Zaida Lentini, Geraldine Restrepo, María E Buitrago, Eddie Tabares

Frontier Plant Sci. 2020 May 8;11:522. doi: 10.3389/fpls.2020.00522.

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 hand-pollinated 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.

See: https://pubmed.ncbi.nlm.nih.gov/32457774/

Figure 1: Flowchart of medium sequence for induction of mature embryos (cotyledonary stage of development), germination, and development of viable plants from open-pollinated ovules cultures. Group 1, consisted of cyathia collected 7–14 DAA, which contained embryos at the initial stage of development. Group 2, included cyathia collected 21–30 DAA, which contained embryos at globular stage or onward. The carpels were cultured with the basal cut end on solid medium. Cultures were kept at 28–30°C in the dark. Once the ovules protruded through the carpel walls (usually about 3–4 weeks after culture initiation), ovules were isolated and placed on fresh medium of the same composition with the adaxial side down on the medium. Ovules were subcultured every 4 weeks on fresh medium according to the medium sequence treatment, until mature embryos (at advanced cotyledonary stage) naturally protruded through the ovule integuments or up to a total of 6 months. Mature embryos were subcultured to induce germination, and subsequently, to develop plants in a 12 h day/night photoperiod with 80–100 μmol.m^{– 2}.s^{– 1} during the day.