AIR2-CT92-1353 European Aesculus Cultivation System

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AIR Cluster V - Speciality Chemicals : Essential Oil : Pharmaceuticals/Cosmetics

	Proposal No:	AIR2-CT92-1353
	Date Prepared:	September 1999
	Source:	Final report September 1997

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Final report September 1997

Summary

The main objective of this proposal was to find out whether Aesculus hippocastanum is a tree which has genetically manifested clones with high escin yield and how easy this tree is to propagate. Also productivity data concerning production of nuts and other aspects of agroindustrial production have been investigated. Results show, that escin yield is not a specific character in single clones. It is mainly depending on environmental conditions. High variability was also observed in most of the other characters under research, which indicates Aesculus as a relative "wild" genus.

Propagation is easy by seeds but needs long time until fruit production. Among the common vegetative methods grafting has been the most effective one to shorten this time. Different types of graft work very well. In vitro propagation seems very difficult due to contamination problems. Productivity was recorded for trees grown near Bologna and in Florence as well as there were first cultures with different training systems established. Clear results have not yet been obtained as the cultivation time was too short.

The ecological impact of growing Aesculus hippocastanum was investigated with naturally growing trees and plantations in experimental plots. In the early developmental stages Aesculus plantlets appear not to be well fitted to the semi-arid Mediterranean climate. The summer water deficiency is the main obstacle. Planting horse chestnuts associated with maquis bushes gives best results with respect to semi-intensive cultivation practice, as the different species start their individual production cycles in different times after planting.

Scientific objectives

The objective is to develop a European Aesculus cultivation system for production of seeds for the pharmaceutical and cosmetics industries. These industries are interested in escin, a mixture of saponins that occur in the seed of the horse chestnut, that can be used in treatment of peripheral vascular disorders. Propagation methods, productivity data, production technology and ecological impact were investigated.

Activities

The determination of escin yield in the seeds was continued in order to confirm previous data and to provide some information on the genetic stability of this character within different genotypes. Recollection of chestnuts from the same trees as in previous years showed similar values for escin yield as in the first and third year. This was observed in all locations. The tendency that there are higher yielding trees in southern Europe, as suggested in the first year, seems to be confirmed.

Analytical HPLC-methods were established to detect escin in horse-chestnut seeds. It was found that the products from hydrolysis of escin gave different HPLC-pattern when compared with the saponin- mixture of escin. To obtain further information about the escin composition of horse-chestnut seeds, samples from three locations in Great Britain (Thetford, Castle Howard and Shodon) and two locations in Italy (Florence and Sardinia) were analysed by HPLC. All horse-chestnut seed analysed by HPLC contained the beta-escin saponin- mixture and no degradation products by hydrolysis. The detected degradation in several samples were caused by the sample preparation and were artefacts. Hence, the isolation of beta-escin from horse-chestnut seed has to be done without any use of acid during the process. Acidic solutions can hydrolyse carbohydrates from the saponin-mixture and degradation products are obtained.

Work on macropropagation techniques has been finished. The examination of a range of different treatments upon the rooting and subsequent growth of Aesculus hippocastanum cuttings as well as clonal variation in rooting performance have been followed up. Some of them are promising for vegetative propagation. This years results showed significant differences in the number of available cuttings from five clones. The pattern of differences closely mirrored that shown in the 1995 collection. This appears to reflect different incident light available to the stumps, where those receiving most light produce most cuttings.

Hedging regimes show clear differences apparent between clones and between treatments in the number of shoots produced in response to hedging. The clones split into two distinct groups, those with epicormic shoots and those with shoots arising from the exposed cambial layer of the sump cut surface. The epicormic shoots were generally more sturdy and vigorous than the cambial ones. No lateral or epicormic buds have produced shoots. This supports the view that Aesculus has a high level of apical dominance.

Work on tissue culture system for rejuvenation in vitro used different sources for starting material. Due to serious problems with contamination among the tested material only the material from in vitro germinated chestnuts gave two uncontaminated cell lines.

Extending root tips were excised from 20 germinating chestnuts from Bush Estate and transferred to Chestnut Multiplication Medium. The cultures were incubated at 2°C with a day length of 16 hours. Some culture were lost to contamination and not all cultures proliferated shoots. Seven proliferating culture lines were produced. These are at present being proliferated further and will be subjected to a rooting test in the future. Somatic embryogenesis gave only one torpedo-stage embryo, but without subsequent development in this culture.

