Sexual Compatibility of Switchgrass (Panicum virgatum L.) with the Panicum species of Northeastern North America
81 Abstract
The characterization of inter-specific gene flow is an important part of ERA. When inter- specific gene flow is possible, the effects of a proposed transgene must be evaluated, not only in relation to transgenic crop itself, but also in relation to other compatible species. Because species level taxonomic divisions are not always a good indicator of two species ability to produce hybrid progeny, it is important to test congeneric species for compatibility when considering the release of a new transgenic variety. This project attempted to assess the inter-specific
compatibility of Switchgrass with four other Panicum species. Panicles were bagged together and the seed yield from switchgrass was assessed. However, technical challenges prevented robust analysis of interspecific sexual compatibility.
82 Introduction
The characterization of interspecific gene flow is an important part of ecological risk assessment (ERA). When interspecific gene flow is possible, the effects of a proposed transgene must be evaluated, not only in relation to transgenic crop itself, but also in other sexually
compatible species (B.-R. Lu & Yang, 2009). Because species level taxa are not always a good indicator of the ability to produce hybrid progeny, it is important to test congeneric species for compatibility when considering the release of a new GE crop. For example floras indicate that P.
virgatum and P. amarum intergrade when found together (Barkworth, et al., 2008; Palmer,
1972). This suggests the possibility of gene flow, and transgene escape. Recent phylogentic work also suggests that P. amarum is the Panicum species most closely related to switchgrass (Huang et al., 2011). In addition, there is at least one personal account that claims the two species hybridize in common garden experiments (Palmer, 1972). However, to this author’s knowledge, no formal study has been published demonstrating that the two species are sexually compatible. In a similar manner, it is important to eliminate the potential for switchgrass to hybridize with P.
capillare, P. dichotomiflorum, and P. miliaceum as these Panicum species are agricultural weeds
(Colosi & Schaal, 1997; Uva et al, 1997). In addition, in recent years members of the genus
Dichanthelium genus have been separated from Panicum (Barkworth et al., 2007). For this
reason, one of the early objectives of this project was to learn whether it was likely that
Dichanthliem species could be sexually compatible with switchgrass. It was determined that
hybridization between switchgrass and Dichanthliem was unlikely in natural settings so that this should not be the focus of forced hybridization experiments. Two factors supported this decision based on two factors. First, the majority of Dichanthliem species are diploid, while switchgrass is predominantly either tetraploid or octoploid (Gould & Clark, 1978), and second, the two genera
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rarely, if ever, release pollen concurrently (Figure 1). This study was designed to address the following questions: 1) Can switchgrass produce hybrid seed when crossed with congeneric species? 2) Is the resulting hybrid seed viable?
Materials and Methods
The Panicum and Dichanthelium species tested for compatibility with switchgrass
included: P. amarum, P. anceps, P. miliaceum, P. capillare, P. dichotomiflorum, D. meridionale,
D. commutatum. Taxonomy follows the treatment in Barkworth et al., (2007). The germplasm
for P. amarum, P. anceps, D. meridionale, and D. commutatum was obtained from the USDA National Genetic Resources Program (GRIN), P. amarum seed was obtained from GRIN and field collected (Hebron, Conneticut). P. miliaceum seed was obtained from commercial bird seed mix (Blue Seal Feeds, NH). Seed for both P. capillare and P. dichotomiflorum was field
collected (Storrs, CT). Plants were grown and panicles bagged in greenhouse conditions from March-September, 2010. Compatibility was tested by bagging panicles from two different species together as described in Martinez-Reyna & Vogel (1998). Plants were monitored first for anthesis and then confined to sealed glassine bags (Uline, Inc., Waukegan, IL). Florets which had already extruded anthers were removed. Co-bagged panicles were allowed to mature for 30 to 60 days before being removed and the switchgrass panicle (as female parent) was examined for the presence of mature caryopses. Putative hybrid seed was tested for viability in a
greenhouse mist chamber. Viable progeny were tested to verify their parentage using microsatellite markers developed by Zalapa et al., (2011). For a detailed account of DNA preparation and analysis (see Chapter 4).
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Representatives of each species were also grown in the summer of 2009 on drip irrigation at the University of Connecticut Plant Science Research and Education Facility (Figures 2-8). Anatomical features were measured, including height, blade length, and width. Counts were made of the number of shoots, panicles, and spikelets per plant. Estimates of total seed
production were produced by multiplying the mean number of spiklets per panicle by the total number of panicles per plant.
Results and Discussion Flowering Periods
One of the early objectives of this study was determining the sexual compatibility of switchgrass with other species by bagging panicles together. This was initially viewed as a simple procedure. Because crosses required only that the individuals reach anthesis at the same time. However, attempting the procedure on two different species proved far more complicated. One of the first hurdles to overcome in interspecies crosses was controlling flowering time. The
Panicum species included in this study share a common general flowering period in this region
from July to early October (Barkworth et al., 2007). However, all of the successful crosses in this project were performed in greenhouse conditions as outdoor moisture conditions damaged the glassine bags used in this study. The perennial Panicum species in this project (P. virgatum, P.
