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


Chapter 5. Materials and Methods

5.1 Micropropagation

5.1.1 Plant Material

Tissue culture experiments were carried out with two kinds of mother A. cherimola and A. muricata plant material. The initial material or stock mother plants of these subtropical and tropical semideciduous fruit trees were commercial plants in Colombia and Chile (Table 16).

Table 16. Source of A. cherimola and A.muricata experimental material

Species and Selections or Cultivars

Procedence Country (Region)

Type of Material

Material Code

Selection Code






A. cherimola

cv. Felpa






cv. Bronceada






cv. ?

Colombia (Cundinamarca)










A. muricata

cv. ?

Colombia (Tolima)





cv. ?

Colombia (Cundinamarca)





cv. ?

Colombia (Valle del Cauca)




* one year old plants from seeds of selected native trees

Healthy, one year old, 1 m high and vigorous native Colombian A. cherimola and A. muricata seedling trees (T1), growing in the field, were selected based on previous information from the farmers in terms of plant quality, because in Colombia there are no clonal plantations established with Annona spp.

These plants were transported from three different regions of Colombia to Berlin without expanded leaves. The trunk, cuttings and roots were covered and protected with moistened paper and kept at a room temperature of 18 ± 3°C for four days and coded or tissue culture work purposes (Table 16).

Selected seeds of A. cherimola var. Felpa and Bronceada (T0) from Chile were also used as experimental material (Table 16). Germination treatments were applied in order to break the seed dormancy of these semideciduous species. Seeds with and without coat were immersed in water for 2 days at 20 ± 2°C, not only to improve endosperm hydration and promote embryo emergency but also to select the viable seeds, those seeds which floated were eliminated.


To prevent fungal attack the hydrated seeds were treated with Benomyl<3> 0.03% for 1 hour and then washed with distilled water for 20 minutes.

Temperature and gibberellic acid GA3 were the experimental variables for A. cherimola var. Felpa and var. Bronceda germination seeds, 20 units were used per immersion treatment:

distilled water and stored in darkness 24h / 4°C

250 ppm GA3 solution and stored in darkness 24h / 4°C

500 ppm GA3 solution and stored in darkness 24h / 4°C

distilled water and stored in darkness 20 ± 2°C

250 ppm GA3 solution and stored in darkness 20 ± 2°C

500 ppm GA3 solution and stored in darkness 20 ± 2°C

Afterwards all of them were sown in pots filled with quartz sand and maintained at seedling germination nursery conditions, 23 ± 2°C, 95% humidity and 16 h photoperiod. After germination some seedlings started to grow and develop new expanded leaves. These were transplanted into pots and maintained under hydroponic conditions with a commercial solution of Wuxal<4> 1 ml/l during the duration of this study.

The A. cherimola (T0) and (T1) plants were planted in subtropical greenhouse conditions,18 ± 5° C and 75 % humidity and the A. muricata (T1) plants in tropical greenhouse conditions , 25 ± 5°C and 70 % humidity, at the Fruit Science Department of the Humboldt University, Berlin (Dahlem).

The A. cherimola and A. muricata plants were pruned three times per year and treated permanently with Benomyl 0.03% to reduce the endogenous contaminants of the tissues such as fungi. After eight months of greenhouse adaptation these plants were transplanted into pots with quartz-sand to prevent wilting.

5.1.2 Explant Selection and Isolation

To induce the establishment of A. cherimola and A. muricata, different tissues from the T0 and T1 selections were evaluated. Factors such as source, age and size were evaluated.

To reduce in vitro contamination, all the selected branches were trimmed from the plant and the big open-wide leaves were removed with a scapel in the greenhouse. The branches were segmented into approximately 15 cm long stems and cleaned with running tap water prior to any surface disinfection.


