Bridg, Hannia: Micropropagation and Determination of the in vitro Stability of Annona cherimola Mill. and Annona muricata L.


Chapter 2. Aims in Annona spp.

2.1 Aims

Among of the worldwide aims in A. cherimola and A. muricata are: Development of technical plantations, control of the typical diseases and promotion of healthy high quality trees.

The exploration, collection, conservation, and evaluation of A. cherimola and A. muricata germplasm, not only in natural conditions, but also in repository gardens are in 1999 a register priority as result of the neglected knowledge of these species in those countries where they are part of the native flora. Furthermore the application of plant research and breeding programs on A. cherimola and A. muricata is still in development principally in those countries where they have been introduced.

The characterization of A. cherimola and A. muricata natural genotypes, selection and multiplication of the elite plants by any propagation system that ensure the preservation of interesting genotypes, are calling the attention of Annona cultivators.

The A. cherimola plant and fruit are not cold tolerant, thus natural selection, multiplication and promotion of cold resistant genotypes from the tropical highlands mountains in South America are a priority not only to transport this fruit long distances in containers with controlled atmosphere, but also to promote its technical development on the native countries.

The A. muricata is a fungi disease sensitive plant, thus selection of trees carrying natural resistance against Colletotrichum spp. is desirable in all the geographic regions where it is planted. Otherwise, fruits of A. muricata seedless and tasting pulp are one of the Colombian farmers additional expectations.

If selected genotypes of A. cherimola or A. muricata are propagated traditionally by seeds a high genotype variation is expected. Otherwise if conventional vegetative propagation methods are applied, the dichogamous protogyneous flower behaviour in A. cherimola and A. muricata is promoting a intervarietal crossing. This situation is not only well known in Annona spp., as well as in another fruit species with this type of flowering. The natural conservation of a selected genotype by conventional propagation methods has been until now impossible therefore, micropropagation of A. cherimola and A. muricata by tissue culture is actually the alternative of clonal multiplication.

The in vitro multiplication of A. cherimola and A. muricata selected genotype is hypothetically profitable because the heterozygosity degree is not expected in a clonal multiplication system, the in vitro regenerants could be included in further plant breeding programs. This type of vegetative clonal multiplication might be not only safe but also economic in terms of time. The storing and monitoring of selected fruit tree clonal material in smaller units is possible.

The in vitro multiplication of A. cherimola and A. muricata selected germplasm could rapidly increase the existing ancient genotypes, in danger of extinction, at the tropical high- and low-lands in America. Those trees are possibly carrying the competitive commercial characteristic, desirable today on the world trade market of exotic commercial plants.


2.2 Needs for in vitro Culture in Annona spp.

2.2.1 Genetic Diversity

It is obvious that a great inherited variation of Annona cherimola and Annona muricata exists not only in the centres of origin, South- and Central America; but also in the tropics and subtropics areas where they have been successfully adapted and cultured (Jordan and Botti, 1992).

Selections of A. cherimola and A. muricata differ considerable in phenotype pattern, when they grow at different locations in the same area e.g., the fruit characteristics, plant pattern growth and cropping capacity are modified by the micro-ecosystem conditions (George, 1984; Gardiazabal and Rosenberg, 1988; Sanewski, 1988; Tijero, 1992).

The characterization and use of these non exploited natural variations of A. cherimola and A. muricata will lead the development of improved and fashion-tailored trees (Reginato and Lizana, 1980).

There are in South America native wild ecotypes of A. cherimola and A. muricata, well adapted to the natural extreme conditions. Of them, some should be cold resistant and naturally protected against fungi diseases. The identification and massive multiplication of those ecotypes should be the first priority in terms of plant germplasm conservation (Bridg, 1993a).

Procurement clones, showing greater productivity, vigour, cold tolerance and other physiological traits is a great advantage, which is provided by the selection of elite Annona spp., cultivars (Jordan, 1988).

The application of Plant Tissue Culture methods in A. cherimola and A. muricata, will allow the mean development of economically and rapidly producing, large numbers of clonal plants. The micropropagation procedures have also proven to be useful for the physiological and genetic uniform plant maintenance, the research purposes find on it additional benefits (Davis and Keathley, 1992).

