Responses of freshly collected seeds to KAR1
Freshly collected seeds of 15 seed lots, constituting eight Brassicaceae weed species, were tested for their germination response to KAR1 under a range of temperature and light conditions. Seven of the eight Brassicaceae species tested were stimulated to germinate with 1 µm KAR1 at one or more of the tested temperature and light combinations (P < 0·001, Fig. ). For some seed lots, the difference in germination response with and without KAR1 was as much as 95 %, for example B. tournefortii ‘C’ and L. africanum incubated in darkness at 15 °C (Fig. C, N). Heliophila pusilla was extremely dormant and did not germinate to ≥5 % under any of the conditions tested (Fig. L), and the following general findings apply to species and seed lots other than H. pusilla.
Fig. 2. Germination of eight Brassicaceae weed species in response to 0 or 1 µm KAR1 (as indicated), with and without 12-h alternating light, at six constant temperatures (10–35 °C) and two alternating temperatures (20/10 °C and (more ...)
Light strongly influenced the germination response for all species independently of KAR1 (P ≤ 0·001). Brassica tournefortii and R. raphanistrum germinated to the highest percentages, with and without KAR1, in constant darkness, whilst Sisymbrium spp., L. africanum and C. annua germinated to higher percentages when incubated in alternating light (P < 0·001).
Some species such as B. tournefortii were stimulated to germinate with KAR1 across a range of temperatures, whilst other species such as C. annua and L. africanum were stimulated by KAR1 to germinate beyond control levels only at select temperatures that may otherwise have been sub-optimal for germination. Indeed, KAR1 was more effective at some temperatures than others for B. tournefortii, R. raphanistrum (A), S. orientale (A), S. erysimoides, C. annua and L. africanum (KAR1 × temperature interaction, P < 0·001). The optimal temperature for germination with and without KAR1 was typically ≤20 °C for all species and seed lots, except for Sisymbrium spp., which germinated to higher percentages at 20–30 °C in the light.
With the exception of S. orientale, the trends in how seeds germinated in response to KAR1, temperature and light were consistent across all seed lots of a species irrespective of their collection source (Table ), although the magnitude of the responses varied among seed lots (Fig. ). For example, seeds of all three B. tournefortii seed lots germinated to a higher percentage in darkness, with KAR1 and at the lower temperatures (10–20 °C), but their particular germination profiles were also unique in that more seeds of seed lot ‘A’ germinated in darkness and alternating light than the other two seed lots. In the case of S. orientale, seed lots ‘B’ and ‘C’ were not stimulated to germinate with KAR1 relative to control levels (P = 0·278 and P = 0·926), but the positive stimulation of seed lot ‘A’ to germinate with KAR1 at 35/20 °C in the light (P < 0·001) indicated that this species could respond to KAR1 when fresh, even if particular seed lots did not.
Mimicking the environment can induce a KAR1 response
Seeds of six species were subjected to combinations of dry after-ripening and wet–dry cycling for up to 3 months to assess whether these processes could alleviate dormancy and improve the germination response of seeds to KAR1. Species that were highly sensitive to KAR1 prior to dry after-ripening and wet–dry cycling retained a pattern of germinating best with KAR1 (Fig. ). Overall, all six species tested germinated better with KAR1 than without it, although for S. orientale KAR1 was only beneficial in darkness (in light, P = 0·365). Very few of the wet–dry cycling and dry after-ripening treatments improved the germination of seeds above control levels, and the only species for which a consistent improvement in germination was observed, with and without KAR1, during the 3 month trial was H. pusilla. Heliophila pusilla, which was initially unable to germinate under any combination of temperature, light and KAR1 treatments (Fig. L), became highly responsive to KAR1, particularly in darkness, after just 1 month of dry after-ripening or wet–dry cycling (P < 0·001, Fig. E). Wet–dry cycling and dry after-ripening progressively alleviated dormancy in this species, as seen by the gradual increase in germination without KAR1; after 3 months, 40–60 % of seeds germinated, irrespective of KAR1 and light conditions, when the treatment included ≥1 month of wet–dry cycling.
Fig. 3. Germination response of six Brassicaceae weed species to combinations of 1–3 months of dry after-ripening (DAR) and wet–dry cycling (WD), germinated with or without 1 µm KAR1 (as indicated), with and without 12-h alternating light (more ...)
In contrast to the positive effects of wet–dry cycling and dry after-ripening on H. pusilla, the other species tested either did not respond to these treatments or in some cases the treatments inhibited germination. Rapistrum rugosum, which seemed dormant during initial testing (Fig. M), did not respond to wet–dry cycling or dry after-ripening, and germinated to a maximum of 20 % under all of the treatments tested. Wet-dry cycling noticeably impeded germination when applied to B. tournefortii for ≥2 months, and to Raphanus raphanistrum for ≥1 month (P < 0·001).
