Studies on Gynogenesis Induction in Cassava (Manihot esculenta Crantz) Unpollinated Ovule Culture

Abstract

Cassava (Manihot esculenta Crantz) is an important crop for subsistence farming in tropical and subtropical regions. There is a need to increase the rate of genetic gain to develop varieties adapted to new environmental conditions affected by climate change, which also influences the patterns of pests and diseases. The rate of cassava genetic improvement is limited by the difficulty in obtaining true-breeding types (inbred/homozygous lines). Cassava inbreeding obtained through conventional sequential self-pollination increases exposure of useful recessive traits and breeding value of progenitors. However, it takes 10-15 years to produce homozygous lines through successive self-pollination. Doubled haploid (DH) technology is a functional alternative to progressive self-pollination, and is already widely used in major crops to accelerate inbreeding. This work aimed at developing a protocol for the culture of isolated ovules and the induction of gynogenesis in cassava. Basic groundbreaking studies on cassava embryo sac development are presented. A protocol using unpollinated ovules collected from ovaries 1 day after anthesis is described. In the unpollinated-cultured ovules, the presence of embryos formed probably from the egg cells and not surrounded by the endosperm, was documented by anatomical analyses. This achievement is an important first step in the development of a reproducible gynogenesis protocol for the generation of doubled haploids in cassava. This protocol can also be useful as a starting point to obtain DHs using alternative methods of induction such as pollination of cassava with pollen of distant species or with cassava pollen irradiated with gamma rays.

Vibratome Sectioning and Clearing for Easing Studies of Cassava Embryo Formation

“…One month after culture, the ovules were excised from the carpels and sub-cultured on fresh MS3 medium, and grown under the same environmental conditions as described by Lentini et al (2020b) Table 1). Histology analyses were conducted using 30 ovules per treatment chosen at random according to Lentini et al (2020a). Ovule samples were fixed in FAA, dehydrated in a graded ethanol series from 70 to 100% then anchored onto a 7% agarose block using superglue.…”

“…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).…”

“…Samples were fixed in FAA [38% formaldehyde (Sigma-Aldrich F8775): glacial acetic acid: (Sigma-Aldrich 537020): 75% ethanol; 7:5:88, v/v)] at least for 8 days. Then, samples were washed for a minimum of 24 h in 70% ethanol to elimite most of formaldehyde and acetic acid and stored in 70% ethanol at 4°C (Lentini et al, 2020a;Lentini et al, 2020b, Table 1, Figure 1A). After fixation, samples were dehydrated in a graded ethanol series (85, 96, and 100%-1.5 h each).…”

“…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.…”