Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens

Abstract

Adventitious shoots formed on the proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl(-1) 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm(-2) cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of 'Sweet Gem' were cocultured with Agrobacterium tumefaciens LBA4404, a disarmed strain harboring a binary vector pBI121 carrying the CaMV 35S promoter-β-glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker, for 48 h on MS medium with 1 mgl(-1) BA and 200 μM β-hydroxyacetosyringone. After 48 h of culture, explants were transferred to medium with 1 mgl(-1) BA 250 mgl(-1) carbenicillin, and 100 mgl(-1) kanamycin and cultured in the light. Adventitious shoots formed on the explants after 4 weeks of culture. When subjected to GUS histochemical assay, young leaves obtained from the shoots showed a positive response at a frequency of up to 16%. Preculturing cotyledonary explants on MS medium with 1 mgl(-1) BA for 5 d enhanced the competence of the cells to be transformed by Agrobacterium. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants. The transformed plants were grown to maturity.

In vitro organogenesis in watermelon cotyledons

“…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-dayold (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 F 1 hybrid watermelon (cvs. Sweet Gem and Gold Metal).…”

“…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.…”

Effect of Plant Growth Regulators on Regeneration from the Cotyledon Explants in Watermelon (Citrullus lanatus (thunb.) Matsum. & Nakai)

“…품종의 종자증식 시간의 단축 때문에 많은 관심을 받고 있다 (Choi et al, 1994;Compton and Gray, 1994;Tabei, 1997). 그 러나 이러한 형질전환체 개발이나 유용한 품종 육성은 기내 재 분화 기술의 실질적인 적용방법과 효율성 때문에 제한된 품종 에 국한되고 있다 (Wang et al, 2013).…”