Salt tolerant sugarbeet progeny from tissue cultures challenged with multiple salts

Lines of sugarbeet (Beta vulgaris L.) tolerant of multiple salts was accomplished by an in vitro multiple salt challenge. Petioles were placed on RV medium amended with 5 different salts along with Murashige and Skoog base salts for one month. Surviving shoots were cultured on RV medium to obtain petioles for subsequent challenges. During the first, second and third challenges, organogenically regenerated shoots developed from 5%, 46%, and 80% of the petioles, respectively. After the third multiple salt challenge, tolerant shoots were rooted and transplanted in soil. Salt was added to this soil at 1.0% by weight and plants were observed for 2 months. The ten most salt tolerant plants were vernalized to obtain seed. The R₁ seed and controls were planted in soil containing 0%, 0.61% or 0.77% multiple salts per dry soil weight. Emergence of R₁ seedlings was significantly greater than the controls under salt stress. Multiple salt tolerant R₁ plants were maintained in salt amended soil to the 8–10 leaf stage and appeared as healthy and vigorous as the control growing in salt free soil.Contribution from Missouri Agricultural Experiment Station Journal Series No. 10948. University of Missouri, Columbia, MO, USA Mention of trade names does not constitute a guarantee or warranty of the product by University of Missouri of Holly Sugar Corporation and does not imply their approval to the exclusion of other products.     

Somaclonal variation in soybean plants regenerated from tissue culture

Callus cultures of soybean (Glycine max (L.) Merr.) genotypes PI 88788, PI 438489B, and cultivar Bedford were initiated in vitro from seedling explants consisting of the cotyledonary node plus epicotyl from germinated mature seed. Plants were regenerated from these callus cultures and subsequently evaluated for qualitative variation in three to four subsequent generations. Variant phenotypes observed that have not been previously reported from tissue culture include lanceolate leaves, leaf variegation (chimeral variegated plants), pod variegation on otherwise normal plants, and change in growth habit from indeterminate to determinate. The lanceolate leaf, chimeral variegated plant, and change from indeterminate to determinate growth habit characters were inherited through at least three generations (R₀-R₂), and segregation occurred in each generation. Pod variegation was inherited through the two generations tested thus far and segregation occurred in each generation. No variation was observed in control plants derived from normal seed. Variants appeared more frequently in regenerants from PI 88788 and PI 438489B than from Bedford. These results confirm and extend the finding that certain tissue culture techniques may be used to induce novel plant formation from somatic tissue of soybean.Missouri Agricultural Experiment Station, University of Missouri, Columbia, Missouri, USAMention of tradenames does not constitute a guarantee or warranty of the product by University of Missouri or USDA-ARS and does not imply their approval to the exclusion of other products.     

Somaclonal variation in plants regenerated from cultures of soybean

Plants were regenerated from embryogenic and organogenic cultures derived from immature embryos of nine soybean (Glycine max L. Merr.) genotypes and extensive qualitative variation was noted in different regenerated families. Three lethal sectoral albinos were seen in the regenerated plants (R₀). Variants observed in later selfed generations included twin seeds, multiple shoots, dwarfs, abnormal leaf morphology, abnormal leaflet number, wrinkled leaves, chlorophyll deficiency, partial sterility and complete sterility. The frequency of possible mutations ranged from 0 to 4% in R plants as determined by studies of corresponding R₁, R₂, R₃ and R₄ families. No significant differences were seen in the frequencies of possible mutations for embryogenic as compared to organogenic culture derived plants. Chlorophyll deficiency, sterility and wrinkled leaf traits were followed in two or more generations and showed that these traits were inherited stably. The known traits of this nature are controlled by single recessive nuclear genes. Other traits occurred more randomly and not in all generations. The genetic basis of the random variation is not known at the present time. This study indicates that heritable somaclonal variation does occur in tissue culture derived plants of soybean.Abbreviations R₀ Original regenerated plant - R₁ Selfed seeds of R₀ plants - R₂ Selfed seeds of R₁ plants - R₃ Selfed seeds of R₂ plants This research was supported by funds from the Illinois Agricultural Experiment Station and Agrigenetics Inc.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

Salt tolerance improvement in some wheat cultivars after application of in vitro selection pressure

