Cucurbit Genetics Cooperative Report 14:34-42 (article 13) 1991
S. Malepszy, K. Witkowski and S. Zgagacz
Warsaw Agricultural University, Dept. Genetics & Hort. Plant Breeding, Nowoursynowska 166, 02-766 Warszawa, Poland
In vitro culture techniques can be employed to improve cucumber (5), including selection of mutants for herbicide resistance. That would be desirable due to high cucumber sensitivity to those substances and because to date there is only one case of cucumber resistance to herbicides (10).
This study was aimed at obtaining metribuzin or linuron resistant cucumber mutants by means of mass selection in suspension cultures.
Methods. The experiments were carried out on a callus and cell suspension of two cucumber inbred lines: Borszczagowski (B) and Gy 3 (G). They were obtained by a method described previously (5). The plating density was 5×105 cells/ml, and 2 ml of cell suspension employed in the selection was exposed to the action of 1 µM N-nitroso-N-ethyl-urea. The mutagen was applied in the last phrase of cell suspension culture (before plating) along with and adequate amount of solution into a conical flask. After 12 hours, the culture was filtered, centrifuged and plated. The application of mutagen decreased in inoculation efficiency by approximately 50 %.
Lethal concentrations of herbicides for the callus were established by placing 100 mg of callus tissue on the medium with different concentrations of pure active substance: 150, 80, 40, and 20 mg/l of metribuzin (M) and 100, 50, 25 and 12.5 mg/l of linuron (L). Five calli were spread over the surface of approximately 25 ml of the medium in 80mm Petri plates. After 3 weeks of culture in diffuse light (approximately 800 lux) at day temperatures of 27 ±1°C under a 16 hour photoperiod and at night temperatures of 22 ± 1°C the calli were weighed (to ± 3 mg).
Two concentrations of herbicides (higher and lower) of each growth regulator were used for the selection of resistant colonies (Table 1). Seven days after inoculation the medium was supplemented with agarose and then, after 14 days in culture-growing colonies, resistant variants were observed. Individual resistant colonies were transferred onto agar medium with adequate herbicide active substance added. Linuron-resistant variants were placed on fresh medium supplemented with 40 mg/l metribuzin. After 2 weeks in culture, the herbicides were reduced to 20 mg/l linuron and 85 mg/l metribuzin.
The callus of resistant variants was used in further experiments to determine the resistance level and the presence of cross resistance. The resistance level was examined by comparing an increase in fresh weight of the callus after 3 and 6 days in culture on the medium supplemented with 150 and 300 mgxl-1 M or 50 and 80 mg/l L. Shoot regeneration ability was examined on the media containing 20 or 85 mg/l of active substances L or M respectively, according to the methods described for cucumber (5, 6).
Root regeneration ability was studied on media with the following hormone concentrations (mg/l: IAA-0.5; BAP-0.05; IAA-5, BAP 0.5; 2,4 -D – 1.2, BAP -0.3; NAA-3, BAP-0.05). Five to seven small calli of 200 to 250 mg were tested.
In the selected lines, resistance stability was investigated by comparing the intensity of callus growth obtained from regenerated shoot leaves of the resistant line with the intensity of callus growth of this line before regeneration took place.
In all experiments on callus growth intensity, the result was the average of 4 to 7 independent measurements (callus samples, depending on the experiment, amounted to 60 or 100 mg).
Various methods were applied to induce roots: in vitro cultures, in perlite, peat, or perlite-peat mixtures. Mineral salts and pH were identical to those applied in the cultivation of seed derived cucumbers. Shoot grafting of resistant lines were carried out on Cucurbita ficifolia or young cucumber plants.
Results. The effect of linuron and metribuzin on fresh weight growth in B and G callus cultures was examined to establish adequate selection concentrations. The application of 150 mgxl-1 M, and 50 mgxl-1L brought about a 90% growth reduction after 3 weeks in culture (Table 1), whereas no growth was found after 6 weeks (data not shown). On the basis of the above results, the following concentrations of herbicide active substances for the selection of resistant lines in the suspension were chosen: 120 mg/l and 150 mg/l for metribuzin, 60 mg/l and 40 mg/l for linuron. Plating efficiency (PE) of the control on herbicide-free medium after 3 weeks amounted to 1.8% and approximately 50% reduction of PE was noted when the mutagen was added to the medium.
