Separation of Integument and Nucellar Tissues from Cotton Ovules ( Gossypium hirsutum L.) for both High- and Low-Throughput Molecular Applications

Abstract

Here we present a quick and low-cost method to separate the different layers of tissue from the ovules and young seeds of cotton (Gossypium hirsutum L.) for use in high- and low-throughput molecular applications. This method is performed at room temperature using standard laboratory equipment and does not require embedding of the samples, time-consuming fixation, or micro-sectioning procedures. We show that the three main tissues can be efficiently separated from isolated ovules collected on the day of anthesis. RNA and genomic DNA extracted from tissues separated by this method are of good quality and suitable for a variety of molecular applications to study the early stages of cotton seed and fiber development.

Genetic and DNA Methylation Changes in Cotton (Gossypium) Genotypes and Tissues

“…The outer integument gives rise to fibre initials (visible at 0 dpa) that develop into fibres and the remaining epidermal cells form the epidermis of the seed coat. The separation of fibre initials from surrounding epidermal cells is difficult, and recently, a method has been developed to isolate the outer integument of ovules that enriches for cells (∼30% of epidermal cells) that produce fibre [54] . The comparison of the fibre forming cells on the outer integument to the non-fibre forming inner integument may be useful to understand the changes during fibre development.…”

“…Three-week-old plants were used to collect cotyledon, stems and total root tissues, while 6-month-old mature plants were used to collect fully expanded (mature) leaves, primary roots, 0 dpa and 3 dpa ovules, and 35 dpa fibre (manually separated from seeds). The outer integument (OI) and inner integument (II) were dissected from 0 dpa ovules harvested between 13∶00–15∶00 [54] . Two flowers were combined as one replicate for OI and II, and for each tissue, three or four biological replicates were collected from individual plants.…”

Cytokinin inhibits cotton fiber initiation by disrupting PIN3a-mediated asymmetric accumulation of auxin in the ovule epidermis

“…Ovule microdissection was performed according to the method of Bedon et al (2012). Fifty fresh ovules harvested from the boll at middle part of each locule were separated.…”

Members of the MYBMIXTA-like transcription factors may orchestrate the initiation of fiber development in cotton seeds

“…A few ( GhMML5, 6, 10 ) are also expressed in leaves and/or cotyledons which are rich in either hair or glandular trichomes that are structurally related to seed fibers and share some common regulators like GhHD-1 (Walford et al, 2012). MML gene expression levels were also investigated using hand-microdissected tissues (Bedon et al, 2013) from whole ovules and young seeds to focus just on the fiber initiation process occurring in the epidermis. This circumvents the transcript dilution or confounding with expression from the other layers of ovule tissues.…”