Micropropagation of the Cucumber Hybrids ‘Brunex’ and ‘Bambina’

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Cucurbit Genetics Cooperative Report 17:50-53 (article 13) 1994

G. Sapountzakis, A.S. Tsaftaris
Mediterranean Agronomic Institute of Chania, Greece; Department of Genetics and Plant Breeding, Aristotelian University of Thessaloniki, Greece

The female parthenocarpic cucumber hybrids which are cultivated in greenhouses have considerable economic importance during the winter period in the European market. The plants are very productive and their fruits have desirable quality traits. Handley and Chambliss (1979) cultured axillary buds of the gynoecious cucumber hybrid ‘Carolina’. The aim of the present work was to test different media and growth regulator combinations for micropropagating the hybrids ‘Brunex’ and ‘Bambina’ at low cost. These hybrids are important to Greek farmers.

Seeds of ‘Brunex’ (Bruinsma) and ‘Bambina (De Ruiter Seeds) cucumber hybrids were soaked in a TritonR x 100 (Merck) 0.01% v/v solution (containing 10 g/l NaOCl and 165 g/ NaCl) for 15 min. The seeds were then rinsed and placed under aseptic conditions in petri dishes containing 0.8% w/v agar-agar (Sigma). Dishes were placed in darkness at 25C for 5 days to allow germination. After germination the seedlings were transferred under aseptic conditions to vessels containing Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) solidified with 0.8% w/v agar-agar, and placed in a growth chamber to allow shoot growth until the stage of 6 leaves. All experiments were conducted in a growth chamber maintained at 25C provided with a 16-hours per day photoperiod by cool-white fluorescent light at 1500 lux.

All media used were solidified with 0.8% w/v agar-agar (Sigma). The pH was then adjusted to 5.7, and they were autoclaved at 121C for 20 min. Four experiments were initiated to estimate optimum growth regulator levels and to quantify propagation rates. Eight replications per treatment were used in each experiment.

Experiment 1: To evaluate the need for auxin in combination with different cytokinin levels for culture, segments of shoots with two buds were transferred in test tubes each containing MS medium supplied with 0.0, 0.1, and 1.0 mg/15/benzylaminopurine (BA) and 0.0 and 0.1 mg/1 1-naphthalenacetic acid (NAA), and placed in a growth chamber.

Experiment 2: To refine the optimum level of cytokinin required for growth, shoots were established horizontally in vessels containing MS medium and incubated in a growth chamber. Two week-old newly formed axillary shoots were established in test tubes containing MS medium supplied with 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 10.0 mg./1BA, and placed in the growth chamber.

Experiment 3: To estimate the optimum level of gibberellin required for growth, shoots were established horizontally in vessels containing MS medium and incubated in the growth chamber. One-week-old newly formed axillary shoots were established in test tubes containing MS medium supplied with 0.5 (in the case of ‘Brunex’) or 1,0 (in the case of ‘Bambina’), mg/l BA and 0.5, 1.0, 2.0, 5.0, and 10.0 mg/l gibberellic acid (GA3) (in both cases).

Experiment 4: To quantify the propagation rate under optimal conditions, shoots with six lateral buds were cut in two segments and placed horizontally in vessels containing MS medium supplied with 0.5 mg/l BA and 10.0 mg/l GA3 (in the case of ‘Brunex’) or with 1.0 mg/l BA and 5.0 mg/l GA3 (in the case of ‘Bambina’). Two weeks later, the formed callus was removed from the cultured explants and separated to six segments per each initial shoot, then transferred to new media of the same composition.

Four weeks after the establishment of each experiment, the number of new shoots were counted. Treatment differences in the first three experiments were determined at P = 0.05 as assessed by L.S.D. tests.

Results

Experiment 1: The means of the shoots produced per explant (shoot segment with 2 buds) of the cucumber hybrids ‘Brunex’ and ‘Bambina’ four weeks after cultivation on MS media supplemented with various levels of NAA and BA are presented in Table 1. When NAA was absent from the media more shoots developed than on media containing NAA. On auxin free media the presence of BA increased the number of shoots. In the case of ‘Brunex’ more shoots (3.0 per explant) were produced at the medium supplied with 1.0 mg/l BA. This value differs statistically from all other values except one. In the case of the hybrid ‘Bambina’, more shoots (3.6 per explant) were produced on media supplied with 0.1 and 1.0 mg/l BA. These values differ statistically from all the others. Moreover, they were also higher than the highest value obtained with the ‘Brunex’ hybrid.

Experiment 2: Since the results from the first experiment indicated that better growth is obtained with media lacking auxin, we used media without auxin in conjunction with more levels of BA in the second experiment. This was done to identify the optimum level of this growth regulator in each hybrid. The means of shoots per explant [newly formed axillary shoot (2-weeks-old)] of ‘Brunex’ and ‘Bambina’ four weeks after cultivation on MS media supplemented with various concentrations of BA are presented in the Table 2. It is obvious that the level of BA in the media affects shoot production and that the optimum level of BA for ‘Brunex’ is 0.5 mg/l (5.2 shoots produced per ex-plant). This value differs statistically from all the other values, except the one achieved with medium supplied with 1,0 mg/l BA. In ‘Bambina’, BA level in the media also affects the number of shoots produced. The optimum BA level for this cultivar is 1.0 mg/l (1 1.0 shoots produced per explant). This value differs statistically from all others tested. The number of shoots produced by the hybrid ‘Bambina’ are higher than ‘Brunex’ at all the cytokinin levels. Thus, the cultivar differences observed in the previous experiments were verified in the second experiment.

