# Recurrent Water Deficit and Epigenetic Memory in _Medicago sativa_ L. Varieties
## 2 Materials and Methods  
### Plant Material and Treatments  
_M. sativa_ variety Chiaronia was developed after applying mass selection in an old American variety named African. It is an early maturing variety of good persistence even after the 4 th year of cultivation, and can tolerate cold temperatures better than its maternal genetic material. It is considered to be moderately tolerant to drought, with a quick rate of regrowth after cuttings. It is better adapted in the hottest areas of Greece. The hay yield of Chiaronia ranges between 0.11 and 0.14 t h\({}^{-1}\) under irrigated fields [26]. Variety Lamia was developed after mass selection within a local Hellenic variety named Ypati 84. It belongs to a mid-early maturity group and has maintained high yield and quality even after the 4 th year of cultivation. The rate of regrowth after cutting is very quick and is characterized by wide adaption capacity in various pedoclimatic conditions. The hay yield of Lamia ranges between 0.12 and 0.16 t h\({}^{-1}\) under irrigated fields [26]. The seeds were sown in plastic pot trays containing a commercial growing medium. Five seeds were sown in each pot. The trays were then placed in a glasshouse for germination, and during that period, sufficient water was supplied so that the potting mix was constantly moist but not overwatered. One month after sowing, the seedlings were transplanted into 1 lt plastic pots containing a mix of peat moss and perlite, in the proportion of 4:1. The plants were thinned down to one seedling per pot and were subsequently subjected to two rounds of drought stress. The pots were placed completely randomly in the glasshouse and were rotated three times throughout the experiment.  
The first round (phase I) started one month after transplanting and lasted for 60 days. During this period, 15 plants of each variety served as controls and were being watered with 70% of the substrate's field capacity, whereas 15 plants were subjected to drought stress by withholding water. The watering of control plants took place once every three days. Pots of control plants were weighted along with pots of stressed plants. When pots of stressed plants reached 30% of water pot capacity, the first round of drought stress was completed. The completion of this first phase of drought stress was marked by cutting the aboveground parts of all the plants at the height of 5 cm. After a period of two months, during which all plants were being thoroughly watered regularly and new stems from the crowns emerged, the second round of drought stress (phase II) initiated, which lasted for 13 days due to the fact that the plants were already grown and showed symptoms of drought stress very quickly. Of the former 15 control plants of each variety, eight were randomly chosen to experience water deficit conditions along with the 15 plants that had been already stressed during phase I. These were the treatments: CD2 (controls in phase I, experiencing drought in phase II) and D1D2 (drought-stressed both in phase I and phase II), respectively. Accordingly, the remaining seven plants of the initial controls served again as controls at phase II and are referred to as CC treatment plants (controls both in phase I and phase II). Watering of all plants took place every other day and the water availability conditions were optimal for the CC plants, whereas both CD2 and D1D2 plants were watered to 30% of pot capacity. This set of treatments (Figure 1) was specifically chosen to observe whether any epigenetic memory would occur as a result of the repetition of drought stress. On day 133, samples were taken for the methylation-sensitive amplification polymorphism (MSAP) analysis. Additionally, seven plants for each treatment for each variety were harvested in order to measure the fresh and dry weight of roots and shoots.  
Figure 1: Schematic illustration of the experimental design. Two months after sowing, the first round of drought stress was carried out for 60 days. Then, the second period of water deficit was implemented.