|Bridg, Hannia: Micropropagation and Determination of the in vitro Stability of Annona cherimola Mill. and Annona muricata L. |
According to Murashige and Skoog (1974) any in vitro culture manipulation should start with an elite or known plant material however, there are in nature, dichogamous protogyneous flowering plants such as A. cherimola and A. muricata which promote intervarietal crossing, thereby making the preservation of elite or selected material impossible.
Cordoba (1969), Gardiazabal and Rosenberg (1988), Sawneski (1988), Gazit and Eisenstein (1985), Sanchez and Torres (1992) and Bridg (1993) did not report the expanded existence of A. cherimola and A. muricata elite material either in the countries where they are planted for commercial purposes. Most of the present plant material of A. cherimola and A. muricata comes from seeds and it is well known that seedling populations promote hetereogenity between species (Hartman and Kester, 1894).
There are no examples in woody species, but in the herbaceous ones where an elite material is either a selected phenotype or a specific plant selected for its specific qualities. In forestry and/or fruit trees the elite material would be the bearer of an elite seed, which would be used to initiate a more or less extensive micropropagation program (Deberg and Read, 1991).
To develop the present in vitro propagation protocol for A. cherimola and A. muricata selected plants were used as mother plants. The selection was made in connection with the advice of the farmers from three Colombian regions where these plants are believed to be part of the native flora. The farmers have a large knowledge of the phenotypic characteristics of the trees, the quality of the fruit produced and their origin.
One year old seedling trees (T1) of A. cherimola and A. muricata were planted in Colombia under open field conditions, their semideciduous natural condition promoted the bud sprouting in the new environmental conditions in the greenhouse in Dahlem-Berlin.
Dehydration of A. cherimola and A. muricata (T1) roots was prevented with the defoliation of the leaves, transport in darkness and by protection with moisture paper. The sprouting of semideciduous and deciduous fruit trees after the dormant or stress season is related not only to the endogenous concentration of plant growth regulators, starch and other metabolic products but also to the water content on the internal tissues (Hartman and Kester, 1984).
A good root system assures the sprouting of a deciduous or semideciduous bud once the optimal environmental conditions give the signal in which water transport normalizes the transport of metabolic substances and the latent bud promotes the new vegetative growth of the plant (Westwood, 1978).
Temperature is one of the factors which influence A. cherimola and A. muricata behaviour, because they are not cold tolerant plants. Ambient temperature variations have been demonstrated to be detrimental to the plant and fruit (Boshell, 1982; Lizana
102and Irarrázabal, 1984, Gutierrez et al. 1992). The Colombian plants were transported at room temperature to protect cold injury of the vegetative structures .
Chilean seeds of A. cherimola cv. Felpa and Bronceada (T0) were also used as a material source (Table 16). Cordoba (1969), Bourke (1976), and Sawneski (1988) reported seed germination problems in A. cherimola.
The seeds of tropical tree species from open field conditions are more susceptible to the attack of insects and micro-organisms such as fungi (Hammond et al., 1999). This study confirms the beneficial effect of Benomyl 0.03% as a systemic fungicide on the germination of A. cherimola cv. Felpa and Bronceada seeds. No losses by fungi and other seed pathogens were noted during germination. Wunkhaus and Aquinas (1990) evaluated the potential effect of Mancozeb and Dichloran as systemic fungicides combined with shock temperature treatments on A. cherimola seed germination.
The low percentage of seed germination in several woody species is related to the seed dormancy characteristic of semideciduous and deciduous fruit trees (Hartman and Kester, 1984). The manipulation of some physical factors to break dormancy have been discussed (Westwood, 1978).
The present study shows the beneficial effect of pre-treatment of seeds with cold temperature on seed germination of A. cherimola cv. Felpa 78% and cv. Bronceada 81% when coated seeds were stored at 4°C for 24 hours during the imbibition in water. The group of seeds of A. cherimola cv. Felpa and cv. Bronceada which were not part of this treatment reduced the rate of germination by 41% and 67% respectively at room temperature conditions. The effect of warm water treatment to improve germination did not increase the rate of germination on A. cherimola (Duarte et al. 1974).
The results suggest that warm temperatures used as a pre-treatment to break the dormancy of A. cherimola seeds did not increase the rate of germination. However a pre-cold temperature shock treatment improved the germination rate (Table 20).
Plant growth regulators such as Gibberellic acid (GA3) and benzyl-aminopurine (BA) have, in combination with seed stratification, been tested to break dormancy of A. cherimola seeds (Vargas, 1986). From the results it can be seen that no high concentrations of plant growth regulators are required to break the seed dormancy of this semideciduous subtropical species.
Toll et al. (1975) reported no germination of A. cherimola seeds on GA3 1,000 ppm., treatment. The best germination results were reported on GA3 500 ppm. Duarte et al. (1974) reported the effectiveness of GA3 on seedling growth but not in seed germination. The concentration of 1,000 ppm GA3 was shown to be more effective than 10,000 ppm in terms of the promotion of vegetative growth of A. cherimola plantlets.
Some seed manipulations during sowing have been suggested to improve germination on semi-deciduous and deciduous woody tree species (Hartman and Kester, 1984). Vincent (1974) and Toll et al. (1975) found that scarification, position and direction of the seed polarities are factors which have no appreciable effect on A. cherimola seed germination.
In this study, it was found that the coat of A. cherimola cv. Felpa and cv. Bronceada seeds have an effect on germination. Those seeds which were coatless showed a low germination rate in all experimental cases (Table 20). This was not only because of the presence of phenolics on the endosperm but also because of the
103susceptibility of the seeds to the attack of some fungi species. Otherwise the coat of A. cherimola seeds has been reported to be rich in oils (Jaramillo, 1952) and oils are reserve energy to promote the embryo growth and development during germination.
The ecophysiological relation between seed germination preconditions and natural growth condition of the A. cherimola native plant in the highland mountains in the South American Andes. It grows in a subtropical mild temperature ecosystem which fluctuates during the year, early morning low temperatures have been registered 4 ± 3 °C for 2 hours and during the day the 25 ± 5°C remains constant (Lauer, 1986). The seeds of A. cherimola are exposed not only to high humidity conditions but also to low soil temperatures during the early morning as a consequence of the "helada" (Bridg, 1993a).
The present study suggests that seeds of A. cherimola cv. Felpa and Bronceada could break their dormancy if they are stored in dry conditions free from fungi attack, pre-washed with 0.03% Benomyl for 1 hour to avoid endogenous pathogens which limit the germination when plants are grown in open field conditions. Pre-cold temperature shock 4°C/ 24 hours in water promoted imbibition and improved the seed germination rate.
It is well accepted that the in vitro propagation of a species is influenced by several factors, the foremost are genotype, age and source of the initial tissues, which, in turn are related to their endogenous hormonal status (Bajaj and Pierik, 1984; Meyer, 1983). The day length and temperature had a definitive effect on the formation of new sprouts in To and T1 A. cherimola and A. muricata plants established in the greenhouse. In order to evaluate the effect of the photoperiod on the in vitro establishment (Table 23) some plants were maintained in total darkness for two weeks.