A week after plating, each Petri dish was supplemented with cell suspension medium having 2 % agarose (low gelling – 0.5 ml per plate). After the next 2 weeks, the number of initially resistant variants was established. Callus pieces (at least 2 mm in diameter) were treated as variants (Table 2). Resistant variants were obtained only after mutagen treatment. Their number was not affected by the concentrations of herbicide active substances during resistance selection to L. Resistant selection of M, however, showed a slightly higher number of variants at the lower concentration, and differences between G and B lines were noted. An average of 3.05 and 2.15 variants per plate was isolated in the B line, and 1.6 and 1.5 in the G line at the concentrations of 120 and 150 mgxdm-3 respectively. On the whole, 17 M resistant variants and 15 L resistant variants were isolated in the B line, 12 and 9 respectively in the G line.
An analysis of resistance to a given herbicide was carried out in a selected group of variants only in the B line. Two active substance concentrations- the same as in the selection and two (M) or 0.6 times (L) higher, were employed. The results obtained (Table 3) show that individual variants exhibited a very differentiated resistance level. At the lower active substance concentration, some variants were developing as in the herbicide-free medium. Most variants, however, showed a certain growth inhibition. At the higher concentrations the difference were subject to the kind of herbicide used. All metribuzin-resistant variants exhibited poor growth whereas the reduction level was different in individual lines. In some L-resistant variants, however, callus growth was identical at both concentrations. The measurements taken after 6 weeks in culture more clearly show line reactions than those after 3 weeks.
Shoot regeneration and the ability to form somatic embryos from callus were markedly reduced in most variants. Above all, this affected M-resistant variants (Table 4). Thirty percent of them failed to develop embryos at all, whereas 20% formed only sporadically. All L-resistant variants formed somatic embryos and only one of them failed to develop shoots.
The embryos of some variants were not capable of further growth, and formed neither shoots nor plants. None of the shoots obtained were phenotypically similar to the control (achieved without herbicide selection). Variants shoots were very compact with a large number of small, dark-green leaves and easy branching. They usually formed no roots, but some had a few which failed to develop when transferred to perlite or peat. Flower buds were often noted which, when removed, resulted in shoot death.
Significant phenotypic differences among the lines can easily be seen in the regeneration characteristics of some of them: M1 had thickened, fasciated petioles, cabbage-like growth; branching average compared to line 3; plants aged rapidly; lower leaves became yellow and, when removed, the next leaves grew and took the same color. M2 had small, not fasciated plantlets of average growth formed with characteristic almost triangular shape of hairy leaves; plants developed a relatively good root system. M16 had shoots, with thick fasciated petioles and very small leaves; aged rapidly. L33 regenerated best on the media used; developed most embryos and grew best; bud formation in leaf axils was so intense that, in 10 days, an individual plant formed a multi-shoot rosette. L7 was very compact with hairy leaves and poor branching.
Since rooting was impossible to achieve, grafting on C. ficifolia and young cucumber plants was attempted. This, however, proved unsuccessful, mostly because the shoot and the stock failed to grow together. In the callus of resistant variants, root formation ability was selected for, but none formed a root system. The callus, however, which did not undergo selection, was found to form roots very easily on the media used.
Shoots regenerating from resistant variants were not capable of regular growth, which made it impossible to carry out investigations on inheritance of resistance to L and M. Attempts, however, were made to establish stability of this characteristic. They were of two types. In type 1, the callus of resistant variants was maintained on L- and M-free medium for 6 weeks and transferred to a medium added with the herbicides. The growth intensity was then compared (retesting). In type 2, the callus was obtained from shoot leaves of some variants (R1 generation) and its growth intensity was established on a medium supplemented with an adequate herbicide (Table 5). The investigations of cross resistance showed that all M- resistant variants were also, to a greater or smaller degree, resistant to L and vice versa (Table 6).
Discussion. Different resistance levels of herbicide resistant variants were described in other plant species, for example 2, 4-D resistant Lotus corniculatus (Swanson and Tomes, 1980), paraquat resistant Ceratopteris richardii (3) and in 2, 4-D resistant Citrus sinensis (9).
