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

60

Chapter 6. Results
Micropropagation

6.1 Plant Material

The seed germination of A. cherimola (T0) cv. Felpa (Ch-X) and cv. Broceada (Ch-Y) was observed on the 35th day after sowing, in all the tested combination treatments. However the germination percent varied (Table 20).

Table 20. Effect of gibberelic acid and cold temperature shock on seed germination of Annona cherimola cv. Felpa and cv. Bronceada

Germination

Previous

Percent of Germination

Treatments

Temperature

A. cherimola cv. Felpa

A. cherimola cv. Bronceada

 

shock for 24 h

coated-seeds

coatless-seeds

coated-seeds

coatless-seeds

 

 

 

 

 

 

water solution

4°C

78

34

81

43

250 ppm GA3

4°C

45

35

73

34

500 ppm GA3

4°C

37

17

54

18

 

 

 

 

 

 

water solution

20 ± 2°C

41

30

67

36

250 ppm GA3

20 ± 2°C

33

12

56

28

500 ppm GA3

20 ± 2°C

28

7

45

23

The coated seeds showed a higher germination percent without any pre-treatment of gibberellic acid. The highest percent of seed germination of A. cherimola cv. Bronceada and cv. Felpa was obtained with the control treatments or without gibberellic acid.

The A. cherimola cv. Bronceada germinated in 78% with a previous cold precondition and without the presence of GA3. The same situation occurred for A. cherimola cv. Felpa in 81%. It is important to note that these seeds were coated.

The coatless seeds of A. cherimola and A. muricata showed the lowest percent of germination in all the experimental cases. However those which were pretreated 24h/4°C and maintained in a water solution were more viable 34% cv. Felpa and 43% cv. Bronceada when these were immersed in a gibberellic acid solution. Coatless seeds lost quality potential, changed in colour; texture and hardening were also observed in the endosperm. These seeds were also more sensitive to fungi attack during germination.The giberellic acid GA3-250 ppm broke the seed dormancy more efficiently than GA3-500 ppm which stimulated the lowest rate of seed germination in both varieties.


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The previous low temperature shock 4°C in 24 hours in darkness promoted seed germination if results are compared with those treatments where the seeds were not submitted to cold temperature shock. The germination of A. cherimola cv. Felpa and cv. Bronceada was promoted more by pre-shock with low temperatures than by the effect of the gibberellic acid as a plant promotor.

Otherwise the seedling plant adaptation was successfully achieved, 70% of A. cherimola (T1) (Ch-Z) and A. muricata (T1) (Mr-2; Mr-4; Mr-5) Colombian plants survived the transport conditions. They were adapted to the subtropical and tropical greenhouse conditions.

After one month under greenhouse conditions both the A. cherimola and A. muricata (T1) plants started to break the dormancy of the latent buds and began the formation of new leaves and branches, plants grew well during the course of this study(Figure 9).

Figure 9. A. cherimola and A. muricata Colombian plants
adapted to greenhouse conditions in Berlin


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Endogenous Precondition of Material

Previous results showed that the micropropagation of A. cherimola and A. muricata in vitro are strongly limited by two factors: first the production of phenolics and secondly the presence of endogenous contaminants such as fungi and bacteria.

The presence of endogenous contaminants not only limits the aseptic establishment but also promotes the production of phenolics in the explant. Therefore in vitro establishment under these conditions is difficult to achieve.

To avoid the infection of fungi and bacteria during the micropropagation, the A. cherimola and A. muricata (T0) and (T1) plants were treated in the greenhouse with Benomyl (0.03%) systemic fungicide, two times per month.

Applications of Benomyl reduced the percent of fungi expression of A. cherimola and A. muricata (T0) and (T1) plants during the in vitro culture (Table 21). The expression of in vitro contamination by fungi was less serious if the mother plants were exposed for a long time prior to endogenous systemic fungicides. Four weeks before taking the explants, the greenhouse plants should be irrigated with Benomyl.

The initial in vitro contamination of fungi of 100% reduced after the first month of treatment on woody explants to 91% and after two years to 58%. Benomyl systemic treatments only preconditioned the explants to be in vitro manipulated. Other surface contaminants from the explant should be eliminated with disinfectant solutions and/or with antibiotics, if some bacteria remain after the precondition and disinfection. The treatments should be applied independently to promote the in vitro establishment the A. cherimola and A. muricata plants should be treated with Benomyl at least four weeks before the first inoculation.

Table 21. Effect of Benomyl 0.03% on A. cherimola and A. muricata in the
greenhouse to control in vitro fungi contamination

Species

Explant “branch“

cutting with 1 bud

% of in vitro fungi contamination expressed with the Benomyl applications 1 after

 

 

1st month

1st year

 

 

 

 

 

 

A. cherimola

Vegetative

 

76

 

44

 

Semiwoody

 

88

 

55

 

Woody

 

100

 

58

 

 

 

 

 

 

A. muricata

Vegetative

 

80

 

45

 

Semiwoody

 

91

 

47

 

Woody

 

100

 

77

1 Contaminants from the woody explant
In vitro establishment media formulation : NN-69 + S 30 g/l + GR 3 g/l : pH 5.7 ± 0.1
A. cherimola and A. muricata selected explant, woody branches 1-3 cm
The values represent the mean of three independent observations, n=36


63

6.2 Initiation of Culture

6.2.1 Explant

The aseptic establishment of A. cherimola and A. muricata (T0) and (T1) plants is not only affected by the presence of contaminants in the in vitro culture media. Also the nature of the plant material, the intrinsic potential of the organ source of explants to improve new cell regeneration, size and age are the principal factors which condition the in vitro responses.

The seedlings of A. cherimola (T0) and ne year old sedling trees of A. cherimola (T1) and also A. muricata (T1) influenced the establishment of in vitro morphogenic responses of the selected explants and subsequently in vitro formation of new shoots.

The cuttings either from vegetative, semiwoody or woody branches are the most potential explants, they promote the shoot sprouting of pre-formed buds, the contamination and blackening problems should be treated independently. In addition the aseptic establishment of cuttings was limited by endogenous contamination. Greenhouse treatments with systemic fungicides such as Benomyl only preconditioned the reduction of some in vitro contaminants, therefore an aseptic culture from cuttings, such as the initial culture material should be improved with the application of exogenous tissue disinfectant solutions.

The size of the cuttings showed some differences. Cuttings (1-3 cm) with one bud were in vitro less shoot prolific if they are compared with cuttings (5-9 cm) with three buds. Because if A. cherimola and A. muricata cuttings carry more than one bud, they have more possibilities to promote new in vitro shoots.

Semiwoody branches with two or three buds (5-9 cm) from the second year growth with a semiwoody lignification grade were the best source of explants. These semiwoody cuttings promoted the highest percent of new formed shoots. The base of them was affected by blackening and some contaminants however, the buds reserve organs had the potential to promote new shoots (Figure 10).

Vegetative branches of A. cherimola and A. muricata were used as a source of small explants such as meristems (0.1 - 0.5 cm), buds (1-3 cm) or shoot tips (0.5 -1.5 cm). However these explants were not able to be established under in vitro conditions, blackening was the most generalized response of these explants which died within the first week after in vitro inoculation (Figure 10).

Meristems of A. cherimola and A. muricata were very sensitive, wounding induced severe injury, and the physical answer was the promotion of phenolics. These explants started to turn from green to black on the scalpel tip. Thus the in vitro establishment from these explants is not reported in this study (Figure 10).

Young leaf segments (0.5-1 cm) could be potentially explants to promote new in vitro callus formations on A. cherimola and A. muricata, however the border cells are phenol reactive and a dark brown was observed. Leaves remained green during some weeks but the quality of these explants diminished with the time of culture.


64

Figure 10. First in vitro responses of various explants of A. cherimola and A. muricata
-after one week of culture-

m : meristems (0.1-0.5 cm), b : bud (0.5 - 1 cm), st : shoot tips (0.5 - 1.5 cm), l : leaves (0.5 - 1 cm),
1b cutting with 1 bud (1-3 cm), 3b : cutting with 3 buds (5-9 cm), green : possible new shoot formation
Establishment basic media formulation : NN-69 + S 30 g/l + GR 3 g/l : pH 5.7 ± 0.1

The values represent the mean (± SE) of three independent experiments, n = 36


65

Other experiments (data not shown) in media supplemented with antioxidant substances and auxines to promote callus formation from leaf segments did not give any in vitro response. This might be due to the presence of phenolics in the leaf border. No regeneration from leaf segments of A. cherimola and A. muricata (T1) and (T0) occurred (Figure 11).