This study confirmed the effect of seasons and the light duration of the day, as factors which have a strong influence on the vegetative growth and development of A. cherimola and A. muricata plants established in the greenhouse, as well as during the in vitro culture. These species are sensitive to the photoperiod. Short days, common in winter, influence the leaf angle of the plants and wilting of the branches, vegetative growth is reduced and the principal branch develops an apical dominance, larger leaves were also observed.
The A. muricata explants from winter are not shoot prolific if compared with those taken in summer or spring. The A. cherimola explants showed the same behaviour but appeared to be a little more resistant to the atmospheric variations in the greenhouse in terms of bud sprouting. The A. cherimola is a subtropical species and A. muricata grows only in the most tropical areas where short days and low temperatures are never reported.
Plant Tissue Culture is defined as in vitro culture of any plant cell, tissue or organ in aseptic conditions (Murashige, 1974). However one of the main difficulties in tissue culture of woody tree species is the disinfection of the selected explant, especially when plant material is collected outdoors, as usually plant tissues and organs from these conditions carry endophytic floras, consisting of inter- and intracellular micro-organisms including viruses, viroids, prokaryotes and fungi species (Cassells, 1990).
The experimental A. cherimola and A. muricata Colombian plants from open field conditions carry micro-organisms which limit the aseptic in vitro micropropagation. Several fungi and nematodes species have been reported to live on Annona plants growing in the field (Raski, 1976; Muñoz, 1981; Ochoa, 1989).
104Figure 9 shows losses from A. cherimola and A. muricata during the initial establishment of several explants under in vitro conditions. Hadelman et al. (1987) reported the particular difficulties found when a woody plant from the field is to be used to promote cultures free from contaminants. In vitro contamination has two sources of origin, external when it is present on the surface of the explants or endogenous or intracellular when it is capable to grow in tissue culture medium (Lawson, 1986)
The use of systemic fungicides for preconditioning the field plants is a recommended practice in tissue culture of woody species (Conger, 1986). The fungi contaminants on A. cherimola and A. muricata during the in vitro establishment was reduced if constant applications of Benomyl 0.03% were made (Table 21). The present study shows on A. cherimola and A. muricata that irrigating with Benomyl in the greenhouse is not enough to promote the aseptic in vitro culture of A. cherimola or A. muricata explants, however the reduction of fungi contaminants was reported (Figure 9).
The fungi on in vitro culture are not always related to the external disinfection of preconditioning treatments. Some fungi reproduce themselves by spores and mycelium which could survive the external disinfected material. The contaminants could be the expression of a latency stage or be natural forms which develop new races in a short period of time in a culture media (Cassells, 1990; Montiel, 1991).
The in vitro establishment of A. cherimola and A. muricata is conditioned, not only by the nature of the selected material and environmental growth conditions, but also by the presence of contaminant micro-organisms such as fungi which have been associated with A. cherimola in open field growth (Benitez and del Pilar, 1990). Preconditioning of the plants with Benomyl should be a permanent practice during the time where initial explants are required. The reduction of in vitro fungi expression were related to the frequency of Benomyl irrigations.
In principle each plant segment or explant has the potential to induce new growth and development on aseptic conditions and regenerate a complete plant (Pierik, 1989). The in vitro culture differentiation is dependent on the cell or tissue conditions of the initial explant (De Fossard, 1977).
Meristems of A. cherimola and A. muricata vegetative branches were selected as initial culture explants because many in vitro cultures were obtained from these explants due its rapid capacity to promote cell divisions.
Meristems are used also to assure the establishment of aseptic cultures (Conger, 1986). Micropropagation of A. cherimola and A. muricata through meristems in the present study could not be reported because of the high production of phenolic substances, meristems did not survive to the first in vitro manipulation. It is well known that the Annona spp., are phenol reactive plants after wounding or injury (Martinez-Cayuela et al. 1998).
The micropropagation of many fruit tree species have been promoted from young tissues like hypocotyls from seedling plants, shoot tips from vegetative branches and meristems because they are less prone to blackening than older tissues (Conger, 1986; Torres, 1989). However in woody trees the survival rate of the small explants to disinfection treatments is minimal (Miller and Murashige, 1976)
105Neither A. cherimola nor A. muricata could be propagated by bud culture from vegetative branches due to the hypersensitive reaction of this soft tissue explant. Other woody fruit species such as Feijoa sellowiana have shown the same limitation during establishment, producing a high concentration of phenolics (Bhojwani et al. 1987).
For most woody species a widely applied technique to promote micropropagation is the enhancing of the axillary bud (Conger, 1977), the induction of adventitious buds and callus culture from the tissue of the original explant (Huhtinen, 1976) and the culture of shoots derived from shoot tips and nodal segments as well as the promotion of terminal and axillary buds (Pirre, 1986).
Micro-cuttings (1-3 cm) and macro-cuttings (5-9 cm) were found to be an optimal source of explants from vegetative-, semiwoody- and woody -branches of A. cherimola and A. muricata (Figure 9). Some differences in bud sprouting and production of phenolics and endogenous disinfection from these explants were noted in the present study. The sprouting of new shoots from pre-formed buds and the green and aseptic establishment of A. cherimola and A. muricata explant were related to the plant position and degree of lignification of the mother branch.
Aghion-Prat (1965) and Jaiswal (1986) noted different regeneration gradients in explants isolated from different plant positions. It is also known that explants from the same plant organ show in vitro response variations because there are different ages on the tissues of the same organ (Torres, 1989). This was confirmed also in Pseudotsuga menziesii where shoot initials isolated from positions low down on the tree showed better development in vitro than the terminal buds and grew faster than axillary buds (Evers, 1984).
The semiwoody cuttings from the second year growth were the most potential explants to induce the establishment of A. cherimola and A. muricata. These explants were phenol reactive on the base but size and plant position benefited their in vitro culture and bud sprouting. This response has been described by Stonier (1971) which indicates that the production of phenolic substances decreases right up the base of the explant.
The potential of rejuvenation of one species can be achieved not only by the propagation of vegetative explants, but also by the adventitious shoot formation or new shoot formation from adult shoot tips (Torres, 1989). Woody species have the inclination to inhibit the bud sprouting and produce high concentrations of phenolic substances in woody branches (Chalupa, 1987).
The promotion of new in vitro shoots of A. cherimola and A. muricata from semiwoody cuttings (5-9 cm) with 3 pre-formed buds is reported. This explant is larger, vigorous and carrying pre-formed semideciduous buds with their own supply of energy and growth regulators. The induction of new shoots from these pre-formed buds in the case of A. cherimola and A. muricata is an alternative to improve the rejuvenation of adult trees.
The micropropagation of tropical and subtropical fruits by stimulation of axillary bud proliferation from cuttings has been reported for several species (Litz and Jaiswal, 1990). The influence of the explant length on the regenerative capacity of apolarly placed bulb scale explants of Hyacinth spp., has been discussed (Pierik and Ruibing, 1973).
106Cultures from pre-formed buds without callus induces true-to-type micro-shoots which through repeated subcultures promotes rejuvenation (Pierik, 1989). A. cherimola and A. muricata selections could find on this type of initial explants an alternative to improve vegetative multiplication of its selections with a high success of rooting because of rejuvenation. This process results from the increments of cell division and regeneration rate as shown on Pinus pinaster, Sequoia sempervirens, Vitis vinifera and Malus sylvestris (Hackett, 1985).
