Microprotoplast fusion technique: a new tool for gene transfer between sexually-incongruent plant species

Abstract

Various aspects of a microprotoplast fusion technique and the strategies followed for intergeneric partial genome transfer (one or a few chromosomes) and alien genes from sexually-incongruent donor species to recipient species are described . The essential requirements of the microprotoplast fusion technique are the induction of micronuclei at high frequencies, as well as the isolation and enrichment of sub-diploid microprotoplasts in donor species, efficient fusion of the donor microprotoplasts with normal recipient protoplasts and stable regeneration of plants from fusion products . The results on the production of microprotoplast hybrid plants between the transformed donor lines of Solanum tuberosum and Nicotiana plumbaginifolia carrying various genetic markers, and a recipient line of Lycopersicon peruvianum or Nicotiana tabacum, and on the transfer and expression of alien genes (kanamycin resistance, j -glucuronidase) are presented . The data obtained on microprotoplast hybrid plants between S. tuberosum and L . peruvianum showed that many of the hybrids contained one potato chromosome carrying nptll and GUS, and 24 or 48 L. peruvianum chromosomes (monosomic additions), and that they were male-and female-fertile . Various applications of chromosome transfer by this technique, especially for economically-important traits (e.g . disease or stress resistance) from sexually-incompatible wild species, for construction of chromosome-specific DNA libraries through microdissection and microcloning of chromosomes, or by flow-sorting of chromosomes for genome analysis, are discussed.

Molecular and cytogenetic constitution of plants obtained via two different somatic hybridization methods

“…Despite this common phenomenon in somatic hybrids, many scientists have reported a stable genomic transfer through this technique, especially using ASH (Wolters et al 1994;Rutgers et al 1997;Binsfeld et al 2000;Chetelat and Meglic 2000;Varoto et al 2001). Furthermore, the latter technique has been successfully used for alien chromosome transfer, the production of addition lines and for the introgression of genes between sexually incompatible species (Ramulu et al 1995;Rasmussen et al 1997;Rutgers et al 1997;Binsfeld et al 2000).…”

“…However, in addition to having desirable traits, some of these wild-type plants also have many undesirable traits, some of which are linked to fertility or complete sterility (Yamada et al 1998). This disadvantage may be overcome by partial genome transfer using asymmetric somatic hybridization (ASH; Ramulu et al 1995). In this method, only a part of the wild genome is transferred to a receptor protoplast (Rasmussen et al 1997), thereby reducing the number of undesirable traits incorporated in the receptor genome.…”

Asymmetric Somatic Plant Hybridization: Status and Applications

“…Application of these spindle toxins to synchronized cells generally blocks cells in metaphase and scatters chromosomes in the cytoplasm; subsequently, those decondense into micronuclei. Subsequently, these micronucleated cells are stripped of their cell wall, and the resulting MPPs are ultracentrifuged to subdivide them into classes [32]. These can be further filtered through sequential filters of smaller pore width.…”

Somatic hybridization as a tool for tomato breeding

“…esculentum (recipient) and S. tuberosum (donor) no viable plants were obtained ) . An alternative procedure that does not involve irradiation is microprotoplast/protoplast fusion (Ramulu et al ., 1992(Ramulu et al ., , 1994b . In this technique, donor microprotoplasts with one or a few chromosomes are produced by treatment with spindle toxins, such as amiprophos-methyl (APM) or cremart, followed by high speed centrifugation, and sequential filtration through nylon sieves (Verhoeven & Ramulu, 1991 ;Ramulu et al ., 1992Ramulu et al ., , 1993Ramulu et al ., , 1994a .…”

“…Ramulu, unpublished results) . A high frequency of micronucleation of the donor genotype after treatment with spindle toxins, and efficient plant regeneration from protoplasts of the recipient genotype or from fusion products, are the two most important prerequisites for successful realization of partial genome transfer by this technique (Ramulu et al ., 1994b) .…”