The reaction of the phenolics and remanent contamination of the small explants from A. cherimola and A. muricata could not be established. In tissue culture it is very well known that it is more difficult to induce growth in very small structures such as cells, clumps of cells and meristems, than in larger structures such as leaf or stems of woody species compared with herbaceous plants (Pierik, 1986).

Cutting explants from semiwoody branches of A. cherimola and A. muricata established plants are an optimal initial culture material. They can promote direct bud sprouting. Four new shoots they could be inducted from one bud. If the cutting is large the establishment rate improves if contamination and phenolization are controlled.

6.2.2 Disinfection

The in vitro culture of any cell organ and tissue should be made in aseptic conditions (Murashige, 1974) because it is well known that the plant surfaces are habitats for microorganisms (Campbell, 1985). The precondition in the greenhouse with systemic chemicals helped the plants to reduce the percent of endogenous microorganisms but not completely. Thus they should be also disinfected externally.

The optimal disinfection of a selected explant depends on the penetration degree of the disinfectant agent into all contaminated parts (Pierik, 1989). The selected disinfectant solution is effective only when the concentration is toxic for the microorganisms present on the surface but not toxic for the cells of the tissue.

6.2.3 Disinfectant Solutions

Previous experiments showed that A. cherimola and A. muricata explants are extremely sensitive to calcium hypochlorite and mercury chloride. These solutions in different concentrations limited the in vitro establishment of A. cherimola and A. muricata because the surface cells of these plants are hypersensitive and phenolic oxidation arises in high concentrations (Bridg, 1993).

The sodium hypochlorite was evaluated concentration vs. time and in combination with other agents such as: Benomyl 0.03% and Tween 20. The results showed that sodium hypochlorite reduced, in all the cases, the presence of contaminants in the in vitro culture media (Figure 11).

Sodium hypochlorite 2% did not eliminate all the surface contaminant microorganisms, some bacteria and fungi were observed in the culture media, neither the exposition for 20 minutes nor for 30 minutes were effective.

The effect of sodium hypochlorite 3% during 15, 20 and 30 minutes was evaluated, the effect of this disinfectant varies with the exposition time. In all the cases the reduction of surface contaminants were effective. The exposition for 30 minutes


66

injured the explant, the bud sprouting reduced significantly and a blackening in the culture media was observed.

The concentration of sodium hypochlorite 3% for 15 minutes helped to reduce the presence of exogenous in vitro contaminants. 45% of cultures could be established aseptically, but they were not all aseptic. The endogenous contamination of these tissues became evident after a few subcultures during the multiplication stage. However this treatment was selected to improve the exogenous disinfection of semiwoody branches of A. cherimola and A.muricata plants, nevertheless this should be complemented.

The combination of sodium hypochlorite with Tween 20 as a wetting agent was made to reduce the surface tension of the cutting explant of A. cherimola and A. muricata and to improve the surface contact of this bleach solution. The presence of Tween 20 in the disinfection treatments in combination with sodium hypochlorite 0.5%/20 was not aggressive for the explant, however brown exudates coming from the explant were gentle on the tissue culture media (Figure 11). The microorganisms present on the surface of the semiwoody cutting explant of the A. cherimola and A. muricata explants seems are harbour and low concentrations of sodium hypochlorite had no effect on the explant surface.

Figure 11. In vitro response of A. cherimola and A. muricata semiwoody shoots (5-9 cm) to different disinfectant solutions
- after two weeks of culture-

NaOCl (sodium hypochlorite): concentration “%“ : time “minutes“, tw: Tween 20 “2 drops“,
B: Benomyl 0.03%
Establishment basic media formulation : NN-69 + S 30 g/l + GR 3 g/l : pH 5.7 ± 0.1
The values represent the mean (± SE) of three independent experiments, n = 36


67

The combination of Tween 20 and sodium hypochlorite 3% by 20 min., registered no differences in terms of reduction of the microorganism contamination in in vitro culture. The exposition time of this disinfectant and maybe the presence of Tween 20 made this treatment aggressive and not advantageous in terms of in vitro establishment. The bud sprouting also reduced from 45% in sodium hypochlorite 3% / 15 minutes to 25% in this treatment.

Prewashes with ethanol 70% for some minutes to condition the surface of the cutting explant of A. cherimola and A. muricata to remove the surface air bubbles and improve the action of sodium hypochlorite were ineffective because the A. cherimola and A. muricata cuttings have a middle lignification grade which makes these explants sensitive to ethanol. The ethanol helps to eradicate some microorganisms on the explant surface, but also the alcohol dissolves the epicuticular layer of the cutting explant (Pierik, 1989) and a high concentration of phenolics was observed. In addition the ethanol promotes some dehydration of the explant surface.

The combination treatments of sodium hypochlorite and Benomyl 0.03% were effective in terms of aseptic in vitro establishment. However this external disinfection combined treatment reduced the percent of bud sprouting. In most of the cases the cutting explant stayed latent for some weeks, while the phenolics substances accumulated in the culture media slowly.

The pre-treatment in the greenhouse of the stock plants with systemic fungicides was not enough to avoid all the endogenous contaminants. The disinfection treatments with sodium hypochlorite helped to disinfect the external surface of the explant but the endogenous contamination should be treated independently.

Semiwoody cuttings of A. cherimola and A.muricata with three pre-formed buds were the most viable explants to be established under in vitro conditions. They could survive the sodium hypochlorite treatment (NaOCl 3%/15 m). Their size (5-9 cm) protects the pre-formed buds from the phenolic products which increase in concentration in the establishment medium. The brown exudates on the base of the explant prevent the directly new bud sprouting (Figure 11).

6.2.4 Antibiotics

Contamination in tissue culture can originate from microorganisms present in the intercellular tissues of the explant. They are able to grow in the plant tissue culture medium (Campbell, 1990). For A. cherimola and A. muricata the endophytic presence of Pseudomonas spp., and Pseudomonas saccharophila were corroborated by the Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA) in the multiplication aseptic media. These endophytic microorganisms came from the internal tissues of the semiwoody cutting explant.

The in vitro contamination is one of the most important obstacles to promote the aseptic micropropagation of A. cherimola and A. muricata. The contaminants of these plants could not be easily eliminated with the sodium hypochlorite disinfectant solution. Therefore nystatin, rifampicin and cefotaxime were evaluated.

The effect of these antibiotics supplied to the explant showed effective results in terms of bacteria control expression during the establishment. Nevertheless the new


68

"aseptic" shoots coming from this essay were subcultured in a new medium without any antibiotics and three weeks after in vitro conditions a residual bacterial contamination was observed.

If the antibiotics were added in a liquid double phase into the culture media, they were effective in terms of the elimination of contaminants. The liquid presentation of the antibiotic allowed it to diffuse into the internal tissues of A. cherimola and A. muricata cuttings. The shoots coming from this treatment retained their aseptic condition during the shoot multiplication stage and aseptic culture could be improved.

If antibiotics were added into the culture medium as a media supplement, the antibiotics diffused from the media into the internal explant tissues of the A. cherimola and A. muricata semiwoody cutting explant. This action had a bacteriostatic effect, the endogenous contamination could not be seen in the establishment media with antibiotics but after the subculture to antibiotic free media some shoot cultures showed endogenous contamination again.

Rifampicin was most effective against residual endogenous contamination supplemented in a double phase, followed by Cefotaxime. Nystatin is not recommended because of its low effectiveness on A. cherimola and A. muricata shoot culture initiation (Table 22).

The residual endogenous contamination of the semiwoody cuttings of A. cherimola and A. muricata which could not be eliminated by the sodium hypochlorite exogenous disinfection were controlled with the supplementation of Rifampicin into the establishment media which improved the establishment of aseptic cultures during the micropropagation.

Table 22. Effect of some antibiotics on the bacterial contamination of
A. cherimola and A. muricata


69

6.2.5 Hypersensitivity Reactions

When plant tissues are exposed to stress situations such as mechanical injury the metabolism of phenolic compounds is stimulated. Wounding leads to hypersensitive reactions in the neighboring cells but without showing symptoms of injury themselves, and/or to the premature death of specific cells in the environment of the wound or the place of infection (Deberg and Read, 1990). The A. cherimola and A. muricata were preconditioned in the greenhouse and also the explants were treated with antioxidant substances to prevent phenolics.

