In vitro morphogenesis of Cucumis melo var. inodorus - Springer Link

Plant Cell, Tissue and Organ Culture 65: 81–89, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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In vitro morphogenesis of Cucumis melo var. inodorus Liliane C. Liborio Stipp1 , Beatriz M. Januzzi Mendes2,∗ , Sonia M. D. Stefano Piedade1 & Adriana P. Martinelli Rodriguez2 1 Escola

Superior de Agricultura ‘Luiz de Queiroz’, University of São Paulo, Piracicaba/SP 13418-900, Brazil; de Energia Nuclear na Agricultura, University of São Paulo, Piracicaba/SP 13400-970, Brazil (∗ requests for offprints; Fax: +55-19-429 4610; E-mail: [email protected])

2 Centro

Received 8 August 2000; accepted in revised form 16 January 2001

Key words: histology, melon, organogenesis, somatic embryogenesis

Abstract In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l−1 ) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l−1 ) + thidiazuron (1 mg l−1 ). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem. Abbreviations: ABA – abscisic acid; BA – 6-benzyladenine; GA3 – gibberellic acid; IAA – indole-3-acetic acid; MS – Murashige and Skoog (1962); NAA – ∝-naphthaleneacetic acid; TDZ – thidiazuron; 2,4-D – 2,4-dichlorophenoxyacetic acid; 2-iP – 6-dimethylallylamino-purine Introduction In vitro morphogenesis of Cucumis melo L. has been obtained from cotyledon segments and leaf discs (Kathal et al., 1988; Dirks and Buggenum, 1989) in induction media containing cytokinins (Kathal et al., 1988; Chee, 1991), auxins (Tabei et al., 1991), or a combination of a cytokinin and an auxin (Moreno et al., 1985; Gray et al., 1993). Plant regeneration occurred via organogenesis (Niedz et al., 1989), or somatic embryogenesis (Tabei et al., 1991; Gray et al., 1993). Most of the reports presented in the literature have been done with Cucumis melo L. var. cantalupensis or var. reticulatus, with fewer data available for var. inodorus (Moreno et al., 1985; Oridate et al., 1992; Gray et al., 1993; Vallés and Lasa, 1994; Fic-

cadenti and Rotino, 1995). Several authors report on the genotype specificity of the in vitro response, with differential response among the botanical varieties, and even among cultivars in the same variety (Oridate et al., 1992). Cultivars of C. melo var. inodorus showed a lower capacity to form somatic embryos compared to most of the cultivars of var. reticulatus (Oridate et al., 1992; Gray et al., 1993). Ficcadenti and Rotino (1995) observed more uniformity among cultivars of var. inodorus, compared to those of var. reticulatus. C. melo L. cultivars belonging to var. inodorus produce the yellow type melons and are mainly cultivated in Spain (Vallespir, 1997) and in Brazil, where cultivars of this variety occupy 70% of the cultivated area with melons. The Brazilian melon production is

82 increasing each year, with a large portion being exported to Germany, United Kingdom and The Netherlands (Deulofeu et al., 2000). In vitro plant regeneration of cultivars of C. melo var. inodorus has been obtained by organogenesis from cotyledon segments in medium containing kinetin and IAA (Vallés and Lasa, 1994; Moreno et al., 1995) or BA (Ficcadenti and Rotino, 1995). Somatic embryogenesis in cultivars of var. inodorus has been reported by culturing cotyledon segments in medium supplemented with 2,4-D and BA (Oridate et al., 1992; Gray et al., 1993) or 2,4- D and TDZ (Gray et al., 1993). Although the results show a good percentage of responsive explants, the establishment of these plants in the field was not always successful (Singh et al., 1996). Gray et al. (1993) observed the presence of abnormal embryos with variations in cotyledon number, fusions, general grossly morphology and precocious germination. Gaba et al. (1999) described poor development of adventitious shoot buds for cv. Galia var. reticulatus. These abnormalities could be responsible for lower production of plants. The present study aimed to define efficient morphogenic processes in three cultivars of C. melo var. inodorus, the yellow melon. Histological studies were done to characterize the morphogenic pathway and to assess difficulties observed during the process. An efficient regeneration system is a prerequisite for association with gene transfer techniques and detailed characterization of the process can help optimization of the protocol for specific cultivars. Oridate et al. (1992) stress the importance of knowing the morphogenic responses of various cultivars for using tissue culture techniques in plant breeding.