Somatic embryogenesis through callus initiation has been quantified under salt stress conditions for 8 wheat cultivars currently cultivated in Morocco. The cultivars were classed according to the mean number of somatic embryos formed per immature embryo half and regenerated plants per 100 explants under saline conditions. Regenerated plants from control callus (R_0–0) and callus initiated on 10 g l^–1 NaCl (R_0–10) did not show significant differences concerning plant height, spike length and grain number per ear but, the R₀ plants remained less developed than parent plants. When watered with a solution containing more than 20 g l^–1 NaCl, the seeds of cultivar Te derived from R_0–10 regenerated plants exhibited the best elongation of roots and coleoptiles. Furthermore, a chlorophyll fluorescence test showed a clear improvement in salt tolerance of R_0–10 plants at four to five-leaf stage, compared to R_0–0 plants. It is concluded that plant regeneration from callus initiated on high NaCl levels may be a valid method of selection for salt tolerance.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants

Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA₄₄₀₄ strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was carried out on more than 1300 leaf explants from each cultivar. Generally, `Induka' plants characterized with higher regeneration potential than `Elista'. The highest number of regenerated shoots was obtained on MS medium with 0.4 mg l ^–1 IBA and 1.8 mg l^–1 BA (3.5 and 1.8 shoots/explant for `Induka' and `Elista', respectively). After plant transformation number of regenerated, transgenic shoots was higher for `Elista' (on the average: 8.3 shoots/100 explants). The number of transgenic `Induka' shoots, obtained at the same conditions, was twice lower (4.2). Simultaneously `Induka' plants needed higher kanamycin concentration for transgenic explants selection than `Elista' (25 mg l^–1). Preliminary incubation of A. tumefaciens in LB or MS medium with acetosyringone and IAA resulted in increasing transgenic shoots number (per 100 explants: `Induka' 4.5, `Elista' 8.0–9.5 shoots). After using untreated bacteria for plants' transformation, number of transgenic plants varied (dependently on cultivar) from 3.8 to 7.0/100 explants. Applying LB or MS as basic medium as well as adding tobacco plant extract to these media did not significantly influence transformation efficiency.     

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     

An efficient method for in vitro regeneration from immature inflorescence explants of Canadian wheat cultivars

Fertile, green plants were regenerated from immature inflorescence explants from each of four Canadian wheat cultivars. The cultivars were representative of four classes of Canadian wheat. Explants from immature inflorescences of three size ranges were cultured on two types of media: MSI/MSR, which contains 1650 mg l^-1 NH₄NO₃and sucrose as a carbon source, and BII/BIR, which contains 250 mg l^-1 NH₄NO₃and maltose as a carbon source. Regeneration from all cultivars was significantly better on BII/BIR media than on MSI/MSR media. On BII/BIR media, `AC Karma', `Plenty', and `Fielder' gave the highest number of shoots per 10 explants, where the explants were derived from immature inflorescences 5.1 to 10.0 mm in length. 'Columbus' did not regenerate on MSI/MSR medium, and regenerated poorly on BII/BIR medium. Differences were found between cultivars with regard to the number of regenerant plants produced with the best treatments: `Plenty' produced 16.1 shoots per 10 explants, `AC Karma' 12.4, `Fielder' 6.4, and `Columbus' 2.2.     

Plant regeneration from cotyledonary node explants of mungbean (Vigna radiata (L.) Wilczek)

Sexually-mature mungbean (Vigna radiata (L.) Wilczek) plants were efficiently regenerated from cotyledonary node explants. The explants were capable of directly developing multiple shoots on basal media devoid of any growth regulators. The shoot multiplication was influenced by media composition, growth regulators, age of donor seedling and explant type. The explants with both the cotyledons attached to the embryonic axis excised from 4-d-old seedlings, produced the highest number of shoots (5 or 6) in 100% of the cultures within 2 weeks on B₅ basal medium (BBM) containing BAP or 2-iP, respectively, (at 5x10^–7M) and 3% sucrose. Shoots elongated and developed better using BAP. Increasing micronutrients, carbohydrate and nitrogen levels in the medium above the original formulation of B₅ basal medium appeared to be of no benefit for increasing the number of shoots. The shoots were rooted on basal MS medium or MS containing 10^–6 of NAA, IAA or IBA. This protocol was found applicable to six other cultivars of mungbean. One hundred rooted shoots were successfully established in soil in the glasshouse, where 90% of them survived. The regenerated plants flowered precociously, but produced normal pods and viable seeds.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - 2-iP 6- — -dimethylallyl aminopurine - AdS adenine sulphate - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium - B₅ Gamborg et al. (1968) medium - C medium MS salts + B₅ vitamins     

