistant colonies were recovered. Plants regenerated from these colonies could be recallused on streptomycin-containing medium three years after growth in the absence of the antibiotic.
Ultrastructural studies showed cells of resistant callus grown in the presence of streptomycin to contain chloroplasts with internal thykaloids and grana. Such mutant plants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids.
Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10(-5) in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 μg/ml N-methyl-N'-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.
Streptomycin-resistant mutations were induced in Solanum melongena by exposing seeds to ethyl methane sulphonate (EMS). Seed mutagenesis resulted in a high frequency of chlorophyll-deficient mutations and a low frequency of resistant shoots, both of which retained their resistance on subsequent testing. Reciprocal crosses between streptomycin-resistant and -sensitive plants showed a non-Mendelian transmission of the resistance trait. Streptomycin resistance is the first selectable and maternally inherited organelle marker described in brinjal.
Leaf mesophyll protoplasts of a nitrate reductase deficient, streptomycin resistant double mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type N. rustica. Hybrid colonies were selected for nitrate reductase proficiency and streptomycin resistance. Green, actively proliferating colonies were recovered in the selection medium. Eleven of twenty green colonies, transferred to regeneration medium, produced plants. Regenerated plants were analysed for their vegetative and floral characteristics, Fraction 1 protein polypeptide composition and leaf esterases. All the eleven regenerants were nuclear somatic hybrids possessing the chloroplast of the N. tabacum parent. By developing a double mutant with a negative (auxotrophic) and a positive (resistance) selection marker in a species of interest, this species can be hybridised by somatic cell fusion with wild type species lacking selectable markers.
A major obstacle to our understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.
The inheritance of six polymorphic loci mapping in the rRNA-encoding (rDNA) region of the inverted repeat sequence of chloroplast DNA (cpDNA) was scored in hybrid subclones derived from reciprocal interspecific crosses between the green algae Chlamydomonas eugametos and Chlamydomonas moewusii. In order to enhance the detection of cells that had undergone recombination between parental cpDNAs, hybrids were selected that inherited a chloroplast antibiotic-resistance marker contributed by the mating-type-minus(mt(-)) parent, the parent that normally contributes fewer cpDNA molecules. The major findings of this study can be summarized as follows. (i) The majority of the hybrids (14/17) were recombinant for cpDNA markers in the 10-kilobase-pair rDNA region under study. (ii) Only one allele of each polymorphic cpDNA locus was ever detected in the hybrids, thus suggesting that newly recombined rDNA sequences in one copy of the inverted repeat are rapidly spread to the other by a copy-correction mechanism. (iii) Chloroplast streptomycin-resistance (sr-2) and erythromycin-resistance (er-nM1) loci, although showing little or no genetic linkage, were mapped to the 16S and 23S rRNA gene regions of the cpDNA, respectively, by virtue of their perfect coinheritance with polymorphic markers within these genes. (iv) cpDNA markers associated with a putative intron of the C. eugametos 23S rRNA gene were inherited by all 17 hybrids. Such a result is similar to that observed for certain alleles of the large rRNA gene of yeast mitochondria in crosses between omega(+) and omega(-) strains.
In Chlamydomonas, as in higher plants, the chloroplast genome codes for chloroplast ribosomal RNAs, tRNAs, about 1/3 of the ribosomal proteins, and a number of proteins of the photosynthetic apparatus (Table 1, Fig. 1). Other proteins are coded by the nuclear genome and imported, generally as precursors which are processed to mature form within the chloroplast (Table 1). Because C. reinhardtii can dispense with photosynthetic functions if provided acetate as an alternative carbon source, this species is an ideal model system for investigation of mutations affecting these components. In the case of the large subunit of ribulose bisphosphate carboxylase (RUBISCO; Spreitzer & Ogren, 1985), the 32 kd herbicide binding protein (Erickson et al., 1985), and the beta subunit of the CF0CF1 ATPase (Woessner et al., 1986), analysis of chloroplast genes has extended to the DNA sequence level, and to comparison of mutations and reversions within the same gene.