6.2.6 Darkness Precondition in the Greenhouse

The A. cherimola and A. muricata mother plants were kept in darkness for two weeks, in order to improve the initiation of shoot cultures and to reduce the plant metabolism with the purpose of retarding the hypersensitivity of the branch reaction caused by mechanical injury.

The main response of A. cherimola and A. muricata mother plants in the greenhouse after two weeks of darkness was defoliation of the expanded leaves and partially etiolated, chlorotic cuttings. The culture explants coming from these etiolated cuttings did not promote new in vitro shoot cultures because the lack of vigour was the main limitation.

The selected A. cherimola and A. muricata explants were not strong enough to promote the de novo shoot formation and most of them died by necrosis or vegetative vigour decayment after the inoculation. The presence of phenolics in the culture media was not different in comparison with the explant from the plants of A. cherimola and A. muricata plants growing in normal light conditions (Table 23).

Table 23. Effect of darkness precondition on A. cherimola and A. muricata
plants in the greenhouse

Species

Explant1

(shoot 5-9 cm)

Photoperiod

 

 

in vitro growth expression

% latent response

% hypersensitivity reaction

 

 

12/12 2

24/243

12/12

24/24

12/12

24/24

 

 

 

 

 

 

 

 

A. cherimola

Soft

8.3

0

100

100

97

88.8

 

Semiwoody

25

0

100

100

100

85

 

Woody

20

0

100

100

100

100

 

 

 

 

 

 

 

 

A. muricata

Soft

5.5

0

100

100

100

90

 

Semiwoody

0

0

100

100

100

75

 

Woody

5.5

0

100

100

100

100

Establishment media: NN-69 + sucrose 30 g/l + GR 3 g/l : pH 5.7 ± 0.1
12/12 : 12 hours light + 12 hours darkness during two weeks
24 : 24 hours darkness during two weeks
The values are the mean of the three independent observations, n=36

6.2.7 Antioxidant Substances

The phenols are phytotoxic products that limit the in vitro culture of A. cherimola and A. muricata. At the place were wounding was made a constant production of phenols


70

was observed and a blackening ring around the explant. This black ring comes from the oxidation products promoted by the phenols, which limit the in vitro morphogenic responses of the selected A. cherimola and A. muricata explant.

The effect of some antioxidant substances such as polyvinylpyrrolidone (PVP) and citric acid (CA) in combination with ascorbic acid (AA) were evaluated during the A. cherimola and A. muricata establishment. The manipulation of the antioxidant substances modified its effect on the tissue explant.

The combination of citric acid and ascorbic acid prevented the phenolics compounds more effectively during the immersion time after the disinfection treatment than polyvinylpyrrolidone solutions. However they had a short period of action and the polyvinylpyrrolidone results were more active in terms of the control of phenols over a period of time. It means that PVP could be supplemented in the culture media to improve the establishment and might be the subsequent step of the micropropagation where the A. cherimola and A. muricata show shoot blackening problems (Table 24).

The cutting explants from the vegetative branch have the tendency to produce phenols: meristems, buds, shoot-tips and border of leaves of A. cherimola and A. muricata are sensitive and react to phenols, PVP, CA and AA, in any of the tested cases could stop this reaction (Table 24).

The woody cutting explants of A. cherimola and A. muricata showed, in the culture medium, a remarkable tendency to produce phenol compounds if they are compared to the response of the vegetative cuttings. The broken cells on the base of the woody cuttings promote the metabolisms of phenols and the oxidation of pre-formed phenolic components such as lignine could then not be stopped in culture media without antioxidate substances.

The Polyvinylpyrrolidone supplemented in the culture media partially reduced the production of phenolics on the woody explants of A. cherimola and A. muricata however, these explants could not be established because the phenolics were produced in high concentrations (Table 24).

The semiwoody cuttings are more able to be established under in vitro conditions, these explants, in combination with the antioxidant substances, promote the direct de novo shoot organogenesis from the pre-formed buds. The production of phenols could be controlled because this explant is not at all lignificated and still retains the potential vegetative condition to improve new in vitro shoots if carrying pre-formed buds.

The effect of the antioxidant substances varies with its chemical stability, time of action and way that they are supplied. The polyvinylpyrrolidone PVP seems to be more effective if it is supplemented into the tissue culture media. The tested A. cherimola and A. muricata explants did not show any blackening in the culture media. It is an indicator that PVP in the culture media is a stable component for a long period of time and the cutting explant found it to be to its own benefit (Table 24).

The semiwoody cuttings are more able to be established under in vitro conditions, these explants, in combination with the antioxidant substances, promote the direct de novo shoot organogenesis from the pre-formed buds. The production of phenols could be controlled because this explant is not at all lignificated and still retains the potential vegetative condition to improve new in vitro shoots if carrying pre-formed buds.


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Table 24. Effect of some antioxidants on A. muricata explants
-after two weeks of culture-

 

 

Immediate response

After two weeks of culture

 

 

 

 

Branch

Explant2

immersion of the explants during time of culturing -air-flow cabinet-

supplemented into the establishment basic media 1

 

 

PVP (ppm)

Cit.ac / Asc.ac (mg/l)

PVP (ppm)

Citr.ac / Asc.ac (mg/l)

 

 

2

4

8

50/100

150/200

2

4

8

50/100

150/200

Soft

m

++++

++++

++++

++++

++++

++++

++++

++++

++++

++++

Woody

b

++++

++++

++++

++++

++++

++++

++++

++++

++++

++++

 

st

++++

++++

++++

++++

++++

++++

+++

++++

++++

++++

 

l

++++

++++

++++

++++

++++

++++

++++

++++

++++

++++

 

1b

++++

+++

++

++++

++++

+++

+++

+++

+++

+++

 

3b

++++

++++

+++

++++

+++

+++

+++

+++

+++

+++

Semi-

b

+++

+++

+++

+++

++

++

++

++

++++

++++

Woody

1b

++

+

+

+

-

+

-

-

+

+

 

3b

++

+

+

-

-

+

-

-

+

+

Woody

b

+++

+++

+++

++

++

++

+

+

++

++

 

1b

++

++

++

+

-

++

+

+

++

++

 

3b

++

++

++

+

-

++

+

+

++

++

1 Establishment basic media: NN-69 + sucrose 30 g/l + GR 3 g/l : pH 5.7 ± 0.1
2 Explants: ½ : semi, m : meristems ( 0.1 - 0.5 cm), b : buds ( 0.5 - 1 cm), st : shoot tips
( 0.5 - 2 cm),
l : leaves ( 0-5 - 1 cm), 1b : shoot 1 bud, 3b : shoot 3 buds ( 5-9 cm).
Response: (-) not blackening, (+) low intensity, (++) medium blackening, (+++)
blackening,
(++++) high blackening
The values represent the mean of two independent observations, n=36
(*) The response of A. cherimola explants were not significantly different, data are also
representative

The effect of the antioxidant substances varies with its chemical stability, time of action and way that they are supplied. The polyvinylpyrrolidone PVP seems to be more effective if it is supplemented into the tissue culture media. The tested A. cherimola and A. muricata explants did not show any blackening in the culture media. It is an indicator that PVP in the culture media is a stable component for a long period of time and the cutting explant found it to be to its own benefit (Table 24).

Ascorbic acid in combination with citric acid was not effective, in comparison with PVP, against blackening for a long period of time. This antioxidant mix was highly effective over a short period of time.

The immersion woody and semiwoody cutting explants of A. cherimola and A. muricata did not produce blackening exudates during two hours of immersion. However if citric acid in combination with ascorbic acid was supplemented into the tissue culture media the percent of blackening exudates in the tissue culture media increased in direct proportion to the period of time for establishment. The phenolic production in A. cherimola and A. muricata semiwoody cutting should be prevented during the ex vitro manipulation of the explant and in the in vitro culture medium with polyvinylpyrrolidone.