Further attempts were made to mature the embryogenic cell lines to later stage embryos. A variety of media and concentrations of ABA were used but without success. Further attempts to mature the cell lines were abandoned and the embryogenic cell lines destroyed. The research into somatic embryogenesis with this species has been relatively unsuccessful. Again, a great deal of further research is required into all stages of somatic embryogenesis is required before a reliable technique can be developed.

The development of a successful grafting technique was finished. Wedge grafts on two year old rootstocks as understock gave the best results. The material used came from different locations in England and from Florence. 310 grafts produced in previous years were transported to Italy in autumn 1996 to permit the establishment of clonal archives and for use in cultivation trials. No other work was undertaken.

Productivity data has been collected under same conditions of the previous years. As in 1994 significant linear correlations were found between some traits, for example trunk diameter, plant height, number of inflorences per tree or total seed yield. Correlation between seed size or seed dry weight and escin content were found to be significant negative, which may indicate that escin content is depending on seed physiology or climate. The populations evaluated over four season spanning 1993/1997 showed a considerable variability of all the examined characters. In particular, this variability was large for all productive characters, such as mean seed DW, yield of nuts and escin, suggesting that the selection of superior individuals for productivity may give quit good results and economical advantages.

Conclusion

In conclusion, our results suggests that in horse-chestnut, where natural variation is large and vegetative propagation through grafting is easy, the traditional method of selection is the less expensive and may give very good results. Moreover, in recent years the use of clones and clonal mixture has assumed increasing interest and importance for breeding strategies and for economical and commercial aims. However, the efficiency of selection for superior genotypes depends on the degree of heritability of the selected trait. Therefore it is of fundamental importance to separate the genotypic variance from the phenotypic variance; i.e. the between clones from the within clones component of variance. In order to estimate variance components (genotypic, environmental, interaction) and to compute broad-sense heritability and genetic correlations, the performance of clonal progeny in replicated trials have to be evaluated. In the future this may be achieved employing our selected clones, which have been already propagated by grafting and established in different clonal banks in Italy and Scotland.

First data on the application of training systems on Aesculus hippocastanum were recorded. The trees show a great variability in habit. Statistical differences were found in trunk diameter and sprouting between the two training systems. Horse-chestnut trees are characterised by a very slow growth rate during the first years of their life. Although trees grown at Antelia showed a quite good vegetative development during the initial years after planting, there was the necessity to reduced pruning as much as possible to allow root settling. Moreover, with minimum pruning fruit trees are expected to come earlier into production. Trees showed a great variability in the habit (from totally up-right to open and very branched), leaf size and colour, and number of buds.

In general horse-chestnut trees adapted well to different pruning and training systems. The experience acquired during the years of field trials, through plant observation and elaboration of the collected data, showed that trees with a complete central axis were at best suitable for training operations. The presence of a leader limb allowed the tree to reach a proper trophic- hormonal balance. Free training systems better fitted with the natural growth pattern of tree. Topping and pollard induced abundant shooting under the cut zone, while bending promoted budbreak on the upper side of branches, in both cases shoots had to be thinned out. Inclination of shoots resulted in a good developed vegetation, independently of tree shape; and must be considered essential for the constitution of a strong tree framework. Shooting was successfully induced by bud notching and nicking; shoots appeared well-developed although thin.

Ecological effects on erosion, and local vegetation have been recorded as in the previous years. This year a resumption of growth at the beginning of autumn was observed, which sometimes is encountered with drop of temperature and first rainfall in autumn. The complete data will be assessed. In sites with well developed Mediterranean maquis the Aesculus seedlings are not able to compete with the adult plants of the maquis. The experimental plots show ecological conditions sufficiently suitable to horse chestnut development. All three areas are included in the cold and medium areas of Lauretum (PAVARI) corresponding to the meso-Mediterranean belt. From a pedological point of view, soils are quite stable, with a good structure, abundant litter and a good supply of nutrients. The Aesculus root system develops horizontally, but in the present study plants were not able to oppose erosion processes due to their limited development: roots of plants located in areas subjected to erosion were visible.

Horse chestnut growth season is usually more than 200 days long, but under these peculiar conditions its length was about 150 days, because of its early defoliation. During autumn, which in our climatic condition is the period when hydric erosion is more likely to occur, plants are completely defoliated and therefore not able to mitigate the effect of heavy rainfall. The best site for establishment of a genebank was found to be in Oristano, even if the climate there is warmer and more arid than the other Sardinian sites. Due to an optimal infrastructure there the development of the Aesculus plants there is luxurious, both for the roots and in the aerial parts.