The absence of tissue juvenility on the third year old branch of A. cherimola and A. muricata could be an explanation of why woody- macrocuttings and microcuttings were more difficult to improve in in vitro culture, not only because of the hypersensitivity of the explant, high contamination rate and low percentage of shoot regeneration but also because there was no juvenility. The adventitious shoot formation is far more difficult to induce in woody shoots than from semiwoody or juvenile shoots (Zimmerman, 1988 ; Hackett, 1985).
The potentialities of cell division and regeneration decreases from the apical to the basal region according to the lignification grade and age of the branch (Bajaj and Pierik, 1984; Meyer, 1983). The micropropagation of A. cherimola and A. muricata from semiwoody cuttings has the advantage of promoting new growth of selected trees under in vitro conditions and to improve their rejuvenation. These explants are physiologically relatively juvenile and for tissue culture purposes more able to be propagated because they could be manipulated under sterile conditions. The bud sprouting from semiwoody cuttings can be built up to form new shoot structures which leads to organ regeneration (Evans et al. 1984).
The micropropagation and the disinfection of one species is strongly dependent on the explant size, source, disinfectant concentration and time of sterilization (Pierik, 1989). The disinfection of A. cherimola and A. muricata was tested with several concentrations of sodium hypochlorite in combination with Benomyl to avoid all the external in vitro contaminants. These treatments were complemented with the effect of Rifampicin as a selected antibiotic to control the bacteria contamination characteristic in plants of tropical and subtropical origin (Young, 1984).
Sodium hypochlorite (NaClO) is one of the common disinfectant solutions that is used in tissue culture to promote surface sterilization and improve the establishment of aseptic cultures (Pierik, 1989). NaClO 3%/15 minutes was the most viable combination in time and concentration to avoid some external contaminants on A. cherimola and A. muricata semiwoody cuttings.
The A. cherimola and A. muricata semi-woody cuttings were irreversibly damaged or even killed by too high concentrations or a long exposure time in sodium hypochlorite (Figure 10). In general, woody tree species are sensitive to high concentrations of disinfectant agents (Deberg, 1986; Gordon and Brown, 1988).
In tissue culture it is difficult to interpret results with respect to endogenous contamination. When a woody species is tissue culture propagated, it is very difficult to distinguish between endogenous and exogenous bacterial contamination on the culture media (Cassells, 1991). In A. cherimola and A. muricata the endogenous bacterial contamination observed during the multiplication stage which revealed from the shoot explant disinfection, that the applied methods to improve the disinfection of the semiwoody branch was only effective on the surface of the explant.
107In plant tissue culture there are several strategies to avoid internal infections when they can not be eliminated by a simple external disinfection. Since most of the micro-organisms are not present in the meristem, the meristem culture is one of the most common techniques to improve the establishment of aseptic cultures when the principal aim is to produce virus free plants such as in Passiflora edulis cv. Flavicarpa (Roca and Mroginski, 1992; Bridg, 1990).
In A. cherimola and A. muricata the in vitro culture through meristems is not possible because of the hypersensitive reaction of this tissue explant and necrosis. The establishment was promoted with semiwoody cuttings (Figure 11; Figure 12) and these large explants have the potential to carry endogenous micro-organisms. Large explants are more difficult to culture because they are more difficult to disinfect (Roca and Mrogisnski, 1989).
The pre-formed shoots from semiwoody branches of A. cherimola and A. muricata showed an endogenous contamination during the multiplication stage which was very difficult to eliminate. Explants with a thick lignin mantle were difficult to disinfect because not all the surface parts could be penetrated by the sodium hypochlorite solution. Otherwise apparently sterile cultures can arise in a multiplication media if microorganism mutations take place during the establishment (Pierik, 1989).
The endophytic micro-organisms are located in the internal tissues of the plants. In some cases their colonization is permanent and in others it depends on the tissue anatomy where the colonization could be systemic or nearly so. There are the cases which the pathogenic micro-organisms have a mechanism to gain entry into the plant. They may follow a pathogenesis or latency of common plant associated bacteria, e.g., Agrobacterium spp., Bacillus spp., Corynebacterium spp., and Pseudomonas spp., which as intercellular endophytic microorganisms escape the effect of surface sterilants (Madoff, 1981; Campbell, 1990).
Young et al. (1984) found that no single antibiotics were effective against bacteria endogenous contaminants in shoot cultures of woody plants. The combination of different antibiotics have been more effective than any single chemical, nevertheless the risk of promoting somaclonal variations increases with the use of antibiotics (Scowcroft et al. 1987).
Rifampicin, Cefotaxime and Nystatin in the culture media as supplements for a long period of time were evaluated and reduction in the percent of bud sprouting and growth and development in A. cherimola and A. muricata semi-woody explants was observed (Table 22). Ammirato et al. (1990) have questioned the use of antibiotics and did not recommend them for routine use in plant tissue culture (Dodds and Roberts,1982). However antibiotics have been proposed as an alternative method to promote the aseptic establishment of several woody species (Young et al. 1984).
Rifampicin in A. cherimola and A. muricata semi-woody explants inhibited more effectively the contamination caused by Pseudomonas spp. in all the evaluated cases. The concentration of Rifampicin 20 µg/ml was not toxic for the explant. The bud sprouting induced green shoots without callus formation. They were excellent in quality due to the absence of contaminants. The effect of Rifampicin has been also reported by Staritsky et al., (1983) who compared the effects of different antibiotics in the culture media of Cryptocoryne spp., and Cinchona spp. and Rfampicin was the most effective antibiotic which supported the in vitro establishment of these species.
108The 20 µg/ml concentration is recommended by Sigma because this concentration is toxic enough for the micro-organisms. Concentrations as high as 25 µg/ml were noted to be toxic for the plant tissue, effective in terms of elimination of bacterial contamination (Tanaka et al. 1983; Mathias et al. 1987). Rifampicin is a commonly used antibiotic in Plant Tissue Culture because of its wide spectrum (Young, 1984).
The establishment media of Nitsch and Nitsch (1969) supplemented with Rifampicin, controlled the expression of endogenous Pseudomonas spp. Gram (-) bacteria, non pathogenic micro-organisms found in A. cherimola and A. muricata in the micropropagation. The bud sprouting increased in medium supplemented with Rifampicin (Table 22).
Phillip et al. (1981) found that Rifampicin was highly effective against bacterial contamination and did not affect rates of cell division and tracheary element differentiation or DNA synthesis in tuber cell cultures of Helianthus tuberousus. Pollock et al. (1983) found as Rifampicin one of the least toxic antibiotics and Goldberf and Friedman (1981) reported that Rifampicin inhibits prokaryotic RNA synthesis, while DNA is unaffected. The RNA polymerase of nuclear origin in eucaryotic cells is resistant to high concentrations of Rifampicin (Mathias et al. 1987).
The use of antibiotics for in vitro culture of higher plants is not encouraging (Pierik, 1989), because they are harmful for particular species (Torres, 1989) and they can lead to the selection of resistant micro-organisms (George and Sherrington, 1984).
In the present study, the in vitro culture establishment of A. cherimola and A. muricata without Rifampicin as a media supplement could not be achieved. The RAPD analysis on regenerants found no polymorphism on the DNA isolated from in vitro regenerants. The true-to-type propagation from pre-formed semiwoody buds was promoted although the establishment medium should be supplemented with Rifampicin.