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6.2.8 Culture Media

The concentration of two mineral salts: Murashige and Skoog (1962) (MS-62) and Nitsch and Nitsch (1969) (NN-69) were evaluated on A. cherimola and A. muricata semiwoody macrocuttings (5-9 cm) explants with two or three buds. The establishment media composition was supplemented with sucrose and the effect of the support agent was evaluated. Some semisolid, solid and liquid mediums were tested. The effect of Agar and Gelrite were compared (Table 25).

Due to the fact that the in vitro establishment of the A. cherimola and A. muricata was limited by oxidation and contamination, the combined effect of Polyvinylpyrrolidone and Rifampicin were evaluated as media supplements to promote healthy and aseptic cultures (Table 25 ). The semiwoody cuttings of A. cherimola and A. muricata were in vitro reactive to the variation of several factors such as:

Liquid media: 44.4 % of A. cherimola and 36.1% of A. muricata semiwoody cuttings sprouted in liquid Nitsch and Nitsch (1969) basal medium without hormones, an outstanding percent. However in water media without any salt composition the sprouting of the cutting was also high 44.4% for A. cherimola and 38.8 % for A. muricata. There were no significant differences between these treatments. The sprouting in Murashige and Skoog (1962) liquid media yielded the lowest percent of sprouting 25.0 % for A. cherimola and 22.2 % for A. muricata, this media has a significantly high mineral salt concentration. The semiwoody macrocuttings of A. cherimola and A. muricata did not need high concentrations of mineral salts to promote the sprouting of the pre-formed buds.

Carbon source: The presence of sucrose increased discretly the percent of bud sprouting of A. cherimola and A.muricata in liquid NN-69 and MS-62 media (Table 25). The cutting explants in water plus sucrose yielded the highest percent of bud sprouting 52.7 % A. cherimola and 38.8 % A. muricata, as well as the quality of the new in vitro shoots which did not show the tendency to be hyperhydrated, in contrast to the shoots coming from mediums not supplemented with sucrose.

Solid media with Agar and Gelrite: Both A. cherimola and A. muricata are sensitive to Gelrite as a media support agent which, looking at them respectively, showed 30.5 % and 22.2 % of bud sprouting in NN-69, in addition cuttings in mediums supplemented with gelrite proved to be less oxidized on the base than those shoots in mediums supplemented with agar.

Semisolid media with Agar and Gelrite: The explant of A. cherimola in semisolid NN-69 media supplemented with Gelrite and sucrose sprouted 41.6% and the A. muricata 33.3%. The semisolid media influenced the quality of the newly formed shoots, because these shoots showed the tendency to degenerate into a hyperhydrated shoot in the multiplication stage.

Polyvinylpyrrolidone: The concentrations 2, 4 and 10 ppm were evaluated: the A. cherimola showed the minimum control of oxidation in NN-69 media supplemented with 2ppm-PVP. 86.1 % of the explants were affected by the blackening exudates. The 10 ppm- PVP showed the highest percent of the cultures without blackening 47.2% and 4 ppm-PVP 41.6%, there were no significant differences between treatments. A. muricata showed similar results, the explant base oxidation reduced from 77.7% in 2ppm-PVP to 47.2% in 10ppm-PVP and 4ppm-PVP. Both species reduced the percent


73

of bud sprouting when in the culture media not only phenol compounds but also microorganisms were present (Table 25).

Table 25. Effect of the culture medium and supplements on A. cherimola and A. muricata semiwoody cuttings -response after 4 weeks of culture-

Salts and Supplements

A. cherimola

A. muricata

 

sprout

fungi or bacteria

phenols

sprout

fungi or bacteria

phenols

 

 

 

 

 

 

 

 

 

mineral salts

 

 

 

 

NN-69

 

44.4

-

-

38.8

-

-

MS-69

 

25.0

-

-

22.2

-

-

Water

 

44.4

-

-

36.1

-

-

mineral salts and sucrose

 

 

 

 

 

 

NN-69

+ S 30 g l-1

47.2

-

-

41.6

-

-

MS-62

+ S 30 g l-1

30.5

-

-

25.0

-

-

Water

+ S 30 g l-1

52.7

-

-

38.8

-

-

mineral salts and gel-agents

 

 

 

 

 

 

NN-69

+ Ag 6 g l-1

27.7

-

-

19.4

-

-

 

+ GR 3 g l-1

30.5

-

-

22.2

-

-

MS-62

+ Ag 6 g l-1

 

22.2

-

-

22.2

-

-

 

+ GR 6 g l-1

 

25.0

-

-

25.0

-

-

Nitsch and Nitsch (1969) sucrose and two gel agents

 

 

 

 

 

 

NN-69

+ S 30 g l-1 + Ag 6 g l-1

33.3

100

100

33.3

100

100

 

+ S 30 g l-1 + Gr 3 g l-1

47.2

100

88.8

38.8

100

91.6

Nitsch and Nitsch + sucrose + gelrite+ antibiotic

 

 

 

 

 

 

NN-69

+ S 30 g l-1 + GR 3 g l-1 + PVP 2 mg l-1

38.8

86.1

86.1

30.5

80.5

77.7

 

+ S 30 g l-1 + GR 3 g l-1 + PVP 4 mg l-1

58.3

83.3

41.6

38.8

75.0

47.2

 

+ S 30 g l-1 + GR 3 g l-1 + PVP 10 mg l-1

52.7

80.5

47.2

30.5

72.2

47.2

Nitsch and Nitsch + sucrose + gelrite+ antioxidant + antibiotic

 

 

 

 

 

 

NN-69+ S 30 g l-1 +GR 3 g l-1+PVP 4 mg l-1+Rf 20 µg ml-1

72.2

11.1

19.4

63.8

5.55

27.7

Number of independent experiments 3, ** Average of in vitro response per explant, n = 36
NN-69 : Nitsch and Nitsch mineral basal media composition (1969)
MS-62 : Murashige and Skoog (1962)
S : Sucrose (alpha-D-Glucopyranosyl-beta-D-fructo-furanoside; saccharose) (Sigma, S-5390)
Gr : GelriteTM, Phytagel (Sigma, P-8169)
Ag : Agar (Sigma, A-1296)
PVP : Polyvinylpyrrolidone (Av. Mol. Wt. 10,000) (Sigma. P-2307)
Rf : Rifampicin (Sigma, R-7382)
W : Double distilled-de-ionized-autoclaved water
- : (data no shown)

Rifampicin: The addition of Rifampicin to the establishment culture media reduced the percent of contaminated cultures to 11.1 % in A.cherimola and 55.5% in A. muricata. Aseptic cultures without Rifampicin in a double phase were difficult to achieve. Additionally the highest proliferation rate was found 72.2% for A. cherimola and 63.8% for A. muricata.

The percent of bud sprouting increased in a number of cultures where PVP was supplemented. The contamination rate was reduced by an outstanding percent when Rifampicin was supplemented into the culture media. These should be selected to improve the aseptic establishment of A. cherimola and A. muricata.


74

6.2.9 Plant Growth Regulators

The relation auxine:cytokine is profitable for species difficult to propagate (Torres, 1989). The effect of 6-Benzylaminopurine (BA) and Indole-butiric acid (IBA) on A. cherimola and A. muricata bud sprouting was evaluated (Table 26).

The mediums substituted with benzylaminopurine were less prolific in this study. The combination of 8.87 : 2.46µM l-1 benzylamino purine and indole butiric acid promoted 86 % of the bud sprouting of semiwoody cuttings of A. cherimola and A. muricata.