Cross resistance was observed in the cucumber variants since some linuron resistant variants were metribuzin resistant and vice versa. The cross resistance level was differentiated: from very high (M2, M4 and L2, L5, L7) to medium or low (M12, M20, M13 and L6). No close correlation between metribuzin and linuron resistance was noted. Some variants exhibited high (M1, L2) or low (M12, M22) resistance whereas others, despite the high herbicide resistance level in the selection, had low cross resistance (M113, M14, L6). The cross resistance characteristic manifested itself differently in the callus obtained from plant leaves of R1 plants. In the case of L3 line it remained almost unchanged but it was significantly lower in M16 line.
Cross resistance is generally recognized in the case of structural similarity of chemicals. That was true in paraquat resistant tomatoes where the resistance was found to be correlated with diquat resistance (4). Similarly, Chaleff and Ray (1) noted cross resistance of tobacco mutants resistant to chlorsuphuron and methylsuphometuron two structurally similar urea herbicides. Cross resistance to herbicides belonging to different groups is known to react on the same metabolic pathway, e.g., photosynthesis, respiration (2). Linuron and metribuzin belong to different chemical groups. They, however, inhibit photosynthesis and other processes in cells of sensitive plants.
Regenerants from individual lines were significantly phenotypically different. They were, however, much more uniform within the line. A few lines did not regenerate at all or growing embryos failed to develop into plantlets. Only a few lines (7 or 18) were capable of forming poorly rooted plantlets. None of them, however, were able to grow under normal in vivo conditions. They were dwarf, and many had fasciated petioles and modified leaves. Most of them wee characterized by rich branching but failed to grow achieving an average of 4 to 5 strongly twisted internodes. Growth incapability under normal in vivo conditions could not be overcome through grafting, changing environmental conditions (from phytotron to greenhouse) of the application of chemical substances (rooting substances or gibberellins).
The variants obtained seem to be incapable of root organogenesis and development outside culture. Similarly, an attempt to form roots from R1 resistant callus was unsuccessful. No roots developed on any used media which stimulate root regeneration in unselected cucumber cultures. The failure to induce roots in shoots of the variants resistant to growth regulators has been reported in literature. Muller et al (7) described NAA resistant mutants of Nicotiana tabacum incapable of inducing roots. Mutation was conditioned by one dominant nucleus gene. No other abnormality except incapability for inducing roots and modified leaf morphology was noticed in this study.
Some Citrus sinensis lines lost their ability to regenerate (9) and some N. tabacum lines were characterized by reduced variability and fertility, shortened nodules and rich branching (4).
The resistant variants obtained in this study were not always capable of regeneration, all the shoots possessed a strongly modified phenotype and failed to grow outside the culture. Such responses may be explained in various ways: some authors report that reduced regeneration abilities of selected cells are generally attributed to prolonged culture and the application of mutagens (2, 9). Both factors may have affected our variants. It should be stressed, however, that no variants were obtained without mutagen treatment. The last result and callus resistance obtained from R1 shoots suggested genetic background of selected resistance. The improvement in regeneration and in the quality of the obtained cucumber shoots might be achieved in the following ways: 1) selection of young calli or the application of in vivo- in vitro methods (at last in the case of M); 2) use of shorter culture duration on herbicide-containing media; 3) lower dose of mutagen; 4) use of a two step selection procedure as for the selection of Fusarium wilt resistant cucumber (6). Another cause of reduced mutant regeneration abilities may be of genetic nature such as a pleiotropic effect or correlation with unfavorable mutations (7). It does not seem probable, however, that in such a great number of independent variants, all of them have suffered the same damage.
Summary. The selection of metribuzin or linuron resistant mutants was carried out in suspension cultures of two cucumber lines (B and G) exposed to the mutagenic action of N-nitroso-N-ethyl-urea. Numerous variants of different resistance levels and shoot regeneration abilities were obtained, but only from line B. Generally, the shoots failed to take root or formed very weak rhizoids and exhibited inability to develop outside in vitro conditions. The shoots of all the variants were markedly modified but phenotypically differed among the variants. Generally, they were markedly shorter, diminished and formed rosettes of dark-green leaves. In most variants, cross resistance was exhibited.
