L. P. Siu and M. A. Weatherhead.
Department of Ecology and Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong
Axillary shoots were induced from node and shoot tip explants of aseptic Camellia seedlings. Callus formation also occurred on cotyledon and root explants of aseptic seedlings and on node, internode, petiole and leaf explants from young shoots of mature plants. A variety of media were utilised. High contamination rates were found in explants from field-grown material whereas no contamination occurred in explants from aseptic seedlings.
In 1978 12 Camellia species were recorded in Hong Kong with three of them being labelled as "exotics". (Hong Kong Herbarium, 1978) and since then a further species has been added to the list. In this study 3 native species viz. C. granthamiana, C. crapnelliana and C. hongkongensis were investigated. All 3 species are now rare in Hong Kong there being less than 30 mature individuals of C. granthamiana, one small population of C. crapnelliana and 3 small populations of C. hongkongensis. Consequently the local populations are very vulnerable to human disturbance and/or natural disasters. Although these species can also be found in mainland China they are again very limited in distribution (Chang, 1981). Therefore the development of in vitro propagation techniques was regarded as crucial to the conservation of these endangered species.
In vitro culture of camellias was pioneered by Lammert (1950) and subsequently utilised by Bennet (1977, 1978), Ackerman (1978), Yamaguchi et al. (1987) and Lu (1982) amongst others. Many techniques including culture of anthers, meristems, cotyledons, shoot tips and nodes have been described for a variety of species but, as far as we are aware, no work has previously been carried out on the three species studied here.
Two different starting materials were used to conduct the experiments, namely young shoots from mature plants and aseptic seedlings obtained from aseptic seed germination. Explants of nodes, internodes, petioles and leaves were taken from young shoots and shoot tips, nodes, roots and cotyledons were taken from aseptic seedlings.
The basal culture medium was that of Murashige & Skoog (1962) as modified by Samartin & Gonzalez (1986) and 11 different formulations were prepared based on different concentrations (1 or 0.5mg/l) of the cytokinins zeatin (Z), 2-isopentyl adenine (2-IP) and kinetin (K) and 0.1mg/l of the auxins 1-napthaleneacetic acid (NAA) and indole-3-acetic acid (IAA).
Cultures of field grown material
Callus formation without further regeneration occurred in contamination-free node, internode, petiole and leaf cultures of C. crapnelliana and C. hongkongensis, but for C. granthamiana only internode culture gave rise to callus formation (7%) (1.0mg/l K and NAA) as a result of the high contamination rates experienced. In general, callus formation was evident after a two week period in culture and while responses varied with media formulation the medium giving the highest % response in each case is shown in parenthesis below. For C. crapnelliana callus was induced on 25 - 86% (1.0mg/l K and NAA) of nodes, 9 - 65% of internodes (1.0mg/l 2-IP and NAA), 20 - 74% of petioles (1.0mg/l Z and NAA) and 5 - 100% (1mg/l Z and NAA) of leaves in uncontaminated cultures. For C. hongkongensis callus was induced on 100% (0.5mg/l 2-IP and NAA) of nodes, 10 -75% (0.5mg/l 2-IP and NAA) of internodes, 15 - 100% (1.0mg/l Z and NAA) of petioles and 3 - 35% (1.0mg/l Z and NAA) of leaves in uncontaminated cultures. Thus callus was routinely formed on media containing 1mg/l or 0.5mg/l of K, 2-IP or Z but only in combination with 0.1mg/l NAA. However as a result of the high contamination rate it was not possible to discern relationships between response and presence and concentration of the plant growth hormones. The calluses were mainly derived from the outer edges of the cut ends of the explants and this is in accordance with the observations of Ogutuga & Northcote (1970) and Frisch & Camper (1987) on stem callus formation in C. sinensis and C. japonica. There appear to be no reports in the literature on in vitro culture of petioles and leaves of Camellia.