For A. cherimola and A. muricata the first year growth- (vegetative), second year growth- (semi-woody) and third year growth- (woody) were used as the source of explants:

First year branch, soft woody material: meristems (0.1 - 0.5 cm), buds (0.5 - 1 cm), shoot tips (0.5 - 1.5 cm), leaf pieces (0.5 - 1 cm), micro shoots with 1 bud (1 - 3 cm) and macro shoots with 3 buds (5 - 9 cm) were isolated as the primary source of explants

From the second year old branch, semi-woody material: buds (0.5 - 1 cm), stems with one pre-formed bud (1 - 3 cm) and stems with two or three pre-formed buds (5 - 9 cm) were evaluated

Third year old branch, woody material: buds (0.5 - 1 cm), stems with one pre-formed bud (1 - 3 cm) and stems with two or three pre-formed buds (5 - 9 cm) were evaluated

The viability of these explants to promote the micropropagation of A. cherimola and A. muricata was analysed in terms of shoot quality and the potential of in vitro regeneration. The explant responses were observed and noted during the manipulation time and after some days of culture:

Latency: Description of any in vitro response of the explant to the aseptic conditions. The explant did not have the potential to promote new cell divisions and subsequently differentiation. The explant did not react to the in vitro conditions and reduced its quality during the culture time

Died: The explant did not survive after wounding. Manipulation problems, excessive concentration of phenolics and also the presence of contaminants were observed.

Hypersensitive reaction: Production of phenolics or tannins by the explant and toxic accumulation in the culture media, the explant tissues were limited by cell blackening or total necrosis

Green: The explant survived to the first in vitro manipulation, it remained fresh and green. It was not affected by wounding and its growth in the in vitro culture media was promoted. No blackening of the in vitro culture media was observed. These explants had the potential to induce new in vitro organogenesis

Contamination: The explant survived to wounding, had the intrinsic potential to induce new organogenesis, but was affected by endogenous or exogenous contaminants like bacteria and fungi and the aseptic in vitro establishment could not be promoted


5.1.3 Phenolics

As most woody species, the A. cherimola and A. muricata are sensitive to wounding. The phenol metabolism is stimulated immediately after the cells of the isolated explant are separated from the mother plant.

The hypersensitive reaction is a tissue-protected reaction originating in the oxidation of polyphenol-like compounds and tannins by the polyphenoloxidase activity to o-quinones, antimicrobial growth (Martinez-Cayuela, 1986; Martinez-Cayuela et al. 1988; Debergh and Read, 1990).

The product of this reaction is a brown/black exudate coming from the explant. The first reactive areas are the tips of the explant, upon wounding. The phenolic compounds diffuse quickly in the tissue culture medium (Bridg, 1993b) and the concentrations are toxic for the explant (Debergh and Read, 1991).


Some A. cherimola and A. muricata plants were maintained in darkness in the greenhouse for two weeks to promote the reduction of the photosynthetic metabolism and subsequently the rapid stimulation of phenolics on the first in vitro culture days. Besides, shoot sprouting is expected to be stimulated under in vitro conditions.

Use of antioxidants to improve the in vitro culture: The combinations of L-ascorbic acid (AA) (50-150 mg/l), citric acid (CA) (100-200 mg/l) and polyvinylpyrrolidone (PVP) 10,000 Mol. Wt (2, 4, 8 -ppm) were evaluated. Several concentrations vs. exposition time (1, 5, 10 -minutes) were combined with two management methods as follows:

5.1.4 Disinfection

Different disinfectant solutions vs. exposition time were tested on separated segments coming from the 15 cm long selected branches of A. cherimola and A. muricata to improve surface disinfection:

Due to the fact that meristem explants are sensitive, the concentration of sodium hypochlorite was reduced to 0.5-1 % since chlorine solution and vapour can, more easily, penetrate the meristem of actively growing shoots which are less tightly bound by leaflets.

Following the surface disinfection with antiseptic solutions, the A. cherimola and A. muricata selected cuttings source of explants were rinsed in sterile deionized, double distilled autoclaved water five times 5 minutes each, to remove any remaining traces of the disinfectant. The branches were laid on Erlenmeyer flasks (500 ml) with the antioxidant solutions during the explant selection and manipulation in the laminar air flow-cabinet.

