Seed viability determination in parasitic broomrapes (Orobanche and Phelipanche) using fluorescein diacetate staining

Abstract

Fluorescein diacetate (FDA) was investigated as a potential viability stain for seeds of holoparasitic broomrapes Orobanche and Phelipanche (Orobanchaceae), using four weedy and two non-weedy taxa. FDA viabilities were compared with the currently used 2,3,5-triphenyl tetrazolium chloride (TTC) test, and in vitro germination induced by the strigolactone analogue GR-24. Viable FDA-stained seeds were characterised by bright yellow-green fluorescence under 450-490 nm blue light. These viable seeds could be discriminated from non-viable seeds, in which low levels of fluorescence, similar to non-stained control seeds were observed. FDA viabilities were not significantly different from those obtained using the TTC stain; however, viability values for both stains were consistently higher than levels of artificially induced germination. Positive TTC-staining showed continuous variation that was difficult to interpret. Nevertheless, the TTC test does not involve grinding seeds, and therefore probably remains a practical alternative to FDA for screening soil samples contaminated with Orobanche and Phelipanche seeds, which indicates different applications for the two viability tests. Interestingly, the strigolactone analogue GR-24 only induced germination in O. crenata, P. ramosa and O. minor, suggesting a high specificity towards this germination inducer among the species investigated. We suggest that FDA-staining provides a potential alternative to the currently used TTC test for seed viability assays for Orobanche and Phelipanche, and other parasitic weeds which are an obstacle to crop cultivation.

Response of wild and weedy broomrapes to synthetic strigolactone analogue GR24

“…alsatica, O. elatior, O. flava, O. lutea, O. pallidiflora, and O. reticulata did not germinate after treatment with GR24. A number of other wild broomrape species were discovered not to respond to GR24 likewise (Fernández-Aparicio, et al, 2008;Thorogood, et al, 2009). Thus it seems to be questionable to utilize GR24 as the universal standard for germination tests.…”

“…in Fig. 1A (Fernández-Aparicio, et al, 2009;Thorogood, et al, 2009;Matusova et al, unpublished data). P. ramosa germinated up to 99% at exposition to 10 -4 mg*l -1 for population Branovo ( Fig.…”

Independent recruitment of a novel seed dispersal system by camel crickets in achlorophyllous plants

“…All the fecal pellets were collected after 48 h and were examined under a dissection microscope, and the intact seeds were counted. The intact seeds were subsequently tested using 2,3,5‐triphenyl tetrazolium chloride (TTC) viability staining, as previously described for dust‐like seeds (Thorogood et al ., ; De Vega et al ., ; Ren et al ., ). TTC is a white salt that turns from a colorless state to a pink/red formazan in living cells (Copeland & McDonald, ); accordingly, dead and necrotic seeds remain uncolored.…”

Parasitic plant management in sustainable agriculture

“…, 2009; Macías et al. , 2009; Thorogood et al. , 2009); (ii) the progress in population genetic approaches for the study of parasitic plant species using molecular markers (Roman et al.…”

“…The main scientific results included: (i) the identification of germination stimulants, e.g. the strigolactone and sesquiterpene lactone groups and their function, and germination physiology of parasite seeds (Roman et al, 2002;Ferna´ndez-Aparicio et al, 2008b, 2009Lo´pez-Ra´ez et al, 2009;Macı´as et al, 2009;Thorogood et al, 2009); (ii) the progress in population genetic approaches for the study of parasitic plant species using molecular markers (Roman et al, 2002;Vaz Patto et al, 2008, 2009Castillejo et al, 2009a;Satovic et al, 2009); (iii) better understanding of source-sink relations between host and parasite (Abbes et al, 2009) and (iv) the use of molecular biology tools in parasitic plant research, in order to understand these interactions at the molecular and the cellular levels (Swarbrick et al, 2008;Castillejo et al, 2009b;Dita et al, 2009).…”