In vitro organogenesis of Citrus volkameriana and Citrus aurantium

Abstract

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm -3 6-benzylaminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm -3 BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm -3 BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.Additional key words: adventitious bud, BAP, epicotyl, hypocotyl, internodal segment, sour orange, volkamer lemon.

In vitro organogenesis of rangpur lime

“…Hypocotyl segment attached to a cotyledon fragment was described by Ananthakrishnan et al (2003) as an efficient explant for improving squash in vitro organogenesis. The use of this alternative explant improved in vitro organogenesis of sour orange and volkamer lemon (TAVANO et al, 2009). Moreover, Tavano et al (2009) detected that in this explant the adventitious buds development occurred always at the hypocotyl end.…”

“…The seed coat was removed and the seeds were treated with sodium hypochlorite solution (0.5% active chloride, 20 min), followed by three rinses with sterile distilled water. Epicotyl segments-derived explants (0.8 cm) were extracted from seedlings (12 -15 cm in height) germinated in test tubes (25 x 150 mm) containing MT solid medium (15 mL; MURASHIGE; TUCKER, 1969) incubated at 27°C, in the dark (3 -4 weeks) and transferred to 16 h photoperiod (65 µmol m -2 s -1 ) for 10 d. Hypocotyl segments-derived explants were extracted from seeds germinated in Magenta boxes containing MT solid medium (40 mL) incubated in the dark for 20 d. The hypocotyl-derived explants consisted of the entire hypocotyl segment (0.8 cm), the hypocotyl segment attached to a cotyledon fragment as described by Tavano et al (2009), and the hypocotyl segment divided longitudinally.…”

“…The success in in vitro citrus culture is related to genotype (KHAN et al, 2009), explant type (TAVANO et al, 2009), culture media composition (COSTA et al, 2004;SILVA et al, 2008), and incubation conditions (MOLINA et al, 2007). Most citrus in vitro organogenesis protocols are based on the culture of young tissue, mainly epicotyls and intermodal segments (GARCIA-LUIS et al, 1999;MOURA et al, 2001) cultivated in culture media supplemented with cytokinin (BORDÓN et al, 2000;ALMEIDA et al, 2002).…”

“…Recently, Tavano et al (2009) improved the protocol of in vitro organogenesis for sour orange and volkamer lemon using an alternative explant consisted of hypocotyl segment attached to a cotyledon fragment. The authors related an explant responsiveness improvement from 42 to 77% for volkamer lemon, and from 59 to 75% for sour orange.…”

Short communication. N-(2-chloro-4-pyridyl)-N-phenylurea(4-CPPU) enhances in vitro direct shoot organogenesis of Citrus aurantium L. epicotyl segments compared to other commonly used cytokinins

“…Among rootstocks, transgenic plants have easily been obtained for Carrizo citrange (Peña et al, 1995), however, other rootstocks, such as C. limonia, C. volkameriana and sour orange, proved to be recalcitrant (Gutiérrez et al, 1997;Azevedo et al, 2006). These difficulties may be related not only to gene transfer process but also to in vitro organogenesis (Tavano et al, 2009). The low in vitro organogenesis rate results in low genetic transformation efficiency.…”