Using alfalfa (Medicago sativa L.) cell suspensions to model physiological and molecular processes occurring in field grown alfalfa plants

Newton Edson Kalengamaliro, Purdue University

Abstract

Alfalfa is the most important forage legume grown in the US. However, production potential is limited by poor plant persistence due to defoliation and freeze stress induced by harvesting and winter temperatures, respectively. Defoliation reduces alfalfa taproot sucrose concentrations and starch reserves. These are used to provide the carbon necessary for herbage regrowth and plant survival. Injury to alfalfa plants exposed to freezing temperatures results in significant losses in herbage yield as well as plant persistence. Our objectives were to (1) understand how sucrose deprivation of cultured cells influenced cell growth, carbohydrate metabolism, and regulation of sucrose concentration responsive genes, (2) determine if genetic differences in defoliation stress tolerance and winter hardiness between dormant and nondormant alfalfa was retained by suspension cells derived from these contrasting cultivars. In Exp. 1 and 2, 8 d-old cell suspensions grown in B5g media were subcultured into two B5g media formulations containing either sucrose or mannitol. Cell suspensions were derived from cultivars that had reduced herbage growth in autumn and were winter hardy (fall dormant) or from cultivars with extensive fall herbage growth, but that winter killed (fall nondormant). In sucrose-containing media, cells from nondormant plants exhibited higher cell growth rates, greater starch concentrations, and higher dark respiration rates when compared to cells from dormant plants. Mannitol-containing media reduced cell growth rates, starch concentrations, and dark respiration rates, but increased α-amylase activities increased significantly. The relative abundance of β-amylase RNA increased in cells from sucroserich media. Five differentially expressed sucrose-starvation-related transcripts were isolated and their nucleotide sequences determined. In Exp. 3, 10-d-old cell suspensions grown in B5h liquid media were acclimated at 2°C for 14 d and exposed freezing temperatures in a stepwise fashion at the rate of –5°C per hour. Acclimated cells of dormant cultivar 5262 survived freezing temperatures as low as –25°C, whereas unacclimated dormant cells were killed. Acclimated nondormant cells of 5929 survived 0 and –5°C but were killed thereafter. Increased sugar and starch concentrations, changes in polypeptide composition are some of the features of these suspension cells that are consistent with known differences between dormant and nondormant plants in the field.

Degree

Ph.D.

Advisors

Volenec, Purdue University.

Subject Area

Agronomy|Botany|Range management

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