The tips of the A. cherimola and A. muricata became white after the disinfection treatment and they had to be removed with a scapel before any in vitro inoculation these white ends or tissue translucent tips were the first negative explant response to the disinfection applied treatment. The respective explant was separated from the branch in sterile conditions in the laminar air-flow cabinet, previously sterilized with UV-light lamps, switched on the night before. All the inoculations were made with sterile instruments. The meristems were separated under a binocular microscope. The sterilized explants were inoculated according to the selected media taking care of its natural polarity and subsequent orientation in the culture media.

5.1.5 Effect of Antibiotics

The A. cherimola and A. muricata endogenous contaminants are difficult to eliminate. Some previous A. cherimola cultures showed the presence of bacteria during the multiplication step. Then a review of the establishment disinfection conditions was made and previous results showed that the establishment media must be supplemented with antibiotics.

The Nystatin is a well known antifungal agent coming from Streptomices spp., and it is recommended in tissue culture because of its fungicidal activity in aqueous solutions. Cefotaxime is one of the most effective antibiotics against Gram (-) and Gram (+) bacteria species and Rifampicin, characterized by its wide spectrum against Gram bacteria and myco-bacteria, is the most recommended antibiotic for tissue culture purposes.

These antibiotics were selected because of their differences in spectrum action, 40 µg/ml Nystatin (Sigma N-9767) , 95 µg/ml Cefotaxime (Sigma C-7039) and 20 µg/ml Rifampicin (sigma R-7382) were evaluated during the establishment of A. cherimola and A. muricata selected explants. These were manipulated at room temperature conditions with 0.22 µm sterile millipore cellulose acetate membrane (Sigma F´9643) filters:

5.1.6 Mineral Basal Composition

The Nitsch and Nitsch (1969) (NN-69) and Murashige and Skoog (1962) (MS) were tested to promote induction of new cell divisions in A. cherimola and A. muricata greenhouse explants during the micropropagation in vitro stages (Table 17).

Table 17. Mineral media basal formulation


Concentration in mg/l













MgSO4 * 7H2O



CaCl2 * 2H2O












MnSO4 * 4H2O



ZnSO4 * 7 H2O



Na2MoO4 * 2H2O



CuSO4 * 5H2O



CoCl2 * 6 H2O






FeSO4 * 7H3O



Na2 * EDTA









Folic acid









Nicotinic acid



Pyridoxine hydrochloride



Thiamine hydrochloride



According to the micropropagation objectives during the establishment-, multiplication- or rooting- steps, the selected basal media formulations were supplemented with factorial combinations of some growth regulators such as:


- Auxin

- Gibberellin

To compare in vitro explant responses, double distilled autoclaved water supplemented with sucrose was used during the establishment. For the multiplication and rooting stage the selected mineral salts and organic nutrients with sucrose and support agent and non growth regulators were used as controls.

The effects of casein hydrolysate (Sigma C 7290) (CH) (100-200 mg l-1) on shoot quality growth and development were evaluated during the multiplication step to avoid in vitro chlorosis.

Other supplements such as Sucrose (Sigma 5391) (S) 1% - 3%, Agar (AG) 6 mg l-1, Gelrite (GR) (sigma P 8169) 3 g l-1or Whatman No.1 paper bridges for the liquid medium were evaluated not only on establishment but also on rooting.

The antioxidant substances L-ascorbic acid (Sigma A 2174) (AA) (50 -150 mg l-1l) + citric acid (Sigma C 4540) (CA) (100- 200 mg l-1) and Polyvinylpyrrolidone (Sigma P 2307) (PVP) 10,000 Mol. Wt. (1-10 ppm) were added to avoid necrosis of the selected explant on the pre-defined medium during establishment.

The pH of all mediums was adjusted at 5.7 ± 0.1 with an electronic pH meter after adding agar or gelrite into the solution and prior to autoclaving at 120°C and at a pressure of 1.2 kg/cm2 for 15-20 minutes.

The media were distributed into different glasses: Test-tubes (25 x 150 mm) with 15 to 20 ml medium for the biggest explants and closed with magenta sigma caps. Single or multiple shoots were grown in wide-necked baby-food jars (55 x 95 mm) containing 30 ml medium with magenta sigma B-caps. Disposable plastic petri dishes (90 x 15 mm) with 1.5 ml media were used to establish meristems and leaf young segments as well as small shoot tips.


