In vitro organogenesis in watermelon cotyledons

Organogênese in vitro em cotilédones de melancia

FISIOLOGIA VEGETAL

In vitro organogenesis in watermelon cotyledons

Organogênese in vitro em cotilédones de melancia

Maria Graziela Zagatto Krug; Liliane Cristina Libório Stipp; Adriana Pinheiro Martinelli Rodriguez; Beatriz Madalena Januzzi Mendes

Universidade de São Paulo, Centro de Energia Nuclear na Agricultura, Laboratório de Biotecnologia Vegetal, Caixa Postal 96, CEP 13400-970 Piracicaba,SP, Brasil. E-mail: [e-mail], [e-mail], [e-mail]

ABSTRACT

The objective of this work was to study the in vitro organogenesis of Citrullus lanatus, by the induction of adventitious buds in cotyledon segments cultured in medium supplemented with cytokinin. Explants were collected from one, three and five-day-old in vitro germinated seedlings, considering the distal and proximal cotyledon regions. The data obtained showed that in vitro organogenesis of watermelon occurred with higher efficiency, when cotyledon segments from the proximal region collected from three-day-old seedlings were cultivated in medium MS, supplemented with BAP (1 mg L^-1) and coconut water (10%). The histological study showed that the organogenesis occurs directly, without callus formation, on epidermal and subepidermal layers of the explants. Adventitious shoots were characterized by the development of shoot apical meristem and leaf primordia. The formation of protuberances, that do not develop into adventitious buds, was also observed.

Index terms:Citrullus lanatus, histological study, adventitious bud.

RESUMO

O objetivo do trabalho foi estudar a organogênese in vitro de C. lanatus, pela indução de gemas adventícias, em segmentos de cotilédones, cultivados em meio de cultura suplementado com citocinina. Os explantes consistiram de segmentos das regiões distal e proximal de cotilédones, coletados de plantas germinadas in vitro com um, três e cinco dias de idade. Os dados obtidos mostram que a organogênese de melancia, in vitro, ocorre com maior eficiência em segmentos da região proximal dos cotilédones, coletados de plântulas com três dias de idade e cultivados em meio de cultura MS, suplementado com a combinação BAP (1 mg L^-1) e água de coco (10%). Pelo estudo histológico, verificou-se que a organogênese ocorre diretamente, sem a formação de calo, na epiderme e subepiderme do explante. As gemas adventícias foram caracterizadas pela presença de meristema apical e primórdios foliares. Observou-se, também, o desenvolvimento de protuberâncias que não se desenvolvem em gemas adventícias.

Termos de indexação: Citrullus lanatus, estudo histológico, gema adventícia.

Introduction

Cucurbitaceae are an economically important family of plants, with species commercially cultivated in tropical and subtropical regions. Species such as watermelon, melon, cucumber and squash are widely cultivated in all regions of Brazil. Among cucurbit species watermelon is the most cultivated in Brazil, with a total of 1,450,324 t of fruits, in an area of 77,409 ha (FNP Consultoria e Agroinformativos, 2003). However, this production is hampered by serious disease problems, especially those caused by virus infections. In the State of São Paulo eight virus diseases were identified in the culture. The PRSV-W can be considered the most important virus that infects watermelon. The control of all these diseases can only be achieved by the use of tolerant or resistant varieties.

Cucurbits breeding programs can highly benefit of biotechnological tools, such as gene transfer technology, which allows the introduction of foreign genes into a germplasm, without modifying the genetic background of elite varieties. This method has been used to develop resistance to cucurbit potyviruses in different cucurbit species (Tricoli et al., 1995; Fuchs et al., 1998; Compton et al., 2004). However, a breeding program associated to biotechnological tools depends upon the development of an efficient in vitro plant regeneration system.

In vitro plant regeneration of watermelon has been reported using protocols for adventitious shoot regeneration from cotyledon segments (Srivastava et al., 1989; Compton & Gray, 1993). The system is based on three culture steps (Dong & Jia, 1991): a bud induction phase, culturing the explants in medium supplemented with cytokinin (Compton & Gray, 1993); an elongation phase, transferring the shoot buds to medium with a lower concentration of cytokinin (Dong & Jia, 1991); and, a rooting phase, using a culture medium supplemented with auxin (Compton & Gray, 1994; Dabauza et al., 1997).