Material and methods

Explant preparation Cotyledon segments: seed coats were removed and surface sterilization of the de-coated seeds was done in a commercial bleach solution (0.6% NaOCl) for fifteen minutes, followed by three rinses with sterilized distilled water. Under a laminar flow hood, the cotyledons were separated, the borders and the embryo axis were discarded (excepted for somatic embryogenesis experiments), and four explants (3–5 mm × 3–5 mm) from the central region of each cotyledon half were obtained. Leaf discs: in the greenhouse, seeds were germinated in pots containing a mixture of earth and substrate. Young true leaves were collected, washed in tap water and surface sterilized as above. Leaf discs (6 mm-diameter) were obtained from the leaf lamina, avoiding the major veins. Organogenesis Cotyledon segments and leaf discs were introduced in to MS medium supplemented with one of the following plant growth regulators: BA (0; 0.25; 0.5; 0.75; 1.0; 1.25 mg l−1 ), kinetin (0; 0.1; 0.5; 1.0; 1.5 mg l−1 ), 2-iP (0; 0.1; 0.5; 1.0; 1.5 mg l−1 ) or TDZ (0; 0.25; 0.5; 0.75; 1.0 mg l−1 ) + NAA (0.1 mg l−1 ). The experimental design was completely randomized with five replications, each replication consisting of one Petri dish (100 × 15 mm) with eight cotyledon segments or leaf discs. Cultures were maintained at 27 ± 2◦ C, under a 16-h photoperiod (63 µmol m−2 s−1 ). After six weeks, the explants were scored with the aid of a stereo microscope, for the number of explants forming shoot buds or multiple shoot primordia. Shoot buds developed in medium containing 1 mg l−1 BA were isolated and transferred to MS basal medium with either 1 mg l−1 GA3 , or 0.2 mg l−1 BA. All media were supplemented with 3% sucrose, 0.8% agar (Sigma) and the pH was adjusted to 5.8 before autoclaving (121◦ C, 20 min).

Plant material Somatic embryogenesis Mature seeds of C. melo var. inodorus, cvs. Yellow Queen, Yellow King (SVS) and the hybrid AF-222 (Agroflora) were used as explant sources for induction of organogenesis (both cultivars and the hybrid) and somatic embryogenesis (cvs. Yellow Queen and Yellow King, only). Leaves of seedlings germinated in the greenhouse were also used as explant sources for organogenesis experiments.

Cotyledon explants were introduced in MS medium supplemented with one of the following plant growth regulator combinations: 2,4-D (0; 2.5; 5.0; 7.5; 10.0 mg l−1 ) + BA (0.1 mg l−1 ), or 2,4-D (0; 2.5; 5.0; 7.5; 10.0 mg l−1 ) + TDZ (0.1 mg l−1 ). The experimental design was completely randomized, with five replications, where each replication consisted of