The substitution of Benomyl in the culture media to control the presence of endogenous contaminants only and in combination with Rifampicin (Table 27) were effective and 100% of aseptic cultures could be register during the establishment of A. cherimola and A. muricata. Benomyl after the autoclave is degraded to 2-benzimidazole carbamic acid methyl ester which has been determined to be the active component of this fungicide (Maxweell and Brody, 1971).
0.4% of Benomyl was used as a media supplement by Haldelman et al (1987) to eliminate persistent contamination and obtain aseptic shoot tip material from Camellia spp. This fungicide was combined as well with Rifampicin to improve the traditional disinfection methods. Consequently a reduction of bud sprouting was observed.
However antibiotics and fungicides have not been recommended for routine proceedings in plant tissue culture (Dodds and Roberts, 1982) and a long period or exposure time of Benomyl is not desirable in a micropropagation protocol because it might induce phytotoxicity and off-type plants (Thurston et al., 1979).
The establishment of A. cherimola and A. muricata found in this Benomyl and Rifampicin combination is an alternative to improve the promotion of aseptic cultures. However, the bud sprouting was low 0.6 ± 0.5 in a medium supplemented also with antioxidants. Due to the low percentage of new aseptic shoots, Benomyl was excluded from the tissue culture establishment medium and only media supplemented with
109Rifampicin were recommended to produce aseptic shoots in a 3.0 ± 0.9 sprouting rate for A. cherimola and A. muricata.
Most of the hardwood species produce phenolic compounds after wounding (George and Sherrington, 1984). The Annona spp., as well as other fruit tree species are limited by the excessive hypersensitivity of the cells to wounding or mechanical injury ( Nair et al. 1983, 1986). During fruit conservation and postharvest, high losses were reported due to phenolic brown exudates (Martinez-Cayuela, 1986). The phenolic substances promote tissue blackening which have been reported as an inhibitor of new in vitro morphogenic responses in Annona spp., (Jordan et al. 1990; Bridg, 1993).
A. cherimola is a species rich in tannins, hydroxyphenolics, cathechin, epicatechin and biflavanes (Martinez-Cayuela, 1987). To control phenolics on A. cherimola, Jordan et al. (1991) compared the effect of some antioxidant substances such as citric acid, ascorbic acid, amino-oxiacetic acid, glutathione and cysteine and reported the effect of Polyvinylpirrolydone on internode explants of A. cherimola.
Young soft tissues of A. cherimola and A. muricata are hypersensitivite, the meristems, shoot tips, segments of leaf, buds and small shoots with one bud, produced after the isolation of a necrotic exudates in in vitro culture. The in vitro establishment of these small and vegetative explants is limited by the phenolic substances in the culture medium coming from the explant, they are not protected by lignin or its cuticular mantle is thinner. Furthermore the surface of these explants is covered by some microscopic trichomes which are a physical barrier and could prevent the action of NaClO. The presence of contaminants in the in vitro culture medium also promoted necrotic exudates on the base of the tissue explant of A. cherimola and A. muricata.
The A. cherimola and A. muricata plant species were characterized by the production of blackening where wounding took place, which released the phenolic content of broken cells. This oxidation at the beginning affected the neighboring cells which were not affected by wounding and did not show symptoms of injury themselves. The phenolic promotes the premature death of specific cells in its own environment (Debergh and Read, 1991).
The hypersensitivite reaction of semi-woody and woody cuttings with pre-formed buds of A. cherimola and A. muricata could be totally controlled. The use of antioxidant substances such polyvinylpyrrolidone (PVP) 4 ppm were effective if it was supplemented in the culture media (Table 23). The PVP in the selected establishment media adsorbs the active phenol-like substances and seems to be more effective than charcoal (Johansson, 1983). Jordan et al. (1991) reported also the reduction of the total phenolic concentration of the A. cherimola internode explants after 40 days in culture on media supplemented with PVP only and in combination with casein hydrolysate.
Annona explants, after wounding, produced phenols which could not be stopped by ascorbic acid and citric acid over a long period of time in in vitro culture media. They are the most common antioxidant compounds, however they are very labile and are also easily to be oxidized themselves and it is well known that oxidation products are phytotoxic (Martinez-Cayuela, 1982; Debergh and Read, 1991).
The production of phenolic substances is promoted when the oxidation of pre-formed phenolic components of the explant such as phytoalexin or lignin (quinones) or by the synthesis of monomeric or polymeric phenolic derivates begin on the cultured explant (Rhodes and Wooltorton, 1978).
110The present study shows that phenolization of semiwoody cuttings of A. cherimola and A. muricata explants could be controlled well if they are immersed previously in a combined solution of citric and ascorbic acid during the manipulation time to prevent oxidation on the surface of the explant during the inoculation time (Table 24).
The semiwoody cutting explants of A. cherimola and A. muricata have the potential to induce new in vitro shoots because they are carrying buds. The mechanical injury caused by the scapel, promotes phenolization on the base and tip of the cutting where the cut was made. The leaf scales of the pre-formed buds protect the meristems of the phytotoxic oxidation products.
The lignification grade in semiwoody cuttings is less intensive than woody cuttings. During the establishment the semiwoody cuttings should be protected from the oxidation reaction with a long-phase antioxidant such as Polyvinylpyrrolydone on establishment, the absence of phenols promotes quality shoots under in vitro conditions.
If the meristems of the pre-formed bud from the axillary branching are stimulated, the formation of four new shoots per bud could be induced by direct shoot organogenesis which promotes the genetic stability of the selections during the in vitro establishment of A. cherimola and A. muricata. The results suggest that the micropropagation of this species begins with a genetically stable shoot material.
A. cherimola and A. muricata are subtropical and tropical plants which grow well on the tropical high-lands with rich mineral soils of volcanic origin (Bridg, 1993) but not on soils with high salt concentrations (Morton, 1987).
Because most plants react positively to the Murashige and Skoog (1962) mineral salt and vitamin combination during in vitro culture (Conger, 1986), it was tested during the establishment of A. cherimola and A. muricata. The mineral basal composition of Nitsch and Nitsch (1969) has been reported by Jordan (1998) on A. cherimola during the induction and promotion of in vitro morphogenic responses.
The A. cherimola and A. muricata responded better during establishment to the mineral salt formulation of Nitsch and Nitsch (1969) than to the Murashige and Skoog (1962) salt combination which is a middle salt rich medium in comparison with the Nitsch and Nitsch (1969) formulation.
This study shows that the A. cherimola as well as the A. muricata did not respond well when cultured in Murashige and Skoog (1962) medium. In accordance with Witjaksono et al. (1999) most woody species did not respond well to this salt high media formulation.
A. cherimola as well as A. muricata did not require a high content of mineral salts to promote the in vitro sprouting of the pre-formed buds (Table 25). Other species like those of the genus Sorbus that are also deciduous trees and shrubs require high concentrations of salts, not only during the establishment, but also during the multiplication (Chalupa, 1987). The sensitivity of A. cherimola and A. muricata to salt high conditions has been reported by Ebert (1998).