Transformation of tomato using an Ri plasmid vector

An intermediate vector, pAMNeo10, was constructed containing the replication origin and carbenicillin-resistance gene of pBR322, an homology region to allow insertion into the T_L-DNA of pRiA4 in Agrobacterium A4T, and a chimaeric kanamycin-resistance gene (nop. neoΔ) for identification of T_L-DNA::pAMNeo10 transformed roots. Roots produced by inoculating stem explants of Lycopersicon esculentum, L. hirsutum × L. esculentum (KNVF Rootstock) and L. peruvianum with an exconjugant stain, A4T (pRiA4::pAMNeo10), were resistant to kanamycin at levels that completely inhibit the growth of transformed roots produced with wild-type A4T. When transformed by the exconjugant strain, roots of the three tomato hosts were resistant to different levels of kanamycin, and, in the case of L. peruvianum, regenerated plants were tolerant to much higher levels (10×) of kanamycin than the transformed roots from which they were derived. Kanamycin-resistant transformed roots expressed aminoglycoside phosphotransferase activity, and Southern blotting confirmed the presence of the intermediate vector sequence in transformed roots and in shoots of regenerated plants. T_R-DNA was shown to be present in most transformed roots and regenerated shoots by testing for agropine and mannopine. The application of Ri plasmid vectors to the study of foreign gene expression in plants is discussed.     

Efficient in vitro regeneration of leek (Allium ampeloprasum L.) via flower stalk segments

Summary A new simple, efficient and rapid in vitro method for mass clonal propagation of leek (Allium ampeloprasum L.) plants, using small (5 mm) flower stalk (peduncle) explants, was established. Adventitious shoots were produced from single subepidermal cells. A wide variation in the percentage of regenerating explants and number of regenerated shoots per explant between individual plants within one cultivar was observed. The concentration of the growth regulators 6-benzylaminopurine and -naphthalene-acetic acid influenced the percentage of regenerating explants and the average number of regenerated shoots per explant. A combination of 10 mg.l^–1 6-benzylaminopurine and 10 mg.l^–1 -naphthalene-acetic acid, resulted in a maximum percentage of regenerating explants and a high average number of regenerated shoots per explant. The percentage of regenerating explants and the average number of regenerated shoots per explant decreased with increasing flower stalk length (age). The basal explants gave both the highest percentage of regenerating explants and average number of regenerated shoots per explant. An average of 300 shoots per flower stalk was obtained for all plants, making this new in vitro method a powerful tool in hybrid leek breeding.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

In vitro regeneration and propagation of chickpea (Cicer arietinum L.) from meristem tips and cotyledonary nodes

Summary Two representative cultivars ofCicer arietinum, the desi-type cv.Annigeri and the kabuli-type cv.ICCV6, were regenerated in vitro and clonally propagated from cotyledonary nodes and meristem tips. The explants were dissected from 1-wk-old seedlings aseptically germinated on WH medium. In both cultivars, all nodes cultured on B5 medium supplemented with 4.4μM 6-benzylaminopurine developed up to seven shoots per node within 3 wk. Meristem tips were much better suited for multiple shoot formation. Cultured on DKW-C-a medium supplemented with 4.4μM 6-benzylaminopurine and 0.05μM indole-3-butyric acid, 96% of the meristem tips produced up to 10 shoots per explant. A new method in improving clonal propagation was subdividing the meristem tips. Doing so, multiple shoot formation was considerably enhanced: up to 90 shoots per original explant could be obtained with cv.Annigeri, and up to 50 with cv.ICCV6. Indole-3-butyric acid proved to be the best rooting factor. From several media tested, the best root induction and development was achieved on WH medium supplemented with 2.5μ M indole-3-butyric acid: 72% rooting with cv.Annigeri and 68% rooting with cv.ICCV6. With both cultivars there were no differences in rooting capacity between shoots of nodal origin and those derived from meristem tips. The plantlets obtained were transferred into soil and kept under greenhouse conditions. The survival frequency was 28% with cv.Annigeri and 23% with cv.ICCV6. R₀ plants remained smaller than seed-grown controls and produced only a few fertile seeds. There was no difference between R₁ plants and controls in growth, development, and seed set.     