Table 26. Effect of BAP and IBA on shoot bud organogenesis of A. cherimola and A. muricata semiwoody cuttings -response after four weeks of culture-

Species

Growth Regulator

( µM l-1 )

% of cultures with induced shoots *

Number of new shoots per bud *

 

 

 

 

A. cherimola

BA 2.20 : IBA 0.49

75.0 ± 3.2

1.5 ± 0.6

 

4.44 : 1.48

80.3 ± 3.5

1.7 ± 0.7

 

8.87 : 2.46

86.0 ± 2.1

3.0 ± 0.9

 

BA 2.20 -

10.2 ± 2.3

1.0 ± 0.2

 

4.44 -

15.3 ± 4.6

1.0 ± 0.7

 

8.87 -

17.7 ± 5.9

1.5 ± 0.8

 

- : -

72.2 ± 2.1

2.0 ± 0.2

 

 

 

 

A. muricata

BA 2.20 : IBA 0.49

80.5 ± 1.1

1.7 ± 0.6

 

4.44 : 1.48

83.3 ± 2.8

2.0 ± 0.4

 

8.87 : 2.46

86.1 ± 1.5

3.0 ± 1.0

 

BA 2.20 -

13.8 ± 1.5

1.5 ± 0.2

 

4.44 -

13.8 ± 2.0

1.4 ± 0.8

 

8.87 -

19.4 ± 1.4

1.4 ± 0.3

 

- : -

63.8 ± 2.3

2.3 ± 0.6

The values represent the mean (± SE) of three independent experiments. n = 36
BA (6-Benzylaminopurine): IBA (4,-3-Indolyl]butyric acid)
Culture media : Nitsch and Nitsch (1969), Sucrose 30 g/l, Gelrite 3g/l, pH 5.7 ± 0.1

6.2.10 Shoot Induction

The semiwoody macro cuttings of A. cherimola and A. muricata with two or three pre-formed buds were induced to promote direct bud sprouting of shoots in Nitsch and Nitsch (1969) basal media formulation, supplemented with BA and IBA, Rifampicin, PVP and gelrite.

The combined effect of antibiotics, fungicides, antioxidants, support agent and plant growth regulators was evaluated to prove that to overcome the contamination and oxidation of the A. cherimola and A. muricata explants the establishment of these woody fruit species in in vitro conditions could be easily achieved. The reported results (Table 27) obtained with A. muricata (Figure 11) were not significantly different for A.


75

cherimola (Figure 12).

The selected chemical composition media of the establishment is able to be used for both species. The addition of Benomyl 0.03 mg/l as a media supplement to control contamination was very effective. The media that were supplemented with Benomyl did not stimulate the new formation of shoots from the pre-formed bud of the macro-cutting. The presence of Benomyl is effective against contamination but is also a shoot culture inhibitor.The other supplements such as antibiotic and antiphenol substances saturated the establishment media that seems to be toxic for the selected explant. No contamination, no phenolization and also no shoot proliferation was reported in this essay.

The high number of shoot per node in A. cherimola and A. muricata was obtained in the control treatment with water, without hormones, and supplemented with antibiotics and polyvinylpyrrolidone (Table 27).

The A. cherimola and A. muricata gelrite (GR) supplemented media were more shoot proliferant (27.3 explants producing shoots / 36 cultured explants) than those media supplied with agar-agar (Ag) (21.8 explants producing shoots / 36 cultured explants).

There were no significant differences between the explants proliferating in gelrite and the explants proliferating in the liquid medium supplemented with sucrose (26.8 explants producing shoots / 36 cultured explants) (Table 27).

The surface of the stem, the cambium was also stimulated and clusters with a whitish color were observed but no regeneration responses were obtained with this new source of multiplication material. Until four new shoots emerged simultaneously all over the pre-formed bud. This result confirmed the tetrapotential dormant meristem of this semideciduous subtropical and tropical trees (Figures 12; 13).

6.2.11 Effect of Closures

Closures prevent drying out and infection, while on the other hand a change of gases with the "outside air" must be possible to avoid a shortage of O2 and to prevent an accumulation of gases produced, such as CO2 and ethylene. The accumulation of ethylene affected the development of A. cherimola and A. muricata new shoots.

The in vitro shoots also developed new leaves, which were induced to fall down during the establishment 45.9 % for A. cherimola and 38.9 % for A. muricata when the magenta sigma cups of the test-tubes were totally closed with parafilm (Figure 14).

The polypropylene magenta sigma caps performed as diffusible membranes for gases (with the exception of water vapour). If they were closed completely with parafilm during the establishment and multiplication of the A. cherimola and A. muricata the humidity increased in the test-tube and some new in vitro shoots were hyperhydrated, 23.3 % for A. cherimola and 18.3 % for A. muricata (Figures 14).

The tendency of the A. cherimola and A. muricata shoots to induce hyperhydrated shoots during the establishment of the test-tubes completely closed with parafilm, was observed also during the multiplication stage.


76

Table 27. Effect of some supplements on the phenolization and contamination of
Annona muricata (T1) semiwoody cuttings (5-9cm) with pre-formed buds to promote the in vitro establishment - after four weeks of culture-

SALTS

BA :IBA

S

G

Ag

Rf

PVP

Cit.ac

/

Asc.ac

Bmyl

Nr. explants producing shoots *

Mean** shoot number per bud

Nr.

non aseptic explants

Nr. phenolized explants

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NN-69

-

-

-

-

-

-

-

-

22

1.6 ± 0.4

36

36

 

8.87:2.46

-

-

-

-

-

-

-

25

2.0 ± 0.5

33

36

 

8.87:2.46

30

-

-

-

-

-

-

25

1.6 ± 0.3

30

36

 

8.87:2.46

30

3

-

-

-

-

-

26

2.3 ± 0.2

32

36

 

8.87:2.46

30

3

-

20

-

-

-

28

2.6 ± 1.0

9

33

 

8.87:2.46

30

3

-

-

4

-

-

28

2.6 ± 0.8

35

23

 

8.87:2.46

30

3

-

-

-

150:200

-

25

1.3 ± 0.5

36

28

 

8.87:2.46

30

3

-

20

4

-

-

33

3.0 ± 0.9

6

8

 

8.87:2.46

30

3

-

20

-

150:200

-

30

1.3 ± 1.2

5

23

 

 

 

 

 

 

 

 

 

 

 

 

 

NN-69

8.87:2.46

30

-

6

-

-

-

-

18

0.6 ± 0.1

32

36

 

8.87:2.46

30

-

6

20

-

-

-

20

0.6 ± 0.5

4

36

 

8.87:2.46

30

-

6

-

4

-

-

23

1.0 ± 0.4

33

23

 

8.87:2.46

30

-

6

-

-

150:200

-

22

1.0 ± 1.1

29

26

 

8.87:2.46

30

-

6

20

4

-

-

25

1.8 ± 0.8

15

14

 

8.87:2.46

30

-

6

20

-

150:200

-

23

1.0 ± 0.2

10

22

 

8.87:2.46

30

-

6

-

-

-

0.3

5

1.0 ± 0.2

0

12

 

8.87:2.46

30

-

6

20

-

-

0.3

8

0.6 ± 0.2

0

8

 

8.87:2.46

30

-

6

-

4

-

0.3

4

0.6 ± 0.0

0

0

 

8.87:2.46

30

-

6

-

-

150:200

0.3

3

0.6 ± 0.5

0

0

 

8.87:2.46

30

-

6

20

4

-

0.3

10

0.6 ± 0.2

0

0

 

8.87:2.46

30

-

6

20

-

150:200

0.3

2

0.6 ± 0.1

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

W

-

-

-

-

-

-

-

-

30

2.3 ± 1.1

36

36

 

-

30

-

-

-

-

-

-

36

3.0 ± 0.4

36

36

 

-

30

-

-

20

-

-

-

27

3.0 ± 0.1

12

36

 

-

30

-

-

-

4

-

-

30

3.0 ± 0.5

36

15

 

-

30

-

-

-

-

150:200

-

20

1.3 ± 1.2

36

23

 

-

30

-

-

20

4

-

-

20

2.3 ± 0.5

10

10

 

-

30

-

-

20

-

150:200

-

25

1.6 ± 1.3

5

22

 

8.87:2.46

-

-

-

-

-

-

-

29

2.3 ± 0.8

36

28

 

8.87:2.46

-

-

-

20

-

-

-

28

1.6 ± 0.6

5

35

 

8.87:2.46

-

-

-

-

4

-

-

30

1.6 ± 0.8

36

10

 

8.87:2.46

-

-

-

-

-

150:200

-

22

0.6 ± 0.3

36

22

 

8.87:2.46

-

-

-

20

4

-

-

25

1.6 ± 1.0

30

9

 