Besides growth regulators, seedling age at the time of explant preparation (Srivastava et al., 1989; Compton & Gray, 1994), explant dissection method (Compton & Gray, 1993; Compton, 2000), and culture conditions (Choi et al., 1994) also influence adventitious shoot development. The development of tissue culture systems, for commercially important Brazilian cucurbit species, is restricted to the analysis of in vitro morphogenesis of Cucumis melo var. inodorus (Stipp et al., 2001).

The objective of the present work was to implement an efficient organogenesis protocol, followed by plant regeneration for watermelon cultivar Crimson Sweet, for further studies of watermelon genetic transformation via Agrobacterium.

Material and Methods

Citrullus lanatus cv. Crimson Sweet mature seeds were used as explant sources. Seeds were surface-sterilized for 30 min, in commercial bleach solution (2.5% NaClO), rinsed three times in distilled water, and soaked overnight in sterile distilled water, in the dark. Seed coats were then removed and the embryos surface-sterilized for 20 min, in 20% commercial bleach solution (2.5% NaClO), followed by three rinses in sterilized distilled water.

The embryos were placed in plastic boxes containing MS culture media, for germination at 27 176;C, in the dark. The cotyledons were then excised and 1 to 2 mm removed from the borders, so that the cotyledon central region was cut transversally into two halves, proximal and distal, and these were used as explants (Figure 1 a). To investigate the influence of seedling age on shoot organogenesis, explants were collected from one, three and five-day-old seedlings, and cultivated in MS medium supplemented with 6-benzylaminopurine - BAP (2.5 mg L^-1). In order to study the effect of different growth regulators on shoot induction, cotyledon segments collected from three-day-old seedlings were cultured in Petri dishes (100 x 15 mm) containing MS basal medium, supplemented with BAP (0, 1.0, 2.0 or 3.0 mg L^-1), the combination of BAP (1 mg L^-1) and coconut water (10%), thidiazuron - TDZ (0, 0.25, 0.5, 0.75 or 1.0 mg L^-1), or the combination of BAP (0, 1.0, 2.5, 5.0 or 7.5 mg L^-1) and 1-naphthaleneacetic acid - NAA (0.1 mg L^-1).

The experimental design was completely randomized with 10 replications, each one consisting of a Petri dish with eight explants. Cultures were maintained at 27 176;C, under a 16 hour photoperiod (63 µM m^-2 s^-1 ). After 4 weeks, the explants were scored with the aid of a stereomicroscope, for the number of explants forming multiple shoot buds, and for the number of shoots per responsive explants. The statistical analysis was performed by ANOVA on (x + 0.5)^1/2 transformed data. Tukey's multiple range test was used for means comparison.

Developed buds were isolated and transferred to MS basal medium supplemented with kinetin (0.2 mg L^-1). Three cm long shoots were transferred to rooting medium consisting of MS basal medium, supplemented with indole-3-butyric acid - IBA (1.0 mg L^-1).

All media were supplemented with sucrose (2.5%), agar (0.8%) and the pH was adjusted to 5.8, before autoclaving (121 176;C, 20 min).

Explants cultivated in organogenesis induction medium, containing BAP (1.0 mg L^-1) and coconut water (10%), were sampled for histological study after 21 days in culture, according to Rodriguez & Wetzstein (1998). 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 cellosolve, ethanol, propanol and butanol, followed by infiltration, in a series of butanol:infiltration medium, and 100% infiltration medium at 4 176;C, overnight. Polymerization was done at room temperature, for 24-48 hours in Historesin (Leica, Heildelberg). Serial sections (5 µM) were prepared in a rotary microtome (Leica RM2155) with a steel carbide knife. The sections, floated in water drops, were dried on a hot plate (40 176;C) stained with acid fuchsin (0.1%), and then rinsed in distilled water and counterstained with toluidine blue (0.05%), for general observations and documentation under an inverted Zeiss Axiovert 35 microscope.

Results and Discussion

Cotyledon segments collected from three-day-old seedlings responded with adventitious bud formation at a much higher percentage (Table 1) than explants collected from either one or five-day-old seedlings (Figure 1 b-d). Seedling age is reported to be an important factor for obtaining high frequency adventitious shoot regeneration in watermelon (Choi et al., 1994; Compton & Gray, 1994). For watermelon organogenesis, explants were collected from seedlings younger than five-day-old (Choi et al., 1994; Compton, 2000), as young cotyledons are physiologically very active and respond efficiently to exogenous hormones (Dong & Jia, 1991).