2.2.2 Increment of Natural Types

Selection and propagation of productive, healthy A. cherimola and A. muricata trees from ancient germplasm and establishment of orchards to promote the natural conservation and observation of these species is important in order to impulse the knowledge of them. Otherwise, the development of a successful micropropagation method should be enable to improve the clonal propagation of superior A. cherimola and A. muricata earliest natural selections in danger of extintion (Cordoba, 1969).

Nevertheless characterization of selected types of A. cherimola and A. muricata in order to identify either genetic variation or define genetic identity between selections, applying conventional biological methods, with a high probability is a time consuming


process with a high probability of fail information (Anderson and Richardson, 1990; Castro and Maia, 1984; George et al. 1986). The application of the new developments in molecular biology based on the DNA technique would identify selections of A. cherimola and A. muricata in a short period of time with a high probability of success (Ronning and Schnell, 1995).

2.2.3 Long Juvenile Periods

The Annonas are slow growing semi-deciduous trees (Samson, 1986) and with exception A. muricata, drop their leaves during the cool season and remain bare and dormant for several months. The length of the juvenile period varies between selections, however the first important fruit production begins after eight years. The juvenile period is extremely variable and greatly influenced by seedling rootstock and type of scion (Jordan and Botti, 1992).


The out-breeding of the Annona spp., which turn in heterozygosis, is a consequence of the dichogamous protogyneous flower behaviour (Sainte-Marie, 1987). The manual pollination is a common practice in productive orchards of A. cherimola and A. muricata and the human manipulation can also promote plant variation promoting genetic combinations uncommon in the nature (Richardson and Anderson, 1996).

In general the commercial varieties of this genus are coming from openly pollinated seeds (Clark, 1992; Farre et al. 1977). Although this is advantageous for certain broad breeding objectives, it is difficult to „fix“ specific characters in a reasonably short period of time (Vargas de la Fuente, 1986).

The Annona cherimola and Annona muricata are traditionally propagated by seeds, because this is the cheapest and most reliable method for establishing commercial plantations. However, commercial orchards derived from seedling populations of selected material are unsatisfactory because of the high degree of heterozygosity promoted (George and Nissen, 1987). The variations in fruit form, color, cold hardiness, pulp and timber quality are wide-spread by seeds. Consequently clonal propagation of both scions and rootstocks from superior selections is highly desirable (George, 1984).

Propagation of Annona by grafting has been made, but with considerable limitations in costs and time. Graft incompatibilities do exist and grafted plants are influenced by the variability imparted by the rootstock. The A. cherimola genotypes selected for invasive root systems, for example, cannot be propagated by grafting. Similarly if A. cherimola cold-resistant scions are grafted on untried seedling material, they survive at low temperatures but eventually die later on, due cold injury (George, 1984).

2.2.4 Tree Improvement

The Annona spp., as well as other woody tree species are problematic, when vegetative propagation of mature trees is desired, once a tree is old enough to determine its merits,


it becomes very difficult or even impossible to root cuttings on a large scale (Gupta et al., 1981).

Proportional with the aging of the orchards the A. cherimola and A. muricata looses of rooting ability, difficulties with hibernation of rooted cuttings are increasing and changes from plagiotropic to orthotropic growth are accelerating in contrast, the in vitro propagation of mature trees by cuttings is suitable (Gupta et al., 1981). Beyond this, the in vitro culture itself can exhibit a rejuvenation effect (Mullins et al., 1979).

The main application of the plant tissue culture techniques in A. cherimola and A. muricata would be the further propagation of a large number of selected superior mature trees. The massive promotion of genotypes not only with invasive root systems, that are difficult to root by conventional methods, but also those which are superior and cold resistance furthermore, the flowering genotypes with desirable fruit in size and pulp qualities, are the primary goals for improvement.

The micropropagation systems allow to test the Annona mature genotypes, which can be used to promote a high-performance multi-clonal varieties. The genetic variation typical in Annona seedling progenies could be avoided. The micropropagation procedures have also proven to be useful for maintain physiological and genetical uniformity of the plant material (Barlass, 1990).

