CHAPTER 3: STUDIES ON Barringtonia racemosa
3.2 Results and Discussion
3.2.1 The development of in vitro shoots
gluconate, the active ingredient in Hibitane, has wide efficacy against an array of bacteria, fungi and viruses and is widely used in the medical field (Anon, 2007).
Table 3.5: Different treatments used for decontamination of Barringtonia racemosa embryonic axes. Treatments 3 - 14 were followed by 10 min soak in 1% NaOCl and Treatment 15 utilised 2% Hibitane for 10 min followed by 10 min in 1% NaOCl with three rinses in sterile distilled water after each step. n=20. Mean values represented by the same alphabetical letters within each column are not significantly different (Chi- squared test, p < 0.05).
Contamination (%)
No. Treatment Treatment
Time (min) Fungal Bacterial
1 1% NaOCl 5 100 a -
2 1% NaOCl 10 100 a -
3 30 60 bc 40 ab
4 60 70 b 30 a
5
Previcur (2.5 ml l-1) + Impact (0.5 ml l-1)
120 50 bc 30 a
6 30 60 bc 40 ab
7 60 50 bc 50 abc
8
Celest (0.33 ml 100 ml-1)
120 50 bc 40 ab
9 30 70 b 30 a
10 60 50 bc 50 abc
11
Heritage (0.08 g 100 ml-1)
120 40 bc 50 abc
12 30 30 c 70 bc
13 60 30 c 70 bc
14
Celest
(0.33 ml 100 ml-1) and Heritage
(0.08 g 100 ml-1) 120 5 d 80 c
15 Celest, Heritage followed
by Hibitane 120 5 d 5 d
After decontamination (using Treatment 15), germination (root and shoot production) of B. racemosa whole axes occurred after 28 days on quarter-strength MS medium and
after 35 days on half- and full-strength MS medium with no germination occurring on WPM (Table 3.6). Although the chi-squared test showed no significant difference (p >
0.05) in percentage germination amongst the different strengths of MS medium tested, there was a significant difference in the average number of shoots produced per explant for these treatments (Table 3.6, p < 0.05). Embryonic axes placed on full-strength MS medium produced 1.3 shoots per explant while those germinated on quarter-strength MS produced 2.3 shoots per explants (Figure 3.1), the latter therefore being selected as the best medium for germination.
Even though explants were decontaminated, and only 5% appeared bacterially contaminated immediately following treatment, using the protocol Treatment 15 (above), 40 - 53% whole axes cultured for germination showed bacterial contaminants (Table 3.6). The high contamination frequency is suggested to have contributed to the low percentage of germination recorded for zygotic axis explants on the MS-based media (Table 3.6). It is probable that the Hibitane did not penetrate sufficiently to affect bacterial inoculum located deep within the embryo tissues.
Table 3.6: The effects of different basal media on germination of B. racemosa embryonic axes. MS - Murashige and Skoog, 1962, WPM - Woody Plant Medium, Lloyd and McCown, 1981. n=15. Mean values represented by the same alphabetical letters are not significantly different within columns (Chi-squared test, p < 0.05).
Basal
medium % Germination
Avg. no. of shoots produced/
explant
Time taken for germination
(days)
% Contamination
MS 56 a 1.3 b 35 43 a
½ MS 30 a 1 b 35 53 a
¼ MS 40 a 2.3 a 28 50 a
WPM 0 b 0 c 0 50 a
Figure 3.1: Multiple shoot formation from B. racemosa zygotic axis germinated on quarter-strength MS medium. Scale bar = 3 mm
Germination of axes using only the central core region and eliminating the contaminated exterior reduced the percentage of contaminated explants, and 60% of the explants germinated on quarter-strength MS medium (results not shown). Therefore, when culturing axes onto medium containing BAP or 2-iP to promote adventitious shoot formation or shoot multiplication, the explants were trimmed such that only the axis core was cultured.
The inclusion of BAP or 2-iP in the culture medium had a significant effect on shoot multiplication (Table 3.7). Media supplemented with 10 mg l-1 BAP or 2-iP produced an average of 1.7 and 2 shoots per axes, respectively. Exposure to 50 mg l-1 BAP or 2-iP, however, resulted in no shoot production, which has also been shown for Zantedeschia aethiopica axes (Ngamau, 2001).There was no significant difference in the percentage of explants producing shoots across the treatments as well as among media supplemented with BAP or 2-iP (Table 3.7, p > 0.05). The time required for shoot formation was however, affected by the inclusion of PGRs in the medium. Explants
exposed to BAP-containing medium took a considerably shorter period for shoot formation compared with those on 2-iP containing medium (Table 3.7).
Callus formation occurred only on the surface of explants placed on media supplemented with 0.1 and 10 mg l-1 2-iP, no callus forming on explants exposed to different concentrations of BAP (Table 3.7). This was somewhat unexpected as the inclusion of BAP has been found to be beneficial for indirect formation of somatic embryos when zygotic axes of a variety of species were utilised as the primary explant (e.g. George, 1996); however, this PGR had no effect in the present study on either shoot multiplication or callus formation.
Table 3.7: The effects of two cytokinins, BAP and 2-iP, on shoot formation from embryonic axis cores. Explants were placed on MS medium supplemented with 30 g l-1 sucrose and various concentrations of BAP or 2-iP. (+) = callus, (-) = no callus. n=20.
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)
2-iP BAP
% explants producing
shoots
Avg. no. of shoots produced/
explant
Callus formation
Time taken to form
shoots (days)
0.1 35 a 1.3 ab + 56
1.0 40 a 1 b - 42
10 45 a 1.7 a + 42
50 0 b 0 c - 0
0.1 30 a 1 b - 28
1.0 25 a 1 b - 28
10 35 a 2 a - 21
50 0 b 0 c - 0
The use of in vitro generated shoots as explants for cryopreservation was decided against because of their thick and leathery nature. In addition, the lack of success
obtained with cocoa vegetative tissue as well as that of other tropical woody species (see Chapter 2), did not encourage the use of such material for cryogen exposure in the present study. Therefore, it was decided that somatic embryos might be more feasible specimens for conservation of B. racemosa germplasm.