Together with seedling age, explant type has shown to be important for morphogenesis induction, since competent cells for adventitious shoot formation in cucurbits seem to be restricted to specific cotyledon regions (Choi et al., 1994; Compton, 2000; Ananthakrishman et al., 2003). For watermelon, it has been shown that organogenic competent cells concentrated at the proximal region of the cotyledon (Compton & Gray, 1993), since most of the adventitious buds develop at the explant basal region (Compton, 2000). Regardless of the seedling age, a higher percentage of explants forming adventitious buds was obtained in explants collected from the cotyledon proximal region (Table 1) (Figure 1 c-d). Efficient response from the distal portions of the watermelon cotyledons was reported only by Choi et al. (1994), with a F₁ hybrid watermelon (cvs. Sweet Gem and Gold Metal).

The composition of induction media is also important for adventitious bud development; the presence of cytokinin is critical for shoot induction and for differentiation from cotyledonary explants of watermelon (Dong & Jia, 1991). BAP has been reported as highly effective for cucurbit organogenesis (Srisvastava et al., 1989; Compton & Gray, 1993), inducing multiple adventitious shoot bud differentiation. Chatuverdi & Bhatnagar (2001) stated that 2-isopentenyladenine (2iP) acts synergistically with BAP, improving organogenesis rates in watermelon cv. Sugar Baby. The combination of BAP (1 mg L^-1) with 10% coconut water favored the morphogenesis, resulting in a higher number of explants with shoots and of plantlets developed (Table 2). Although no significant differences were detected between this treatment and 2 mg L^-1 BAP, the use of a lower growth regulator concentration (1 mg L^-1 BAP + coconut water) seems to be more adequate, in order to avoid somaclonal variation.

The use of either TDZ or the combination of BAP and NAA caused a drastic decrease in the frequency of explants with shoot buds. Dong & Jia (1991) reported an improvement in shoot bud development in watermelon cotyledons, when combining cytokinin and auxin in the induction media, but both Compton & Gray (1993) and Srisvastava et al. (1989) detected an inhibition of shoot organogenesis, when NAA or indole-3-acetic acid (IAA) was added to the induction medium. TDZ has been reported to stimulate organogenesis in several crops (Zhang et al., 2001; Fratini & Ruiz, 2002), but this does not seem to be the case for watermelon, as Compton & Gray (1993) also reported a lower number of explants with shoot buds, using culture media supplemented with this growth regulator.

An accurate determination of the number of buds developed per explant is very difficult, because the organogenesis occurs in clusters and not as individual buds (Figure 1 d-f). Transferring the explants with multiple shoots to elongation and rooting media allowed the development of complete plants ready for acclimatization (Figure 1 e-g, Table 2).

Although a high number of multiple shoot buds apparently formed on the explants, very few buds were found when a histological study of cultures was done (Figure 1 h-i), in a similar way to the one reported for other cucurbit species (Gaba et al., 1999; Stipp et al., 2001). Adventitious shoots developed directly from the explant epidermal and subepidermal layers, without callus formation. These adventitious shoots were characterized by the development of shoot apical meristem and leaf primordia with trichomes (Figure 1 h-i). Protuberances with trichomes (Figure 1 j) which do not develop into buds were formed, indicating that these structures could be leaf primordia. Structures from which only leaves are developed have been described in cucurbit organogenesis (Gaba et al., 1999; Stipp et al., 2001; Yalcin-Mendi et al., 2003). This explains the apparently large number of buds observed during induction, resulting in a low number of plants after transfer to elongation medium.

Conclusion

In vitro morphogenesis of watermelon cotyledon segments can be obtained from explants collected from the proximal region of three-day-old cotyledons of in vitro germinated seedlings, on a culture media supplemented with BAP and coconut water.

Acknowledgements

To Fapesp for first author scholarship; to CNPq for third and fourth authors research fellowships.

