Chapter 7: Physiological responses and endogenous cytokinin profiles in relation to
7.3 Results and discussion
7.3.2 Effect of roscovitine and INCYDE on endogenous cytokinin content
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Figure 7.2: Total isoprenoid and aromatic cytokinin content (%) in aerial (A) and underground (B) parts of the micropropagated „Williams‟ bananas. 1 = meta-topolin; 2 = benzyladenine; 3 = benzyladenine + INCYDE; 4 = benzyladenine + roscovitine; 5 = benzyladenine + INCYDE + roscovitine; 6 = meta-topolin + INCYDE; 7 = meta- topolin + roscovitine; 8 = meta-topolin + INCYDE + roscovitine.
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Figure 7.3: Relative abundance (%) and distribution pattern of the isoprenoid and aromatic cytokinins in the micropropagated „Williams‟ bananas. A = aerial and B = underground regions. 1 = meta-topolin; 2 = benzyladenine; 3 = benzyladenine + INCYDE; 4 = benzyladenine + roscovitine; 5 = benzyladenine + INCYDE + roscovitine; 6 = meta-topolin + INCYDE; 7 = meta-topolin + roscovitine; 8 = meta- topolin + INCYDE + roscovitine.
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The importance of exogenous application of CKs for in vitro shoot development cannot be overemphasized, particularly in the micropropagation of bananas (Bairu et al., 2008). The response of explants observed in terms of the growth and development in vitro are regulated by the interaction and balance between the applied PGRs (type and concentration) and the endogenously produced ones (George, 1993; Krikorian, 1995).
Factors such as the exogenous application of CKs are known to affect the biochemical pathways that regulate endogenous CK levels (Krikorian, 1995; Blagoeva et al., 2004a). Since aromatic type CKs were used in the current study, as expected, the aromatic CKs were several fold more abundant compared to the isoprenoid CKs which are generally more common in plant tissue. When compared to the lower levels that ranged from approximately 0.2-39% for the total isoprenoid CK pool in the regenerants, the aromatic CKs were more abundant with the highest level (99.8%) observed in BA- treated plantlets (Figure 7.2). Ivanova et al. (2006) reported a similar scenario whereby the application of exogenous BA to the media resulted in an alteration from isoprenoid to aromatic CKs in the newly-formed Aloe polyphylla shoots compared to those grown on CK-free and Z-supplemented media. As only aromatic CKs were used in the current study, the detection of isoprenoid CKs indicates the occurrence of the de novo CK synthesis pathway in the regenerated banana in vitro. However, the high concentration of the exogenously applied aromatic CK probably suppressed the isoprenoid CK synthesis pathway in the cultured explants. In addition, CKX have been shown to exhibit preferences for the isoprenoid CKs while the aromatic CKs are more resistant (Zalabák et al., 2012). Interestingly, the current findings show that roscovitine and/or INCYDE when combined with mT or BA (with the exception mT + INCYDE) possibly prevented the breakdown of isoprenoid CK during the in vitro growth of „Williams‟ bananas (Table 7.1 and 7.2).
Cytokinin uptake, metabolism and transport within the explants are vital parameters for the growth and development in PTC (Auer, 1997). In the current study, the wide disparity between the aerial and underground parts is probably a function of the CK transport mechanism. In the eight treatments, the concentration of total CK detected in the underground parts was higher than in the aerial parts. Plantlets regenerated from
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mT + roscovitine had the highest total CK content (661215 pmol/g FW) with the underground parts having approximately a 68-fold more than the aerial parts. A general trend observed was that the addition of roscovitine and/or INCYDE with mT improved the total CK pool in both aerial and underground parts of the regenerants. A similar pattern was observed in the aerial parts when BA was combined with roscovitine and INCYDE; however, both compounds reduced the total CK content in the underground sections as well as the sum total in the plantlets. It is noteworthy to highlight that the decrease in total CK pool was mainly due to the reduction in the quantity of 9- glucosides which are generally detrimental to plant growth. This reduction in 9- glucosides levels could be attributed to the presence of either roscovitine or INCYDE when acting individually with BA.
Often, most of the physiological activity of Z as a free base has been attributed to tZ while the cis isomer is regarded as an inactive or weakly active form of CK (Kamínek et al., 1987; Haberer and Kieber, 2002). Although a precise role for cZ-type CKs remains to be fully elucidated, recent studies have been identifying their potential functions in plants (Dwivedi et al., 2010; Gajdošová et al., 2011). In accordance with previous findings (Blagoeva et al., 2004b; Dwivedi et al., 2010) where olomoucine and its analogues such as roscovitine enhanced the cZ levels in vitro, roscovitine stimulated the production of more cZ in both aerial and underground regions of the regenerated plantlets compared to the controls (Table 7.1 and 7.2). Although, cZ was less abundant in the underground region, the concentrations were several folds higher than the tZ in the plantlets. In addition to the potential role of cZ and/or its derivatives in regulation of physiological processes, Gajdošová et al. (2011) postulated that these compounds may be relevant under growth-limiting conditions connected to developmental processes or external signals.
In BA-supplemented treatments, 9-glucosides account for approximately 60% of total CK content (Figure 7.4A and B). In terms of their distribution, an estimated 80% were detected in the underground part of the plantlets (Figure 7.5B). The quantity and
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distribution of CK derivatives are important information which may explain some observed physiological disorders in PTC (Aremu et al., 2012).
Figure 7.4: Total cytokinin pool (%) of the different cytokinin forms in aerial (A) and underground (B) parts of the micropropagated „Williams‟ bananas. 1 = meta-topolin;
2 = benzyladenine; 3 = benzyladenine + INCYDE; 4 = benzyladenine + roscovitine; 5
= benzyladenine + INCYDE + roscovitine; 6 = meta-topolin + INCYDE; 7 = meta- topolin + roscovitine; 8 = meta-topolin + INCYDE + roscovitine.
It is generally known that most 9N-glucosides, particularly BA9G are formed and stored in the basal region of plant and transport difficulty has been implicated in problems such as acclimatization failure (Werbrouck et al., 1995), shoot-tip necrosis (Bairu et al., 2011b) and high toxicity (Amoo et al., 2011). In the current study, when compared to BA alone, the presence of roscovitine and/or INCYDE with BA produced a substantial
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quantity of the 9-glucosides in the aerial parts (Figure 7.5A and B). The enhanced transportation of 9-glucosides could be a regulatory mechanism to alleviate the detrimental effect in the underground region of the plantlets.
Figure 7.5: Relative abundance (%) and distribution pattern of the different cytokinin forms in the micropropagated „Williams‟ bananas. A = aerial and B = underground regions. 1
= meta-topolin; 2 = benzyladenine; 3 = benzyladenine + INCYDE; 4 = benzyladenine + roscovitine; 5 = benzyladenine + INCYDE + roscovitine; 6 = meta-topolin + INCYDE; 7 = meta-topolin + roscovitine; 8 = meta-topolin + INCYDE + roscovitine.
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7.3.3 Effect of roscovitine and INCYDE on photosynthetic and phenolic contents