High contamination rates (up to 100%) were evident in all types of culture and media formulation using field grown explants. Contamination was particularly evident in those cultures of C. granthamiana and it is conceivable that this may have adversely affected callus formation and other forms of differentiation. It is however consistent with the reports of other workers e.g. Haldeman et al. (1987) experienced contamination rates of at least 90% when culturing field grown shoots of C. sinensis and C. japonica.
Cultures of aseptic seedling material
Cultures initiated from aseptically grown seedlings were considerably more successful than those initiated from field grown material and no contamination was observed. In nodal and shoot tip culture C. granthamiana explants gave the best results for axillary shoot induction with 88% of explants responding on medium containing 1.0mg/l Z and 0.1mg/l NAA and 80% responding on medium containing 0.5mg/l K and 0.1mg/l NAA. Responses on other media were lower but a positive response was only observed on media containing either K, Z or 2-IP and NAA. A similar pattern was evident for explants of C. crapnelliana where 67% of node and shoot tip explants gave rise to axillary shoots on medium containing 0.5mg/l K and 0.1mg/l NAA. Again responses on other media were lower but only positive in the presence of K, Z or 2-IP and NAA. For C. hongkongensis the results were less satisfactory with axillary shoots being induced on a maximum of 40% of explants. However the response in terms of media composition was similar with medium containing 0.5mg/l 2-IP and 0.1 mg/l NAA giving the strongest response. Weaker responses were obtained on media containing K and Z in the presence of NAA.
Cotyledon and root cultures were only initiated from aseptic seedlings of C. granthamiana due to a lack of sufficient experimental material of the other species. The cotyledon explants produced callus after 4 weeks in culture and the percentage response was greater on media with a high cytokinin : auxin ratio. 1mg/l Z, 2-IP and K each in combination with 0.5mg/l NAA gave 85%, 79% and 67% callus response respectively whereas 0.5mg/l 2-IP, Z and K again in combination with 0.5mg/l NAA gave 60%, 25% and 25% respectively. Initially the calluses were whitish yellow but they turned brown quickly without further growth or development.
Root explants became green and elongated after 1 week in culture and callus was induced after 3 weeks. The pattern of response in terms of plant growth hormones was the opposite of that observed in cotyledon cultures with the greater responses being found at a lower cytokinin : auxin ratio. 64%, 52% and 50% of cultures responded on media containing 0.5mg/l 2-IP, K and Z each in combination with 0.1mg/l NAA respectively whereas 50%, 42% and 25% responded on 1mg/l Z, 2-IP and K each in combination with 0.1mg/l NAA. Neither organogenesis nor embryogenesis occurred in the root explants or the callus and both became brown in a short time.
Although tissue culture techniques are widely used for the propagation of woody species only limited research has been reported on micropropagation of Camellia species. As described above, for field grown material, callus formation without further regeneration occurred in node, internode, petiole and leaf cultures of C. crapnelliana and C. hongkongensis but only internode cultures gave rise to callus in C. granthamiana. Similar results have been reported by Ogutuga & Northcote (1970) and by Frisch & Camper (1987) who observed stem derived callus in cultures of C. sinensis and C. japonica. While the high contamination rate did not allow a meaningful interpretation of the results it was evident that media containing NAA were more satisfactory than those containing IAA. The effects of the different cytokinins could not be quantified. Another factor which may affect callus formation and subsequent differentiation in field grown explants could be that such explants are mature and thus could exhibit some inhibition to further development.
Axillary shoot induction from nodal and shoot tip explants of C. crapnelliana and C. granthaminana gave satisfactory results particularly on media containing 0.5mg/l K or Z and 0.1mg/l NAA. In contrast the highest numbers of axillary shoots induced on explants of C. hongkongensis were on media containing 2-IP and NAA. However these axillary shoots did not develop further and became translucent with a high mortality rate.
It is evident from the results that juvenile material of all three species is more sensitve to the action of auxins and cytokinins than mature material and it has been found (Bonga, 1982) that this physiological factor makes success in the in vitro propagation of woody species rare. Nevertheless the successful shoot initiation from aseptic seedlings of these three indigenous, rare and endangered species is a promising result for both conservation and commercial purposes.
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