The shoot cuttings of A. cherimola and A. muricata used in this study to promote the direct bud sprouting of these species under in vitro culture seems to have all the energy and nutritional requirements to induce new growth. Then the culture media
111should only provide a low mineral balance that permits the shoot cutting to be in an osmotically balanced environmental condition.
Furthermore 3.0 ± 0.1 was the mean shoot number per bud in A. cherimola and A. muricata cutting in media supplemented with sucrose 30 g/l. Establishment culture media without sucrose reported a low percent of bud sprouting 1.6 ± 0.6 mean of shoot number per bud. The sucrose in all the tested media in this study is shown to improve the quality of the new in vitro sprouted shoot. The shoots from media supplemented with sucrose in any case showed a hyperhydratation or anomalies during their development.
The osmotic relation between the tissue cells of the semiwoody cuttings of either A. cherimola or A. muricata was promoted by the presence of sucrose which is a very important component in any nutrient medium and its addition is essential for in vitro growth and development. Sucrose is a disaccharide used in in vitro culture because this sugar is also synthetized and transported naturally by the plant. The sucrose present in the culture medium is rapidly broken down to fructose and glucose by hydrolysis because of extracellular enzymes.
A. cherimola and A. muricata shoot cuttings sprouted well in medium without plant growth regulators, because the shoot explants with two or three pre-formed buds themselves have a endogenous plant growth regulator concentration to promote the new shoots.
6-benzyl amino purine 8.87 µM and indol-butiric acid 2.46 µM are required for the semiwoody macro-cutting of A. cherimola and A. muricata to stimulate bud sprouting and shoot growth and cell elongation. The relation of cytokinin : auxin is very significant for A. cherimola and A. muricata in terms of shoot quality.
Liquid media and semisolid media of NN-69 for A. cherimola and A. muricata establishment promote bud sprouting, however these new shoots showed the tendency to be hyperhydrated during multiplication. The hyperhydrated shoots of A. cherimola and A. muricata showed a poor epidermal development and glassy appearance which restricts its multiplication because leads to metabolic and morphological derangements in the in vitro explant.
Species such as Malus spp., Picea sitchensis, Gladioulus grandiflorus and Prunus avium which have been been cultured in a liquid medium, have reported vitreous shoots, aerenchyma disorders, unorganized cortex, thin cell walls, abnormal stomata and low epicuticular wax deposits (John, 1986).
The A. cherimola and A. muricata hyperhydrated shoots in the multiplication stage produced some phenols in the culture media and a light green color was observed suggesting that chlorophyll content was reduced. Liquid media could promote extra-protoplastic water on the tissues as an effect of high enviromental humidity.
The incubation temperature used during the micropropagation of A. cherimola and A. muricata cuttings was 25 ± 3 °C and the total closure of the test tube with parafilm (Figure 13) was not favorable for the explant. The gas circulation was stopped, the water vapor increased and the relative humidity in the in vitro test tube around the explant promoted an abnormal epicuticular development. Furthermore A. cherimola as well as A. muricata in natural conditions do not grow well in high humidity conditions (George and Nissen, 1992).
112In accordance to Pàques and Boxus (1987) vitrification and hyperhydratation limit in most of the cases the industrialization of micropropagation protocols thus in the present study the A. cherimola and A. muricata hyperhydrated shoots were eliminated
Solid media of Nitsch and Nitsch (1969) either with agar or gelrite did not promote vitrification or hyperhydratation of shoots on A. cherimola and A. muricata. The presence of a support agent contributes to the regulation of the humidity which affects the availability of the water status (Deberg, 1983), because the water in a solid medium is bounded.
In this study the A. cherimola and A. muricata in all the micropropagation did not respond well to a high concentration of Agar which not only limited the bud sprouting, but also the new shoot formation and rooting.
The A. cherimola and A. muricata in this study responded well to a culture medium supplemented with Gelrite which was added in a half concentration of agar and showed a clear medium which permitted the evaluation of contaminants more easily.
The A. cherimola and A. muricata reduced the percent of phenolics on a Gelrite supplemented medium on establishment. Pierik (1989) showed that gelrite gels are more or less free from contaminating materials which could promote phenolic compounds. In comparison with Agar which is seaweed derived, gelrite is a natural anionic heteropolysacharide that forms rigid, brittle agar-like gels in the presence of soluble salts. Gelrite is a polysaccharide comprised of glucuronic acid rhamnose, glucose and O-acetil moieties (Pierik, 1989).
The cuttings with pre-formed buds of A. cherimola and A. muricata semi-woody tissue, showed regeneration differences on NN-69 medium combined with Benzyl amino purine (BA) and Indole-butiric acetic acid (IBA) in terms of shoot sprouting.
The A. cherimola and A. muricata shoots that developed in NN-69, not supplemented with plant growth regulators, showed a high percent of bud sprouting (Table 27) because larger explants such as macro-cuttings used to promote new shoots from pre-formed buds, have a starch reserve which promotes the meristem activity if in the culture condition sugars, minerals and plant growth regulators are added.
A. cherimola and A. muricata in NN-69 supplemented with BA and IBA promoted the formation of quality shoots, the balance cytokinin : auxin during the establishment benefit the promotion of healthy shoots, probably due by the regulation of endogenous plant hormones on the bud.
86 ± 2.1 % of A. cherimola and 86.1 ± 1.5 % A. muricata buds were sprouted successfully in NN-69 media supplemented with BA 8.87 mM and IBA 2.46 mM. This study showed four healthy new shoots formed from pre-formed buds on semiwoody cuttings of A. cherimola and A. muricata in the presence of plant growth regulators.
The bud sprouting and in vitro plantlet differentiation of A. muricata from hypocotyl segments have been reported using BA (Bejoy and Hariharan, 1992). However they failed to elongate. To promote shoot elongation the BA (8,9 mM) was combined with NAA (0,54 mM) and further growth into elongated shoots was observed. Even then, all shoots buds did not develop into elongated shoots.
113The endogenous growth regulators, age and plant location condition the in vitro response of the cutting explant in A. cherimola and A. muricata as well as in other woody species (Torres, 1989).
The new shoots of A. cherimola and A. muricata promoted in the highest frequency in a high cytokinin and low auxin concentration (Table 26). In tissue culture there are plants which in principle need neither cytokinins nor auxines for the formation of adventitious shoots (Pierik, 1989).
Most of the plants require cytokinin for shoot formation and benzyl-aminopurine is the cytokinin which shows the most efficient application in the promotion of adventitious shoots. The citokinin:auxin ratio is very important for the shoot formation in many different plant species (Pierik, 1989). Jordan et al. (1991) reported from single nodes of seasonal buds the sprouting and forming of leaflets in the presence of NAA 0.5 mg/l and BA 2.0 mg/l with the presence of some calli.
A. cherimola and A. muricata promoted not only direct bud sprouting form pre-formed buds of semiwoody cuttings but also clumps of white cells on the surface of the explant. The cambium tissue of cuttings was stimulated (Figue 11; Figure 12 ) in medium NN-69 supplemented with BA and IBA reported concentrations (Table 25). Callus induction and shoot regeneration were tried on A. cherimola and A. muricata from this meristematic cambium with high levels of auxines such as 2,4-D and NAA , this study shows no regeneration from meristematic cambium callus on A. cherimola and A. muricata.
