Comparison of stem morphology and anatomy of two alfalfa clonal lines exhibiting divergent cell wall composition

Authors: Gronwald, John; Bucciarelli, Bruna

Submitted to: Journal of the Science of Food and Agriculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/13/2012
Publication Date: 8/30/2013
Citation: Gronwald, J.W., Bucciarelli, B. 2013. Comparison of stem morphology and anatomy of two alfalfa clonal lines exhibiting divergent cell wall composition. Journal of the Science of Food and Agriculture. 93:2858-2863.

Interpretive Summary: Alfalfa is an important forage for livestock and has potential for development as a cellulosic feedstock. A major factor limiting the use of alfalfa for both purposes is the cell wall composition of stems. Pectin, cellulose, and lignin are major components of the cell walls of alfalfa stems. Increasing pectin and cellulose, and decreasing lignin in cell walls of alfalfa stems would increase both the nutritive value of this forage and its bioconversion efficiency to ethanol. In previous research, two alfalfa lines were selected that exhibited environmentally stable differences in cell wall composition of stems. One line, 1283, had consistently higher pectin but decreased cellulose and lignin. The other line, 252, had lower pectin but increased cellulose and lignin. To learn more about factors responsible for these differences, we examined stem anatomy of the two lines. Histochemical staining of stem cross-sections of the two lines revealed the proportions of secondary xylem and pith. Secondary xylem contains high levels of cellulose and lignin while pith contains high levels of pectin. For both first-cut and second-cut harvests, the proportions of pith and secondary xylem were inversely related in stems of the two lines. For the first-cut harvest, significant differences in the proportions of the two tissues were measured in the two alfalfa lines. Compared to 252, 1283 exhibited a higher proportion of pith and a lower proportion of secondary xylem. These changes are consistent with the increased pectin but decreased cellulose and lignin found in cell walls of 1283 stems. Although similar trends for the proportions of secondary xylem and pith were measured in the two lines in the second-cut harvest, they were not statistically significant. The results indicate that the proportions of secondary xylem and pith are not environmentally stable in the alfalfa lines examined and hence cannot be the sole basis for the differences in cell wall composition in stems. The knowledge gained in this study has advanced understanding of stem anatomy in alfalfa. Selection for increased pith and decreased secondary xylem in alfalfa stems in a breeding program has potential for improving the value of this forage as a livestock feed and cellulosic ethanol feedstock.

Technical Abstract: In previous research, two alfalfa clonal lines (252, 1283) were identified that exhibited environmentally stable differences in stem cell walls. Compared to stems of 1283, stems of 252 have a higher cell wall concentration and greater amounts of lignin and cellulose but reduced levels of pectic sugars. These results suggest greater deposition of secondary xylem and a reduction in pith in stems of 252 compared to 1283. We examined stem morphology and anatomy of first-cut and second-cut harvests of genotypes 1283 and 252 in the field. For both harvests, stems of 1283 were thicker and had a higher leaf to stem ratio compared to stems of 252. Stem cross-sections of both genotypes were stained for lignin using acidic phoroglucinol, and the proportion of stem area that was pith and secondary xylem was measured using ImageJ. For both genotypes, the proportions of these stem tissues were inversely related. Stems of 252 exhibited a greater deposition of secondary xylem and a reduction in pith compared to stems of 1282 for the first-cut harvest, but this difference was not statistically significant for the second-cut harvest. The results indicate that the proportions of secondary xylem and pith are not environmentally stable in stems of the two genotypes and hence cannot be the sole basis for the differences in cell wall concentration and composition.