Microcomputer tomography (microCT) as a tool in Pinus tree breeding: pilot studies
New Zealand Journal of Forestry Science
Pinus breeding programmes yield high numbers of seeds, but a non-destructive method to determine seed viability is still lacking. With the long reproductive cycle (up to 28 months) of Pinus species, determining when fertilisation occurs can assist when applying tissue culture methods like somatic embryogenesis (SE). For SE, pre-cotyledonary zygotic embryos are ideal for culture initiation (some weeks after fertilisation), while mature zygotic embryos are extracted and used for in vitro
... tion during organogenesis. Achieving automated viability assays in extracted seeds would also be helpful, as would being able to find numbers and condition of seeds in immature or unopened cones. For such applications, microcomputer tomography (microCT) was a candidate technology. MicroCT was used to scan immature cones of Pinus radiata D.Don at four-weekly intervals after pollination to determine time of fertilisation. After harvesting of mature cones, a sample of 30 seeds of each of three Pinus species (P. radiata, P. patula Schiede ex Schltdl. & Cham. and P. pinaster Aiton) were scanned together in one microCT scan, and the same seed was allowed to germinate on moist filter paper after scanning. In addition, a mature unopened cone of P. radiata was also microCT-scanned to determine the potential for determining seed viability in situ, and the seed was subsequently extracted for examination. Findings: Fertilisation evidently occurred between weeks 64 and 68 after pollination in P. radiata. The microCT data for extracted seeds of the three species identified some seeds with large voids, which suggest nonviability (low to zero germination potential), and that was confirmed by the germination test. In addition to demonstrating in situ seed viability in the unopened P. radiata cone, the seed extraction revealed that some non-viable seeds remained trapped in the basal part of the dried cone. Conclusions: This case study demonstrates the feasibility of using microCT scans as a partially non-destructive method in Pinus tree breeding programmes to confirm fertilisation and determine seed viability. However, scanning the seed after extraction from mature cones can help to determine viability which is often hard to gauge from seeds' external appearance. Future studies need to narrow the time window of fertilisation by scanning a wider variety of genotypes between weeks 64 and 68 after pollination.