2.3 Biotechnology Applications on Annona spp.

2.3.1 Micropropagation

In vitro propagation from Annona spp., has been described by some authors with limited success. The most common limiting problems have been explant selection, avoid or control of phenolics, difficulties to eliminate exogenous and endogenous contaminants without induce tissue damages, low rate of shoot multiplication, sporadic rhizogenesis and hardening (Table 13).

Haploid plants have been reported from anther culture of A. squamosa (Nair et al. 1983). Triploids have been obtained from endosperm culture of mature A. squamosa seeds, shoot organogenesis was induced from callus, rhizogenesis was not successful.

The cytological analysis of roots and leaf tips of regenerated plants has been reported (3n =21) (Nair et al., 1986). Direct shoot organogenesis of A. squamosa has been reported from seedling leaf explants, root organogenesis was not reported (Nair et al., 1984).

Micropropagation from nodal explants with axillary buds from Annona hybrid (A. squamosa x A. cherimola) have been reported, rooted plants and field transfer have been promoted (Nair et al. 1984). From hypocotyl and seedling petioles of A. cherimola shoot organogenesis have been reported (Jordan, 1988) and rooting has been sporadically promoted.

Direct shoot and root formation in A. cherimola hypocotyls were studied and the outcomes were reported with regard to the regeneration potential under in vitro conditions. Explants, culture conditions and hormone levels required to induce morphogenic responses for examination of plant regeneration have been reported


(Jordan et al., 1990). Adventitious shoot buds developed directly from hypocotyl explants and the rhizogenesis potential of A. muricata shoot have been studied (Bejoy and Hariharan, 1992).

The high activity of the polyphenoloxidase on A. cherimola (Martinez-Cayuela et al., 1988a, b) and the presence of phenolic compounds on the in vitro culture medium coming from the explant in general are limiting the in vitro propagation of A. cherimola (Bridg, 1993b). The morphogenic responses of A. cherimola is affected by phenols (Jordan et al., 1991). Fruits from A. cherimola and A. muricata species after picking are affected by phenols, the tissues take a brown colour and the fruit is quality reduce (Vargas de la Fuente, 1986).

Table 13. In vitro approaches on Annona spp.



Regenerated Organs








Annona spp.




shoots, roots*

Bapat and Narayanaswamy, 1977





Nair et al. 1984a




rooted shoots

Rasai et al. 1994, 1995


A. cherimola


shoots, roots*

Jordan, 1988




shoots, roots*

Jordan et al. 1991



zygotic embryos


Jordan et al. 1992





Tazzari et al. 1990





Bridg, 1993b


A. muricata


shoots, roots *

Encina et al. 1994




rooted shoots*

Bejoy et al. 1992




adventitious bud, shoots, roots *

Lemos and Blake, 1998

Brazil & U.K.

A. squamosa


shoots, roots *

Lemos and Blake, 1996





Nair et al. 1983





Nair et al. 1984




shoot, roots *

Sreelata et al. 1986





Nair et al. 1986


* root formation as regeneration potential under in vitro conditions

The regeneration of Annona spp., from explants of clonal origin has not been reported (Jordan and Botti, 1992). On A. squamosa root and organogenesis has been induced form anther, endosperm and leaf explants, as well as in A. cherimola hypocotyl, petiole, internode and nodal cuttings. Hypocotyl and nodal cuttings of Atemoya (Rasai et al. 1994) and A. muricata have proved to be suitable for in vitro culture. Shoots from hypocotyl and nodal segments of some Annona spp., have been successfully rooted (Rasai et al. 1995). The effect of arbuscular mycorrhizal fomation have been investigated on A. cherimola rooted in vitro plantlets (Azcoaguilar et al. 1994; 1996).


From hypocotyls of A. muricata seedlings adventitious bud and shoot proliferation were achieved, rooting and acclimatization have been studied (Lemos and Blake, 1996).Adventitious shoot regeneration from internodal explants of mature plants of A. muricata from meristem culture have been reported, rhizogenesis was not achieved (Lemos and Baker, 1998).

2.3.2 Genetic Variation

Genetic variation among cherimoya cultivars has been studied using isozyme markers, and DNA analysis by Polymerase Chain Reaction and Restriction Fragment Length Polymorphic (PCR-RFLP), Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphic (AFLP).