High efficiency plant regeneration and genetic fidelity of regenerants by SCoT and ISSR markers in chickpea (Cicer arietinum L.)

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l^?1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l^?1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l^?1 BAP along with 0.05 mg l^?1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l^?1), GA₃ (1.0 mg l^?1) and BAP (1.0 mg l^?1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l^?1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation.

     

Shoot regeneration and free-radical scavenging activity in <Emphasis Type="Italic">Silybum marianum</Emphasis> L.

The morphogenic potential and free-radical scavenging activity of the medicinal plant, Silybum marianum L. (milk thistle) were investigated. Callus development and shoot organogenesis were induced from leaf explants of wild-grown plants incubated on media supplemented with different plant growth regulators (PGRs). The highest frequency of callus induction was observed on explants incubated on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ 6-benzyladenine (BA) after 20 days of culture. Subsequent transfer of callogenic explants onto MS medium supplemented with 2.0 mg l⁻¹ gibberellic acid (GA₃) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) resulted in 25.5 ± 2.0 shoots per culture flask after 30 days following culture. Moreover, when shoots were transferred to an elongation medium, the longest shoots were observed on MS medium supplemented with 0.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, and these shoots were rooted on a PGR-free MS basal medium. Assay of antioxidant activity of in vitro and in vivo grown tissues revealed that significantly higher antioxidant activity was observed in callus than all other regenerated tissues and wild-grown plants.     

RETRACTED ARTICLE: Influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types

A comparative performance of two explants types (CN and Nodal) for their efficiency to induce multiple shoot regeneration in Clitoria ternatea has been carried out. Thidiazuron (TDZ) in different concentrations (0.05–2.5 μM) was used as a supplement to the Murashige and Skoog’s (MS) basal media. Explant type apart, two factors viz. concentration and exposure duration to TDZ played an important role in affecting multiple shoot regeneration. Cotyledonary node explants produced the best results at 0.1 μM TDZ, while in nodal explants the highest rate of shoot formation was achieved on MS medium supplemented with 1.0 μM TDZ. In both the explants, shoot multiplication increased when the regenerated shoots were subcultured on hormone free MS medium after 4 weeks of exposure to TDZ. Among the two, cotyledonary node explants produced considerably higher number of shoots at a comparatively lower concentration of TDZ than nodal explants. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA) and were successfully established in pots containing garden soil with 88 % survival rate. All the regenerated plants showed normal morphology and growth characteristics.     

In vitro clonal propagation of annatto (Bixa oreliana L.)

Summary A protocol for in vitro propagation of Bixa orellana is described. Plants were regenerated from shoot apex and nodal explants on B5 medium supplemented with 4.9 μM 2-isopentenyl adenine. The multiplication factor of shoot apex explants was higher (nine shoots per explant) than that of the nodal explants (five shoots per explant). Regardless of the position of the nodes, all the nodal explants gave similar responses. However, the size of the nodal explant was an important factor in producing multiple shoots: 0.5 cm nodal explants produced the maximum multiple shoots. Regenerated shoots from shoot apex explants rooted best on MS medium supplemented with 0.05 μM α-naphthalene acetic acid (NAA). whereas shoots regenerated from nodal explants needed 2.7 μM NAA for rooting. Eighty per cent survival of in vivo transferred plants occurred on the best potting substrate, coco peat. Since the multiplication factor was nine per explant, this protocol can be use for commercial microprogation. However, the regeneration capacity declined after 10 subcultures. Approximately, 3350 rooted plants could be generated in 10 mo. after eight subcultures, from one shoot with a shoot apex and four nodes.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l⁻¹ activated charcoal, 1.5 mg l⁻¹ phosphinothricin, and 300 mg l⁻¹ cefotaxime. Phosphinothricin at 1.5 mg l⁻¹ was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l⁻¹ phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l⁻¹ phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.