Received on April 22, 2004 and accepted on March 15, 2004

• ANANTHAKRISHNAN, G.; XIA, X.; ELMAN, C.; SINGER, S.; PARIS, H.S.; GALON, A.; GABA, V. Shoot production in squash (Cucurbita pepo) by in vitro organogenesis. Plant Cell Reports, v.21, p.739-746, 2003.
• CHATURVEDI, R.; BHATNAGAR, S.P. High-frequency shoot regeneration from cotyledon explants of watermelon cv. Sugar baby. In Vitro Cellular and Developmental Biology - Plant, v.37, p.255-258, 2001.
• CHOI, P.S.; SOH, W.Y.; KIM, Y.S.; YOO, O.J.; LIU, J.R. Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens Plant Cell Reports, v.13, p.344-348, 1994.
• COMPTON, M.E. Interaction between explant size and cultivar affects shoot organogenic competence of watermelon cotyledons. HortScience, v.35, p.749-750, 2000.
• COMPTON, M.E.; GRAY, D.J. Adventitious shoot organogenesis and plant regeneration from cotyledons of tetraploid watermelon. HortScience, v.29, p.211-213, 1994.
• COMPTON, M.E.; GRAY, D.J. Shoot organogenesis and plant regeneration from cotyledons of diploid, triploid, and tetraploid watermelon. Journal of the American Society for Horticultural Science, v.118, p.151-157, 1993.
• COMPTON, M.E.; GRAY, D.J.; GABA, V.P. Use of tissue culture and biotechnology for the genetic improvement of watermelon. Plant, Cell, Tissue and Organ Culture, v.77, p.231-243, 2004.
• DABAUZA, M.; BORDAS, M.; SALVADOR, A.; ROIG, L.A. Plant regeneration and Agrobacterium-mediated transformation of cotyledon explants of Citrullus colocynthis (L.) Schrad. Plant Cell Reports, v.16, p.888-892, 1997.
• DIRKS, R.; BUGGENUM, M. In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L. Plant Cell Reports, v.7, p.626-627, 1989.
• DONG, J.Z.; JIA, S.R. High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad.). Plant Cell Reports, v.9, p.559-562, 1991.
• FNP CONSULTORIA & AGROINFORMATIVOS. Agrianual 2004: anuário da agricultura brasileira. São Paulo, 2003. p.366-368.
• FRATINI, R.; RUIZ, M.L. Comparative study of different cytokinins in the induction of morphogenesis in lentil (Lens culinaris Medik.). In Vitro Cellular and Developmental Biology - Plant, v.38, p.46-51, 2002.
• FUCHS, M.; TRICOLI, D.M.; CARNEY, K.J.; SCHESSER, M.; McFERSON, J.R.; GONSALVES, D. Comparative virus resistance and fruit yield of transgenic squash with single and multiple coat protein genes. Plant Disease, v.82, p.1350-1356, 1998.
• GABA, V.; SCHLARMAN, E.; ELMAN, C.; SAGEE, O.; WATAD, A.A.; GRAY, D.J. In vitro studies on the anatomy and morphology of bud regeneration in melon cotyledons. In Vitro Cellular and Developmental Biology - Plant, v.35, p.1-7, 1999.
• RODRIGUEZ, A.P.M.; WETZSTEIN, H.Y. A histological and morphological comparison of the initiation and development of pecan somatic embryogenic cultures induced with NAA or 2,4-D. Protoplasma, v.204, p.71-83, 1998.
• SRIVASTAVA, D.R.; ANDRIANOV, V.M.; PIRUZIAN, E.S. Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv. Melitopolski). Plant Cell Reports, v.8, p.300-302, 1989.
• STIPP, L.C.L.; MENDES, B.M.J.; PIEDADE, S.M.D.S.; RODRIGUEZ, A.P.M. In vitro morphogenesis of Cucumis melo var. inodorus Plant, Cell, Tissue and Organ Culture, v.65, p.81-89, 2001.
• TRICOLI, D.M.; CARNEY, K.J.; RUSSELL, P.F.; McMASTER, J.R.; GROFF, D.W.; HADDEN, K.C.; HIMMEL, P.T.; HUBBARD, J.P.; BOESHORE, M.L.; QUEMADA, H.D. Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and zucchini yellow mosaic virus. Bio/Technology, v.13, p.1458-1465, 1995.
• YALCIN-MENDI, N.Y.; IPEK, M.; KACAN, H.; CURUK, S.; SARI, N.; CETINES, S.; GABA, V. A histological analysis of regeneration in watermelon. Journal of Plant Biochemistry & Biotechnology, v.12, p.147-150, 2003.
• ZHANG, C.L.; CHEN, D.F.; ELLIOT, M.C.; SLATER, A. Thidiazuron-induced organogenesis and somatic embryogenesis in sugar beet (Beta vulgaris L.). In Vitro Cellular and Developmental Biology - Plant, v.37, p.305-310, 2001.

Publication Dates

• Publication in this collection
  11 Nov 2005
• Date of issue
  Sept 2005

History

• Received
  22 Apr 2004
• Accepted
  15 Mar 2004