Clonal propagation of Betula pendula Roth and Pinus sylvestris L. by stem cuttings and tissue culture
Clonal propagation of Betula pendula and Pinus sylvestris using stem cuttings and tissue culture was studied. Cuttings of both species rooted with a high level of success and rooted cuttings showed high levels of survival. Single-internode cuttings, with one axillary bud and leaf, were most successful with Betula pendula. particularly if such cuttings were collected from elongating shoots. Pinus sylvestris cuttings were also successful if collected from elongating shoots. Although exogenous auxin increased speed of rooting and number of roots formed by cuttings it had little effect on the number of cuttings which formed roots and the subsequent survival of these rooted cuttings. Growth and biomass distribution of rooted cuttings of Betula pendula and Pinus sylvestris was similar to that of seedlings. Successful propagation of both species depended on ortets being in a "juvenile" phase of growth. An attempt to produce "juvenile" cutting material from "adult" genotypes of Betula pendula was made by inducing the formation of epicormic shoots. Although these shoots had "juvenile" morphology, and cuttings derived from these shoots had similar rooting characteristics to cuttings taken from young seedlings, cuttings collected from epicormic shoots demonstrated plagiotropic growth of shoots in comparison with the orthotropic habit of cuttings taken from young seedlings. These results suggest that only partial rejuvenation occurred. Methods of enhancing the yield of cuttings from young seedling ortets of Betula pendula and Pinus sylvestris are discussed. A method to propagate Betula pendula by callus culture was developed. Callus nodules, initiated from stem internodes, were induced to undergo morphogenesis with the development of shoots. Although only half of the cultured nodules regenerated shoots, "morphogenic" nodules formed an average of 100 shoots of 4mm in length or greater when subcultured on a medium with BAP (benzylaminopurine) and NAA (napthaleneacetic acid). Other growth substances tested were less successful or unsuccessful at regenerating shoots. These shoots readily rooted outwith aseptic culture in an intermittent spray unit. Level of rooting and survival of rooted shoots was very high. Successfulb morphogenesis depended on the culture of "juvenile" tissues. Rate of growth of tissue-culture plants was the same as genetically similar seedlings, however tissue-culture plants were found to have slightly thinner stems after one season of growth. A cytological investigation of tissue-culture plants revealed the presence of polyploids which were identified through chromosome counts as allotetraploids with 4n=56 chromosomes in comparison with the normal diploid 2n=28 karyotype. These allotetraploids were readily identified by their unusual leaf morphology and very slow growth. No other ploidy levels were found. The frequency of occurrence of these allotetraploids varied with the type of auxin incorporated in the culture medium. NAA was associated with a level of tetraploidy of 11.0 percent whereas IBA (indolebutyric acid) was associated with a level of 3.8 percent. BAP was used in combination with both these auxins. The higher level of tetraploidy, associated with NAA, was more than compensated by a greatly enhanced level of shoot regeneration on callus nodules cultured on a medium with this auxin in comparison with nodules cultured on a medium with IBA. Presence of allotetraploids was of little concern because of their ease of identification and low level of occurrence. Vegetative material taken from genotypes of Betula pendula greater than one year old developed callus tissues but this callus showed little capacity to undergo morphogenesis. A few shoots did develop from callus initiated from shoot internodes collected from a four-year-old genotype. Only one of these shoots formed roots. Study of this plantlet suggested that it was not "juvenile" although its origins were adventitious.