83 one Petri dish with nine explants (eight cotyledon segments and the embryo axis). Cultures were maintained in the dark at 27 ± 1 ◦ C. After six weeks, cultures were transferred to MS basal medium and maintained at 27 ± 2 ◦ C, and 16-h photoperiod (4 µmol m−2 s−1 ). Embryos were transferred to germination medium, which consisted of MS supplemented with 1 mg l−1 GA3 . The optimal concentration of TDZ (0.1 mg l−1 ) was confirmed in an experiment, where combinations of 2,4-D (5 mg l−1 ) + TDZ (0; 0.025; 0.05; 0.075; 0.1; 0.125; 0.15 mg l−1 ) were tested, with five replications per treatment. In this experiments cultures were maintained in the dark. The effect of culture conditions was also determined, with explants being cultured in MS medium supplemented with 2,4-D (0; 2.5; 5.0; 7.5; 10.0 mg l−1 ) + TDZ (0.1 mg l−1 ) for two weeks in the dark, followed by four weeks in the dark or under a 16-h photoperiod. All media were supplemented with 3% sucrose, 0.8% agar (Sigma) and the pH was adjusted to 5.8 before autoclaving (121 ◦ C, 20 min). Microscopical analysis Cotyledon segments cultivated in organogenesisinduction medium containing 1 mg l−1 BA were sampled after 0, 4, 7, 12, 17 and 20 days in culture. Somatic embryos in different stages of development obtained in medium supplemented with 2,4-D (5 mg l−1 ) + TDZ (0.1 mg l−1 ) were also sampled. Samples were fixed in paraformaldehyde (3%) and glutaraldehyde (2%) in cacodylate buffer (0.2 M, pH 7.2) under refrigeration, for five days. Dehydration was done at room temperature in a series of 100% methyl celosolve, ethanol, propanol and butanol, followed by infiltration at 4 ◦ C, overnight. Polymerization was done at room temperature, in Historesin (Leica, Heidelberg). Serial sections (5 µM) were prepared in a rotary microtome with a tungsten-carbide knife, the sections floated in water drops and dried on a hot plate (40 ◦ C), and stained with acid fuchsin (1%), rinsed in distilled water and counter stained with toluidine blue (5%), for general observations. Explants and somatic embryos were collected and prepared for observation under the SEM, according to Rodriguez and Wetztein (1998). The material was sputter coated with gold and observed under the SEM (Zeiss DSM-940A), operating at 10–15 kV.

Table 1. Multiple shoot bud formation from mature cotyledon segments of C. melo var. inodorus treated with various BA concentrations BA (mg l−1 )

Yellow Queen

Yellow King

AF 522

0.00 0.25 0.50 0.75 1.00 1.25

0.00 (0.0) a 1.24 (15.5) b 6.56 (82.0) c 6.98 (87.25) c 7.37 (92.12) c 5.98 (74.75) c CV=10.17%

0.00 (0.0) a 1.73 (21.62) b 6.98 (87.25) cd 6.95 (86.87) cd 7.59 (94.87) c 5.78 (72.25) d CV=7.77%

0.00 (0.0) a 1.97 (24.62) b 6.97 (87.12) c 7.39 (92.37) c 7.59 (94.87) c 5.77 (72.12) c CV=10.83%

Each value represents the mean (%) of 5 replications, a total of 40 explants per treatment. Means having a common letter are not significantly different at 0.05 level by Duncan’s multiple range test.

Statistical analysis All the experiments were performed at least twice. The results of different experiments were √ similar. The statistical analysis was performed on x + 0.5 transformed data. Mean number of responsive explants were compared by ANOVA. Duncan’s multiple range test (p = 0.05) was used for means comparison.

Results and discussion Organogenesis Six weeks after induction, cotyledonary explants in medium with kinetin, 2-iP and TDZ + NAA failed to show an organogenesis response in vitro. In treatments with kinetin and 2-iP, the explants enlarged and became green. In the following weeks, a white and translucid callus with loose cells formed in most of the explants. Shoot buds were not observed in any of the above mentioned treatments. Cellular aggregates formed in only five percent of the explants in medium with 1 mg l−1 2-iP. In explants in TDZ + NAA callus and root formation was observed. BA-induced cotyledonary explants also enlarged and became green, but this was followed by a more intense cell proliferation on the cut edges of the explants, after two weeks in culture. In these regions structures similar to adventitious buds and leaf primordia became apparent in the following weeks. The best results were observed between 0.5 and 1.0 mg l−1 BA (Table 1). Adventitious shoot buds from cotyledon (Figure 1a) and leaf explants (Figure 1e) of all three

