Sunflower (Helianthus annuus L.) organogenesis from primary leaves of young seedlings preconditioned by cytokinin

Abstract

Leaf tissue is widely used for regeneration of many different plants, but an efficient regeneration method using leaf tissue of sunflower is not yet available. In this report, adventitious shoots were obtained from primary leaves of 7-day-old sunflower seedlings. If seeds were germinated on a medium without cytokinin, only 25 % of the leaf explants produced shoots after 3 weeks of culture on a shoot induction medium (SIM) containing 1.5 mg L -1 benzylaminopurine (BA), giving an average of three shoot primordia per leaf. Shoot induction was enhanced up to sixfold when seeds were germinated on media containing BA or 6-(c, c-dimethylallylamino)purine at 5 mg L -1 and higher, or kinetin at 15 mg L -1 and above, with an average of 12-19 shoot primordia per leaf. Shoots further developed and elongated following transfer to a medium containing 0.1 mg L -1 gibberellic acid. The highest number of developed shoots was obtained from leaves preconditioned with 5 mg L -1 BA, with 1.9 developed shoots per explant. Histological analysis of leaves from seedlings germinated on a medium containing 5 mg L -1 BA revealed increased cell division primarily in the leaf surface tissue on the adaxial side of the mid vein. After the leaves were excised and plated on SIM, these cells continued to divide and differentiate to form adventitious shoots, suggesting that the cytokinin-containing germination medium preconditioned leaves for shoot organogenesis. An in vitro micrografting approach was developed using the elongated shoots as scions, and 57 % of grafted shoots survived to form plants.

Use of cytokinin pulse treatments and micrografting to improve sunflower (Helianthus annuus L.) plant recovery from cotyledonary tissues of mature seeds

“…This situation could be similar to the previous observation of a rapid BA uptake, and a continuous increase of endogenous cytokinins after immature sunflower embryos was placed on a medium containing 1.5 mg L −1 BA for shoot organogenesis (Charrière et al 1999). The rapid initiation of shoot meristems was previously observed in epidermal and subepidermal tissues of sunflower explants such as intact cotyledons from mature embryos (Sujatha et al 2012), lower hypocotyls of immature embryos (Bronner et al 1994), and primary leaves from 7-d-old seedlings preconditioned with cytokinin (Zhang and Finer 2015). After shoot meristem formation was induced, shoot development continued in the absence of exogenous cytokinins as also described in bindweed (Convolvulus arvensis; Christianson and Warnick 1983) and mangosteen (Garcinia mangostana; Lakshmanan et al 1997).…”

“…The axillary shoots could compete with the grafted shoots, leading to reduced growth of grafted shoots if axillary shoots were not removed (Mneney and Mantell 2001). As previously found with shoots from leaf tissues (Zhang and Finer 2015), shoot size had minimal effects on graft survival (Table 3). In contrast, higher micrografting survival from longer shoots was observed in citrus (Navarro et al 1975), cotton (Luo and Gould 1999), and cashew (Puthra and Anil 2002).…”

“…Micrografting was used to recover regenerated shoots of citrus (Citrus sinensis; Peña et al 1995a, b), cotton (Gossypium hirsutum; Luo and Gould 1999), lentil (Lens culinaris; Gulati et al 2001), and king protea (Protea cynaroides; Wu et al 2007), in which rooting was also problematic. Shoots from sunflower leaf tissues were also successfully micrografted to form whole plants (Zhang and Finer 2015). In the present study, micrografting was used to efficiently recover whole plants from shoots regenerated from cotyledon tissues.…”

“…As a result of inefficiencies in sunflower regeneration, transgenic approaches for basic research and improvement of this important oilseed crop have been somewhat limited. Thus far, shoot organogenesis in sunflower has been reported using shoot tips (Paterson 1984), cotyledon tissues from mature embryos (Power 1987;Dhaka and Kothari 2002;Sujatha et al 2012) and germinated seedlings (Knittel et al 1991;Ceriani et al 1992;Baker et al 1999), immature embryos (Müller et al 2001), and leaf tissues (Konov et al 1998;Zhang and Finer 2015). Due to year-round availability and high-shooting response from mature embryos and seedling materials, these tissue sources are preferred over immature embryos for sunflower regeneration.…”

“…Plants were more successfully recovered following grafting of regenerated shoots onto decapitated greenhouse-grown seedlings (Paterson 1984;Konov et al 1998), but the graft survival frequency was not reported. More recently, micrografting of leaf-derived sunflower shoots was reported (Zhang and Finer 2015), and this approach may have value for higher efficiencies in whole-plant recovery from cotyledon-derived shoots.…”