Plant regeneration from protoplasts of Capsicum annuum L. cv. California Wonder.

Plant regeneration from mesophyll protoplasts of pepper, Capsicum annuum L. cv. California Wonder has been demonstrated via shoot organogenesis. Protoplasts isolated from fully expanded leaves of 3-week-old axenic shoots when cultured in TM medium supplemented with 1 mg l-1 NAA, 1 mg l-12,4-D, 0 5 mg l-1 BAP (CM 1) resulted in divisions with a frequency ranging from 20-25 %. Antioxidant ascorbic acid and polyvinylpyrrolidone (PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed in TM medium containing 2 mg l-1 NAA and 0·5 mg l-1 BAP (CM Π) and MS gelled medium containing 2 mg 1-1 NAA and 0 5 mg 1-1 BAP (CM III), respectively. Regeneration of plantlets was possible via caulogenesis. Microshoots, 2-5 percallus appeared on MS gelled medium enriched with 0·5 mg l-1 IAA, 2mg l-1 GA and l0mg l-1 BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mg l-1 NAA and 0·5mg l-1 BAP. Protoplasts isolated from cotyledons failed to divide and degenerated eventually.

Embryogenesis in flowering plants: Recent approaches and prospects.

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy. Several genes have been identified and cloned from the carrot system. These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood. Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using a Cuscuta haustorial (organspecific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes.

A simple method for the isolation of plant protoplasts.

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described· The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplastreleasing enzymes that are stable and efficient at higher temperatures· Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H2O), all dissolved in distilled water· The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division· Plant regeneration was demonstrated for Nicotiana tabacum cv· Thompson from protoplasts isolated by this method· Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.