8.87:2.46

-

-

-

20

-

150:200

-

19

1.3 ± 0.5

4

17

Number of independent experiments 3. ** Average number of new formed shoots per bud (± SD)
NN : Nitsch and Nitsch mineral basal media composition (1969)
S : 30 g/l Sucrose (alpha-D-Glucopyranosyl-beta-D-fructo-furanoside; saccharose) (Sigma, S-5390)
G : 3 g/l GelriteTM, Phytagel (Sigma, P-8169)
Ag : 6 g/l Agar (Sigma, A-1296)
BA : 8.87 µM (6-Benzylaminopurine) (Sigma, B-9395)
IBA : 2.46 µM (4,-3-Indolyl]butyric acid) (Sigma, I 5386)
PVP : 4 mg/l Polyvinylpyrrolidone (Av. Mol. Wt. 10,000) (Sigma, P-2307)
Asc. ac : 150 mg/l L-Ascorbic acid (Sigma, A-2174)
Cit.ac : 200 mg/l Citric acid (Sigma, C-4540)
Bmyl : Benomyl 0.03 mg/l
W : Double distilled- de-ionized-autoclaved-water
Rf : 20 µg/ml Rifampicin
- : non supplemented


77

Figure 12. Various stages during the in vitro establishment of A. cherimola

  1. A. cherimola shoot with a non manipulated node showing the location of a pre-formed bud complex with four meristems
  2. Bud sprouting of semiwoody cuttings after the control of phenols and contaminants under in vitro culture
  3. Direct sprouting of four new shoots per bud on semiwoody cuttings of A. cherimola after four weeks of culture
  4. Semiwoody branch of A. cherimola in medium supplemented with Polyvinylpyrrolidone and Rifampicin
  5. Adventitious bud formation in Nitsch and Nitsch (1969) medium with BA and IBA
  6. Organogenic potential of A. cherimola in optimal establishment conditions
  7. Sequence of the formation of multiple shoots without callus formation
  8. Multiple shoots in optimal development and size to be subcultured in multiplication medium


78

Figure 13. Various stages during the in vitro establishment of A. muricata

  1. A. muricata shoot with a non manipulated node with four meristems
  2. Bud sprouting of semiwoody cuttings after the control of phenols and contaminants under in vitro culture
  3. Direct sprouting of four new shoots per bud on semiwoody cuttings of A. muricata after four weeks of culture
  4. Detail of the multiple shoot formation
  5. Organogenic potential of A. muricata microcutting
  6. Adventitious callus formation from the meristematic cambium in A. muricata
  7. Formation of callus in A. muricata subcultured on NN-69 + 2,4-D (4,53 µM)
  8. Formation of multiple buds on A. muricata callus (no regeneration is reported)

A. cherimola and A. muricata during in vitro conditions needed to diffuse some gases produced during organogenesis. It is important to note that the recipients, where the plants were micropropagated, were greatly aromatic. During the in vitro root organogenesis a total closed recipient is not recommended, because the purpose is to induce in the explant a gradual adaptation for an improved successful adaptation to the ex vitro conditions

Figure 14. Effect of magenta caps and parafilm during the establishment of
A. cherimola and A. muricata in test-tubes

Establishment media: NN-69+ S 3 % + GR 0. 3 % + B 8.87µM + IBA 2.46 µM + Rf 20 µg/ml
Number of independent experiments 3, Average of number of new formed shoots per bud (± SD)
Total cultured explants: n=36

It is convenient to initiate growth in medium prepared in test-tubes 25 x 150 mm being not totally closed. Only the magenta Sigma caps were useful. The test-tubes make it easy to monitor the new shoot formation and allow individual treatments of explants.

The incubation conditions for A. cherimola and A. muricata cuttings were 16 h light and 8 h darkness and 26 ± 2 °C as temperature standard. Incrementes of temperature to 30°C or higher can result in condensation on the inner surface of culture vessels and the cultures. This can lead to hyperhydration, a condition where internal leaf air spaces become water-filled and leaf and culture morphology become abnormal. The A. cherimola and A. muricata the semiwoody cuttings taken in summer were more in vitro reactive than cuttings from winter.

Effect of Season


80

Previous experiments inducing the establishment of A. cherimola and A. muricata showed that the season had an influence on bud sprouting in in vitro conditions. The A. cherimola and A. muricata selections showed different responses in different seasons. In winter the number of shoots per bud reduced significantly in both species. Thus semiwoody shoots with 3 buds taken from A. cherimola and A. muricata semideciduous selections were evaluated in summer and winter (Figure 15).

The bud sprouting and subsequently the number of shoots per bud were more effective in summer. From A. cherimola the cv. Felpa (Ch-X) 3.45 ± 0.46 was more prolific than the cv. Bronceada (Ch-Y) 3.1 ± 0.42 and the Colombian selection (Ch-Z) 2.75 ± 0.50 showed no great difference in the number of shoots per bud in summer 2.75 ± 0.47 than in winter 2.35 ± 0.33 (Figure 14).

The semiwoody cuttings of A. muricata selections (2; 4; 5) were severly affected in terms of bud sprouting in winter. A very low rate of the number of shoots per proliferating bud was registered (Figure 14). The A. muricata selections were more proliferating in summer, this species is also the most tropical of the Annona spp.

Figure 15. Season effect on bud sprouting of A. cherimola and A. muricata selections

Establishment media: NN-69+ S 3% + GR 0.3% + B 8.87µM + IBA 2.46 µM + Rf 20 µg/ml
Number of independent experiments 2, Average of number of new formed shoots per bud (± SD) n=20


81

6.3 Multiplication

The new in vitro sprouting shoots of A. cherimola and A. muricata from the pre-formed established buds were transplanted after five weeks of in vitro establishment in the multiplication media.

The mineral basal formulation reported by Nitsch and Nitsch (1969) used during the establishment was the basic salt combination during multiplication experiments. Additionally the Woody Plant Medium and Murashige and Skoog (1962) salt formulations were evaluated.

The young shoots with a nodal segment coming from the semi-woody A. cherimola and A. muricata cuttings were not phenol reactive in the tissue multiplication media therefore antioxidants were excluded.

6.3.1 Effect of Cytokinins

To promote the new shoot multiplication in A. cherimola and A. muricata different combinations of cytokinins were evaluated in Nitsch and Nitsch (1969) mineral and vitamin combinations in addition to sucrose 30 g l-1, gelrite 3 g l-1 the media pH 5.7 ± 0.1. Temperature and photoperiod factors have been previously described (cf. Materials and Methods). Finally the cultures were not closed with parafilm.

6.3.2 Zeatin

The A. cherimola reported a special sensitivity to different zeatin concentrations. The best results were obtained at 1.36 µM l where 75 % (15/20) of established shoots induced a proliferation rate of 2,4 ± 0,7 new shoots per explant without callus (Figure 16). A. muricata, on the contrary, did not proliferate in Nitsch and Nitsch (1969) media supplemented with zeatin. Only a few small new shoot formations were observed in the best cases. The highest mean proliferation rate of A. muricata was obtained in the treatment which excluded the zeatin. 1.38 ± 0.3 shoots were induced, in addition the shoot mean proliferation rate reduced proportionally when zeatin concentration increased.

6.3.3 Kinetin

The A. cherimola shoots also proliferated in media supplemented with kinetin, the concentration of 2.32 µM is the most proliferating, 60% (12/20) of established shoots induced a proliferation rate with 3.5 ± 0.5 new shoots per explant without callus (Figure 17). A. muricata was not shoot proliferant in kinetin supplemented medium, big-green and more expanded leaves were observed.


82

Figure 16. Zeatin effect on A. cherimola and A.muricata shoot formation

Zeatin (µM l-1)

Mean of 3 replicates per treatment - Number of shoots per treatment = 20
Medium: NN-69 + S 3% + Gr 0.3% pH 5-7 ± 0.1

Figure 17. Kinetin effect on A. cherimola and A.muricata shoot formation

Kinetin (µM l-1)

* Mean of 3 replicates per treatment - Number of shoots per treatment = 20
* Medium: NN-69 + S 3% + Gr 0.3% pH 5-7 ± 0.1


83

6.3.4 Benzyl amino-purine

The cytokinin benzylaminopurine which had a positive effect on shoot induction for A. cherimola and A. muricata semi-woody cuttings in combination with Indole butyric acid were tested singly to induce shoot multiplication.

As a result, neither A. cherimola nor A. muricata showed shoot proliferation in a media supplemented with several concentrations of BA (Figure 18) The explants remained green but no new in vitro response was observed, less than 5% (1/20) of the shoots induced 0.8 ± 0.2 new shoots per explant were obtained.

