Somatic embryo induction via cotyledon explants

CHAPTER 2: STUDIES ON Theobroma cacao L

2.2 Results and Discussion

2.2.5 Somatic embryo induction

2.2.5.2 Somatic embryo induction via cotyledon explants

Table 2.32: The effect of NAA in combination with BAP and TDZ on leaf explants using DKW medium. Types of callus: WF = white friable callus and BS = brown soft callus. Amount of callus scored as: (-) = no callus, (+) = quarter of explant, (++) = half of explant, (+++) = three quarter of explant and (++++) = whole explant. n=50. Mean values represented by the same alphabetical letters are not significantly different (Chi- squared test, p < 0.05).

Growth regulator (mg l^-1)

NAA BAP TDZ

% explants producing

callus

Types of callus

Amount Of Callus

1.0 3.0 - 50 ^ab WF ++

0.5 1.0 - 66 ^ab WF ++

0.1 0.5 - 40^b BS +

1.0 - 1.0 80^a WF ++

0.5 - 0.5 76 ^a BS

+ ++

1.0 - 0.1 80 ^a BS

Table 2.33: Decontamination of cotyledon explants for somatic embryo induction.

n=20. Mean values represented by the same alphabetical letters within each column are not significantly different (Chi-squared test, p < 0.05).

% Contamination Treatment

No. Treatment Time

(min) Fungal Bacterial

1. 1% NaOCl 5 15 ^a 80 ^c

2. 1% NaOCl 10 10 ^a 70 ^bc

3. 1% Hibitane

1% NaOCl

10 10 ^a 65 ^bc

4. 1% Hibitane

1% NaOCl

10 5 ^a 50 ^abc

70% EtOH 1% Hibitane

1% NaOCl

2 10 10

0 ^a 30^ab

70% EtOH 2% Hibitane

1% NaOCl

2 10 10

0 ^a 15 ^a

Preliminary experiments using cotyledonary explants revealed that phenolics exudation constituted a major problem upon culture initiation; in an attempt to counteract this, explants were treated with two concentrations of citric acid, an anti-oxidant. Soaking both axillary bud and nodal explants in an anti-oxidant solution was found to be beneficial in counteracting phenolics exudation earlier in this study, as well as for Prunus avium microshoots (Vasar, 2003) and litchi fruit (Duan et al, 2007). A ten minute immersion in 2% citric acid significantly reduced the browning of the culture medium associated with phenolics exudation by cotyledonary explants (p < 0.05, Table 2.34). This pre-treatment was then routinely applied to cotyledonary explants, prior to any of the following manipulations.

Table 2.34: Effect of citric acid pre-treatment on cotyledon explants. n=20. Mean values represented by the same alphabetical letters are not significantly different (Chi- squared test, p < 0.05).

Citric acid concentration Time % explant browning

5 min 100 ^c

10 min 60 ^b

5 min 30 ^ab

10 min 5 ^a

Aguilar et al. (1992) obtained somatic embryos from cotyledon explants by culturing them on medium containing 3.0 mg l^-1 BAP in combination with 1.0 mg l^-1 NAA. In the present study, however, provision of NAA in combination with BAP or TDZ did not induce embryo production (Table 2.35). In addition, neither type of callus produced by the explants (white friable and brown soft) was embryogenic, nor was there any significant difference in the percentage of explants producing callus. The protocol used to generate somatic embryos from cotyledon explants by Aguilar et al. (1992) was not successful in this study. This may have been because the material used in the present study was of a different genotype compared with material used in that study: protocols established are usually species, and in most cases, genotype specific (Maximova et al., 2002).

Table 2.35: Effects of NAA in combination with BAP and TDZ in DKW medium on embryo formation. Types of callus: WF = white friable callus and BS = brown soft callus. Amount of callus scored as: (-) = no callus, (+) = quarter of explant, (++) = half of explant, (+++) = three quarter of explant and (++++) = whole explant. n=50. Mean values represented by the same alphabetical letters are not significantly different (Chi- squared test, p < 0.05).

Growth regulator (mg l^-1)

NAA BAP TDZ

% explants producing

callus

Types of callus

Amount Of Callus

1.0 3.0 - 100 ^a BS

+++

0.5 1.0 - 86 ^a BS

+++

0.1 0.5 - 100 ^a BS

1.0 - 1.0 100 ^a BS +++

0.5 - 0.5 80 ^a BS

1.0 - 0.1 100 ^a WF

Therefore, procedures used for leaf explants were applied to cotyledon explants including the three-step procedure (see above) established by Li et al. (1998). The effect of MS medium supplemented with 2,4-D and 2,4-D in combination with TDZ was investigated as these combinations gave rise to yellow/cream embryogenic callus when using leaf explants (see above). The inclusion of more than 2.0 mg l^-1 2,4-D into the culture media when using cotyledon explants was associated with low proportions of explants producing callus (Table 2.36). In no case, however, was embryogenic callus formed on the growth-regulator supplemented MS medium. Instead, non-embryogenic white friable, translucent crystalline, compact white and brown soft callus resulted.

