Selection of Glyphosate-Tolerant Tobacco Calli and the Expression of this Tolerance in Regenerated Plants

From nonmutagenized haploid suspensions of Nicotiana tabacum L. cv Wisconsin 38 cells, 51 cell lines capable of growth in the presence of 1 millimolar glyphosate (N-phosphonomethyl glycine) were initially isolated at a frequency of 2.3 × 10⁻⁸. Eighteen cell lines retained tolerance when grown on selective medium for 3 years. Tolerance persisted for at least 14 months in six cell lines cultured in the absence of glyphosate. Some plants regenerated from four glyphosate-tolerant cell lines were tolerant. Glyphosate-tolerant tissue was isolated from some sensitive as well as some tolerant regenerated plants. Six of the tolerant cell lines were also tolerant to the herbicide amitrole (3-amino-1,2,4-triazole). Five cell lines selected for amitrole tolerance were glyphosate tolerant. Some plants regenerated from three of these five cell lines were glyphosate tolerant and glyphosate-tolerant tissue was obtained from several of these regenerated plants. Amitrole uptake in suspension cultures of several variants was assessed in terms of influx rate constants. This parameter was not sufficiently different indicating that altered membrane properties could not account for the herbicide tolerance.     

Stability of salt tolerance at the cell level after regeneration of plants from a salt tolerant tobacco cell line

Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells ( Nicotiana tabacum/gossii ). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants.
The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line.     

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

Regeneration of plants from tissue- and cell suspension cultures of Symphytum officinale L. and effect of in vitro culture on pyrrolizidine alkaloid production

Primary calluses were induced from various organs of Symphytum officinale L. (comfrey) plants on solid MS and B5 medium supplemented with plant growth regulators. The callus was further subcultured on B5 medium. Cell suspension cultures were derived from B5 grown calluses by transfer to liquid B5 medium. Calluses as well as cell suspension cultures could be induced to regenerate whole plants on solid MS medium. Plants regenerated from short term cultures were identical with plants from which cultures were initiated in morphology and chromosome number. Production of pyrrolizidine alkaloids ceased on prolonged subculturing of suspensions although polyamines, which might act as precursors, were still detectable. However, regenerated plants produced the original alkaloids.     

Characterization of alfalfa (Medicago sativa L.) plants regenerated from selected NaCl tolerant cell lines

Selection of stable, NaCl tolerant alfalfa (Medicago sativa L.) cell lines was accomplished by a step-up selection procedure, whereby cell lines originally selected for tolerance at 0.5% NaCl were subsequently selected at 1.0% NaCl. Sodium chloride tolerant cell lines retained tolerance following four subcultures (16 weeks) on control media (0% NaCl). Plants were regenerated from selected NaCl tolerant cell lines of three initial genotypes, one diploid (2n=2x=16) and two tetraploids (2n=4x=32). In addition, plants were regenerated from control cell lines maintained on 0% NaCl media for the same duration. Plants regenerated from NaCl tolerant cell lines were characterized by extensive somaclonal variation compared to plants regenerated from control lines. Morphologically, all plants regenerated from NaCl tolerant cell lines are abnormal and many (44.7%) were extreme dwarfs (maximum height of 5 cm). The grossly aberrant phenotypes prevented an in-depth characterization of many of the plants regenerated from NaCl tolerant cell lines. Most plants regenerated from NaCl tolerant cell lines had unbalanced polyploid chromosome sets with the most extreme cytogenetic variant having 106 chromosomes. In contrast, 98.5% of the plants regenerated from control cell lines were euploid (85% were tetraploid, 15% were octoploid). Isozyme phenotypes of the plants from NaCl tolerant cell lines were also extensively altered, compared to plants from control cell lines. In vitro NaCl tolerance was maintained following plant regeneration for nine of the 12 regenerants tested. Importantly, whole plant NaCl tolerance was expressed in two of the seven regenerated plants tested at the whole plant level; however, only one of these plants has flowered and is both male and female sterile; the other plant has never flowered. Although NaCl tolerant alfalfa cell lines are efficiently selected, the extensive somaclonal variation that accompanied the selection was a deterrent to successful recovery of heritable NaCl tolerance.     

