Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization

Abstract

Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10(6) mature pollen protoplasts were fused with 7·10(6) mesophyll protoplasts using a PEG/Ca(2+) method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l(-1)). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l(-1)). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.

Haploids: Constraints and opportunities in plant breeding

“…Earlier attempts to produce gametosomatic hybridization by the fusion between male-gametophyte protoplast and somatic protoplast were successful (Choi et al, 1992;Lee and Power, 1988a,b). Gametosomatic hybridization has been reported in genus Nicotiana and Petunia (Choi et al, 1992;Desprez et al, 1995;Giddings and Rees, 1992;Lee and Power, 1988a,b;Pental et al, 1988). Isolation of ovules from the ovary is a very cumbersome procedure, with risk of damage to ovary and contamination from somatic tissue.…”

Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

“…Variations on this method have been used to introduce a variety of desired traits into crops, including stress resistance (Hennig et al, 2015), pathogen resistance (Kumari et al, 2020), seedlessness (Wu et al, 2005), male sterility (Bruznican et al, 2021), and increased photosynthetic efficiency (Takahata and Takeda, 1990). This can be performed to create a symmetric cell fusion, in which the complete nuclear genomes of the two species are combined (Narasimhulu et al, 1992;Laiq et al, 1994;Ling and Iwamasa, 1994;Desprez et al, 1995;Kirti et al, 1995); an asymmetric cell fusion, in which DNA fragments or partial chromosomes from one species are introduced into the other (Zhou and Xia 2005;Sigeno et al, 2009); or a cybrid, in which chloroplast or mitochondrial genomes from one species are introduced into cells of another (Kochevenko et al, 2000;Guo et al, 2004). Not only intra-genus, one especially interesting case was a monocotdicot (Triticum aestivum and Arabidopsis) protoplast fusion in which regenerated calli and green plants resembling that of wheat were obtained (Deng et al, 2007).…”

Development and isolation of the male gametophyte of Torenia fournieri

“…Since sporopollenin is well known for its resistance to enzymatic degradation, pollen protoplast isolation techniques are based on applying osmotic shock (Wever and Takats 1971;LaFountain and Mascarenhas 1972;Zhou et al 1986, Zhou 1988Tanaka 1988;Wagner et al 1989aWagner et al , 1989bKranz et al 1991aKranz et al , 1991bTheunis et al 1991;Southworth and Morningstar 1992;Dumas and Russell 1992;Yang and Zhou 1992;Kranz and Lörz 1993;Faure et al 1994;Ueda and Tanaka 1994). Protoplasts obtained from such experiments are often used in fusion studies in order to hybridize plant species that are not able to breed in a natural way as a result of different incompatibility systems (Pirrie and Power 1986;Lee and Power 1988;Pental et al 1988;Zenkteler 1990;Desprez et al 1995). Sperm cells of plant species with tricellular pollen can easily be isolated by making the pollen grains burst by osmotic shock or grinding (Cass 1973;Russell and Cass 1981;MatthysRochon et al 1987).…”