Hanna Laukkanen

Detection of Intracellular Bacteria in the Buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization

ABSTRACT Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified asMethylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.

Two endophytic fungi in different tissues of scots pine buds (Pinus sylvestris L.).

Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant.

Chitinase production in pine callus (Pinus sylvestris L.): a defense reaction against endophytes?

Abstract. In shoot tip-derived tissue cultures of Scots pine (Pinus sylvestris L.), browning and subsequent degeneration of the culture is accompanied by lipid peroxidation and lignification of cells, which are characteristic features of a plant defense reaction. Since chitinases are enzymes acting primarily in plant defense, their expression was studied in pine callus in order to elucidate the defense reaction. Chitinases were present diversely in tissue cultures originating from shoot tips and embryos of P. sylvestris, in contrast to Pinus nigra embryogenic callus, where production of chitinases or browning was not detected. Because endophytic microbes had earlier been detected in buds of Scots pine, their subsequent presence in the tissue cultures was considered a potential cause of the defense reaction. Therefore, the presence of endophytes in the tissue cultures was examined by in situ hybridization. Endophytes were found to colonize heavily in 45% of the tissue cultures of P. sylvestris and to form biofilms, while the P. nigra callus was not found to contain any microbes. The endophytes seemed to propagate uncontrollably once a tissue culture of P. sylvestris was initiated. Regardless of the high level of chitinase production in the callus, the control of the endophytes presumably becomes inadequate during the tissue culture of P. sylvestris.

Effect of exogenous polyamines on scots pine callus in vitro

Summary Callus cultures were started from buds of mature Scots pines ( Pinns sylvestris L.) and exogenous putrescine, spermidine and spermine were added to the growth medium in order to test their capacity to reduce browning and poor callus growth. Spermine (1 mmol/L) inhibited callus growth as measured in terms of dry weight. The effect of 1 mmol/L putrescine was studied more closely using two types of growth medium (Krogstrup and modified Murashige and Skoog), and its influence on callus growth and development was determined by measurements of dry weight, enzyme activities (arginine decarboxylase 4.1.1.19, ornithine decarboxylase 4.1.1.17 and phenylalanine ammonia-lyase 4.3.1.5), ethylene production, synthesis of soluble proteins and levels of free polyamines in calli. It was found to increase ethylene production in the callus, while its effect on protein synthesis, ADC and PAL activities depended on the growth medium, and changes in these reactions were seen in poorly growing callus in the form of decreases in ADC activity, while PAL activity and the concentrations of two small polypeptides increased. High values of free putrescine were measured in calli in the presence of putrescine, but the concentrations of free spermidine and spermine did not clearly change. The results suggest that exogenous polyamines affect metabolism in pine callus but cannot prevent browning and deterioration.