SummaryPolygalacturonase-inhibiting protein (PGIP) i s a cell wall protein purified from hypocotyls of true bean (Phaseolus vulgaris L.). PGlP inhibits fungal endopolygalacturonases and is considered to be an important factor for plant resistance to phytopathogenic fungi (Albersheim and Anderson, 1971; Cervone eta/., 1987). The amino acid sequences of the N-terminus and one internal tryptic peptide of the PGlP purified from P. vulgaris cv. Pinto were used to design redundant oligonucleotides that were successfully utilized as primers in a polymerase chain reaction (PCR) with total DNA of P. vulgaris as a template. A DNA band of 758 bp (a specific PCR amplification product of part of the gene coding for PGIP) was isolated and cloned. By using the 758-bp DNA as a hybridization probe, a lambda clone containing the PGlP gene was isolated from a genomic library of P. vulgaris cv. Saxa. The coding and immediate flanking regions of the PGlP gene, contained on a subcloned 3.3 kb Sall-Sall DNA fragment, were sequenced. A single, continuous ORF of 1026 nt (342 amino acids) was present in the genomic clone. The nucleotide and deduced amino acid sequences of the PGlP gene showed no significant similarity with any known databank sequence. Northern blotting analysis of poly(A)+ RNAs, isolated from various tissues of bean seedlings or from suspension-cultured bean cells, were also performed using the cloned PCR-generated DNA as a probe. A 1.2 kb transcript was detected in suspension-cultured cells and, to a lesser extent, in leaves, hypocotyls, and flowers. A cDNA clone containing part of the coding region for

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Thin cell layer explants of tobacco were floated in vitro on the surface of liquid culture media. The initial exogenous concentrations of indolyl-3-butyric acid, and kinetin, the initial medium pH, and the explant density were varied. Various patterns of de novo and direct differentiation without any intermediate callus (flower, vegetative bud, root) as well as the absence of morphogenesis and callus formation without any subsequent organogenesis were separately controlled on 100% of the explants. On the same exogenous combination of glucose, indolyl-3-butyric acid, and kinetin, changes in initial medium pH changed the pattern of morphogenesis. For a given initial exogenous indolyl-3-butyric acid concentration, vegetative buds were obtained at either pH 6.1 or 7.8, whereas a mixture of flowers and vegetative buds was obtained at pH 6.8. Furthermore, changes in explant density changed the morphogenetic response. It is suggested that the effects of the initial medium pH and explant density on morphogenesis may be related partially to modifications of the physicochemical properties of the cell wall and (or) plasmalemma.

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein purified from hypocotyls of true bean (Phaseolus vulgaris L.). PGIP inhibits fungal endopolygalacturonases and is considered to be an important factor for plant resistance to phytopathogenic fungi (Albersheim and Anderson, 1971; Cervone et al., 1987). The amino acid sequences of the N-terminus and one internal tryptic peptide of the PGIP purified from P. vulgaris cv. Pinto were used to design redundant oligonucleotides that were successfully utilized as primers in a polymerase chain reaction (PCR) with total DNA of P. vulgaris as a template. A DNA band of 758 bp (a specific PCR amplification product of part of the gene coding for PGIP) was isolated and cloned. By using the 758-bp DNA as a hybridization probe, a lambda clone containing the PGIP gene was isolated from a genomic library of P. vulgaris cv. Saxa. The coding and immediate flanking regions of the PGIP gene, contained on a subcloned 3.3 kb SalI-SalI DNA fragment, were sequenced. A single, continuous ORF of 1026 nt (342 amino acids) was present in the genomic clone. The nucleotide and deduced amino acid sequences of the PGIP gene showed no significant similarity with any known databank sequence. Northern blotting analysis of poly(A)+ RNAs, isolated from various tissues of bean seedlings or from suspension-cultured bean cells, were also performed using the cloned PCR-generated DNA as a probe. A 1.2 kb transcript was detected in suspension-cultured cells and, to a lesser extent, in leaves, hypocotyls, and flowers.(ABSTRACT TRUNCATED AT 250 WORDS)

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Thin cell-layer explants (TCLs) have been proposed as favorable tissues for the study of root, vegetative shoot and flower formation. We tested the effects of pH, light quality, light quantity, and IBA and kinetin concentrations on the morphogenesis of TCLs cultured individually on a liquid medium. Alterations of the amounts of exogenously supplied IBA and kinetin were sufficient to induce the formation of roots, vegetative shoots and flowers on TCLs cultured on otherwise identical media. The type and number of organs formed were sensitive to the intensity of light (55, 75, 100 and 120 muEinsteins m-2 sec-1) under which TCLs were grown. Evidence was obtained that the effects of light on TCL morphogenesis were associated with photochemical degradation of IBA in the medium. Evaluation of the organogenesis that occurred in TCLs cultured on a medium containing a range of IBA and kinetin concentrations showed that the number and type of organs formed, and overall growth, were dependent upon the initial concentrations of auxin and cytokinin. We have developed the TCL culture system into a sensitive and reproducible bioassay for the study of morphogenesis. The advantages of using the TCL morphogenesis bioassay for the identification and study of molecules (e.g. cell wall oligosaccharides) that may regulate morphogenesis are discussed.

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