In accordance to Pierik (1989), Torres (1989) and Murashige (1976) the year`s season has an effect on the in vitro manipulation. A. cherimola and A. muricata explants coming from actively growing plants in summer are more able to induce morphogenic responses than those coming from winter.
The A. cherimola and A. muricata, as other semideciduous woody species, show different rates of growth during the year (Sawneski, 1988; George, 1984; Torres and Sanchez, 1992) and under in vitro conditions they were affected also by the year`s season during establishment and multiplication. The semiwoody explants reduced the sprout percentage in winter and in summer they were more shoot proliferant (Figure 14). In open field conditions the A. cherimola have the tendency to burst in spring and loose the leaves in winter (Gardiazabal and Rosenberg, 1988).
The deciduous trees as well as the deciduous trees such as Malus spp., Pyrus spp. and Prunus spp., have an intrinsic biological cycle which under the most optimal environmental conditions promote leaf fall. The A. cherimola and A. muricata during in vitro culture in terms of bud sprouting were affected by the year`s season. The percentage of shoot sprouting from pre-formed buds reduced in winter more significantly for A. muricata than for A. cherimola (Figure 14).
The A. muricata is the most tropical of the Annonas and it is extremely affected by the reduction of the photoperiod. The light intensity has a strong influence on the metabolism of the plants and active growing. The endogenous plant growth regulators level in semideciduous and deciduous trees have a different balance during short days and low temperatures (Hartman and Kester, 1984).
During the tissue culture establishment A. muricata needs more time to promote bud sprouting than A. cherimola which is the most subtropical of the Annona species
114and adapted to some temperature variations on the highland mountains in natural conditions.
The proliferation of new shoots includes the processes of first dedifferentiation and second differentiation which possibly lead to the re-determination and rejuvenation of the new formed cells (Roca and Mroginski, 1991; Conger, 1977).
The organogenic potential of A. cherimola and A. muricata are different, although these species showed the same shoot sprouting behaviour during establishment in the multiplication stage the A. cherimola was shown to be more proliferant than the A. muricata which was not proliferant in in vitro conditions but elongated well in a Benzyl-aminopurine supplemented media.
The establishment Nitsch and Nitsch media supplemented with 8.87 µM Benzylamino-purine (BA) and 2.46 µM Indole-butiric acid (IBA) showed the highest sensitivity of A. muricata to BA levels (Table 26).
Lemos and Blake (1996) reported the effect of BA 9.5 mg/l in combination with NAA concentrations on A. muricata where the increments of BA concentrations inhibit the formation of buds per explant. This result is also confirmed in this study as well as the fact that the presence of a cytokinin on the multiplication culture media had no significant effect in terms of number of shoots per explant neither in A. cherimola nor in A. muricata.
The A. muricata in vitro shoot explants have the capability to induce bud elongation and become useful shoots in media supplemented with BA 2.22 µM. In contrast with Lemos and Blake (1988) the increments of BA concentration did not increase the number of shoots per explant in A. muricata.
The elongation of A. muricata shoot explants in media supplemented with BA was compared to Gibberellic acid (GA3) supplemented media. A. muricata shoots in media with GA3 were intermediate in length and the multiplication rate per shoot reduced from 1 explant : 4 new cuttings with bud in BA medium to 1 explant : 2 new cuttings in BA medium.
The GA3 as media supplemented has been reported by Jordan et al (1991) as an inhibitor of the de novo bud promotion in A. cherimola and in A. muricata. It has been suggested as an inhibitor of bud development (Lemos and Blake, 1996). The use of gibberellins in in vitro culture has been reported as not essential for most species or higher plants. Gibberellins promotes elongation of internodes as plant growth regulators but inhibits adventitious shoot formation and adventitious root formation in most of the cases (Boggetti et al. 1999)
The comparative effect of Thidiazuron (TDZ) and Benzylamino purine (BA) on shoot proliferation of A. cherimola and A. muricata was evaluated, although TDZ has recently been used in tissue culture on recalcitrant tissues because of its cytokinin-like activity (Huetteman and Preece, 1993).
TDZ stimulated shoot organogenesis in A. cherimola and A. muricata (Figure 18) but on the base of the explant multiple adventitious leaves were observed in 0.45 µM, 1.36 µM and 2.27 µM (Figure 11). The reason why TDZ as a pyridil-urea compound has a high activity in low concentrations in woody plants to promote adventitious shoots is until now not clear (Huetteman and Preece, 1993).
115TDZ has been reported as a persistent growth regulator in tissue culture and since most tissues are transferred to fresh medium without a regulator the cytokinin activity of TDZ continues, because little is known about the cytokinin plant receptors (Huetteman and Preece, 1993).
The A. cherimola and A. muricata in this study were shown to be very sensitive to TDZ supplemented medium but the new shoot organogenesis was interfered by a non normal leaf regeneration on the base of the shoot explant. The TDZ supplemented medium was avoided because the principal aim was to produce in vitro true-to-type plants.
Although TDZ has been used to promote adventitious shoot formation in many species, this study did not report positive effects on the shoot quality regeneration in A. cherimola and A. muricata. There is no information (as far as it is known) about the endogenous cytokinin shoot concentration of woody species when they are exposed to synthetic cytokinins in culture media.
The TDZ was developed by Schering AG (Germany) for utilization as a defoliant for Gossypium hirsuum L. TDZ is up to 10,000 times a cytokinin activity and in several woody species caused a dramatic increase in the number of shoots and proliferation rate. In concentrations less than 0.1 µM it is capable of inducing adventitious shoots in Poplar spp. calluses but also axillary shoot formation is severely inhibited in Poplar spp, when thidiazuron is present in all the treatments then it is necessary to transfer shoot-regeneration cultures to TDZ free media for shoot development (Pierik, 1989).
The A. cherimola and A. muricata new shoots proliferated also in Zeatin (Z) which is reported to be 10 times more powerful than Kinetin (K) and the synthetic cytokinin Benzyl-amino purine (BA). A. cherimola is bud proliferating in media supplemented with Zeatin (1.36 µM), whereas A. muricata is not bud proliferating in Zeatin supplemented media. These species are not shoot proliferating in media supplemented only with Zeatine.
Encina et al. (1994) reported the effect of 1.36 µM of zeatin on A. cherimola shoot formation, at 0.66 µM wide and longer leaves were reported and in high zeatin concentrations aberrant shoots were observed. This study showed a reduction in the proliferation rate in a zeatin high concentration medium. High quality shoots were observed in a 1.36 µM concentration with a means of shoot number of 2.5 ± 3 per explant. Also A. cherimola was shoot proliferating in a kinetin supplemented media which at 2.32 µM shows 3.5 ± 5 shoots per explant.
The synergistic effect of zeatin and kinetin were tested on A. cherimola shoot explants which were high shoot proliferants in Z 1.36µM + K 4.65 µM., 6.8 ± 0.7 new shoots were induced in this concentration but shoots should only be exposed to this concentration for a short period of time because if several subcultures are promoted in a Z+K supplemented medium no root formation could be induced in the next micropropagation. The A. cherimola shoots elongated well in a BA supplemented medium which is a less powerful cytokinin (Krikorian, 1989b).