In a further experiment to test whether Brassicaceae seeds would change in dormancy state and sensitivity to KAR1 during mimicked environmental changes, seeds of the same six species (and seed lots) were stratified at 5 °C (cold stratification) or 20 °C (warm stratification) for up to 3 months (Fig. ). Four species germinated to higher levels with KAR1 following warm stratification, namely B. tournefortii, R. raphanistrum, S. erysimoides and H. pusilla (P < 0·001). For these species, the highest germination response in both alternating light and darkness was typically recorded following 4 or 8 weeks of warm stratification; germination levels either stayed the same or declined following 12 weeks of stratification.
Fig. 4. Germination response of six Brassicaceae species to warm (20 °C) and cold (5 °C) stratification on 1 % (w/v) water agar media for up to 12 weeks. Stratified seeds were subsequently incubated at 20/10 °C for germination, with and (more ...)
Compared with warm stratification, cold stratification was generally less effective at alleviating dormancy and changing the response of seeds to KAR1. Germination following warm stratification was higher than that following cold stratification for all species (P < 0·001) except S. orientale (P = 0·288); however, cold stratification did improve germination in two particular circumstances; more B. tournefortii seeds germinated with KAR1 (relative to time zero in darkness: P = 0·027 at 2 months, P = 0·002 at 3 months) and more seeds of R. raphanistrum germinated following up to 8 weeks of cold stratification when tested in alternating light (P < 0·016, irrespective of KAR1), and when tested without KAR1 in darkness (P < 0·014). In the case of R. rugosum and S. erysimoides, fewer seeds germinated with KAR1 following cold stratification (P <0·001).
Dormancy state and KAR1 response vary seasonally and between sites
A 2 year seed burial trial aimed to assess whether germination of Brassicaceae weed seeds in response to KAR1 varies seasonally, and whether it depends on the particular seed lot and burial site. Overall, all four species were stimulated to germinate with KAR1 above control levels; however, the degree of stimulation was strongly seasonal, and more apparent when seeds were incubated in darkness than in alternating light [Figs and ; P < 0·001 for all factors and seed lots, except S. orientale ‘B’ for which light was not significant (P = 0·158)]. Brassica tournefortii and R. raphanistrum were highly responsive to KAR1 when freshly collected and throughout the first few months of burial, whereas Sisymbrium spp. were initially very dormant, but became responsive to KAR1 within 2 months of burial. By the first winter season (June 2009), the relative benefit of KAR1 declined for the majority of seed lots, but was often restored as seeds cycled back into dormancy later in the year. In the first year of the trial, germinability varied seasonally and between species, peaking in autumn for S. erysimoides (April 2009), winter (June 2009) for B. tournefortii and S. orientale, and in spring (September 2009) for R. raphanistrum. In the second year, germinability for the majority of species and seed lots peaked in the winter (June 2010).
Fig. 5. Germination in 12-h alternating light of seed lots recovered from seed burial trials at Perth, Northam and Merredin over a 2 year period. Seeds were germinated with or without 1 µm KAR1 (as indicated). Seed lots ‘A’ and ‘B’ (more ...)
Fig. 6. Germination in constant darkness of seed lots recovered from seed burial trials at Perth, Northam and Merredin over a 2 year period. Seeds were germinated with or without 1 µm KAR1 (as indicated). Seed lots ‘A’ and ‘B’ (more ...)
Light during incubation reduced the stimulatory effect of KAR1 for all seed lots (light × KAR1 interaction, P <0·001), except S. orientale ‘B’ (P = 0·047). For B. tournefortii and R. raphanistrum, KAR1 stimulated as much as an extra 70 % of seeds to germinate in darkness in the first and final summers of the trial (Fig. ; December 2008 and 2010), whilst in alternating light the benefit of KAR1 reached a maximum of 40 % in the second year of the trial (Fig. ). Seeds of Sisymbrium spp., which germinated more readily in light than in darkness overall, were also stimulated to germinate with KAR1 to the greatest degree in darkness. Interestingly, S. erysimoides became highly sensitive to KAR1 in the light after 2 months of burial, and retained this sensitivity throughout the trial, even though germination without KAR1 fluctuated seasonally. For S. orientale, KAR1 did not trigger more seeds to germinate in any season when the seeds were incubated in alternating light.
Of the four species included in the trial, B. tournefortii and R. raphanistrum were buried at each of three sites, enabling us to explore how subtle differences in location and climate can alter the response of seeds to KAR1 (see also Fig. ). Both R. raphanistrum seed lots germinated best and similarly following burial at the drier sites of Northam and Merredin (compared with Perth, P < 0·001; compared with each other, seed lot ‘A’ P = 0·072, seed lot ‘B’ P = 0·135). The germination response of B. tournefortii seeds with and without KAR1 was also moderated by site differences, and, as with R. raphanistrum, germination was lowest following burial at the wettest site, Perth.
Testing two seed lots for B. tournefortii, R. raphanistrum and S. orientale enabled us to assess how different seed populations vary in their response to the same environment. Although actual germination percentages varied between the paired seed lots (P < 0·001), key trends such as how seeds responded to KAR1 relative to control levels, with and without light, were consistent for each species.