Mendelian inheritance was demonstrated on five clonally propagated varieties of A. cherimola by the linkage of isozymes (Lee and Ellstrand, 1987). Cultivar identification by isozyme variation patterns in 15 varieties of A. cherimola and 1 Atemoya (A. cherimola x A. squamosa) has been reported (Ellstrand and Lee, 1987).

The allelic segregation of 13 isozyme loci in hand fertilized heterozygous A. cherimola trees was demostrated (Pascual et al., 1993). Gametic selection appears to be the main contributer, although zygotic selection seems also to play a part (Perfectti and Pascual, 1996).

Isozymes have been used as genetic markers to characterize more than characterized 200 A. cherimola and Atemoya (A. cherimola x A. squamosa) plants, however the isozyme analysis is limited by the relatively small number of loci (Perfectti and Pascual, 1998 a,b).

The phylogenetics of A. reticulata, A. glabra, A. muricata, A. montana and Atemoya (A. cherimola x A. squamosa) were studied by PCR-RFLP. A basic information in the phylogenetic studies of Annona spp. could be made using a large number of experimental units (Rahman et al. 1997).

The RAPD have been applied to identify the species A. reticulata, A.muricata, A. cherimola, A. squamosa and some interspecific hybrids of Atemoya, this molecular marker technology showed to be fingerprinting efficient method to identify genotypes within between Annona species with an expected Mendelian fashion.

The genetic diversity of 19 A. cherimola cultivars classified into five types based on their skin texture was taxonomically identified and differentiated by AFLP, this molecular marker technologie provides a better resolution, AFLP enables to analyse the phylogentic relationships between A. cherimola cultivars (Rahman et al. 1998).

RAPD markers offer the potential of generating large number of markers representing a random sample of the genome, thereby presents an advantage over isozyme markers (Ronning et al. 1995).

2.3.3 Chromosome Number

In Annona spp., cytological and karyological studies have been limited due the tendency to form chromosome clumps. Hutchinson (1923) described for Annona karyotypes and


outstanding its fixed predominance for taxonomic and phylogenetic relations. Bowden (1948) figured the chromosome complements of nine species of Annonaceae, some of the species showed one pair of large chromosomes and one small pair, e.g., Asimina triloba and Annona muricata. In Annona cherimola the two small chromosomes are also reported, one large pair of chromosomes and other pair almost as large can be recognized. The two small chromosomes frequently stuck to or were hidden by larger chromosomes (Table 14).

Bowden (1948) reported the chromosome number (x = 9) and (x = 8) for most of the species of Annona genus except A. glabra (x = 7) because it may be tetraploid (4n = 28) (Table 14). Sreelata et al. (1986) analyzed the roots and young leaf tips of A. squamosa cytologically and reported a triploid (3n=21) chromosome number. The Annona spp. Have a ruminate type of endosperm which is characteristic of the Annonaceae family (Bhojwani and Bhatnagar, 1978).

The Annonaceae species shows distinctive cytological characteristics such as chromosome number, range of chromosome size and fixation image (Bowden, 1948) but discrepancies in counting A. cherimola and A. muricata chromosomes may have been caused by the failure to observe the two small chromosomes which are often obscured by the larger chromosomes. Nair et al. (1983) reported that haploid A. squamosa could be regenerated from anther callus. Regenerated plants had the haploid (n = 7) chromosome number and survived transplantations

Table 14. Reported chromosome number in A. cherimola and A. muricata











A. cherimola Mill. (cherimoya)

2n = 14



Kumar and Ranadive, 1941


2n = 16



Bowden, 1948


2n = 16



Darlington and Wylie, 1956






Annona muricata L. (soursop)

2n = 16

Colombian seed plants

Berlin Botanical Garden, Germany

Bowden, 1948


2n = 16

Colombian seed plants

Walter R. Lindsay, Canal Zone Experiments Gardens, Panama

Bowden, 1948


2n = 16

Colombian seed plants

F.G. Walsingham, Atkins, Institution of the Arboretum, Soledad, Cienfuegos, Cuba

Bowden, 1948


n = 7



Kumar and Ranadive, 1941


2n = 14



Darlington and Wylie, 1956

Simmonds, 1954

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