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

Abstract

Regeneration of sunflower plants from tissue culture can be problematic due to inefficiencies in shoot elongation and subsequent rooting. To minimize exposure to cytokinins, which can negatively impact shoot elongation and rooting, a cytokinin pulse treatment was evaluated by placing cotyledonary tissues from sunflower seeds onto a shoot induction medium containing 1.5 mg L −1 6-benzylaminopurine and 0.2 mg L −1 1-naphthaleneacetic acid for 0, 2, 4, 8, 12, and 16 d, prior to transfer to an elongation medium containing 0.1 mg L −1 gibberellic acid. The 16-d pulse treatment gave the highest numbers of shoot primordia, but shoot development was minimal and only 0.4 well-developed shoots could be obtained per explant. The 4-d pulse treatment followed by 14 d of culture on elongation medium led to the production of 5.5 well-developed shoots per explant. For plant recovery, well-developed shoots obtained with the 4-d pulse treatment followed by a 14-d elongation treatment were micrografted onto in vitro seedlings, with graft survival of over 60%. Short pulse treatments (4-and 8-d), followed by culture on elongation medium, generated well-developed shoots that seemed to be more responsive to micrografting than shoots obtained with the 16-d pulse treatment. The use of the 4-d pulse treatment with micrografting of well-developed shoots significantly improved the efficiency of whole-plant recovery of sunflower.

Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata)

“…Transgenic shoots that failed to form roots were recovered by in vitro grafting adapted from a method for sunflower (Zhang and Finer 2016 ). Sterilized mature seeds were germinated under a sterilized folded paper towel saturated with sterile RODI water in Magenta TM GA7 vessels (Sigma-Aldrich, St Louis, MO, USA), with ten seeds in each box.…”

In Vitro Micrografting of Horticultural Plants: Method Development and the Use for Micropropagation

“…In Citrus, side grafting by inserting the shoot apices into inverted T-cuts of rootstocks has been successfully used [70]. Side insertion was also applied when longer shoots or nodal sections were used as scions [19,77,78]. The major methods applied in micrografting are illustrated in Figure 1.…”

“…Scions can be grafted onto either the hypocotyls or epicotyls of in vitro germinated seedlings. Hypocotyls are usually preferred because epicotyls may contain axillary meristems that compete with the scions and may grow following the grafting, thus adding extra steps of identifying and removing rootstock-derived shoots [78]. In micrografting of cashew, Thimmappaiah et al [39] noted that side grafting of shoots onto the hypocotyl resulted in higher grafting success (100%) than top-wedge grafting on epicotyls (80%), attributing this to better cambial contact established when side-grafting to hypocotyls.…”

“…Micrografting has been applied to overcome the inability of many organogenesisderived regenerants to readily produce roots [77,78,93,113]. The poor rooting ability observed in regenerated shoots of sunflower was resolved by micrografting shoots regenerated from leaves onto in vitro-germinated seedlings using a side insertion method [77].…”