Protocol for Rescuing Young Cassava Embryos

Abstract

Embryo rescue (ER) in cassava breeding has several relevant applications, from the recovery of broad crosses to the recovery of seeds from the standard pollination program. Cassava fruit setting may drop from 100%, during the 1st week after pollination, to less than 40% during the 2nd week after pollination due to the abscission of fruits depending on genotypes. Therefore, the availability of an ER protocol for early stages of embryo development, in particular during the first 2 weeks after pollination (prior the cotyledonary stage), could have practical implications for cassava breeding. Until now, attempts to recover cassava immature embryos at stages of development earlier than the cotyledonary stage failed. The earliest successful rescue reported in cassava is from embryos excised 32-36 days after anthesis (DAA). However, limited information was available regarding embryo development in cassava. This work studied and documented the stage of embryo development in histological sections of handpollinated ovules fixed from 1 to 30 days after anthesis (DAA). At 7 DAA, zygotes were just at the first stages of cell division (pro-embryo stage). At 14 DAA, embryos were at the pre-globular stage. Embryos at the early globular stage were observed in sections fixed at 21 DAA, and at the proper globular stage at 24 DAA. Samples at 30 DAA contained cotyledonary embryos that easily developed after ovule culture into viable plants using existing protocols. A second contribution of this work is the development of a protocol for the recovery of fully developed plants from immature embryos rescued and cultured in vitro as early as 7-14 DAA. Since embryos collected at this age are at the pro-embryo to pre-globular stage, ovary/ovule culture was necessary. A method is described whereby ovules were cultured to allow the development of pro-embryos and pre-globular stage embryos into the cotyledonary stage. Subsequently, these mature embryos were excised from the ovules to induce germination and the recovery of fully developed plants.

Vibratome Sectioning and Clearing for Easing Studies of Cassava Embryo Formation

“…Fifteen cyathia (45 ovules) per date (7, 14, 21-24 DAA) and treatment (as described below) were collected and analyzed. This sample size proved to be adequate for studies of cassava ES (Lentini et al, 2020a) and embryo development (Lentini et al, 2020b).…”

“…Day of pollination and anthesis day were assumed to be the same (Lentini et al, 2020b). Day after anthesis (DAA), instead of day after pollination (DAP), was used as reference to predict the developmental stages of zygotic embryo formation.…”

“…Handpollinated cyathia were used to collect ovules with embryos at different stages of development. Female cyathia were selected, hand-pollinated, collected (at varying DAA), and transported to the laboratory as described by Lentini et al (2020b). The isolating bags were kept on the hand-pollinated cyathia until samples were collected to prevent undesirable pollinations.…”

“…Therefore, several histology sections are required in order to reconstruct a particular developmental stage. Cassava ovules are about 2.5-5.0 mm long and 1.0-2.0 mm wide, depending on the stage of development (Lentini et al, 2020a;Lentini et al, 2020b). In contrast, the average size of angiosperm ovule is about 0.5 mm with a variation from 0.15 mm to 2 mm (Endress, 2011).…”

“…Reproductive biology is the basis for genetic improvement and a comprehensive understanding of it is essential for plant breeding, whether by conventional or biotechnological methods. Fundamental studies on plant reproductive biology include anatomical analyses by microscopy using diverse histological methods (Lentini et al, 2020a;Lentini et al, 2020b). Cassava (Manihot esculenta Crantz) is a major staple food in tropical regions and has diverse uses as basic material for processed food, animal feed, and industrial products.…”