Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

Abstract

For 18 sugarcane cultivars, four,.distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 106 protoplasts per gram fresh weight of cells [gfwt-~]) compared to heterogeneous cell suspension cultures (0.1 × 106 protoplasts gfwt -~). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cuitivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 106 protoplasts gfwt -~. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 106 protoplasts gfwt-~. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Qll0 and Q138.

Protocol optimization and histological analysis of in vitro plant regeneration of RB92579 and RB93509 sugarcane cultivars

“…Regarding the results described in the literature and here observed the morphological variations of callus and shoot regeneration pathways are frequently reported in studies of in vitro shoot induction of sugarcane. The association of organogenic and embryogenic callus on the same sugarcane explant and the plant regeneration through indirect organogenesis from friable callus has been described in some studies (GNANAPRAGASAM & VASIL, 1992;TAYLOR et al, 1992;RODRIGUEZ et al, 1995). In another study, FRANKLIN et al (2006) reported the shoots induction of sugarcane from globular structures associated with the original explant and suggest that somatic embryos in globular stage may lead shoots induction from indirect organogenesis in the presence of cytokines, confirming the results observed here.…”

“…The somatic embryogenesis induction has been obtained from explants of sugarcane inoculated in medium supplemented with various auxin types (HO & VASIL, 1983;FITCH & MOORE, 1990;BRISIBE et al, 1994). In other study, TAYLOR et al (1992) described the induction of organogenic and embryogenic callus in 18 cultivars, as well as to obtain cell cultures in suspension. Plant regeneration from embryogenic callus obtained from immature leaves was observed by DIBAX et al (2011), NIEVES et al (2008, VICKERS et al (2005) and BLANCO et al (1997) and using MS medium (MURASHIGE & SKOOG, 1962) supplemented with 2,4 dichlorophenoxyacetic acid (2,4-D) and LIU (1993), using different combinations of 2,4-D, kinetin (KIN), benzylaminopurine (BAP) and calcium pantothenate.…”

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation - Review of Sugarcane (Saccharum spp.) and Pineapple (Ananas comusus (L.) Merrill) Cases

“…Many authors (Jimenez et al, 1991;Lorenzo et al, 2001;Lourens. & Martin 1987;Taylor et al, 1992) indicate that such differences disappear during the course of field growth and the first clonal multiplication, as observed in our experiments. Bioplantas for mass production of in vitro sugarcane plants by means of somatic embryogenesis.…”

Morfogênese in vitro e susceptibilidade de calos de variedades nacionais de cana-de-açúcar (Saccharum officinarum L.) a agentes seletivos utilizados em sistemas de transformação genética

“…A indução de calos embriogênicos já foi obtida em explantes de cana-de-açúcar inoculados em meio suplementado com diversos tipos de auxina (Ho & Vasil 1983, Fitch & Moore 1990, Brisibe et al 1994. Taylor et al (1992) descreveram a indução de calos organogênicos e embriogênicos em 18 cultivares, bem como a obtenção de culturas de células em suspensão. A regeneração de plantas a partir de calos embriogênicos obtidos através do cultivo de explantes de inflorescência imatura foi observada por Blanco et al (1997) em meio suplementado por 2,4-D (ácido 2,4 diclorofenoxiacético) e por Liu (1993), utilizando diferentes combinações de 2,4-D, cinetina, 6-BA (6-benzilaminopurina) e pantetonato de cálcio.…”