Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales, Chlorophyta)

Abstract

Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15-20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 x 105 cells ml-before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10-15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 ps duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18-24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.

Development and Characterization of Somatic Hybrids of Ulva reticulata Forsskål (×) Monostroma oxyspermum (Kutz.)Doty

“…The hybrids regenerated in this study were asymmetric since the ploidy content was not additive of both the genomes of the fusion partners. The previous study on protoplast fusion between U. pertusa and E. prolifera resulted in hybrids with no change in chromosome number (Reddy et al, 1992 ) whereas Kapraun ( 1989 ) reported parasexual fusion products of Enteromorpha with diploid and tetraploid levels. The occurrence of a non-additive chromosome count is a common phenomenon and reported in several studies of somatic hybridization in land plants (Chen et al, 2004 ; Minquin et al, 2008 ; Tu et al, 2009 ; Wang et al, 2011 ) but this study is the first in macroalgae.…”

“…The hybrids generated in this study showed heterozygous vigor (heterosis) displaying some traits superior to both the fusion partners. Earlier reports on the somatic hybridization of seaweeds have largely dealt with morphology and pigmentation, albeit showing a few improved traits such as growth (Reddy et al, 1992 ) and fatty acid composition (Reddy and Fujita, 1989 ) but lacked the evidence for the molecular level of recombination. Also, most of the studies reported the development of chimeric thalli after protoplast fusion indicating independent segregation of the two genomes without recombination.…”

Seaweed protoplasts: status, biotechnological perspectives and needs

“…Although they were able to produce several green and red chimeric thalli, the genetic nature of somatic hybrids has not been reported. Since then, several attempts have been made to accomplish interspecific and intergeneric protoplast fusions in several algae employing both PEG and electrofusion methods Cheney 1990;Fujita and Saito 1990;Reddy et al 1992;Cheney and Kurtzman 1992;Dai et al 1993;Mizukami et al 1995). In most cases, the frequency of heterokaryon formation and subsequent recovery of somatic hybrids or cybrids following the protoplast fusions was very low.…”

Analysis of viability and cell types of macroalgal protoplasts using flow cytometry

“…Unlike methods used for identifying higher plant heterokaryons, differential labelling of one of the populations would not be required in this case, because a marker, phycoerythrin, occurs naturally in these red algae. Conversely, techniques already applied to higher plants could be used for heterokaryons described for the intergeneric fusion between protoplasts of the chlorophytes Ulva and Enteromorpha (Reddy et al, 1992). Somatic fusion is a particularly significant technique for genetic engineering in seaweeds due to the lack of effective transformation techniques; traditional selection methods for heterokaryons (visual inspection and micromanipulation) are tedious at best.…”