Selection for NaCl tolerance in cell cultures of Medicago sativa and recovery of plants from a NaCl-tolerant cell line

Medicago sativa lines with a high incidence of regeneration were established as suspension cultures and used to select for NaCl tolerant lines. Attempts were then made to regenerate plants from these lines. Regeneration was severely depressed in NaCl tolerant calli and the only plants that were successfully regenerated were from one callus of M. sativa cv. Regen S which grew in 62.5 mM NaCl. Plants from this callus, and new calli derived from the recovered plants, have shown a tolerance to NaCl comparable to calli and plants from the initial seed stock rather than an improved level of tolerance.     

Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants

Efficient and reproducible selection of transgenic cells is an essential component of a good transformation system. In this paper, we describe the development of glyphosate as a selective agent for the recovery of transgenic embryogenic corn callus and the production of plants tolerant to Roundup^® herbicide. Glyphosate, the active ingredient in Roundup^® herbicide inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and thus prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites in plants. A maize EPSPS gene has been cloned, mutated to produce a modified enzyme resistant to inhibition by glyphosate, and engineered into a monocot expression vector. In addition, a bacterial gene which degrades glyphosate (glyphosate oxidoreductase, or GOX) was also cloned into a similar expression vector. Stably transformed callus has been reproducibly recovered following introduction of mutant maize EPSPS and GOX genes into tissue culture cells by particle bombardment and selection on glyphosate-containing medium. Plants have been regenerated both on and off glyphosate selection medium, and are tolerant to normally lethal levels of Roundup^®. Excellent seed set has been obtained from both self and outcross pollinations from both sprayed and unsprayed regenerated plants. Progeny tests have demonstrated normal Mendelian transmission and tolerance to the herbicide for some of the transgenic events.     

Isolation and characterization of isonicotinic acid hydrazide-resistant mutants of Nicotiana tabacum

Summary Twenty stable variant lines resistant to isonicotinic acid hydrazide (INH), an inhibitor of the conversion of glycine to serine in the glycolate pathway, were isolated in cell cultures initiated from allodihaploid Nicotiana tabacum. Plants were regenerated from 13 of these lines and explants were tested for resistance. For some lines virtually all of the regenerated plants scored as resistant; for others a mixed population of sensitive and resistant plants were obtained. One or more plants from 5 lines were fertile, presumably as a result of spontaneous diploidization of cells in the plant or culture. Callus initiated from the seed progeny of these plants was resistant to INH confirming the characteristic as a stable mutation. Seedlings from all INH-resistant plants were small and slow-growing, but the slow-growth trait could be separated from resistance in backcrosses of hybrids. In one case (line I21) crosses with sensitive lines show the resistant trait in that line to be dominant.     

In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants

A stable salt-tolerant potato cell line, able to grow on media containing 60–450 mM NaCl (i.e. low to high salinity) was selected. Callus grown on 120 or 150 mM NaCl showed higher fresh weights than the rest of the treatments. Replacing NaCl by KCl or Na2SO4 showed that reductions in fresh weight were mainly due to the presence of Na+ ions. When PEG 6000 was added to the medium instead of salt, the salt tolerant cell lines were unable to overcome the PEG-induced water stress. Whole plants, regenerated from salt tolerant callus, exhibited salt stress tolerance as evidenced by their higher fresh and dry weights when watered with 90 mM NaCl, and they also produced more tubers per plant under salt stress. Salt-tolerant plants differed phenotypically from control plants both in terms of leaf shape, tuber flesh and skin colour, which was reddish. In addition, DNA fingerprinting by RAPDs, with 70 different primers, confirmed that the salt tolerant regenerants also differed genotypically from the control, salt sensitive Kennebec potato plants from which they had been selected. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of salt tolerant alfalfa (Medicago sativa L.) plants regenerated from salt tolerant cell lines

Salt tolerant cell lines have been selected from Medicago sativa, by a single step selection process on tissue culture medium containing 1% NaCl. Plants regenerated from these lines show improved salt tolerance compared to parent plants. The regenerated plants are vigorous, have flowered and are self fertile. The cellular salt tolerance characteristic can be passaged through the regenerated plants, since callus cultures initiated from immature ovaries of the salt tolerant regenerated plants are salt tolerant without additional selection on 1% NaCl. Several of these second generation callus cultures have been regenerated to produce vigorous plants which maintain the salt tolerance characteristic. The tolerance phenotype appears dominant in seeds obtained from self fertilization of the tolerant plants. The regenerated salt tolerant plants are therefore a valuable source as genotypes in plant breeding for salt tolerance and isolation, identification and manipulation of genes which confer salt tolerance in alfalfa.Abbreviations SH Schenk and Hildebrandt medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.     