Experiment 3: Keeping the BA at the optimum level (i.e., 0.5 mg/l for “Brunex’ and 1..0 mg/l for ‘Bambina’) we examined whether or not we could increase the number of shoots produced by adding different amounts of GA3 to the medium. the means of shoots produced per explant [newly formed axillary shoot (1-233k-old)] ‘Brunex’ four weeks after cultivation on MS media supplemented with 0.5 mg/l BA and various concentrations of GA3 are presented in Table 3. Likewise, data of ‘Bambina’ four weeks after cultivation on MS media supplemented with 1.0 mg/l BA and various GA3 concentrations are also presented. The level of GA3 affects the number of the shoots produced in both hybrids. Better results for ‘Brunex’ were obtained at high GA3 levels (5.0 and 10.0 mg/l). These two values (6.4 and 6.6 shoots per explant, respectively) differ statistically from all other values. For ‘Bambina’ the best shoot production as between 5-10 mg/l GA3 , but the optimum level was 5.0 mg/l. The value of 15.0 shoots per explant differs statistically from all the other values, except the one achieved at the medium supplied with 10.0 mg/l GA3 . At all the GA3 levels, the number of shoots produced in ‘Bambina’ were higher than those of ‘Brunex’ indicating differences in performance between the two genotypes. IOn both cases, the increase in the number of produced shoots is due to the elongation of some small shoots. It is hypothesized that without the GA3 treatment these shoots of small size would have gone uncounted.

Table 1. Means of shoots per explant from ‘Brunex’ and ‘Bambina’ cucumber explants four weeks after cultivation on MS media supplemented with various levels of NAA and BA

BA (mg/l)
0.0
0.1
1.0
NAA

(MG/1)

 0.0 1.62 2.75 3.00
0.1 1.75 2.12 1.75

Table 2. Means of shoots per explant [newly formed auxiliary shoot (2-weeks-old)] produced from explants of ‘Brunex’ and ‘Bambina’ (in parentheses) cucumber four weeks after cultivation on MS media supplemented with various concentrations of BA.

BA (mg/l)

0.1

0.5

1.0

2.0

5.0

10.0
Shoots produced 3.5
(4.6)		 5.2
(6.7)		 4.6
(11.0)		 2.6
(5.5)		 1.7
(2.0)		 0.7
(1.0)

Table 3. Means of shoots per explant [newly formed auxiliary shoot (1-week-old)] from “Brunex’ and ‘Bambina’ (in parentheses) cucumber explants four weeks after cultivation on MS media supplemented with BA and various concentrations of GA3 .

GA3

0.5

1.0

2.0

5.0

10.0
Shoots produced 4.5¹
(10.0)2		 3.5
(8.0)		 3.6
(6.9)		 6.4
(15.0)		 6.6
(14.0)

1 ‘Brunex’ supplemented with 0.5 mg/l BA
2 ‘Bambina’ supplemented with 1.0 mg/l BA.

Experiment 4 The optimum level of BA and GA3 for propagation was calculated. In ‘Brunex’ four weeks after culture in MS medium supplemented with 0.5 mg/l BA and 10.0 mg/l GA3 , a cluster of shoots were produced (from shoot segments with six buds). After their separation into isolated propagules, an average of 48 shoots were produced from each initial explant. Elongation and rooting of the new shoots was carried out on MS medium free of growth regulators within four weeks. Similarly, in ‘Bambina 52 shoots were produced from each initial explant. These shoots were developed into plantlets.

In conclusion, the data indicate that:

  1. The presence of auxin (NAA) in the culture media decreased the shoot propagation rate.
  2. The cytokinin (BA) level in the culture media affected the number of shoots produced. The optimum level was 0.5 mg/l and 1.0 mg/l for the hybrids ‘Brunex’ and ‘Bambina’, respectively.
  3. The presence of gibberellin (GA3 ) in high levels (5.0-10 mg/l) increased the number of shoots produced.
  4. In the case of ‘Brunex’, the highest propagation rate was achieved when the medium was supplemented with 0.5 mg/l BA and 10 mg/l GA3 . From every shoot with six buds, 48 new axillary shoots were produced within four weeks.
  5. In the case of ‘Bambina’, the highest propagation rate was achieved when the medium was supplemented with 1,0 mg/l BA and 5 mg/l GA3 . From every shoot with six buds, 52 new axillary shoots were produced within four weeks.
  6. Elongation and rooting of the produced new shoots can be carried out on MS medium free of growth regulators within four weeks.

Literature Cited

  1. Handley, L.W. and O.L. Chambliss. 1979. In vitro propagation of Cucumis sativus L. HortScience 14:22-23.
  2. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.