Evaluation of new approaches in breeding Pinus radiata

The objective of our first experiment was to explore the possibility of production of haploids via in vitro culture of unfertilised mega gametophytes, study the effect of various cultural treatments on the process of initiation of somatic embryogenesis from the fertilized immature mega gametophyte, in Pinus radiata. The experiment was conducted in two parts. In the first part of experiment one unfertilized mega gametophytes of Pinus radiata were cultured on media with various concentrations of 2,4-D and BA (6-Benzylaminopurine. Two types of response were observed from the cultured mega gametophyte. The mega gametophytes cultured 2 months before fertilization gave rise to embryonic initials and the mega gametophytes 1 month before fertilization gave rise to organogenic shoots. The culture media had a significant effect on the developmental response of the mega gametophytes. The nuclear size of these structures was much smaller that of a diploid nucleus suggesting a possible haploid nature of these structures. In a second part of our first experiment immature fertilized mega gametophytes and mature embryos from a large number of clones at different developmental stages were cultured on three different media having three different concentrations of 2,4-D, the concentration of BA and Kinetin was kept constant. The effect of different storage treatment on the embryogenic capacity of the five best performing clones was also investigated. The study revealed that the culture medium, maturity stage and genotype of the explant have significant effects on initiation of SE (somatic embryogenesis). Freshly excised explants exhibited a higher initiation frequency than cold stored ones, and there were significant difference in the initiation frequencies among the six storage treatments. Storage at 1°C rather than 5°C was found to be a better storage treatment in terms of initiation of embryogenic tissue (ET) from the stored explants. High frequency of initiation of ET was observed from a large number of genotypes and very early stage somatic embryos were observed in the proliferation medium. We were not successful in obtaining fully mature somatic embryos from any of the cultures because of the specific cultural requirement, which could not be optimised due the constraint of time. A large no of calli were produced from mature embryos but none of them was embryogenic. RAPD (randomly amplified polymorphic DNA) fingerprints have been used to estimate genetic and taxonomic relationships in crop plants. The use of this technique is not extensive in forest trees, In this study RAPD analysis was performed on four elite clones of Pinus radiata in addition to embryogenic callus derived from immature embryos after one and three months of proliferation. RAPD-PCR methodology was optimised and an efficient protocol is presented. Seven primers out of 40 screened, were selected on the basis of number and frequency of polymorphism produced. With these, a total of 137 bands were reproducibly obtained. The polymorphism were scored and used in band sharing analyses to identify genetic relationships. The results show that RAPD analysis allows one to discriminate among all four clones tested and therefore, can be recommended as a convenient tool for DNA fingerprinting and calculation of genetic distances in Pinus radiata. We were also able to demonstrate that RAPDs may be used to detect and quantify somaclonal variations in somatic embryogenesis (SE) derived populations. RAPDs, will thus allow direct cloning of normal and variant DNA to detect the source of somaclonal variation. Two separate experiments were designed to explore the presence and the possibility of inducing gametophytic apomixis in Pinus radiata. First experiment was aimed at identification of a source of genetically determined apomixis (formation of embryo without fertilization). For the first experiment, female cones from 40 genotypes were bagged (to prevent pollination). For the second experiment female cones from 11 genotypes were bagged and treated with combination of plant growth regulators and irradiated pollen, in order to identify a method to induce apomixis in Pinus radiata. The results indicate that pollination is essential to enable the female cones to continue their development to maturity. The treatments with plant growth regulators (PGRs) did not have any significant effect and the cones treated with the PGRs aborted as did the unpollinated cones (control). The interesting finding was that some of cones which were pollinated with irradiated pollen continued there development to maturity. Microscopic examination of the developing mega gametophyte revealed a developing embryo inside the seed. DNA analysis was performed using RAPD-PCR to determine the origin of the embryos produced with irradiated pollen. Most of the progeny showed bands from male and female parents but some of the progeny showed only maternal bands, which could be an indication of haploid or doubled haploid nature of these embryos, while some showed some bands which were not present in either the female or the male parents.