Propagation for Cultivars

Determining Self and Cross Fertilization

Based on these inferences, using molecular methods and the results from the field fecundity studies, we will select populations with high levels of genet diversity and high fecundity as potential stock populations for habitat restoration. We will further use the same molecular markers to estimate potential selfing rates. The preliminary conclusions of the genetic population analysis are as follows. The “Ansley, MS”, “Weeks Bay, AL”, and “Heron Bay, AL” populations of S. alterniflora and the “Waveland, MS”, “Popps Ferry, MS”, and “GCRL, MS” populations of J. roemerianus exhibited the lowest values of LD and the highest values of Ne. Relatively low levels of LD and high Ne are indicative of diversity within populations. In turn, this suggests these six populations may be better able to adapt to a broader range of environmental conditions because they carry more genetic variation, and it also suggests higher levels of outcrossing. These populations of each particular species appear to be appropriate candidates for restoration efforts based on the molecular data. Based upon these results of the DNA findings we will now make paired crosses and selfs to determine any advantage of outcrossing the most diverse individuals of S. alterniflora and J. roemerianus at the MSU facility (Figures 3 and 4).

Figure 3 and 4: S. alterniflora (left) and J. roemerianus (right) cross and self-fertilization experiment in MSU greenhouse.

Based upon the results of these different DNA findings we have also made paired crosses and selfs to determine any advantage of outcrossing the most diverse individuals of S. alterniflora at the MSU facility. Ten S. alterniflora populations were maintained in a cool greenhouse at the R.R Foil Plant Research Facility of MSU. Twenty individuals of ten salt marshgrass populations were maintained in circulating water pools until ambient conditions promoted flowering. Upon flowering in fall 2018, S. alterniflora plants were subjected to selfing, individual specific crosses between population individuals, and open pollination. Seed of the S. alterniflora was collected and threshed in early 2018. There was a 30-day after-ripening period followed by a six week stratification treatment to enhance reliability of germination. Most seed was placed in an accelerated aging chamber to determine the overall robustness of the seed produced under stress conditions. Open-pollinated seed were used to test general combining ability (a measure of how well an individual crosses with other members of the population based on progeny performance) and specific crosses between individuals of different and distant populations will assess superior hybrids. The “Weeks Bay, AL” population of S. alterniflora was precocious in flowering and is likely to be crossed only among its own individuals.

Open pollinated mean seed yield ranged from 40 g/plant in “Popps Ferry, MS” population to 822 g/plant for the “Pascagoula River, MS” population (Table 5). Salt marshgrass is protogynous (female flowers are receptive before anthers on the same inflorescence emerge). This strategy encourages outcrossing. Forced selfing (by bagging fertile inflorescence before “silks” emerge) caused substantial decrease in seed production among nine of the ten populations. Only “Ansley, MS” maintained seed production at levels that were similar to open-pollinated levels. This is important, because while molecular techniques indicate a high degree of genetic variability among individuals and these populations, there is a negative impact due to selfing. The inconsistency with molecular diversity techniques is likely due to the S-Z system of gametophytic self-incompatibility. This system is well reported in grasses, is controlled by two multiallelic genes. It allows seed production from only the most unrelated parents. If pollen is similar to either allele in the female parent, then no seed is produced. The result is high diversity among individuals in the population and highly genetically diverse seed production, but extreme reduction in seed production when forced to self. The fact that any seed is produced during selfing is a testament to the extreme genetic diversity of the individuals in this subset of the field population.

Accelerated aging was performed on a subset of the seed of each population (Table 6). This is a well-known seed test among domestic crops used to determine storability and vigor. This test uses high humidity (100%) and high temperature (45°C) for 72 hrs to age seed. During the aging period the seeds take up water and are stressed at high temperatures and seed moisture. High vigor seed deteriorate slower than low vigor seed and seed lots can be separated into various vigor levels. The objective was to obtain highly vigorous progeny from individual populations. Germination of the accelerated aging test seedlots were extremely compromised. Initially we thought the conditions were too severe, but it became apparent with an attempt at replication we were at the limits of stored seed viability. Most literature indicates saltmarsh grass seed is recalcitrant (losses viability rapidly when allowed to dry). Still some results were obtained. The Fort Pike, Pascagoula River, and Grand Bay/Heron Bayou populations had 7, 4 and 3 percentage of their respective, populations identified as extremely vigorous. Unfortunately these individuals died. Crosses among individuals from subsequent screenings are probably not diverse enough because of limited numbers) to generate a seed source for sustained propagation. However, rhizomes from the individuals from eight to ten populations incorporated together could be the base for propagated material for transplanted restorations. Replications of these experiments was not possible as seed viability of salt marshgrass was lost within 45 days. Salt marshgrass seed cannot tolerate prolonged desiccation. Literature indicates seed must be stored in seawater (or the equivalent) at temperatures less than 10°C; this will allow seed to remain viable for eight to ten months.

Finally, J. roemerianus has been flowering for about 3 weeks during spring 2018 (Figure 6). The Grand Bay – Heron Bayou population is precocious in flowering and will likely fail to nick with the balance of the populations. Seed heads of J. roemerianus were bagged for maturing in summer 2018. Additional seeds of J. roemerianus were collected in late spring (April/May 2018) around the Gulf Coast Research Laboratory and are being stored prior to germination. These additional seedlings will provide further plants for genetic analysis as necessary to determine local (site scale) variability in an annual cohort. Seeds of S. alterniflora were also collected in fall (Nov/Dec 2018) and seed germination of both species will commence in spring 2019.

Table 5 and 6: Spartina alterniflora (salt marshgrass) seed yield (left) and seed exposed to accelerated aging conditions (100% RH, 45°C for 72 hrs) yield (right) by location 2017 production year. Germination conditions were 30/20° with an 8/16 hr (light dark) photoperiod.