The effect of parafilm on the closures was evaluated in terms of shoot development, some media were completely closed and then compared with others which were only closed with magenta caps.

5.1.7 Rooting

The well developed shoots of A. cherimola and A. muricata (3-4 cm long) were excised to improve rhizogenesis under ex vitro and in vitro conditions.

To promote in vitro rhizogenesis 1/4 -, 1/2-, and full strength, Nitsch and Nitsch (1969) medium supplemented with different concentrations and combinations of sucrose (0, 1, 2, 3 %) and plant growth regulators (IBA, NAA and IAA) previous concentration were evaluated.

The ex vitro rhizogenesis was stimulated with a commercial growth regulator powder, Rhizopon<5> in two presentations (AA) and (B). The base of the micropropagated shoots were treated with:

Rhizopon (AA-powder), 0.5 mm for 2 seconds and the excess was shaken off

Rhizopon (B-tablet), the shoots were immersed in a water solution, 0.5 mm for 24 hours

In both cases the shoots with roots were planted in sterile autoclave quartz sand. Rooting development was improved in a water foggy chamber with 90% humidity and 25± 3°C temperature.

5.1.8 Hardening

The well developed regenerants of A. cherimola and A. muricata with in vitro-formed roots were removed from the culture media and transplanted into ex vitro nursery conditions in pots containing quartz-sand and misted every five minutes. They were covered with a funnel to permit transpiration and hardening, 90% humidity reduced slowly to 70% in 30 days, 25 ± 3 °C and 16-h photoperiod remained constant.

The A. cherimola and A. muricata in vitro shoots were rooted under ex vitro foggy conditions in the greenhouse with Rhizopon (-AA) or (-B) and were transferred after 1 ½ months hardening to subtropical greenhouse conditions and maintained there under hydroponic conditions.

5.1.9 Physical Factors

According to the objectives of the experiment, the cultures were maintained at 50-60% relative humidity, 25 ± 3°C temperature, 16-h photoperiod promoted by cool light white lamps (45 µE m-2 s-1) intensity or total darkness.


5.1.10 Statistical Analysis

Statistical analysis was performed on the results of each experiment and the data was analysed through the Statgraphics Programme for Windows (ANOVA). Analysis of variance was calculated (Steel and Torrie, 1985). In total 36 or 20 experimental explants were used depending on the objectives of the experiment which were repeated three times

5.2 Random Amplified Polymorphic DNA (RAPD)

5.2.1 Plant Material

Two kinds of material were used as a source of genomic DNA to compare the RAPD on A. cherimola and A. muricata (T0) and (T1) plants (Table 15):

DNA from mother plants established in the greenhouse and,

DNA from in vitro regenerant clones of the established plants in the greenhouse

5.2.2 DNA Extraction

Genomic DNA was isolated from young leaves of A. cherimola and A. muricata modifing Rogers and Bendich (1985) method. In this procedure, the precipitation of soluble nucleic acids complex was induced in combination with a high concentration of natriumchloride NaCl 0.7 M + CTAB. The reduction of NaCl to 0.4 M permitted the precipitation of CTAB<6>/nucleic acid complex while the solution of polysaccharides remained.

Two grams of fresh weight (FW) border young leaves were ground to a fine powder in liquid nitrogen (-70°C) to construct the nuclei acid complex. Grinding was continued with the addition of pre-warmed buffer 5 ml of CTAB 2% [2% (W/V) CTAB; 1,4 M NaCl; 100 mM Tris/Base (pH 5.8); 20 mM Ethylenediaminetetraacetic acid (EDTA)].

The homogenate was incubated at 65°C for 15 minutes, with occasional swirling in polyallomertubes and extracted with the same volume amount of chloroform : isoamyl alcohol (24:1). Samples were centrifuged at 6000 rpm in JA 13.1 for 10 minutes at room temperature to extract the denatured proteins which can be collected in the interphase.