Table 1. The growth of fresh callus of Borszczagowski and Gy3 cucumber lines after 3 weeks of culture on media supplemented with various concentrations of active substances of metribuzin or linuron. The values were expressed as percentage control.
Line |
Herbicide active substance |
Concentration (mg/l) |
Callus growth (% of control) |
| Borszczagowski | Metribuzin | 150 | 23.80 |
| Gy 3 | 36.59 | ||
| Borszczagowski | 80 | 51.84 | |
| Gy 3 | 53.77 | ||
| Borszczagowski | 40 | 61.00 | |
| Gy 3 | 74.63 | ||
| Borszczagowski | 20 | 91.28 | |
| Gy 3 | 78.33 | ||
| Borszczagowski | Linuron | 100 | 12.55 |
| Gy 3 | 12.68 | ||
| Borszczagowski | 50 | 21.71 | |
| Gy 3 | 23.27 | ||
| Borszczagowski | 25 | 57.31 | |
| Gy 3 | 50.72 | ||
| Borszczagowski | 12.5 | 71.78 | |
| Gy 3 | 77.04 |
Table 2. Number of linuron and metribuzin resistant cucumber variants isolated 3 weeks after plating of cells on media containing herbicide active substances.
Line |
Herbicide |
Concentration of herbicide active substance (mg/dm3) |
Number of petri dishes |
Number of variants |
| Borszczagowski | Metribuzin | 120 | 5 | 5,2 |
| “ | “ | 120 | 2 | 2 |
| “ | “ | 150 | 4(1*) | 3 |
| “ | “ | 150 | 3 | 3 |
| “ | Linuron | 40 | 4 | 4 |
| “ | “ | 60 | 5(1*) | 4 |
| “ | “ | 60 | 3 | 3 |
| Gy 3 | Metribuzin | 120 | 3 | 0 |
| “ | “ | 120 | 4 | 4 |
| “ | “ | 150 | 5 | 5 |
| “ | “ | 150 | 3 | 3 |
| “ | Linuron | 40 | 3(1*) | 2 |
| “ | “ | 60 | 4 | 4 |
| “ | “ | 60 | 3 | 3 |
| Borszczagowski | Control | – | 5 | 0 |
| “ | “ | – | 5 | 0 |
| “ | Linuron | 40 | 4 | 4 |
| “ | Metribuzin | 120 | 4 | 4 |
| Gy 3 | Control | – | 4 | 0 |
| “ | “ | – | 5 | 0 |
* Number of infected dishes.
Table 3. Callus fresh weight (mg) of line B variants resistant to linuron or metribuzin after 3 and 6 weeks of culture on a medium with or without herbicides.z
Variant |
Herbicide concentration (mg/dm3) |
Fresh weight (mg) after 3 weeks |
Fresh weight (mg) after 6 weeks |
| Metribuzin | |||
| M1 | 150 | 279 | 710 |
| “ | 300 | 195 | 249 |
| M2 | 150 | 128 | 228 |
| “ | 300 | 104 | 132 |
| M4 | 150 | 221 | 433 |
| “ | 300 | 202 | 342 |
| M6 | 150 | 279 | 601 |
| “ | 300 | 236 | 365 |
| M8 | 150 | 246 | 383 |
| “ | 300 | 184 | 272 |
| M12 | 150 | 248 | 350 |
| “ | 300 | 160 | 176 |
| M13 | 150 | 243 | 478 |
| “ | 300 | 154 | 188 |
| M14 | 150 | 278 | 667 |
| “ | 300 | 123 | 107 |
| M16 | 150 | 326 | 517 |
| “ | 300 | 256 | 321 |
| M20 | 150 | 200 | 502 |
| “ | 300 | 121 | 156 |
| M21 | 150 | 161 | 327 |
| “ | 300 | 127 | 152 |
| M22 | 150 | 149 | 258 |
| “ | 300 | 141 | 218 |
| Linuron | |||
| L1 | 50 | 187 | 359 |
| “ | 80 | 137 | 148 |
| L2 | 50 | 308 | 586 |
| “ | 80 | 309 | 621 |
| L3 | 50 | 260 | 302 |
| “ | 80 | 131 | 111 |
| L5 | 50 | 354 | 507 |
| “ | 80 | 171 | 578 |
| L6 | 50 | 334 | 435 |
| “ | 80 | 243 | 390 |
| L7 | 50 | 145 | 276 |
| “ | 80 | 163 | 218 |
| Control | 0 | 306 | 840 |
z Initial size of inoculum was 100 mg. Data are means of 5 replications.
