In Vitro Seed Germination and Rootstock Establishing for Micrografting of Theobroma cacao L.

Micrografting has been successfully implemented in several plant species of Citrus, Eucalyptus, Havea, Malus, Olea, Prunus and other genera. This technique is employed for plant rejuvenation, true-to-type propagation, recovery of virus-free plants, testing of post cryopreservation viability and for regeneration of plants. The procedure involves grafting of a meristematic shoot or of an embryo onto epicotyl or hypocotyl cambium layer of a young seedling grown aseptically. This study tested the possibility of establishing cacao (T. cacao L) seedlings in vitro with the purpose of using them as rootstocks. Seeds from cacao pods (accession TARS 16542) provided by TARS in Mayaguez, PR were shipped overnight to Fort Collins, CO. The cacao seeds were extracted, cleaned of mucilage, sterilized and cultured aseptically. The highest germination was observed when seeds were sterilized in isopropyl alcohol (70%; 10 min) followed by sodium hypochlorite (1%; 20 min), rinsed with sterile water and placed on a solid, half strength MS medium supplemented with cysteine (100 mg/L), at 30^oC, in dark conditions. The seed germination varied from 60 to 100%. The main impediments for the in vitro germination and seedling development were excretion of phenolic compounds as well as contamination that occurred at various stages of plant development. These factors decreased the number of usable rootstocks. Increasing the concentration of sodium hypochlorite and sterilization time (5%; 40 min) lowered contamination but drastically inhibited the seed germination. After 4-5 days, the seedlings were transferred to double stacked Magenta boxes and placed in a growth chamber (26^oC) with an 18 h photoperiod. After 5-6 weeks of cultivation under light conditions, seedlings (ca. 10-12 cm long) were ready for grafting. Shoots 4-6 mm long were grafted into the hypocotyl cambium layer of the rootstocks. After 5-7 day cultivation period under dimed light, the grafted plants were transferred to light conditions (18 h) and 8 weeks later to pots. Over 50% of micrografts developed canopies. Future studies will focus on evaluating factors that might increase the percentage of plants developed from grafts. Additionally, the feasibility of using micrografting for plant regeneration from cryopreserved meristematic shoots of cacao will be tested.