A. cherimola and A. muricata during the multiplication stage showed chlorotic shoots in cytokinin supplemented media, these shoots lost their vigour and reduced the multiplication rate. The formulation of Nitsch and Nitsch (1969) (NN-69) is not a high salt concentration formulation in comparison with that reported by Murashige and Skoog (1962) (MS-62) and Woody Plant Medium (WPM).
116The MS-62 showed the highest concentration in ammonium and nitrate (20.6 and 39.4) respectively. In contrast WPM showed the lowest concentration of these ions in its formulation (5 mM and 9.8 mM), the NN-69 has a concentration of these ions (9 mM and 18.4 mM). Therefore these ions were substituted in the formulation of NN-69 to increase the ammonium and nitrate concentration and support the A. cherimola and A. muricata during their multiplication with a media rich in nitrogen. The other elements such as potassium, magnesium, calcium, sodium, phosphate, sulphate and chloride showed in MS-62, WPM and NN-69 concentrations that did not vary at all.
The catabolic metabolism of ammonium during the photorespiration and the anabolic inorganic nitrogen assimilation are related to the electron transport systems in chloroplast and mitochondria organelles. If there is a non-balance of these ions in the cell it promotes the production of free radicals and ultimately the death of the plant (Miflin and Lea, 1977; Skotut et al. 1978: Krogmann et al. 1959; Puritch and Baker, 1967).
In the present study the A. cherimola and A. muricata shoot multiplication was stimulated with high levels of ammonium and nitrogen ions (Table 28) in order to preserve the quality of the new in vitro produced plant material. The variation of the amount in Nitsch and Nitsch (1969) media formulation from 9 mM to 20.6 mM NH4- and 18.4 mM to 39.4 mM NO3- promoted shoots without chlorosis on a high cytokinin supplementation.
The casein enzymatic hydrolisate is a nitrogen complex which is used in plant tissue culture to promote growth and as a nitrogen source because it is rich, not only in aminoacids, but also in reduced nitrogen. In this study the supplementation of casein in the culture media increased the number of shoots without chlorosis in the culture media, but not all.
Rooting has been occasionally reported in Annona spp., but regeneration from explants of clonal origin has not been reported (Jordan and Botti, 1992). A low rate of rhizogenesis was reported by Tazzari et al. (1990) on A. cherimola cuttings micropropagated from adult cuttings.
In this study A. cherimola and A. muricata micropropagated shoots in preliminary rhizogenesis experiments (rooting culture media supplemented with cytokinins and dark stimulation for five days) were tested and did not show any in vitro root organogenesis, also no callus formations were observed on the base of the explant.
The in vitro media during the micropropagation have been supplemented with sucrose which is a carbon source, essential for in vitro growth and development, because phosynthesis is insufficient, due to growth taking place in conditions unsuitable for photsynthesis or without photosynthesis (in darkness). Green tissues are not sufficiently autrophic in vitro (Pierik, 1989).
The organogenic potential of plant explants under in vitro conditions is closely associated with the content of natural and exogenous applied plant growth regulators (PGRs) (Terzi and LoSchiavo, 1990; Wenck et al. 1990). Unfortunately, little is known about this topic due to the complexity and sophistication of the techniques required for PGR analysis. Only a few research teams have found links between the embryogenic capacity of plant tissues and a specific endogenous hormonal content (Rajasserakan et al. 1987; Ivanova et al. 1994; Wenck et al. 1988), but all these results were obtained in herbaceous plants and not in woody species (Centeno et al. 1997).
117A. cherimola and A. muricata shoots were obtained from the multiplication stage with three subcultures in a rich cytokinin medium during six months after its inoculation. All the media were supplemented with sucrose 30 g/l to improve the photosynthesis of the new vegetative plant organs. The endogenous synthesis of PGRs is related to the photosynthetic activity of the vegetative explant.
Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin ribosied, dihidrozeation, dihydrozeatin riboside, N6-isopentenyl adenine (iP) and N6- isopentenyladenine riboside] have been evaluated in Coryllus avellana, results suggest that the endogenous hormonal balance is a very important factor defining the in vitro potential of tissue explants in plant culture (Centeno et al. 1997).
A. cherimola shoots were multiplicated in a zeatin:kinetin combination medium and A. muricata in a benzyl-amino-purine supplemented medium, the selected shoots to improve rhizogenesis were cultured in a root precondition medium with 20 g/l sucrose and a PGR supplementation because the shoot explants were cultured in a high cytokinin combination medium.
Previous results indicated that rhizogenesis could be stimulated and root formation promoted after this precondition, then mineral salt composition, carbon source concentration and the effect of some auxines were evaluated to promote either in vitro or ex vitro roots.
The effect of indole-acetic-acid (IAA), naphtalene-acetic-acid (NAA) and IBA (indole-butiric-acid) were evaluated. No roots were observed except with 1% IBA from Rhizopon ® applied on the base of the explant in ex vitro conditions. The effect of 0.3% IBA in talcum powder has been reported also on Betula spp (Meier-Dinkel, 1992). A. cherimola and A. muricata had rooted shoots in ex vitro conditions in 90% high humidity.
To promote in vitro rooting, shoots were treated with IBA 1% (4. 90 µM l-1) with the reduction of macroelements to ¼ and ½ of its concentration. The media used the mineral basal composition of Nitsch and Nitsch (1969), reduction of sucrose concentration was observed also as one of the factors which are related to rhizogenesis in A. cherimola and A. muricata selections.
The precondition of the rooting media in darkness for five days to promote rhizogenesis by the intensive activity of the IBA in darkness conditions to prevent photo-degradation of this PGR, reported no significant differences among treatments, suggesting that this species needs hormone free media precondition but not darkness when the media are supplemented with IBA.
Bejoy and Hariharan (1992) reported 1-2 roots induced on 80% of the A. muricata explants a root organogenesis when the shoots were treated with 14,8 mM IBA. Similarly, on 4.9 mM IBA 85% shoots rooted but with only 1-3 roots. Root organogenesis on A. squamosa has been reported when the explants were tried with 98 mM IBA (Nair et al. 1984).
In this study 4.90 µM IBA promoted in A. cherimola and A. muricata roots after 40 days either in vitro and ex vitro. 3.6 roots per shoot were the registered promedium after the root emergence after 60 days. Encina et al. (1994) reported rooting in A. cherimola at 500 µM IBA. In this study this concentration did not promote rhizogenesis.
118The other auxins such as IAA and NAA had no effect on A. cherimola and A. muricata root organogenesis.
The sucrose and concentration of macroelements salts of Nitsch and Nitsch (1969) in the culture medium should be reduced. Carbohydrates are responsible for inducing changes from the pentose phosphate to the glycolitic pathway in plant tissues and this could explain its effect on rooting (Haissing, 1982).
In accordance to Encina et al. (1994) A. cherimola reduced the percent of rooting progressively with the increments of sugar in the culture media. A. cherimola and A. muricata are sensitive to the accumulation of sugar in the culture media. High carbohydrates in the culture media have been reported as root inhibitors in A. cherimola (Encina et al. 1994), however low concentrations are essential to promote roots in A. muricata (Lemos and Blake, 1996).