It has been known for over 25 years now that plant cell and tissue cultures undergo genetic erosions and show changes of various types, especially in chromosome numbers and ploidy level (Partanen 1963; D’Amato 1965; Murashige and Nakano 1966). However, till the mid 1970’s such changes were considered undesirable and were therefore discarded because the main emphasis was on clonal propagation and genetic stability of the cell cultures. Extensive studies conducted during the last decade have shown that the cell cultures, especially on periodical subculturing, undergo various morphological and genetic changes, i.e., polyploidy, aneuploidy, chromosome breakage, deletions, translocations, gene amplification, inversions, mutations, etc. (see Nagl 1972; Meins 1983; D’Amato 1985). In addition, there are changes at the molecular and biochemical levels (Day and Ellis 1984; Landsman and Uhrig 1985; Ball and Seilleur 1986) including changes in the DNA (Berlyn 1982; Cullis 1983), enzymes (Brettell et al. 1986b), gliadin (Cooper et al. 1986), etc. Since the publication of Larkin and Scowcroft (1981) there has been an upsurge of interest, and the attitude toward these changes has shifted to using the somaclonal variations for plant improvement; they are intentionally induced and much sought after.
Ribosomal proteins from chloroplasts of Nicotiana tabacum L. (cv. Petit Havana) and of SRl, a mutant derived from it, with uniparentally inherited streptomycin resistance, were characterised by two-dimensional gel electrophoresis. From the 67 proteins identified, one has an altered electrophoretic mobility when isolated from the mutant. Streptomycin resistance of the SRl mutant therefore seems to be the consequence of a mutation in the chloroplast DNA coding for a chloroplast ribosomal protein.
The SR180 cell line has been isolated in a callus culture derived from a haploid Nicotiana sylvestris (n = X = 12) plant by its ability to proliferate on a selective medium containing 2,000 μg/ml streptomycin sulphate. From the cell line diploid plants have been regenerated. The SR180 selfs are resistant to streptomycin. Streptomycin sensitivity in F1, and a 3∶1 (sensitive to resistant) segregation in F2 indicate that resistance in the SR180 mutant is the result of a recessive Mendelian mutation.
The chloroplast gene for 16S rRNA was cloned from two maternally inherited streptomycin-resistant mutants ofNicotiana differing in degree of resistance at the whole plant and isolated chloroplast level. A single-nucleotide change in the 16S rRNA gene was detected for each mutant: a C to T transition at nucleotide 860 (Escherichia coli coordinate C912) which is an often mutated site, and a novel transition of C to T at nucleotide 472 (E. coli coordinate C525). The novel mutation is located in the phylogenetically conserved "530 loop".
Induced autotetraploids with high pollen and seed fertility have been developed in a medicinally important Solanaceous plant: Hyoscyamus muticus (2n=28). The colchiploids are vigorous and the increase in all the determinate parts - plant height, size and thickness of leaves, cell, pollen, seed and flower size - along with an increase in alkaloid content has been achieved. The overall improvement in the colchiploids has the advantage of enhancing nearly 1.5 times the production potential of the economic product - the total alkaloids. Although the seed is not the ultimate economic product in this species, it is still required for propagation. The seed fertility in the colchiploids improved because of reduction in quadrivalent frequency and subsequent balanced anaphase separation.
Somatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82-120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33-78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.
The effects of streptomycin on the germination of tobacco seeds were studied by germinating seeds on 1% agar in tap water containing 0-10 mg/ml streptomycin sulfate. Low concentrations of the drug (0.25-0.75 mg/ml) inhibited the formation of green colour in the seedlings. Higher drug concentrations inhibited the appearance of cotyledons, hypocotyles and radicles, and reduced the size of the seedling. A test for streptomycin resistance was developed for germinating tobacco seedlings. In this tobacco seeds are germinated on 1% agar in tap water with 0.25 mg/ml streptomycin. Seeds from St-S genetic lines produced yellow seedlings, while seeds from a St-R mutant produced green seedlings. Genetic studies indicate that, in the St-R701 line which was derived from a spontaneous mutation in diploid callus culture, the streptomycin resistance is cytoplasmically inherited.
This paper describes a simple and rapid high performance liquid chromatographic method using two different polarity columns in series for the quantitative analysis of the major tropane alkaloids atropine and scopolamine. The problems associated with the simultaneous determination of the two alkaloids in plant extracts have been overcome. The method has been successfully applied to the analysis of three species of Solanaceous plants.