Figure 18. Benzyl-amino-purine effect on A. cherimola and A.muricata shoot formation

Benzyl-amino-purine (µM l-1)

* Mean of 3 replicates per treatment - Number of shoots per treatment = 20
* Medium: NN-69 + S 3% + Gr 0.3% pH 5-7 ± 0.1

6.3.5 Kinetin and Zeatin

In order to increase the mean proliferation rate in A. cherimola and A. muricata, the combination kinetin and zeatin were evaluated (Figure 19).

A. cherimola reported, in a concentration of 4,65 µM kinetin + 1,36 µM zeatin, 100% shoot proliferation (20/20) with 6,7 ± 1,3 new shoots as a mean proliferation rate (Figure 19). The individual shoots coming from this experiment were also shoot proliferant in the subsequent multiplication subcultures.

The A. muricata established shoots did not induce new proliferation shoots. The control medium without citokinin showed the better quality shoots, increasing in size.


84

Figure 19. Effect of Kinetin combined with Zeatin on A.cherimola shoot proliferation

Kinetin (µM l-1) + Zeatin (µM l-1)

* Mean of 3 replicates per treatment - Number of shoots per treatment = 20
* Medium: NN-69 + S 3% + Gr 0.3% pH 5-7 ± 0.1

6.3.6 Thidiazuron

Thidiazuron is a synthetic cytokinin with a benzyl-amino-purine activity but 10,000 times more powerful. Several concentrations were tried to promote the proliferation of A. cherimola and A. muricata. As a result, at the base of shoots some roseta formations were observed (Figure 22) with organogenesis of multiple leaves which did not induce any regeneration response.

6.3.7 Gibberellic acid

Due to the fact that the A. muricata were not shoot proliferating in Nitsch and Nitsch (1969) supplemented with cytokinins, shoot elongation was stimulated in order to induce proliferation through in vitro cuttings in this media formulation supplemented with Gibberellic Acid (GA3) (Figure 21).

Elongation was performed with 1 cm in length A. muricata and A. cherimola shoots. The A. cherimola did not increase in length and some shoots lost their quality when they remained in this medium supplemented with GA3

A. muricata increased up to 3.5 cm in four weeks of culture with a mean promedium of 5 ± 3 leaves per shoot and 3 ± 1 internode. The quality of the shoot was optimal and no bud sprouting or adventitious formations were observed.


85

Figure 20. Effect of thidiazuron on A. cherimola and A.muricata shoot formation

Thidiazuron (µM l-1)

* Mean of 3 replicates per treatment - Number of shoots per treatment = 20
* Medium: NN-69 + S 3% + Gr 0.3% pH 5-7 ± 0.1

Figure 21. Effect of gibberellic acid on A. cherimola and A. muricata shoot elongation

Giberellic acid (µM)

* Mean of 3 replicates per treatment - Number of shoots per treatment = 20
* Medium: NN-69 + S 3% + Gr 0.3% pH 5-7 ± 0.1


86

Figure 22. Several in vitro responses of A. cherimola and A. muricata during the multiplication stage - after four weeks of culture-

1. 2. 3. 4. A. cherimola shoot proliferation on Nitsch and Nitsch (1969) and 2.32 µM K and 1.6 µM Z
5. A. cherimola showing proliferation rate, the same situation is reported for A. muricata
6. 7. A. muricata shoot elongation
Effect of thidiazuron 2.27 µM on A. muricata base of the shoot, as well as in A. muricata, no adventitious shoot formation is reported from this type of organogenesis


87

6.3.8 Shoot Quality

During the shoot multiplication in a Nitsch and Nitsch (1969) medium, supplemented with zeatine and kinetine, necrotic spots on the leaves and poor growth were observed in all the A. cherimola selections after three months of culture before they started to loss the green colour and showed chlorosis. The shoots lost their quality and it was not possible to rescue them. A subculture in a fresh medium was tried as a first strategy for not loosing the micropropagated material, but without success.

On the base of many chlorotic explants a white exudate was observed. The presence of some residual bacterial contamination Pseudomonas saccharophila<7> was found. The explants with residual contamination during the multiplication were eliminated and a new establishment with reported strategies (cf. 5.1.2 Endogenous disinfection) was reviewed, healthy explants were obtained after these experiments.

6.3.9 Chlorosis

Healthy explants of A. cherimola, after the third subculture in a defined Nitsch and Nitsch (1969) basic salt medium formulation supplemented with zeatine 1,36µM + kinetin 4,65 µM, started to be chlorotic.

Preliminary experiments, varying the concentration of macro and micro elements as reported by Nitsch and Nitsch (1969), showed that the chlorosis in A. cherimola shoots was related to the concentration of nitrogen ions (data not shown).

The effect of ammonium nitrate, potassium nitrate and ammonium carbonate ions supplied in the Nitsch and Nitsch (1969) salt formulation medium were evaluated. In this series of experiments the other media components such as: macroelements, iron, microelements, vitamins, sucrose and gelrite were maintained standard as well as the kinetin and zeatin concentrations (cf. Materials and Methods).

The total concentration of (NH4- mM) and (NO3- mM) reported in the media of: Lloyd and McClown (1980), De Gref and Jacobs (1979), Murashige and Skoog (1962) and Nitsch and Nitsch (1969) were compared.

The effect of casein hydrolysate, natural complex of nitrogen (Sigma C 7290) in concentrations of 100 - 200 mg l-1 was evaluated and compared with the different nitrogen medium formulations described before (Table 28).

The results showed chlorosis and leaf defomation with some necrotic spots in all the tested media where the ions NH4- and NO3- were not included (Table 28). All this material was eliminated because the quality was poor.

The casein hydrolysate, as an external nitrogen source, promoted growth and the number of shoots without chlorosis, suggesting that the observed chlorosis was due to a deficiency of nitrogen in the mediums (Table 28)

The substitution of the concentration reported by Nitsch and Nitsch(1969), NH4- 9.0 mM and NO3- 18.4 mM by the concentration reported by Murashige and Skoog (1962) NH4+ 20.6 mM and NO3- 39.4 mM was enough to maintain the A. cherimola cultures green and proliferating (Table 28) (Figure 23).


88

With the optimal (NH4+ / NO3-) balance the number of new shoots preserved their quality. Thus ammonium nitrate reported by Nitsch and Nitsch (1969) was substituted by the ion concentration reported by Murashige and Skoog (1962).

Table 28. Effect of different nitrogen sources during the shoot multiplication
of A. cherimola on Nitsch and Nitsch (1969) basic medium *

CHa

Supplemented on NN-69

Total Ion * concentration reported by

% cultures without chlorosis **

(mg l-1)

NH4+mM

NO3- MM

 

 

 

 

 

 

0

0

0

-

4.0 ± 2.6

 

5.0

9.8

WPM b

41.3 ± 3.3

 

6.0

19.8

Dg-J c

22.4 ± 2.5

 

9.0

18.4

NN-69 d

8.3 ± 1.3

 

20.6

39.4

MS e

100.0 ± 0.0

 

 

 

 

 

100

0

0

-

20.0 ± 3.1

 

5.0

9.8

WPM

55.4 ± 5.2

 

6.0

19.8

Dg-J

25.2 ± 1.8

 

9.0

18.4

NN-69

33.1 ± 3.4

 

20.6

39.4

MS

100.0 ± 0.0

 

 

 

 

 

200

0

0

-

41.6 ± 4.2

 

5.0

9.8

WPM

60.2 ± 3.3

 

6.0

19.8

Dg-J

32.4 ± 2.5

 

9.0

18.4

NN-69

36.3 ± 1.3

 

20.6

39.4

MS

100.0 ± 0.0

Only the NH4+and NO3- concentration in “mM“ vary on the original mineral salts reported formulation “ Nitsch and Nitsch, 1969“.
The mediums were supplemented with sucrose 3%, Gelrite 3 g l-1 and Zeatine + Kinetine as growth regulators
The values represent the mean (± SE) of three independent experiments, twenty explants were used for each experiment
a CH : casein-enzymatic hydrolysate (N-Z-Amine A) total nitrogen 13.1% and amino nitrogen 6.5% approx.
b Lloyd and McClown (woody plant medium) (1980), c De Gref and Jacobs (1979), d Nitsch and Nitsch (1969), eMurashige and Skoog (1962)

6.3.10 Subcultures

The continued production of multiple shoots in A. cherimola and A. muricata was evaluated during the subcultures in the same multiplication medium. The percentage of abnormal plantlets or plantlets not growing well was very low and not significant. Hyperhydration and chlorosis were totally avoided. Callus formations were not observed in any case. There were no significant differences between selections during the multiplication rate (Figure 24).