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Figure 1. Organogenesis in Cucumis melo var. inodorus. (a–d) adventitious shoot buds from cotyledon-segment explants, in medium containing BA; (a) initial development of buds and leaf primordia; (b) histological section showing proliferation of meristematic regions from the explants; (c–d) histological sections of adventitious buds with a meristematic shoot apex (point) and leaf primordia (lp) with leaf hairs (lh); (e–f) organogenesis from leaf-disc explant in medium containig BA; (e) initial organogenic stage on the edge of the explant; (f) histological section showing the formation of protuberances with a continuous epidermis (point) and leaf hair (lh), possibly a leaf primordium; (g) prolific culture initially induced from cotyledon segments, after two weeks in elongation medium; (h) plantlets obtained from cultures as in Figure 1g, in elongation medium. Bars = 100 µm (c–d); 200 µm (b,f); 1 mm (a,e).

85 Table 2. Multiple shoot bud formation from leaf discs of C. melo var. inodorus treated with various BA concentrations BA (mg l−1 )

Yellow Queen

Yellow King

AF 522

0.00 0.25 0.50 0.75 1.00 1.25

0.00 (0.0) a 0.47 (5.87) b 2.31 (28.87) c 2.31 (28.87) c 3.16 (39.50) c 1.96 (24.50) c CV=24.71%

0.00 (0.0) a 0.68 (8.50) b 1.25 (15.62) b 1.54 (19.25) bc 3.14 (39.25) c 2.14 (26.75) bc CV=27.13%

0.00 (0.0) a 0.47 (5.87) b 1.53 (19.12) bc 2.3 (28.75) c 3.06 (38.25) c 1.31 (16.37) bc CV=28.87


cultivars of var. inodorus occurred only in medium containing BA as the plant growth regulator. Media containing either kinetin, 2-iP, or combination of TDZ + NAA were not effective for shoot bud induction, with the concentrations tested. The superiority of BA compared to other citokinins for adventitious bud induction was also reported by Singh et al. (1996) and Ficcadenti and Rotino (1995) for cultivars of varieties reticulatus and inodorus. Neither explant type cultivated in absence of BA showed any organogenic response. In general, concentrations above 0.25 mg l−1 , as shown in Tables 1 and 2, gave the best results for all three cultivars and both types of explants. A slight decrease in response was observed with BA concentration higher than 1.0 mg l−1 . Cotyledon explants (Table 1) responded with adventitious bud formation at much higher percentages than leaf discs (Table 2). Other advantages of the cotyledon explants were the constant availability of explant source (seeds) and a lower percent of culture contamination, compared to leaf explants. The percent induction obtained with cotyledon explants was above 80% for all three cultivars when 0.5–1.0 mg l−1 BA was used. Organogenesis of C. melo var. inodorus cultivars have been reported by Ficcadenti and Rotino (1995) using as explants cotyledon segments of 4-day-old seedlings germinated in vitro, in medium containing BA, with or without ABA, or containing TDZ. Vallés and Lasa (1994) used cotyledon segments from 5-day-old seedlings germinated in vitro in MS medium containing IAA + kinetin. After transfer of the explants from induction to elongation medium, shoot elongation occurred only in medium containing 0.2 mg l−1 BA and in cultures