KR103 is a kanamycin resistant cell line of Nicotiana sylvestris, deficient in inducible shoot redifferentiation. The existence of a genetically unaltered inducibility factor in KR103 was tested by fusing protoplasts of this line with those of Nicotiana knightiana, which can not be induced to form shoots in callus culture.
Selection of somatic hybrids was based on screening for kanamycin resistance and green pigmentation, factors originally separated in the parental lines. The hybrid nature of two lines (H1 and H2) was shown by studies on esterase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase isoenzymes.
In the somatic hybrids shoot formation on inductive medium was restored, indicating that the inducibility factor had not been altered in KR103. Genetic complementation and induced regulatory changes due to genome interaction are discussed as possible explanations for the restoration of morphogenic potential.
Chloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F1 and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.
A protoplast mutagenesis and cell selection system was used for the isolation of streptomycin resistant Lycopersicon peruvianum colonies. Protoplasts were treated with the mutagen N-nitroso-methylurea and could be regenerated into fertile plants, carrying the streptomycin resistant character. Several classes of streptomycin resistance could be distinguished. Reciprocal crosses between streptomycin resistant and sensitive plants showed a non-Mendelian transmission of the resistance trait. Streptomycin resistance is the first selectable and maternally inherited cell organelle marker described in tomato.
We sequenced a 82 bp fragment in the chloroplast 16 S rRNA gene of the streptomycin resistant mutant SR1 of Nicotiana tabacum. The sequence contains a transition from cytosine to adenine in position 860 of the 16 S rRNA. We suggest that this mutation is sufficient for the streptomycin resistance phenotype in the SR1 mutant.
Twenty-nine cybrids possessing an Atropa belladonna nuclear genome and a Nicotiana tabacum plastome were selected from two independent protoplast fusion experiments. In contrast to the previously described reciprocal, green and fertile cybrids with a Nicotiana nuclear genome and an Atropa plastome (Kushnir et al. 1987), the plants obtained were totally chlorophyll-deficient. An Atropa nuclear genome and a Nicotiana plastome from these chlorophyll-deficient cybrids were combined with an Atropa or a Scopolia plastome and a Nicotiana nuclear genome, respectively, in control fusion experiments. All of these nuclear genome/plastome combinations gave rise to normal, green plants. Therefore, we conclude that an N. tabacum plastome is incompatible with an A. belladonna nuclear genome.
To isolate resistant cell lines, small pieces of callus (each weighing 50 or 250 mg) were planted on a selective medium containing a borderline streptomycin concentration (0.5 mg ml-1) according to the experiments of Binding4. After 6-8 weeks development of green shoots and proliferation of green callus could be observed in a few cases. Green tissues regularly appeared at a well defined region of inocula.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Genetic recombination between chloroplasts of two flowering plant species, Nicotiana tabacum and Nicotiana plumbaginifolia, after somatic cell fusion is described. The parental lines differed in three cytoplasmic genetic markers. The N. tabacum mutant SR1-A15 was streptomycin-resistant, defective in chloroplast greening, and lincomycin-sensitive. The N. plumbaginifolia mutant LR400 was streptomycin-sensitive, normal green, and lincomycin-resistant. Streptomycin-resistant clones in cell culture are identified by their ability to form a green callus on a selective medium. Streptomycin resistance in the SR1-A15 mutant could not be expressed due to defective chloroplasts. Protoplasts of the two species were fused, and calli grown from the fused population were screened for the expression of streptomycin resistance from the SR1-A15 line as the result of interspecific chloroplast recombination. A somatic hybrid, pt14, expressed a new combination of the cytoplasmic genetic markers. In the pt14 chloroplast genome three N. tabacum and four N. plumbaginifolia parent specific restriction sites have been identified, indicating that the pt14 chloroplast genome contains at least six recombination sites.
Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: evidence for a novel site of streptomycin resistance in the small sub-unit rRNA
- A Gauthur
- M Turmel
- C Lemieux
A. Gauthur, M. Turmel, C. Lemieux, Mapping of chloroplast mutations conferring resistance to antibiotics in
Chlamydomonas: evidence for a novel site of streptomycin
resistance in the small sub-unit rRNA, Mol. Gen. Genet.
214 (1988) 192 -197.