The combined effect of photoperiod, light intensity and GA3 on adventitious shoot regeneration from cotyledons of spinach (Spinacia oleracea L.)

Abstract

Spinach (Spinacia oleracea L.) is a leafy vegetable of the Chenopodiaceae family. It is a rich source of vitamins and minerals (Al-Khayri 1995). Spinach is dioecious and flowers during long days. An efficient regeneration system is a prerequisite for the production of genetically modified spinach plants in crop improvement programs for disease resistance and late bolting. Reports on spinach regeneration through somatic embryogenesis and organogenesis have indicated that the type of explant plays an important role: root segments are superior in somatic embryogenesis while cotyledons are efficient in organogenesis (Komai et al. 1996 a,b;Molvig and Rose 1994;Zhang and Zeevaart 1999). Leaf discs, hypocotyls, root segments, leaf-derived protoplasts, thin cell layers from hypocotyls and roots have also been utilized in organogenesis (Al-Khayri et al. 1991Goto et al. 1998;Knoll et al. 1997;Leguillon et al. 2003;Mii et al. 1992;Molvig and Rose 1994;Sasaki 1989;Xiao and Branchard 1995;Zhang and Zeevaart 1999). Organogenesis has been utilized for the genetic transformation of spinach by Agrobacterium tumefaciens (Al-Khayri 1995;Knoll et al. 1997;Zhang and Zeevaart 1999). Regenerated plants obtained by somatic embryogenesis of explants transformed by root inducing transfer-DNA (Ri T-DNA) of Agrobacterium rhizogenes showed abnormal phenotypes (Ishizaki et al. 2002). In spinach regeneration, the role of GA 3 has been highlighted in most occasions in both somatic embryogenesis and organogenesis (Al-Khayri et al. 1992;Komai et al. 1996b;Molvig and Rose 1994;Xiao and Branchard 1993). The effect of exogenous ethylene on embryogenic callus formation in the presence of GA 3 has also been interpreted to facilitate cell response to GA 3 (Ishizaki et al. 2000).In plant tissue cultures, external factors such as light (wavelength, light intensity and photoperiod) also play an important role, in addition to medium composition (Hughes 1981, Hussey 1986. These factors are not independent but interact in complex ways (Hughes 1981 Abstract The effects of photoperiod, light intensity and gibberellic acid (GA 3 ) on adventitious shoot regeneration from spinach cotyledons were tested. The Analysis of Variance F test showed that the effects of photoperiod and GA 3 on shoot regeneration were significant at high light intensity of 90-100 mmol m Ϫ2 s Ϫ1. The combined effect of optimum shoot regeneration and highest shoot multiplication was observed in explants of short day (SD)-grown seedlings cultured under the SD condition and at high light intensity, with 0.5 mg l Ϫ1 GA 3 in Murashige and Skoog medium supplemented with 1 mg l Ϫ1 6-benzyladenine and 0.4 mg l Ϫ1 a-naphthaleneacetic acid. Scanning electron microscopy observation revealed multiple shoots regenerating directly from the basal end of cotyledon preceding callus. The regenerated shoots developed into normal plantlets and grew well upon transfer to pots. Some regenerated shoots flowered in vitro under the non-inductive SD condition.

A Dark Incubation Period Is Important for Agrobacterium-Mediated Transformation of Mature Internode Explants of Sweet Orange, Grapefruit, Citron, and a Citrange Rootstock

“…Because its effect on shoot regeneration was weak, AgNO 3 was not included in the shoot regeneration medium used for transformation. GA was selected as a potential enhancer of shoot regeneration and growth [36] , [43] – [45] and, because its effect was small but positive on shoot regeneration, GA was included in the shoot regeneration medium used for transformation. BA + NAA was compared to ZR as BA + NAA are two growth regulators commonly used together to regenerate shoots from mature citrus explants [9] , [22] , [23] , [46] .…”

Variability in somatic embryo-forming capacity of spinach

“…These discrepancies may be due to different light requirements of the root and cotyledonary explants for induction of de novo regeneration. However, nearly all differences among treatments in the study of Geekiyanage et al 41 were statistically insignificant, indicating that this may be due to a high variability of the plant material.…”

“…For this reason, in a previous study 21 we used genetically identical spinach donor plants, grown under the same conditions, to study the effect of light on SE regeneration from the root sections, and undoubtedly demonstrated that light, in terms of photoperiod and light intensity, strongly affected embryogenic capacity of the root explants of spinach, with LD conditions and light intensity of 100 µmol m −2 s −1 being optimal. By contrast, Geekiyanage et al 41 found bud regeneration from the cotyledonary explants of spinach cv. Longstanding Bloomsdale Dark Green grown under SD conditions to be more efficient.…”

Genetic Transformation and Genomic Resources for Next-Generation Precise Genome Engineering in Vegetable Crops

“…Although the genotype has generally shown a significant effect, regeneration has been achieved either through organogenesis or embryogenesis depending mainly on the explant type and auxin/cytokinin ratio: cotyledon explants and low ratios facilitate the former, whereas root explants and high ratios favor the latter. In addition, low temperature (14°C), photoperiod, light intensity, and GA 3 content had substantial effects on shoot regeneration ( Geekiyanage et al, 2006 ; Chin et al, 2009 ; Nguyen et al, 2013b ), while low levels of hygromycin (0.5 mg l -1 ) have been found to promote SE ( Milojević et al, 2012 ). An optimized regeneration protocol has been recently published ( Nguyen et al, 2013a ).…”