Table 2.36: The effect of growth regulators in MS medium on cotyledon explants of cocoa. Types of callus: WF = white friable callus, T = translucent callus, CW = compact white callus, BS = brown soft callus. Amount of callus scored as: (-) = no callus, (+) = quarter of explant, (++) = half of explant and (+++) = three quarter of explant, (++++) = whole explant. n=50. Mean values represented by the same alphabetical letters are not significantly different (Chi-squared test, p < 0.05).

Growth regulator (mg l^-1)

2,4-D TDZ

% explants producing

callus

Types of callus

Amount of callus produced

0.5 - 66 ^bcde WF ++

1.0 - 30 ^abcd WF

+ +

2.0 - 46 ^abcde WF +

3.0 - 24 ^abc WF +

5.0 - 10 ^ab WF +

10 - 6 ^a T +

1.0 0.005 74 ^cde WF

1.0 0.01 66 ^bcde WF

1.0 0.05 66 ^bcde

WF CW BS

+ +

1.0 0.1 90 ^e

T CW

+ + +

2.0 0.005 90 ^e WF

2.0 0.01 70 ^cde WF

+ +

2.0 0.05 64 ^bcde WF

+ +

2.0 0.1 80 ^cde

T CW WF

+ + +

Use of 2,4-D in combination with TDZ or 2-iP in DKW medium was beneficial in promoting the formation of yellow/cream nodular callus. This callus-type was embryogenic when 1.5 mg l^-1 2,4-D was combined with either 0.1 or 1.0 mg l^-1 TDZ (Table 2.37). The protocol established by Tan and Furtek (2003) using DKW medium supplemented with 2.0 g l^-1 2,4-D and 100 mg l^-1 2-iP for inflorescence explants, did not induce embryogenesis from cotyledon explants in the present study (Table 2.37).

Table 2.37: Effect of selected growth regulators incorporated in DKW medium, on callus production by cocoa cotyledon segments. Types of callus: WF = white friable callus, BS = brown soft callus and Y = yellow/cream compact nodular callus. Amount of callus scored as: (-) = no callus, (+) = quarter of explant, (++) = half of explant and (+++) = three quarter of explant, (++++) = whole explant. n=40. Mean values represented by the same alphabetical letters are not significantly different (Chi-squared test, p < 0.05).

Growth regulator(mg l^-1)

2,4-D TDZ 2-iP

% explants producing

callus

Types of callus

Amount Of Callus

1.5 0.01 - 75 ^bc BS

1.5 0.1 - 80 ^bc Y

1.5 1.0 - 90 ^c

Y WF

+ +

1.5 - 1.0 15 ^a WF

1.5 - 10 40 ^ab

WF BS

+ +

1.5 - 50 10 ^a WF

+ +

2.0 g l^-1 - 100 50 ^abc T ++

The exposure of cotyledon explants to the three step procedure as described by Li et al.

(1998) gave rise to globular somatic embryos (Figure 2.7). The explants were initially incubated on 1.5 mg l^-1 2,4-D with 1.0 mg l^-1 TDZ for four weeks with subsequent transfer to WPM with and without 1.0 mg l^-1 kinetin, for a further four weeks with final incubation on DKW medium without growth regulators gave rise to globular embryos (Table 2.38). In addition, globular embryos formed on medium supplemented with 1.5 mg l^-1 2,4-D and 0.1 mg l^-1 TDZ with subsequent transfer onto the secondary callus medium which contained either 1.0 mg l^-1 BAP or 0.5 or 1.0 mg l^-1 kinetin.

Figure 2.7: Globular embryos formed on cotyledon explant. Scale bar = 1 mm

Table 2.38: Effect of placing cotyledon explants on DKW medium containing 1.5 mg l^-1 2,4-D in combination with either 0.1 or 1.0 mg l^-1 TDZ for 4 weeks and transferring explants onto seven different treatments based on WPM medium. n=50. Mean values represented by the same alphabetical letters within each column are not significantly different (Chi-squared test, p < 0.05).

Growth regulator (mg l^-1)

1.5 mg l^-1 2,4-D + 0.1 mg l^-1 TDZ

1.5 mg l^-1 2,4-D + 1.0 mg l^-1 TDZ

BAP Kinetin

% explants producing yellow callus

% yellow callus producing

globular embryos

% explants producing yellow callus

% yellow callus producing

globular embryos

- - 80 ^b 0 â 80 â 60 â

0.1 - 50 ^a 0 ^a 10 ^c 0 ^b

0.5 - 30 ^a 0 ^a 30 ^bc 0 ^b

1.0 - 50 ^b 30 ^b 50 ^ab 0 ^b

- 0.1 50 ^b 0 ^a 10 ^c 0 ^b

- 0.5 50 ^b 40 ^b 60 ^a 0 ^b

- 1.0 50 ^b 30 ^b 60 ^a 30 ^a

Due to time constraints, the globular embryos formed in this study could not be advanced towards germination. However, once this has been achieved, it is suggested that these structures could form suitable explants for cryopreservation.

Dalam dokumen Development of explants potentially suitable for cryopreservation of the recalcitrant-seeded species Theobroma cacao L. and Barringtonia racemosa (L.) roxb. (Halaman 114-122)