Physiological aspects of glyphosate degradation in Alcaligenes spec. strain GL

Alcaligenes spec. strain GL (IMET 11314) is able to grow on glyphosate (N-[phosphonomethyl]glycine) and other phosphonates as sole source of phosphorus. Degradation of glyphosate to inorganic phosphate and sarcosine by this strain is subject to several regulatory principles. While uptake and dephosphonation of glyphosate are regulated by P_i starvation, the intensity of glyphosate degradation is also controlled by the cellular ability to utilize the C-skeleton derived from glyphosate. Depending on the external concentration of glyphosate, the liberated sarcosine is differentially metabolised. Utilization of the sarcosine moiety and complete incorporation of 3-[¹⁴C]-label of glyphosate into cellular material occur only in cultures adapted to higher concentrations (5 mM) of the herbicide. At low concentrations of glyphosate (1 mM) only the P_i required by the growing cultures is utilized but not the sarcosine. Initially high rates of glyphosate uptake obtained after P_i-starvation decrease in the presence of low glyphosate concentrations. It is suggested that uptake and metabolism of glyphosate are connected with the expression of the sarcosine metabolizing capacity of the Alcaligenes cells.Abbreviation AMPA aminomethylphosphonic acid     

In Vitro selection of groundnut cell lines fromCercosporidium personatum culture filtrates and regeneration of resistant plants through cell culture

Cell suspensions derived from immature leaves of the groundnut (Arachis hypogaea L.) were cultured in the presence and absence ofCercosporidium personatum pathotoxic culture filtrates. Cell viability and reactions of cell lines were determined after exposure to various concentrations (25–100%, v/v) of the filtrates. Cell lines have been selected for resistance to the toxin(s) produced byC. personatum. Selected cell lines were used for plant regeneration on regeneration media containingC. personatum culture filtrates. Plant regeneration frequency was found to be low in long-term cultures, whereas it was high in short-term cultures. The selfed progeny of the plants regenerated from the resistant cell lines showed resistance to the pathogen in the field. Six out of 82 plants exhibited enhanced resistance in the R₂ generation. The culture filtrate stimulated callus proliferation as well as plant regeneration at lower concentrations, a response that could prove to be very useful for obtaining disease resistant plants throughin vitro selection.     

Selection of ethionine-resistant variants with increased accumulation of methionine from embryogenic protoplasts of the forage legume <Emphasis Type="Italic">Astragalus adsurgens</Emphasis>

In vitro selection was carried out to obtain ethionine-resistant plants with increased contents of free methionine in the vegetative tissues of the forage legume Astragalus adsurgens Pall. Three-week-old cell colonies were derived from protoplasts mutagenized with N-methyl-N-nitrosoguanidine from embryogenic callus and were selected with 0.6mM ethionine. Four colony lines were isolated and their resistance to ethionine was 7–8 times that of the wild-type callus. No plant regeneration occurred on these colony lines in the differentiation medium containing ethionine. Only one colony line (R-1) regenerated plants through somatic embryogenesis in the absence of ethionine. Stem and leaf segments from the regenerated plants showed the same potential to produce callus in the presence of ethionine as in the absence of ethionine. The formed callus kept continuously growing in ethionine-containing medium. Free amino acid analysis revealed that colony line R-1, its regenerated plants and callus from the regenerated plants accumulated methionine at levels at 5–9 times higher than in wild-type. These results suggested that ethionine resistance and methionine over-accumulation were also expressed at plant level. Thus, the obtained resistant colony line that could regenerate plants with over-accumulation of methionine might provide an alternative approach to improve the nutritional quality of this forage.     