The supernatant was transferred to a fresh tube with 1 ml vol. CTAB 5% [5% (W/V) CTAB; 350 mM NaCl] buffer, pre-warmed. The mix was incubated at 65°C for 10


minutes. Chloroform-isoamyl alcohol was added in an equal volume and swirled. The mix was centrifuged for 10 minutes, 6000 rpm JA 13.1 at room temperature.

The genomic DNA was recovered carefully in corex tubes and mixed with 1 vol. precipitation buffer [1% (W/V) CTAB; 50 mM Tris/Base (pH 8), 10 mM EDTA], extraction, closed with parafilm and swirled. After some hours the precipitation of CTAB/nucleic acids complex were visible.

The nucleic acids complex re-suspended in the solution was recovered by centrifugation at 6000 rpm (JA 13.1) for 10 minutes, at room temperature. The precipitate was washed with 2 ml HS-TE Buffer [1M NaCl; 1 mM Na2EDTA; 10 mM Tris/HCl (pH 8)]and warmed for10 minutes at 65°C to be dissolved and separated with 2 Vol. absolute Ethanol (30 minutes in -70°C), centrifuged 10 minutes, 6000 rpm (JA 13.1), 4°C. The supernatant was removed with ethanol and the dried pellet was dissolved in autoclave bi-distilled water.

For the DNA-RNA-CTAB complex, 0.3 ml 5 M ammoniumacetate pH 7,5 (final concentration 2,5M) was added and incubated for 20 minutes at 0°C. Precipitation of RNA was induced by centrifugation for 10 minutes,13000 rpm, 4°C, supernatants were taken and distributed in new Eppendorf tubes and complete with 2 Vol. abs. ethanol. DNA concentration was calculated fluorometrically.

5.2.3 DNA Amplification

The DNA amplification of A. cherimola and A. muricata was supported by the Polymerase Chain Reaction (Table 18) and synthetic oligonucleotide 10-base primers with 40-60% of Guanosine and Cytidine (GC) distributed uniformily. These are available commercially from the following companies:

CARL-ROTH, Karlsruhe, Baden-Württemberg. Germany

B-11, C-04, C-05, C-07, C-11, C-19, G-19, J-19, L-04, P-02, P-05, P-10, P-13, P-20, Q-04, Q-05, Q-06, Q-07, Q-08, Q-09, Q-10, Q-11, Q-12, Q-13, Q-14,Q-17

Operon Technologies, Alameda, CA. U.S.A.

OPA-16, OPA-17, OPA-18.

In a DNA-Thermal Cycler (Perkin-Elmer, serial P19516) programmed to 45 cycles, the PCR reactions (25 µl) were improved (Table 19).

5.2.4 DNA Separation

Amplification products were separated by electrophoresis in ethidium bromide stained gels: 1.5 % (w/v) agarose gel soluble in “TBE“ [Tris-borate (TBE) 5X: 54g Tris base, 27.5 g boric acid, 20 ml 0.5 M EDTA (pH 8.0) 1x (0.5 x: 0.045 M Tris borate + 0.001 M EDTA] to provide adequate buffering power.


Table 18. Polymerase chain reaction mix


25 µl reaction mixture

Final concentration 25 µl reaction mixture




Annona spp., DNA template

2.5 µl

25 ng

PCR-Buffer 1

2.5 µl

1x (incl. 1.5 mM MgCl2)