initiated from cotyledon segments (Figure 1h), with a mean of 2 plants per explant (2 cm in height). This rate of plant recovery is very low, considering the apparent presence of multiple shoot buds in most of the explants, as shown in Figure 1g. Tabei et al. (1991) observed that shoot elongation was influenced by the type of auxin in the induction medium. Elongation failed in shoots induced on medium with NAA, while only a few shoots formed on medium containing 2,4- D elongated to form plantlets. Gaba et al. (1999) also reported on the small number of plantlets obtained from multiple adventitious protuberances. Histological analysis of the organogenesis induction and development showed the initiation of numerous meristematic regions (Figure 1b). Although multiple shoot buds apparently formed on the explants, with a large number of leaf primordia being observed with the naked eye and under the stereo microscope (Figure 1a and 1e), very few shoot buds were found in histological sections of cultures initiated from cotyledons (Figure 1c–d). When present, these were only observed after 20 days in induction medium. In leafdisc derived cultures, shoot buds apparently formed (Figure 1e), but histological cuts showed the formation of protuberances on the surface of the explants, with a continuous epidermis, where leaf hairs were present, indicating that these structures could be leaf primordia. However, a shoot apical meristem was never observed associated with these structures (Figure 1f). Gaba et al. (1999) recently described the presence of meristematic protuberances which do not develop to form shoot buds, forming instead hairy leaf primordia which develop into many larger leaves covering the proximal end of cotyledon explants. The meristematic protuberances observed here (Figure 1b) are very similar to those presented by Gaba et al. (1999). These authors also comment that a similar phenomenon has also been noted in other cucurbit systems. Through histological sections it could be observed that the apparent buds are in fact protuberances and leaf primordia without a subtending shoot apical meristem. According to these authors, this is a notable phenomenon in melon and has been infrequently reported in tissue culture. Lack of a shoot apical meristem is the reason for the low regeneration of plants and the use of histological analysis was decisive for the characterization of this aberrant organogenic pathway, reported in cv. Galia var. reticulatus by Gaba et al. (1999). Our results confirm the occurrence of this phenomenon in three

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Figure 2. Somatic embryogenesis in Cucumis melo var. inodorus. (a) cotyledon-segment explant after two weeks in induction medium showing enlargement of the explant and initial formation of callus; (b) globular-stage somatic embryos developed after 6 weeks in induction medium; (c–f) longitudinal histological sections of different morphological types of somatic embryos, most of them showing procambial diferentiation (pc), protoderm (pd), but lack of shoot apical meristems, except for the embryo in Figure 1d, which shows the formation of a shoot apex (sa); (g–h) scanning electron micrographs of somatic embryos showing fused (g) and single (h) somatic embryos with hypocotyl (hc) elongation and well developed cotyledons (co). Bars = 200 µm (c–g); 1 mm (a,b,h).

87 Table 3. Effect of 2,4-D concentrations associated with 0.1 mg l−1 TDZ on the number of cotyledons forming somatic embryos in 2 cultivars of C. melo var. inodorus 2,4-D (mg l−1 )

Yellow Queen

Yellow King

0.0 2.5 5.0 7.5 10.0

0.0 (0.0) a 7.98 (88.67) b 8.59 (95.44) b 7.39 (82.11) b 3.74 (41.56) c CV=5.86%

0.0 (0.0) a 7.59 (84.33) b 8.39 (93.22) b 7.30 (81.11) b 3.11 (34.55) c CV=7.1%

Each value represents the mean (%) of 5 replications, a total of 45 explants per treatment. Means having a common letter are not significantly different at 0.05 level by Duncan’s multiple range test. Table 4. Effect of 2,4-D (5 mg l−1 ) associated with different concentrations of TDZ on the number of cotyledons with somatic embryos in 2 cultivars of C. melo var. inodorus TDZ (mg l−1 )

Yellow Queen

Yelow King

0.000 0.025 0.050 0.075 0.100 0.125 0.150

0.00 (0.0) a 1.74 (19.3) b 6.97 (77.4) c 8.38 (93.1) c 8.19 (91.0) c 6.97 (77.4) c 2.60 (28.9) b CV=10.49%

0.00 (0.0) a 1.12 (12.4) b 2.65 (29.4) c 8.39 (93.2) c 7.95 (88.3) c 7.97 (88.5) c 2.31 (25.7) b CV=15.86%


different cultivars of var. inodorus, confirming that this pathway can occur in different C. melo cultivars. Somatic embryogenesis The use of 2,4-D associated with TDZ was effective for somatic embryogenesis induction. Concentrations of 2.5 to 7.5 mg l−1 2,4-D appeared to be the most efficient for both cultivars studied (Table 3). After four weeks in culture the explants had enlarged (Figure 2a), with callus proliferation and globular structures formed throughout the surface of the explant (Figure 2b). After six weeks, somatic embryos had developed from globular structures. The association of 2,4-D and TDZ was essential for embryogenic induction, with best levels of TDZ between 0.05 and

