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

This communication provides a protocol for stable transformation of Opuntia callus cultures. Itis a summary of ten years research from 2006 to 2016 of more than 340 experiments toobtain stable transformation and regeneration of five clones of Opuntia ficus-indica. Althoughregeneration was not achieved, stable transformation was achieved as evidenced byexpression of a fluorescent marker gene six months after inoculation by soaking explantsderived from unopened floral buds in Agrobacterium tumefaciens EHA 101. As cactus hadtoo much auto fluorescence to permit use of even enhanced green fluorescent proteins, thefluorescent marker TdTomato with red/orange emission was used. To avoid consumerobjections from antibiotic selective marker systems, the Phosphomannose Isomerase (PMI)gene that screens for growth on mannose was used. Explants that fluoresced grew well up to10 g L-1 mannose, while explants that did not fluoresce senesced and died when cultured on2 g L-1 mannose. The optimal basal media were found to be either Murashige-Skoog with 30g L-1 sucrose, with either double the standard Ca concentration or Woody Plant media with anadditional 2,000 mg L-1 KNO3. Standard liquid shake, temporary immersion system or solidagar with 3 g L-1 gel rite was examined and the solid gel rite media was used. Previousreports that reported stable transformation of meristems by needle injection could not berepeated, possibly because those experiments were conducted with non-intron GUS thatwould have permitted the Agrobacterium to properly transcribe and splice transcripts for theuidA gene. Two independent reports of somatic embryogenesis Bouamama et al. (2011) andGomez 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 produceshoots. The hormone combinations that resulted in the greatest “structure” from immaturefloral 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 NAA0.25 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 hormonecombination with (PIC) on floral explants was 0.01 TDZ/0.06 ZA/0.02 mg L-1 PIC. It issuggested that the most promising approaches to obtain shoots from these types ofstructures will come from transient or stable expression of the WUSCHEL and/orBABYBOOM regulatory genes in order to stimulate shoot development.