amarum, and P. anceps) underwent biannual flowering when grown year round in greenhouse
conditions. This allowed crosses with these species to be attempted twice per year; from March to May and July to September. This is consistent with previous switchgrass studies indicating that it has a facultative short-day response to photo-period (Van Esbroeck et al., 2003). Not surprisingly, the annual species were capable of just one robust period of flowering each growing
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season. Two of these, P. capillare and P. dichotomiflorum, appeared to be sensitive to short day photoperiods; only they produced flowers in the late summer and early fall. P. miliaceum, however, behaved in a day-neutral manner flowering at maturity regardless of season. Although complex, it is possible to orchestrate other species to flower at the same time as switchgrass. Pollen Viability and Anthesis
The challenge of timing was further complicated by the short-lived nature of switchgrass pollen, and presumably, that of the other Panicum species as well (Ecker et al., 2012; Ge et al., 2011). Unlike species in which pollen remains viable for days or weeks, or can be stored for extended periods (e.g. tomato; Song & Tachibana, 2007), switchgrass pollen survives for only around 60 min when harvested fresh at anthesis (see Chapter 2). In addition, all of the Panicum species in this project have asynchronous anthesis among the florets on a panicle. Switchgrass florets, for instance, go into anthesis starting at the top and outermost florets anthesis then proceeds down and inward on the panicle. This can be useful in that the opening of the
uppermost florets serves a signal that other florets on the same panicle will soon reach anthesis and extend receptive stigmas. That said, floret emergence pattern varies among the species in this study and caused difficulties when bagging receptive florets of two different species together. The pattern in P. capillare is particularly difficult as florets reach anthesis as soon as they exit the leaf sheath and well before the structure of the whole panicle has fully expanded or matured.
P. miliaceum produced panicles in which the florets were almost entirely synchronized and the
flowering period only lasted one or two days. This may be a reflection of the fact that a
commercial cultivar was used. The behavior of weedy genotypes may be different. P. amarum,
P. anceps, and P. dichotomiflorum all exhibited floret maturation patterns similar to that of
86 Panicle Size and Growth Rate
Another challenge in this project was the size and growth rate of panicles, and associated plant parts, in the different species. Switchgrass is a large plant, up to 3m in height producing panicles that can be as large as 55cm long and 20cm wide (Barkworth et al., 2007). While stature was not a significant problem for crosses with P. amarum, differences in overall size with the other species were problematic. P. anceps, P. capillare, and P. dichotomiflorum all grow to maximum heights of between 1.3-2m making them one half to two thirds the size of switchgrass (Figure 2). This makes bringing the species flowering panicles together difficult because the peduncles of these grasses are rigid and prone to breaking when bent. Therefore, some crosses required small platforms to raise the height of smaller plants to that of switchgrass.
Another problem was that the point in time at which the florets on a panicle began to mature, when trimming and bagging must take place, the structure of the panicle itself is not yet mature. In fact, the panicles of these species were, still expanding in both length and width. In addition, the peduncles of these immature panicles were still growing and at rates that differed by age and species. Thus, the glassine bags protecting the two panicles often burst.
Morphological Measurements and Seed Set Estimates
Although the morphological measurements did not prove necessary for distinguishing putative hybrids, they are presented here as part of the scientific record. The heights, lengths, and widths of the leaf blades of all eight species fell within the ranges of treatments currently found in the literature (Figures 2-4). Notably, P. capillare, and P. dichotomiflorum grew much larger than in the ruderal environments in which I have generally observed them; this was likely due to the high nutrient and moisture conditions of the drip irrigation system. The species in this study produced an average of 15 to 40 shoots per plant (Figure 5). Only D. oligosanthes produced
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substantially more with well over 100 shoots per plant. Finally, the mean number of panicles per plant and spikelets per panicle were used to estimate maximum seed yield if all flowers were fertilized, a likely outcome since a number of the species are self fertile (Figures 6-8). Not surprisingly, P. capillare and P. dichotomiflorum were by far the most fecund with an estimate of over 200,000 seeds per plant (Figure 8). This is consistent with observation that they are highly morphologically plastic and economically important weeds (Uva et al., 1997). In contrast,
P. miliaceum produced many fewer seeds than its weedy counterparts despite also being a
commercially important weed. This may reflect natural differences between species or it could be because the seed is from a commercial cultivar rather than a weedy genotype (Colosi & Schaal, 1997).
Detection of Putative Hybrid Seed
This project failed to produce evidence of interspecific hybridization between switchgrass and six other species. This is partly due to the numerous complications with forced hybridization methods discussed above. The failure to produce hybrids between switchgrass cultivar
‘Blackwell’ (8x) and P. amarum(4x) was likelydue to mismatches in ploidy(Triplett et al., 2012; Zalapa et al., 2011) (Table 1).
One hybrid cross in this project did produce putative hybrid seed. Seed was produced between a GRIN switchgrass accession from North Carolina and P. amarum (Table 1). However, the validity of identification of the P. amarum parent was called into question as it came from a local CT inland site which contained a population with individuals similar morphologically to P.
virgatum (open panicles) and P. amarum (compressed panicles). Seeds produced by this cross
were viable and produced putative hybrid plants. DNA was extracted from parents and progeny, and 18 SSR markers were applied, and it was determined that the putative hybrid offspring were
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in fact from the expected (data not shown). Unfortunately, subsequent attempts to replicate this finding with other lines of P. amarum parent were unsuccessful. Two GRIN accessions were grown to maturity, but they were not morphologically consistent with description of P. amarum.
Future Directions
This project was unable to produce a credible test of the sexual compatibility between switchgrass and other members of the Panicum genus. Future studies should testing
compatibility from other species by exposing individual swichgrass florets to fresh pollen. This approach, while more labor intensive, might yield more authoritative results. Evidence from several sources and this project suggest that P. virgatum and P. amarum might be sexually compatible and might hybridize in the wild. Further studies are needed to verify this so that scientists and policymakers engaged in the ERA of transgenic switchgrass can take transgene flow between species into account.
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