0.125 mg l−1 for both cultivars (Table 4). Similar combinations of growth regulators were used by Gray et al. (1993), who found that medium with 5 mg l−1 2,4-D and 0.1 mg l−1 TDZ was the best combination for somatic embryo induction in cultivar Male Sterile A147 (C. melo var. reticulatus). This combination was tested for 51 other cultivars and the response varied according to the genotype. The results of induction under different light regimes (Table 5) showed that six weeks in the dark gave a better response for both cultivars at all 2,4-D concentrations, when compared to two weeks in the dark followed by four weeks at a 16-h of photoperiod. In this study, differences between cultivars were observed, with Yellow King showing a higher number of responsive explants. The combination of 2,4-D + BA in the induction medium failed to induce somatic embryos in the two cultivars. After 4 to 6 weeks, abundant formation of a non-compact whitish callus associated with roots was observed. Homma et al. (1991) demonstrated that 2,4D (2 or 4 mg l−1 ) associated with 0.1 mg l−1 BA was efficient for somatic embryogenesis. However, Gray et al. (1993), working with cv. Male sterile A147 observed a low frequency of somatic embryogenesis in induction medium with 2,4-D + BA and observed that 5.0 and 0.1 mg l−1 , respectively, was the best combination. However, interaction of 2,4-D and TDZ gave better results. Although somatic embryogenesis induction in melon cultivars, var. inodorus has been reported as less efficient than in cultivars of var. reticulatus (Oridate et al., 1992; Gray et al., 1993), the results obtained here for both cultivars, Yellow King and Yellow Queen, show a high response to treatments with the combination of 2,4-D + TDZ. After transfer to basal medium, the development and germination of the somatic embryos, however, was difficult, with a very low conversion into plants. Several different morphological types of somatic embryos were observed in culture. Some showed slight morphological alterations, with different sizes of cotyledons, some degree of cotyledon or embryo fusion (Figure 2g). Somatic embryos were observed in different developmental stages: globular, heart and torpedo shape. A few embryos showed an elongated morphology, with short cotyledons, with an elongated axis (Figure 2h). Kageyama et al. (1991) report that normal somatic embryos have a long thin hypocotyl, as opposed to abnormal embryos, which have a short hypocotyl.

88 Table 5. Effect of light during the induction period on the number of cotyledons forming somatic embryos in 2 cultivars of C. melo var. inodorus on different concentrations of 2,4-D and 0.1 mg l−1 TDZ 2,4-D (mg l−1 )

0.0 2.5 5.0 7.5 10.0 Mean

Yellow King

Yellow Queen

light

dark

Mean

light

dark

Mean

0.00 7.23 12.79 5.19 1.82 6.17 a

0.00 15.15 35.41 20.12 9.80 19.07 b

0.00 a 10.84 b 22.72 c 11.49 b 5.10 b

0.00 5.41 7.28 7.23 1.10 4.87 a

0.00 8.42 18.63 15.30 3.96 10.80 b

0.00 a 6.84 b 12.33 c 10.9 bc 2.36 d


Histological analysis of somatic embryos showed that most of the somatic embryos had well-developed cotyledons, initial procambial development, however lacked well-defined shoot and root apices (Figures c-f). The development of the shoot apex can occur after the development of the cotyledons (Cutter, 1971); however, in this system the development of a shoot apex and germination was rarely observed. The results obtained here showed that the cultivars of var. inodorus studied showed difficulties in shoot apical development, in both the organogenic and embryogenic systems. More studies are necessary to understand the reasons that lead to the lack of development of shoot buds and somatic embryo apical meristems.

Acknowledgements The authors acknowledge FAPESP for financial support and scholarship to LCLS and CNPq for a research fellowship to BMJM. SVS do Brasil Sementes Ltda – Seminis Vegetable Seeds and Agroflora are acknowledged for providing the seeds and NAP/MEPA, ESALQ/USP, for the use of the electron microscopy facilities.

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