|Bridg, Hannia: Micropropagation and Determination of the in vitro Stability of Annona cherimola Mill. and Annona muricata L. |
A. cherimola and A. muricata are semideciduous native trees from the tropical highlands of South America and tropical areas of the Caribbean Islands. Both have developed a commercial promise in the fruit trade market, because of their edible fruits and phytochemical products.
The knowledge of these fruit trees has been scattered thus, exploration, collection, conservation and evaluation of A. cherimola and A. muricata natural genotypes is a priority in Colombia, Peru, Ecuador, Venezuela and El Salvador, countries where they are believed to be part of the native Flora. Furthermore, the promotion of technical plantations with healthy and high quality trees are the principal worldwide aims for these species.
If a selected genotype of A. cherimola and A. muricata is propagated by seeds a high genotype variation is expected, when conventional vegetative propagation methods are applied, the dichogamous protogyneous flower behaviour promotes an intervarietal crossing. Therefore the conservation of selected ecotypes by the application of conventional propagation methods has been until now impossible, in those regions where they have been introduced because of their qualities such as Spain, Australia, Asia, California and Chile.
The aim of the present study was to review the botanical and cultural aspects of A. cherimola and A. muricata and develop a reproducible micropropagation protocol preserving the genetic stability of the selections or promote true-to-types genotypes in order to open an alternative for this plant species.
Preformed axillary bud sprouting excised from the side branches of four year old plants have been developed with a yield of 4 new shoots per bud in 20 days. The position of the buds on the branches had an effect on the bud break and establishment of cultures under in vitro conditions, therefore semiwoody cuttings with three buds were the most suitable explants for multiple shoot proliferation when cultured on a Nitsch and Nitsch (1969) medium containing 8.87 µM of benzylaminopurine and 2.46 µM of indole-butiric acid.
The effect of Benomyl, Rifampicin and some antioxidants like Polyvinylpirrolydone, ascorbic acid and citric acid are discussed. There were no significant differences during the establishment of A. cherimola and A. muricata in terms of in vitro requirements, time and yield of bud new shoots formation.
To improve shoot proliferation and multiplication the effect of benzylaminopurine, kinetin, zeatin and thidiazuron were compared. The NN-69 media supplemented with 2.32 µM kinetin and 1.36 µM zeatin was the proliferant for A. cherimola. A. muricata, it showed no shoot proliferantion but elongated well in 1.44 µM Gibberellic acid. Both species improved the formation of eight new shoots in 60 days of culture with one subculture after 30 days.
A. cherimola and A. muricata multiplied shoots lost their quality and started to be chlorotic in relation to the number of subcultures on the same multiplication media. The variation of ammonium nitrate, potassium nitrate and ammonium carbonate
Xsupplied as salts on the Nitsch and Nitsch (1969) media were evaluated as well as the supplementation of casein hydrolisate and coconut water. The supplementation of 20.6 mM NH4- and 39.4 mM NO3- improved the promotion of quality and green shoots available for rooting.
The indole-3-butiric acid 4.90 µM promotes rhizogenesis under in vitro and ex vitro conditions in both cases a previous precondition of the shoots was required. To improve in vitro rooting the concentration of Nitsch and Nitsch (1969) macro-salts should be reduced to ¼ with a supplementation of 1 % of sucrose and 3% gelrite. The ex vitro rooting is succesfully in quartz-sand substract without plant growth regulators and 90% of relative humidity.
Pattern band comparison by Random Amplified Polymorphic DNA markers was applied to determine the genetic stability of micropropagated shoots of A. cherimola and A. muricata 4 Colombian and 2 Chile selections. 29 primers were screened, of them 5 primes gave clear reproducible bands. No variation in RAPD banding patterns among the tested shoots. These results verify the genetic stability of the in vitro regenerants and tested the true-to-type propagation.
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