Alves, S. A. O. et al. 2
J. Biotec. Biodivers. v. 2, N.2: pp. 1-6, May. 2011
overcome the problem caused by fatal yellowing disease. However, crosses between Elaeis oleifera x Elaeis guineensis can produce seeds with embryos that present pre and post-zygotic barriers, resulting in seeds with abortive embryos. On the other hand, these hybrid embryos can be rescued if were removed before that the abort can occur and these hybrid embryos must be cultivated in vitro to develop a complete plant (Asano and Imagawa, 1999; Alves, 2007).
The aim of this research was to rescue in vitro embryos of four varieties of interspecific hybrids of oil palm ( Elaeis oleifera x Elaeis guineensis ) in different media.
MATERIAL AND METHODS
This research was carried out at laboratory of Biotechnology and Genetic Resources of Embrapa Amazônia Oriental, Belém, Pará, Brazil.
Culture of oil palm embryos
Plant Material
The embryos from hybrid seeds of oil palm used in this experiment were originated from the Embrapa Amazônia Ocidental germplasm bank, Amazonas, Brazil. It was used four varieties: (1) Cj-2141; (2) CI-2061; (3) Cj-502 and (4) Cj-494. These varieties were chosen in agreement with the performance in the field, as high production of bunches and oil. All the embryos were originated from crosses ( E. oleifera x E. guineensis ).
Disinfection
The seeds were washed with distilled and sterile water for five times until do not exist any type of visible dirt. After this washing, the seeds were placed in a laminar air flow chamber followed by immersion in 70% alcohol for 2 minutes and after, the seeds were immerged in a sodium hypochlorite solution 1% (NaClO) for 20 minutes, and after the embryos were washed for four times with distilled and sterile water. After the disinfection the embryos were removed from seeds and inoculated on the regeneration medium.
Regeneration culture medium
In order to select the best culture medium to regeneration of embryos, four varieties were cultured in following treatments: (T1) half strength MS medium (½ MS) (Murashige and Skoog, 1962), (T2) MS, (T3) MS + 0.5mg L 1- naphthaleneacetic acid (NAA) and 6-
benzylaminopurine (BAP) and (T4) ½ MS + 0.5mg.L NAA and BAP. All media were supplemented with 0.17g.L NaH 2 PO 4 , activated charcoal (0.25%), sucrose (3%) and solidified with phytagel (0.2%).
The pH was adjusted to 5.8 before phytagel to be added. The media were autoclaved at 121°C for 15 min. The embryos were excised from the seeds and it was inoculated in flasks of 300 ml with 50 ml culture medium. The embryos were cultivated for eight weeks. In the first week, the embryos were cultivated in the dark. The evaluation was made through scores. It was attributed one score to each answer of embryo. This score varied from 0 to 6 (Figure 1). The description of score is: Zero (0) - attributed to embryos without development; One (1) -for embryos that was observed swelling; Two (2) for embryos that obtained curvature beginning; Three (3) for embryos partially curved; Four (4) for embryo completely curve; Five (5) for embryo with beginning of shoot and root development; and Six (6) for embryos with aerial part and root developed.
It was considered as the best treatment those that show the high percentage of score six during the period of 60 days of culture.
Figure 1 - Pattern of development of the embryos used to determine the evaluation score (range from 0 to 6).
RESULTS
After the first week of culture, it was observed just swelling of zygotic embryos (Figure 2A). From 17 to 21 days it was observed the curvature process of embryo (Figure 2B). Beginning of the root and aerial part of seedling was observed at 30 days of culture (Figure 3C). After 40 days, the differentiation was too clear with formation of expanded cotyledon, root and leaves (figure 2D).