1. Seed anatomy and morphology of Thlaspi arvense (pennycress) and preliminary germination results

2. Terms used on slides Testa – seed coat and can act as a barrier for germination Embryo – living portion of seed that grows into the seedling Endosperm – In the case of Pennycress this is an envelope or sack that surrounds the embryo and acts as a barrier for germination. Cotyledons – embryonic leaves that emerge from soil after germination Radicle – embryonic root Micropylar end – point on seed where radicle emerges GA 3 – gibberellic acid (a germination promoter) KNO 3 - potassium nitrate (promotes germination in some species) Rukuni & Taylor, Cornell, Geneva

3. Figure 1. Germination of pennycress at 20/30 o C under various treatments. GA 3 was used at 100 µM, and KNO 3 was at 0.2%. Excised embryos (Ex emb) germinated 100% after 4 days when GA was added, but in the Mid and Late it took 18 days without GA. *Punct; punctured testa and endosperm. Rukuni & Taylor, Cornell, Geneva

4. Figure 2. Germination of pennycress at 20/30 o C or 10/20 o C under various treatments. Chilling was done at 5 o C for 7 days prior to the germination test, and KNO 3 was at 0.2%. Rukuni & Taylor, Cornell, Geneva

5. SUMMARY The anatomy and morphology of T. arvense seeds resembles that of the model species Lepidium sativum and Arabidopsis thaliana, which are frequently used to study germination and dormancy physiology. These three species belong to the Brassicaceae family, also known as the Cruciferae or Mustard family. The seeds consist of a seed coat, a single cell-layer of endosperm (endospermic seeds), and a dicotyledonous embryo, but other brassica species may not have an endosperm (non- endospermic seeds). The embryo consists of the cotyledons (embryonic leaves), the radicle (miniature root) and an embryonic shoot between the cotyledons (not visible in pictures). Rukuni & Taylor, Cornell, Geneva

6. In these endospermic brassica seeds, dormancy is normally classified as combinational, in the sense that the endosperm and seed coat act as physical barriers to germination and the embryo itself has physiological dormancy. Physiological dormancy is known to decline under suitable after-ripening conditions, normally at ambient conditions (temperature and relative humidity). After-ripening is a little understood phenomenon and many factors affect the length of the after- ripening period, and these factors include the genotype of plants, the seed maturation environment and the post- harvest seed storage conditions. Rukuni & Taylor, Cornell, Geneva

7. In order to germinate, seeds have to first overcome physiological dormancy of the embryo, and when the embryo has acquired the ability to grow, it also has to gain the growth strength or vigor to overcome the restrictive physical forces exerted on it by the seed coat and endosperm. A good example of physiological dormancy is illustrated in Figure 1, where excised embryos take about 4 days to germinate when gibberellic acid (GA) is added but takes 18 days without GA for the Mid and Late seed lots. In many cases, seed pre-treatments like GA, cold stratification (chilling) or potassium nitrate do not overcome physiological dormancy (Figure 1), but after-ripening will overcome dormancy in time. Rukuni & Taylor, Cornell, Geneva

8. Of the two outer layers, the seed coat normally ruptures first and then the endosperm follows. Puncturing the seed coat and endosperm, and adding of GA (Figure 1) promoted germination, further proof that these two seed tissues are a physical barrier to germination, though the more dormant Mid seed lot had limited germination due to the deeper physiological dormancy. The endosperm has the ability to inhibit germination even when the testa has ruptured. In some species, enzymes that digest the endosperm are known to exist, and endosperm weakening through digestion has to occur before germination proceeds. In endospermic seeds, the endosperm is the major physical germination barrier. Rukuni & Taylor, Cornell, Geneva

9. After-ripening relieves dormancy, and non-dormant seeds germinate in a wider range of environmental conditions, especially various soil temperatures. The behavior of all pennycress seed lots demonstrates that the seed lots have varying degrees of dormancy. Figure 2 shows the unpredictable germination behavior of dormant or partially dormant pennycress seed lots under different temperature and pre-treatment (chilling or potassium nitrate) regimes. The most dormant is the Mid then the Late, and the least dormant is the Alberta. Rukuni & Taylor, Cornell, Geneva

10. It is possible to enhance germination in such seed lots, but the degree of dormancy determines the success of these treatments, and more chances of success lie with the least dormant. A treatment that might work with one seed lot might not necessarily be the best for another seed lot, this being influenced by the physiological status of the seeds. Ad- hoc seed enhancements could be used, but more reliable techniques need more time to develop, and this begins with appropriate seed production and handling methods, seed conditioning and sanitation and seed storage under suitable conditions (temperature and relative humidity) to maintain longevity. Rukuni & Taylor, Cornell, Geneva

11. Cardinal conditions that promote after-ripening need to be established and these will determine how long the seeds need to be after-ripened before long-term storage. After- ripening durations may vary with the degree of dormancy even for seed lots of the same variety or landrace harvested in the same or different years or at various locations. Therefore, a periodic monitoring system needs to be employed to ascertain when seeds have after-ripened and also to avoid seed aging after seeds have fully after-ripened. With this in mind, it is apparent that a more in-depth seed physiology study needs to be commissioned to support long- term efforts to domesticate T. arvense for biofuel production. Rukuni & Taylor, Cornell, Geneva