89

Figure 23. Effect of nitrogen concentration in [µM] on in vitro chlorosis of A. cherimola and A. muricata

Figure 24. Effect of subculture number on the proliferation of
A. cherimola and A. muricata selections - after three four weeks of subculture


90

6.4 Rooting

To promote rhizogenesis in A. cherimola and A. muricata previous negative results have suggested that the in vitro shoots multiplied in vitro should be preconditioned before the application of any root organogenesis treatment.

The number of subcultures and the cytokinin synergistic combination in A. cherimola seemed to inhibit the potential of the shoots to promote new roots in vitro. Also A. muricata showed the same limitation, because the gibberellic acid is known as a root inhibitor in tissue culture.

6.4.1 In vitro

The root organogenesis of A. cherimola and A. muricata multiplied shoots (3 -5 cm long) was very difficult. The A. cherimola was rooted by 43.2% with IBA 4.90 µM and A. muricata 36.4% respectively. The in vitro medium was ¼ macro salt NN-69 concentration and 1% sucrose.

The concentration of macro-elements reported by Nitsch and Nitsch (1969) was reduced to ¼ of its normal concentration and supplemented with a low concentration of sucrose 1%. For both species the first roots began to emerge after 40 days without callus formation (Table 29).

Table 29. Evaluation of some auxins to improve the rhizogenesis on
A. cherimola and A. muricata

s

Growth Regulator µM l-1

Promedium in % of rooted shoots ± SD After 40 days of in vitro culture

 

 

A. cherimola

A. muricata

 

 

 

 

 

1/4

-

-

0

0

 

IBA

2.46

0

0

 

 

4.90

43.2 ± 3.6

36.4 ± 5.3

 

 

9.80

0

0

 

 

24.60

0

0

1/4

NAA

2.69

0

0

 

 

5.37

0

0

 

 

10.74

0

0

 

 

26.85

0

0

1/4

IAA

2.85

0

0

 

 

5.71

0

0

 

 

11.42

0

0

 

 

28.54

0

0

* The values represent the mean (± SE) of five independent experiments.
Twenty explants were used for each experiment


91

The root induction of A. cherimola micropropagated explants were tested with the total macro-elements salt concentration (1X) and (1/2) with IBA, NAA and IAA combinations. In all the experiments rhizogenesis was not reported (data not shown).

The A. cherimola and A. muricata are not root prolific under in vitro conditions. The base of the multiplied explant did not improve any callus or rosette formation when they were treated with NAA and IAA or IBA in a high concentration. The shoot explants showed the tendency to improve the production of phenolics which were not present during multiplication.

The concentration of IBA 4.90 µM promoted the rhizogenesis of A. cherimola explants only if the mineral basal composition was reduced to ¼ concentration. The A. cherimola and A. muricata rhizogenesis were evaluated also in the clonal selections (Table 30).

Table 30. Evaluation of in vitro rhizogenesis of A. cherimola and A. muricata selections - shoots conditioned one month in a hormone free media-

Species

Cultivar or selection

¼ NN-69* + 1% S* + 3% GR* IBA 4.90 µM l-1

 

 

 

A. cherimola

Ch-X

41.8 ± 1.5

 

Ch-Y

39.2 ± 3.1

 

Ch-Z

36.4 ± 5.2

 

 

 

A. muricata

Mr-2

32.4 ± 5.3

 

Mr-4

29.8 ± 2.6

 

Mr-5

31.6 ± 8.2

* The values represent the mean (± SE) of three independent experiments.
wenty explants were used for each experiment
¼ macro elements reduction, S -sucrose, GR -gelrite

6.4.2 Ex vitro

Rooting micro-cuttings under semi-sterile conditions can be an easy way to obtain A. cherimola and A. muricata plantlets. With a pre-dip in 4.90 µM IBA. Rooting percentages of 51.3 % for semiwoody coming shoots were obtained for A. cherimola and 58.5% for A. muricata .

The ex vitro root organogenesis requires careful attention to the physiological condition of the microcuttings and to the rooting humidity atmospheric condition. The first roots appeared after 40 days and then intensive growth and development of the plants proceeded. The highest number of rooted plants in all the selections was obtained when they were treated with Rhizopon (Table 31).


92

Table 31. Effect of Rhizopon on ex vitro root formation of
A. cherimola and A. muricata selections

Species

Selection (code)

Rhizopon **

control *

NAA 10% (solution) *

IBA 1% (pulver *)

 

 

 

 

 

A. cherimola

Ch-X

0

51.3 ± 2.7

0

 

Ch-Y

0

46.7 ± 3.3

0

 

Ch-Z

0

41.6 ± 3.8

0

 

 

 

 

 

A. muricata

Mr-2

0

56.7 ± 5.1

0

 

Mr-4

0

52.1 ± 7.4

0

 

Mr-5

0

40.8 ± 1.2

0

* The values represent the mean (± SE) of three independent experiments.
Twenty explants were used for each experiment
** Rhizopon ® (Hazerswoude-Holland)

To compare the efficiency of the in vitro and ex vitro rhizogenesis in A. cherimola and A. muricata the number of roots and root growth were compared (Table 32).

Table 32. Comparison of the ex vitro and in vitro root organogenesis
Of A. cherimola and A. muricata

Species

IBA

4.90 µM

Root emergence in days

Number of roots per shoot

(mm/week)Root growth rate

(%)Rooting efficiency

Callus formation

 

 

 

 

 

 

 

A. cherimola

in vitro

40

3

2.4

21.2

-

 

ex vitro

40

5

4.0

45.6

-

 

 

 

 

 

 

 

A. muricata

in vitro

40

3

2.0

25.0

-

 

ex vitro

40

7

4.3

55.5

-

6.5 Hardening

The A. cherimola and A. muricata micropropagation success required that the new regenerant shoots, which are heterotrophic under in vitro conditions with a very high humidity (90-100%), began to be autotrophic under ex vitro conditions.


93

Figure 25. Morphogenic events related with the the root organogenesis of cherimola and A. muricata under in vitro and ex vitro conditions

1. In Vitro rooting of A. cherimola after 70 days on ¼ NN-69 salts supplemented with 10% surcrose
2. In Vitro rooting of A. muricata after 70 days on ¼ NN-69 salts supplemented with 10% surcrose
3. Ex vitro root formation in A. cherimola induced by Rhizopon IBA 1% after 50 days
4. Ex vitro root formation in A. muricata induced by Rhizopon IBA 1% after 50 days
5. Ex vitro adaptation of A. cherimola
6. Sequence of ex vitro growth in quartz-sand pots in A. muricata in vitro regenerant
7. A. muricata ex vitro root developmen
A. muricata regenerant after four months (autotrophic plant)


94

After two months in the rooting micropropagation stage, the regenerants were transferred to sterile sand-quartz pots. The acclimatization was promoted in a moderate low humidity (60-70%) in nursery conditions with 23 ± 5 °C.

To increase the acclimatization rate, the pots were covered with an inverted funnel in order to facilitate the O2 and CO2 mobilization. About 40% of the A. cherimola and A. muricata regenerants survived in the ex vitro conditions.

Once the A. cherimola and A. muricata in vitro plants were acclimatized, they did not promote a new shoot growth formation. They seemed to be dormant, the growth in terms of elongation and new leave formation was observed slightly after two months of adapting to the greenhouse conditions.

At the time of acclimatization the shoots elongated, and the leaves expanded and turned deep green. The leaves coming from in vitro culture were weak and showed a slight green colour possibly due to the low photosynthetic activity and had a tendency to loose water with extreme facility.

The new ex vitro formed leaves were stronger and after two months they were very green showing the new successful photosyntesis activity. It is important to notice that the acclimatization process is dependent on the apical meristem, which stimulates the new leaf formation. If the apical meristem died, because of dehydration or browning, there was no chance to induce an ex vitro new growth development. The regenerant could not be adapted and died.

After its perfect adaptation to ex vitro conditions the A. cherimola and A. muricata grew vigorously and after six months they were pruned for the first time. The well adapted plants are still growing with full vigour and uniformity in the greenhouse (Figure 25).


Fußnoten:

<7>

Analysis made by the „Biologische Bundesanstalt für Land- und Forstwirtschaft . BBA“


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