DNTP-Mix 2

2.0 µl

each 5 µl, 100 µM

50 mM Mg-Ac2

1.5 µl

3 mM

Taq DNA polymerase 3

0.2 µl

1 u/µl 4

Primere- [5‘rarr 3‘] 10 nucleotides

1.5 µl





Sterile deionized water

14.8 µl





Final volume

25.0 µl





Mineral parafilm oil

50.0 µl

by PCR tube

1 PCR-Buffer: 20mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.5% Nonidet P40, 0.5% Tween 20 and 50% glycerol
2 dNTPs: Mix solution:in mM of 2‘-deoxyadenosine 5‘-triphosphate sodium salt (dATP), 2‘- deoxycytidine 5‘-triphosphate sodium salt (dCTP), 2‘-deoxyguanosine 5‘-triphosphate sodium salt (dGTP), 2‘-deoxythymidine 5‘-triphosphate sodium salt dTTP
3 Thermus aquaticus DNA Polymerase (recombinant): Purified from an E.coli train carrying a Thermus aquaticus DNA polymerase overproducing plasmid. Catalyses 5‘rarr 3‘ synthesis of DNA and possesses low 5‘rarr 3‘ exonuclease activity. Reported to have reverse transcriptase activity
4 Unit Definition: 1 unit of the enzyme Taq DNA polymerase catalyses the incorporation of 10 nmoles of deoxyrribonucleotides into an acid insoluble form in 30 min at 70°C

Table 19. DNA amplification steps applied on Annona spp.









Initial denaturation


4 min




20 sec


Primer annealing


1 min




20 sec






Number of cycles










6 min






5.2.5 DNA Separation

Amplification products were separated by electrophoresis in ethidium bromide stained gels: 1.5 % (w/v) agarose gel soluble in “TBE“ [Tris-borate (TBE) 5X: 54g Tris base,


27.5 g boric acid, 20 ml 0.5 M EDTA (pH 8.0) 1x (0.5 x: 0.045 M Tris borate + 0.001 M EDTA] to provide adequate buffering power.

5.2.6 RAPD Analysis

The amplification products were visualized and photographed under standard conditions used for ethidium bromide-stained gels on an UV-transiluminator, and identified with reference to the Lambda DNA/ EcoRI+HindIII marker (MBI Fermentas, Germany)

The polymorphic reproducible bands in the agarose-gels were scored as (1) present or (0) absent. PCR reactions were performed five times to establish reproductibility of results.

5.3 Karyotype Observations on Annona spp.

For the analysis of mitotic chromosomes of A. cherimola and A. muricata mother plants and in vitro regenerants, the youngest and whitest translucent light root-tips were selected. Each sample consisted of 10 root-tips, 0.5-1.0 cm.

Several treatments were tried for arresting the A. cherimola and A. muricata chromosomes at metaphase because chromosomes can be visualised in their most condensed form in mother plants and in vitro regenerants : Ice-cold water 4°C/ 24 h and alcohol- acetic acid 3:1 48 h; Bromonapthatlene (6ml/l) 4 h then glacial acetic acid 30 min and 1 N HCl 60°C 10 min; Hydroxyquinoline 0.003 M 4 h then alcohol-acetic acid 3:1 48 h stain aceto-carmine 0.5%+ ferric-III-chloride 10% 1:100 48 h

The root-tips were taken with forceps and immediately subjected to a suitable treatment. They were incubated for the appropriate time and temperature as described above. Five root-tips per plant and at least 10 cells per root-tip were analysed.

The root tips were stained with Orcein-HCL filtered and with Aceto-carmine. Also feulgen 45% acetic acid was used. This colours the cytoplasm and enables the determination of whether the cell is intact. This background colours are also helpful to determine the chromosomes in Annona spp. cells.

A fluorescence method was also applied to see the chromosomes of A. cherimola and A. muricata. The probes were stained with 2µg ml-1 diamino-2-phenylindole (DAPI, Sigma) in PBS (pH 7.4) for 10 min. and analyzed under a fluorescense microscope (Orthoplan, Leitz, Germany) using the epifluorescence optics of filter A (excitation filter, 340 to 380 nm; dichroic mirror, 400 nm; and barrier filter, 430 nm).



Benomyl (DuPont) : Fungicide 0.03 %


Wuxal Top N (Hoechst Schering AgrEvo): N (140-), P2O5 (45-), K2O (70-). B (0,11-), Cu (0,08-), Fe (0,18-),

Mn (0,15-), Mo (0,011-), Zn (0,055-), - g/l.


Rhizopon (Hazerswoude-Holland) AA: Indol-butiric-acid 1%

Rhizopon (Hazerswoude-Holland) B : Naphtalene-acetic-acid 10%


CTAB : hexadecyltrimethylammonium bromide

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