The effects of light quality emitted by light-emitting diodes (LEDs) on the growth and morphogenesis, and concentrations of endogenous phenolic compounds of Protea cynaroides L. plantlets in vitro, were investigated. Plantlets were cultured under four light treatments: conventional fluorescent lamps (control), red LEDs (630 nm), blue LEDs (460 nm), and red + blue LEDs (1:1 photosynthetic photon flux). Four phenolic compounds extracted from the plantlets were analyzed: 3,4-dihydroxybenzoic acid, gallic acid, caffeic acid, and ferulic acid. The highest rooting percentage was observed in plantlets cultured under red LEDs (67%) compared with 7% under conventional white fluorescent light, 13% under blue LEDs, and 13% under red + blue LEDs. The highest number of roots per plantlet was also found under red LEDs, whereas a significantly lower number of roots per plantlet was obtained under the other light treatments. Furthermore, red light promoted the formation of new leaves in P. cynaroides plantlets. However, the highest leaf dry weight (53.8 mg per plantlet) was found in plantlets irradiated by the combination of red and blue LEDs. Phenolic analyses showed that the lowest concentrations of 3,4-dihydroxybenzoic acid (4.3 mg·g−1), gallic acid (7.0 mg·g−1), and ferulic acid (7.4 mg·g−1) were detected in plantlets exposed to red light, whereas those irradiated by white fluorescent light contained the highest concentration. A significant inverse correlation (r = –0.419) was established between 3,4-dihydroxybenzoic acid and rooting percentage. Strong inverse correlations were also established between 3,4-dihydroxybenzoic acid and number of roots per plantlet (r = –0.768) as well as between ferulic acid and number of roots per plantlet (r = –0.732). These results indicate that the stimulation of root formation in P. cynaroides plantlets under red LEDs is the result of the low endogenous concentrations of 3,4-dihydroxybenzoic acid and ferulic acid.

Analysis of stem extracts identified large quantities of 3,4-dihydroxybenzoic acid and other similar phenolics. The exogenous application of 3,4-dihydroxybenzoic acid on Protea cynaroides explants in vitro significantly increased the root mass at 100 mg l −1 , but not at lower concentrations, while root inhibition was observed at 500 mg l −1 . HPLC analysis of cuttings during vegetative propagation showed a considerable increase in 3,4-dihydroxybenzoic acid levels from initial planting to when root formation took place, indicating for the first time that 3,4-dihydroxybenzoic acid may be an important phenolic compound in regulating root formation in P. cynaroides cuttings. HPLC analysis also identified caffeic, ferulic, gallic and salicylic acids in the cuttings.

The inability to induce rooting of in vitro-established Protea cynaroides microshoots has prevented the production of complete plantlets. A successful shoot-tip micrografting technique was developed using in vitro-germinated P. cynaroides seedlings as rootstocks and axenic microshoots established from pot plants as microscions. Thirty-day old seedlings, germinated on growth-regulator-free, half-strength Murashige and Skoog medium, were decapitated and a vertical incision made from the top end. The bottom ends of microshoots established on modified Murashige and Skoog medium were cut into a wedge ('V') shape, and placed into the incision. The micrografted explants were cultured in a growth chamber with the temperature adjusted to 25 ± 2°C, with a 12-h photoperiod. Best results were obtained by placing the microscions directly onto the rootstock without any pre-treatments. Dipping the explants in anti-oxidant solution or placing a layer of medium around the graft area led to the blackening of the microscion.Abbreviations EDTA Ethylenediaminetetraacetate -BAP 6-Benzylaminopurine -GA 3

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