Organogenesis and fine structure in megagametophyte callus lines of Picea abies

Abstract

Differentiation and fine structure were studied in 63 callus lines originating from the haploid megagametophyte of Picea abies (L.) Karst. Developing cones were collected from 27 trees growing in 13 localities in Finland. Vernalization of cones for 12–42 days at 4°C was optimal for callus initiation from the immature megagametophyte (primary endosperm). Five combinations of media based on the macronutrient elements of Chu et al. (1975; Sci. Sin. 18: 659–668) and the micronutrient elements and vitamins of Murashige and Skoog were tested for callus induction, growth and differentiation. Only about 1.5% of the megagametophytes produced subculturable callus (which may be partly due to the high frequency of lethal genes), although in certain mother trees callus production was as high as 20%. In most of the trees sampled, polyamines could not replace casein hydrolysate and glutamine in induction of megagametophyte callus. About half of the originally haploid, diploid and mixoploid callus lines were able to differentiate. A combination of three polyamines (putrescine 0.25, spermidine 0.1 and spermine 0.025 mM) favoured development of roots. In five callus lines shoots and roots developed in the same piece of callus, but these organs usually had no connection with each other. The fine structure of the callus cells was normal, but their starch stores were rather abundant. Mesophyll cells of needles differentiated from originally haploid callus had chloroplasts with fairly well‐developed grana. Secondary metabolites were observed in the vacuoles of some callus cells and in organ initials. Plasmodesmata were very rare in callus cells but they were characteristic of those of the needles. The electron microscope observations showed that the poor capacity for differentiation of P. abies callus cultures was not due to cytological instability.