Stable transformation of Opuntia ficus indica callus cultures as evidenced by fluorescence of the tandem dimer tomato gene

Abstract

This communication provides a protocol for stable transformation of Opuntia callus cultures. It is a summary of ten years research from 2006 to 2016 of more than 340 experiments to obtain stable transformation and regeneration of five clones of Opuntia ficus-indica. Although regeneration was not achieved, stable transformation was achieved as evidenced by expression of a fluorescent marker gene six months after inoculation by soaking explants derived from unopened floral buds in Agrobacterium tumefaciens EHA 101. As cactus had too much auto fluorescence to permit use of even enhanced green fluorescent proteins, the fluorescent marker TdTomato with red/orange emission was used. To avoid consumer objections from antibiotic selective marker systems, the Phosphomannose Isomerase (PMI) gene that screens for growth on mannose was used. Explants that fluoresced grew well up to 10 g L-1 mannose, while explants that did not fluoresce senesced and died when cultured on 2 g L-1 mannose. The optimal basal media were found to be either Murashige-Skoog with 30 g L-1 sucrose, with either double the standard Ca concentration or Woody Plant media with an additional 2,000 mg L-1 KNO3. Standard liquid shake, temporary immersion system or solid agar with 3 g L-1 gel rite was examined and the solid gel rite media was used. Previous reports that reported stable transformation of meristems by needle injection could not be repeated, possibly because those experiments were conducted with non-intron GUS that would have permitted the Agrobacterium to properly transcribe and splice transcripts for the uidA gene. Two independent reports of somatic embryogenesis Bouamama et al. (2011) and Gomez et al. (2006) in Opuntia were intensively examined, but could not be repeated. However smooth, green structures similar to “nodules” described by McGown et al. (1988) that can be induced to produce shoots were obtained but could not be induced to produce shoots. The hormone combinations that resulted in the greatest “structure” from immature floral explants were Zeatin (ZA) 0.2 to 0.75 mg L-1 with naphthalene acetic acid (NAA) 0.2 mg L-1, or Thidiazuron (TDZ) 0.75 with ZA 0.5 and NAA 0.4 mg L-1, or metatopolin 1.5 with NAA 0.25 mg L-1. Long-term culture on Picloram (PIC) resulted in cultures with a red tinge, thought to be stress-induced betalain production. However, the most promising hormone combination with (PIC) on floral explants was 0.01 TDZ/0.06 ZA/0.02 mg L-1 PIC. It is suggested that the most promising approaches to obtain shoots from these types of structures will come from transient or stable expression of the WUSCHEL and/or BABYBOOM regulatory genes in order to stimulate shoot development.