Expression of 5-Methyltryptophan Resistance in Plants Regenerated from Resistant Cell Lines of Datura innoxia

Seventy-nine 5-methyltryptophan-resistant cell lines have been selected from haploid Datura innoxia Mill. cell cultures by plating suspensions in agar medium containing a growth inhibitory concentration of 5-methyltryptophan. Mutagen treatment increased the frequency of resistance. The eleven variants tested posses an altered anthranilate synthase less sensitive to feedback inhibition by tryptophan. All five of the variants which were analyzed for free amino acids contained elevated levels of free tryptophan (8 to 30 times the wild type level). None of the selected cell lines were auxin-autotrophic. Resistance to 5-methyltryptophan, altered anthranilate synthase, and high free tryptophan (4 to 44 times) were also expressed in leaves of plants regenerated from the variant lines and in cultures reinitiated from the resistant plants. These results show that the amino acid overproduction phenotype can be selected at the cellular level of organization and be expressed identically in whole plants regenerated from the selected cells.     

Aluminum tolerance in maize is correlated with increased levels of mineral nutrients, carbohydrates and proline, and decreased levels of lipid peroxidation and Al accumulation

We investigated the uptake of aluminum (Al) and transport to shoots in two inbred maize lines (Zea mays L., VA-22 and A(4/67)) differing in Al tolerance. Seedlings were grown for 7 days in hydroponic culture with nutrient solution that contained 0, 240, 360, and 480muM Al at pH 4.2. After 7 days of exposure to Al, roots of sensitive maize line (A(4/67)) plants accumulated 2-2.5 times more Al than roots of tolerant line (VA-22) plants. Inductively coupled plasma atomic emission spectrometry (ICP-AES) showed that the tolerant line retained higher concentrations of Ca(2+), Mg(2+), and K(+) compared with the sensitive line. In response to Al treatment, proline (Pro) concentration increased three-fold in roots of tolerant plants, while a slight increase was observed in roots of sensitive-line plants. A substantial carbon surplus (two-fold increase) was observed in roots of the Al-tolerant maize line. Carbohydrate concentration remained almost unchanged in roots of Al-sensitive line plants. Al treatment triggered the enhancement of lipid peroxidation in the sensitive line, while no change in lipid peroxidation level was observed in the tolerant maize line. These data provide further support to the hypothesis that a mechanism exists that excludes Al from the roots of the tolerant maize line, as well as an internal mechanism of tolerance that minimizes accumulation of lipid peroxides through a higher Pro and carbohydrate content related to osmoregulation and membrane stabilization.     

The use of cell membrane stability (CMS) technique to screen for salt tolerant wheat varieties

Cell membrane stability (CMS) technique was used to screen salt tolerant (V1, V2), salt sensitive (V5) and two salt/water deficiency tolerant wheat genotypes (V3 and V4) using 100-250 mM NaCl salinity maintained in pots containing gravel and nutrient solution. The objectives were to study: (i) the reliability of CMS technique for screening wheat under high salinity, (ii) factors that impart stability and/or injury to the cell membrane, and (iii) the relationship of CMS with other physiological parameters affected by the salt stress. Generally, cellular injury increased with increasing salinity levels. In V5, it was the highest (74.2%) at 250 mM, probably due to combined effect of Na+ toxicity and low (54%) relative water content (RWC). In V1, RWC was similar to that in V5 but injury was comparatively low possibly due to low concentration of Na+. The difference between V1 and V2 was significant, either due to the highest concentration of K+ or the lowest reduction in RWC in V2. In V3 and V4, injury was the lowest at all salinity levels and was within the range of values observed earlier for drought tolerance. A significant negative correlation was detected between cellular injury and RWC for V1 and V5 but not for V3 and V4. Cellular injury also showed a significant positive correlation with Na+ and a negative correlation with K+ and grain yield (GY). It appeared that CMS technique is suitable for screening wheat under high salinity levels and for detecting differences that may arise due to cumulative effects of salinity and reduced water contents.     

Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn

Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.     

Chromosome number and nuclear DNA content of plants regenerated from salt adapted plant cells

Plants regenerated from tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells that were adapted to 428 mM NaCl were found to have hexaploid or near-hexaploid chromosome numbers compared to the normal tetraploid, 2N(2C)=4X=48 chromosome numbers of plants regenerated from unadapted cells. Even though cells with chromosome numbers other than hexaploid were found in the cell population only hexaploid plants were regenerated. The hexaploid condition may impart some karyotypic stability that allows more efficient morphogenic activity. The hexaploid condition could not be correlated with several phenotypic alterations associated with plants regenerated from adapted cells, including male sterility and increased salt tolerance.