Table 4. The number of embryoids (after 10 weeks) and shoots (after 15 weeks) obtained from the resistant variants of line B.
Variant |
Number of embryoids |
Number of shoots |
| M1 | 14 | 18s |
| M2 | 19 | 7 |
| M4 | 14 | 15s |
| M6 | 18 | 15s |
| M8 | 1 | 0 |
| M12 | 0 | 0 |
| M13 | 6 | 0 |
| M14 | 0 | 0 |
| M16 | 19 | 9 |
| M20 | 0 | 0 |
| M21 | 0 | 0 |
| M22 | 1 | 0 |
| L1 | 8 | 6s |
| L2 | 7 | 0 |
| L3 | 77 | 53s |
| L5 | 46 | 6 |
| L6 | 6 | 3 |
| L7 | 16 | 5s |
s Grafting was made.
Table 5. The growth of fresh callus on media with L or M. Callus was initiated from leaf explants of some L- or M- resistant variants. Initial callus mass -100 mg, 3 weeks of culture.
Variant |
Herbicide active substance |
Fresh weight (mg) |
| M 1 | M 159 | 250 |
| M 16 | M 150 | 360 |
| 12 | L 50 | 140 |
| L 3 | L 50 | 220 |
Table 6. The ability of M-variants to grow on the media supplemented with L and vice versa. The callus fresh weight of variants after 6 weeks of culture.z
Variant |
Concentration of herbicide |
Weight |
Concentration of herbicide |
Weight |
| M 1 | M 150 | 710 | L 50 | 334 |
| M 300 | 249 | L 80 | 318 | |
| M2 | M 150 | 228 | L 50 | 374 |
| M 300 | 132 | L 80 | 350 | |
| M 4 | M 150 | 433 | L 50 | ++++ |
| M 300 | 342 | L 80 | ++++ | |
| M 6 | M 150 | 601 | L 50 | +++ |
| M 300 | 365 | L 80 | +++ | |
| M 8 | M 150 | 383 | L 50 | ++++ |
| M 300 | 272 | L 80 | +++ | |
| M 12 | M 150 | 350 | L 50 | +++ |
| M 300 | 176 | L 80 | +++ | |
| M 13 | M 150 | 478 | L 50 | +++ |
| M 300 | 188 | L 80 | ++ | |
| M 14 | M 150 | 667 | L 50 | +++ |
| M 300 | 107 | L 80 | ++ | |
| M 16 | M 150 | 517 | L 50 | ++++ |
| M 300 | 321 | L 80 | ++ | |
| M 20 | M 150 | 502 | L 50 | +++ |
| M 300 | 156 | L 80 | ++ | |
| M 21 | M 150 | 327 | L 50 | +++ |
| M 300 | 153 | L 80 | + | |
| M 22 | M 150 | 258 | L 50 | +++ |
| M 300 | 218 | L 80 | + | |
| L 1 | L 50 | 359 | M 150 | 387 |
| L 80 | 148 | M 300 | 154 | |
| L 2 | L 50 | 586 | M 150 | 762 |
| L 80 | 621 | M 300 | 160 | |
| L 3 | L 50 | 302 | M 150 | +++ |
| L 80 | 111 | M 300 | ++ | |
| L 5 | L 50 | 507 | M 150 | 451 |
| L 80 | 578 | M 300 | 88 | |
| L 6 | L 50 | 435 | M 150 | +++ |
| L 80 | 390 | M 300 | +++ | |
| L 7 | L 50 | 276 | M 150 | ++++ |
| L 80 | 218 | M 300 M | ++++ |
z Initial size of inoculum was 100 mg, each value is the mean of 5 replicates. ++++ -very strong growth, +++ -strong growth, ++ – weak growth, -no growth.
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