Direct plant regeneration from cucumber embryonal axis

Abstract

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 µM BA and 1.59 µM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 µM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 µM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.Additional key words: adventitious shoot bud, benzyladenine, Cucumis sativus L., L-glutamine, naphthalene acetic acid. ⎯⎯⎯⎯Cucumber (Cucumis sativus L.) is an important vegetable crop. Cross-incompatibility and narrow genetic base in cucumber pose limitations to improvement by classical breeding (Ziv 1992). Tissue culture method for production of multiple shoots in cucumber using cotyledon explants was described by Gambley and Dodd (1990). A high frequency of variation in regenerated cucumber plants occurs at the callus or somatic embryo stage (Malepszy and Nadolska-Orezyk 1989). In contrast, in the shoots produced by direct regeneration from explants the morphological or physiological variation was much less (Burza and Malepszy 1995, Plader et al. 1998). Therefore, the present protocol was aimed to develop a technique which allows the production of multiple shoots directly from the embryonal axis of cucumber.Seeds of cucumber (Cucumis sativus L.) cv. Poinsett 76 (Indo-American hybrid seeds, Pvt Ltd, Bangalore, India) were soaked in tap water for 15 min disinfected with 70 % alcohol for 1 min and 2.5 % (v/v) commercial bleach Teepol (5.25 % sodium hypochlorite; ) for 15 min followed by three rinses with sterile distilled water. Seeds were further disinfected by soaking in 0.5 % mercuric chloride (m/v) for 3 min and rinsed four times with sterile distilled water. The axenic seeds were inoculated in sterile culture tubes (25 × 150 mm) containing moist cotton and kept in the culture room. The embryonal axis was removed from germinating seeds at different growth periods under sterile conditions and was used as the explant. Intact embryonal axes without the radical region, embryonal axes without apical and radical regions, and embryonal axes in which a cut was made at the apical region without the radical were cultured on Murashige and Skoog's (1962) (MS) medium supplemented with N 6 -benzyladenine (BA; 0 -8.88 µM), kinetin (KIN; 0 -9.28 µM) individually and in combination with -naphthaleneacetic acid (NAA) at different concentrations (0.53 -2.65 µM) (shoot induction medium). All the media contained 3 % sucrose and were gelled with 0.8 % Phytagel (m/v) (Himedia Laboratories, Mumbai, India). Plant growth regulators were added to the media under sterile conditions and the pH was adjusted to 5.8 before autoclaving at 1.06 kg cm -2 (121 ºC) for 20 min. MS medium without growth regulators served as control. Each culture tube containing 20 cm 3 of one of the above concentrations or combinations was ...

Efficient plant regeneration in vitro from cotyledon explants of chieh-qua (Benincasa hispida Cogn. var. chieh-qua)

“…In bottle gourd, AgNO 3 (an ethylene inhibitor) is able to promote shoot regeneration from proximal cotyledon explants 9 . Addition of cytokinins to culture media has been reported to be crucial for the induction of adventitious shoot and proliferation in vitro shoot regeneration of numerous Cucurbitaceae such as cucumber, bottle gourd, squashes, and a Cucurbita interspecific hybrid 3,[7][8][9][18][19][20] . Cytokinin can be used alone or in combination with a low concentration of auxins 4,5,14 .…”

“…An important prerequisite for a successful plant transformation is the development of a yet unavailable efficient plant regeneration system in vitro for chieh-qua 1 . Up to now, there have been a number of studies on in vitro regeneration of several Cucurbitaceae crops including cucumber 2,3 , watermelon 4,5 , melon 6 , squash 7,8 , bottle gourd 9 , figleaf gourd 10 , ash gourd 11 , muskmelon 12 , and chieh-qua 1 . However, only in cucumber, watermelon, squash, and melon, efficient and reliable plant regeneration protocols have been established.…”

Direct shoot regeneration via organogenesis in chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How)

“…Thomas and Sreejesh (2004) have reported that the addition of 0.2 μM NAA with 4 μM 6-benzylaminopurine (BAP) was beneficial for adventitious shoot regeneration in the cotyledon-derived callus of ash gourd. When compared with cucumber (Vasudevan et al, 2004;Vasudevan et al, 2007b), watermelon (Sultana and Bari, 2003;Sultana et al, 2004), melon (Curuk et al, 2002;Muruganantham et al, 2002) and squash (Ananthakrishnan et al, 2003;Kathiravan et al, 2006), chieh-qua also needed a high concentration of 6-BA for shoot induction. Such a high concentration may influence shoot proliferation as well as elongation, and could be one of the reasons for the difficulty of regeneration of chieh-qua.…”

“…Up to now, some efficient plant regeneration protocol has been established in some crops of Cucurbitaceae (Chaturvedi and Bhatnagar, 2001;Soniya and Das 2002;Kintzios et al, 2002;Curuk et al, 2002;Lee et al, 2003;Ananthakrishnan et al, 2003;Sultana et al, 2004;Akasaka-Kennedy et al, 2004;Thomas and Sreejesh, 2004;Kathiravan et al, 2006;Selvaraj et al, 2007;Vasudevan et al, 2007a;Suratman et al, 2010). However, the regeneration of Cucurbitaceae crops is not accomplished like other model plants.…”

Interspecific hybridization of Cucumis anguria and C. zeyheri via embryo-rescue

“…immature embryos. Vasudevan et al 2007 reported positive effects of BA and α-naphthaleneacetic acid (NAA) on the cucumber regeneration from embryonal axis. Table 1.…”