There are differences between the in vitro and ex vitro percentage of rooted shoots in A. cherimola and A. muricata. The number of roots per shoot under in vitro conditions of both A. cherimola and A. muricata was reduced in comparison with the ex vitro rooting. These differences may be caused by their different levels of action of metabolic regulators such as ethylene. The in vitro shoots of A. cherimola and A. muricata probably accumulated more ethylene in the baby food jar than the ex vitro shoots. Ethylene inhibits root formation of apple shoots under in vitro conditions (Mo et al, 1989).
For the Acclimatization of regenerated plantlets with well established root systems plants were washed carefully to remove the gelrite and transferred to pots (9x9 cm) containing quartz sand. Potted plants were acclimatized in a transparent plastic cabinet covered with polyethylene bags at 25 ± 2°C under 16 h photoperiod. After 4 weeks, acclimatized plants were transferred to the greenhouse.
The pre-acclimatization of the A. cherimola and A. muricata explants was made in a sterile medium and then they were transplanted successfully into a non sterile quartz sand. Nair et al. (1984) reported for A. squamosa a pre-transplantation made before acclimatization in the greenhouse on aseptic conditions. Bejoy and Hariharan (1992) transferred the A. muricata micro plants directly into non sterile soil.
Hence the present procedure is simple and the autotrophic development of A. muricata plantlets was satisfactory. Regenerated plantlets were successfully established in the greenhouse after acclimatization. The plants, thus established, did not exhibit altered phenotypes, there were no morphological visible differences between regenerated plants of A. cherimola and A. muricata and mother source explants.
The genetic molecular variability of the DNA sequences in plants could be monitored by RAPD markers because of the wide spectrum of application (Williams et al. 1990). RAPD have been found to be efficient in the detection of genetic variability at the nuclear genome level (Tulseiram et al. 1992; Brown et al., 1993; Munthali et al., 1996).
DNA characterization through RAPD permits us to characterize the relationships and determine likely parentage between species, selections or varieties because they are dominantly inherited markers (Villand et al. 1998). The expected segregation in F1 can
119be inferred, in some cases, from parental phenotypes as Ronning and Schnell, (1995) reported for Annona spp.
The RAPD analysis have been compared with other molecular techniques such as Restriction Fragment Length Polymorphism (RFLP) and isozymes and no differences between results have been found (Munthali et al. 1996; Sabir et al., 1992).
Genetic variation between A. cherimola cultivars has been studied using isozyme markers (Ellstrand and Lee, 1987; Lee and Ellstrand, 1987; Pascual et al. 1983; Perfeccti and Pascual, 1996). However isozyme analysis is limited by the relatively small number of loci. RAPD offer the potential of generating large numbers of markers representing a random sample of the genome, thereby presenting an advantage over isozyme markers (Ronning and Schnell, 1995).
29 primers were tested by RAPD to analyse the DNA amplification of A. cherimola and A. muricata in vitro regenerants comparing results with the DNA amplification of ex vitro mother plants. The Q-12, C-11, C-5, Q-4, OPA-18 and OPA-16 showed repeatable and scoreable bands for both species. Because the number of tested plants was reduced the amplification might be the result of the duplication of some parts of the genome.
The DNA amplification of the A. cherimola and A. muricata experimental plants shows monomorphic bands in all the six tested primers(Figures-36,-37, -38 and -39). Although these results are limited by the number of plants tested, the developed and scorable bands assure the absence of DNA polymorphism in A. cherimola and A. muricata regenerants, coming from the developed micropropagation protocol presented in the present study (Table 36).
Using the RAPD technique, various investigators have reported somaclonal variation in Pinus thunbergii (Goto et al. 1998), Picea glauca (De Verno, 1999), Allium sativum (Al-Zahim, 1999), Acacia nilotica (Garg et al. 1996) and Phalaenopsis spp. (Chen et al. 1998) in vitro cultured plants from somatic embryogenesis, endosperm culture.
The somaclonal variation promoted by the in vitro culture of a selected plant material is related to several factors (Larkin and Scowcroft, 1981). One of them is the natural condition of the initial material mother plants, because there are, in nature, plants that shows a tendency to induce bud mutations and diversifications than others (Krikorian, 1991) and there are no reasons to suppose other behaviour when they are manipulated in vitro , (Conger, 1987; Zimmerman, 1981, Krikorian et al., 1983). The causes of the natural mutations on open grown field plants is not clearly explained (Krikorian, 1991).
The cell organization of the selected explant is conditioning from the beginning the true-to-type and true-off-type in vitro multiplication (De Fossard, 1977). The most stable explants described are: meristems, lateral buds and nodes which could be propagated directly, succeeding somatic embryos and advent buds, while the most unstable explants are cell callus and protoplasts (Roca et al., 1981), because they are not organized structured explants (Scowcroft et al., 1987).
The RAPD results suggest that A. cherimola and A. muricata plants exhibit relative stability during in vitro propagation through the direct bud sprouting induced under in vitro conditions. Genetic stability has been reported as well as in Eucalyptus
120tereticornis micropropagated by enhanced-axillary-branching-derived plants and tested by RAPD (Rani and Raina, 1998).
The discrete polymorphism revealed by RAPD and between A. cherimola and A. muricata selections is of future interest not only for phylogenetical information of this species, but also because there are several diverse and discrete fragment lengths, which can serve as characteristic markers for quality control in plant production systems, and for marker-assisted breeding programs
The use of different primer combinations enables the analysis of the presence or absence of polymorphisms. This is enough to distinguish not only cultivars, but different clones belonging to the same cultivar. Moreover, the results allowed the identification of high certainty cultivars, especially in fruit species, that belong to the same variety within the collection, which will save time and reduce the cost of their conservation.
The nuclear genome analysis has played an important role in early genetics. The basic cytological techniques enable an accurate determination of chromosome number and structure either in meiosis or mitosis cell division phases (Karp, 1991).
The observation of mitotic chromosomes under the light microscope is a rapid and informative method of studying the genomes as a whole. The technique involves the collection, fixation, staining and preparation of chromosome squashes. Karp (1991) commented that although the cytology technique is easy to perform, there are some problems determined by species and its particular genome. The A. cherimola and A. muricata present some difficulties also to visualize the chromosomes by any traditional cytology method.
In a wide range of plant species, aneuploidy and/or changes in the ploidy level of the chromosomes have been considered to play a major role in describing somaclonal variation (Bayliss, 1980; D'Amato, 1985). RAPD and nuclear genome size estimation singly have been effectively used in many studies to detect somaclonal variation on in vitro produced plants (Brown, et al., 1993; Cecchini et al., 1993; Rani, 1995).
In vitro plantlets regenerated from callus and/or meristem cultures exhibited startling differences in the chromosome number in Populus species (Rani et al., 1995; Sommer and Wetzstein, 1984). Chromosome doubling is a common type of somaclonal variation (Novak, 1980) and has not been observed in Annona regenerants in the reported results.
The efficiency of RAPD markers to differentiate the Annona spp is in agreement with the conclusions reached by Ronning and Schnell (1995). Further, this study has demonstrated for the first time that RAPD markers applied to A. cherimola and A.muricata in vitro regenerants screen the quality of the produced plantlets.
This work contributes to the knowledge of the A. cherimola and A. muricata potential fruit species and reports a clonal regeneration protocol with a successful rate of rhizogenesis and without much risk of genetic instability according to the RAPD analysis.
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