The role of meta-topolins on the physiology of micropropagated 'Williams' bananas (Musa spp. AAA)

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Banana production ranks fifth behind cereals as a food crop and has potential, along with other major crops, to feed the world's increasing population. Globally, continuous efforts and techniques including the use of plant tissue culture (PTC) have been devised for increasing the production of several Musa species. The choice of cytokinin (CK) is one of the most critical factors in developing a successful PTC protocol. Since the discovery of topolins as naturally occurring aromatic CKs, they have emerged as genuine alternatives to the long serving CKs (benzyladenine = BA, zeatin = Z and kinetin = KIN) in PTC. Globally, the past 15 years has witnessed a surge in the use of topolins and their derivatives in research laboratories. Topolins have demonstrated great potential during culture initiation and protocol optimization as well as for counteracting various in vitro induced physiological disorders in some species. In terms of general physiology (growth, phytochemical and photosynthetic pigment contents as well as genetic fidelity), the topolins were compared with BA using 'Williams' bananas with minimal residual exogenous CK carry-over effects. The five topolins tested were meta-Topolin (mT); meta-Topolin riboside (mTR); meta-Methoxy topolin (MemT); meta-Methoxy topolin riboside (MemTR) and meta-Methoxy topolin 9-tetrahydropyran-2-yl (MemTTHP). Based on evidence of potential CK- and auxin-like activity of smoke-water (SW) and karrikinolide (KAR1) at low concentrations, a similar comparative study involving both compounds and mT was performed. For a further understanding of banana physiology in vitro, the effect of supplementing either mT- or BA-requiring cultures with roscovitine (a cyclin-dependent kinase and N-glucosylation inhibitor) and INCYDE (an inhibitor of CK degradation) on the endogenous CK profiles was investigated. In addition, greenhouse experiments geared towards improving the acclimatization competence of tissue-cultured banana plantlets via application of different concentrations of SW and vermicompost leachate was conducted. Sterile shoot-tip explants were cultured on modified Murashige and Skoog (MS) media supplemented with 10, 20 or 30 μM of the tested CKs for 42 days while rooting experiments involved the use of classic auxins as well as SW and KAR1. Apart from 10 μM BA and 30 μM MemTTHP treatments, the number of shoots produced with all the CK treatments were significantly higher than the control. Treatment with 30 μM mT resulted in the highest number of shoots (7.3±1.0) which is an indication of the requirement of exogenous CK for increased shoot proliferation in 'Williams' bananas The use of 10 μM MemTTHP had the least root inhibitory effect during the shoot proliferation phase. As an indication of the toxicity of applied CK, MemT- and MemTR-regenerants were the most deformed while mTR-regenerated plantlets demonstrated the best quality across all the CKs tested. In mT- and BA-derived shoots, SW and KAR1 significantly increased the number and length of roots compared to the control. During the rooting phase, topolin treatments produced more off-shoots than BA-treated ones which inevitably improved the overall number of regenerated shoots. Total phenolic levels were highest in 10 μM mT- and 30 μM MemTTHP-treated plantlets detected in the aerial and underground parts, respectively. It is interesting that in the underground parts, 10 μM mT resulted in the production of the highest amount of proanthocyanidins which was approximately five-fold higher than in the control plants. On the other hand, 10 μM MemTTHP-treated plantlets had significantly higher total flavonoids within the aerial parts. In view of the stimulation of secondary metabolites in the majority of the CK-treated plantlets, the current results indicate the role of the type and concentration of applied CK as potential elicitors in PTC. Generally, the maximum photosynthetic pigment content was attained between 40-50 days. The control plantlets had the highest pigment content (1150 μg/g FW) while 10 μM MemTTHP had the best pigment stimulatory effect among the tested CKs. Nevertheless, in vitro propagation of banana devoid of CKs is not a practical option due to low shoot proliferation rates. Scanning electron microscopy (SEM) of the foliar surface showed that the stomatal density was highest in 10 μM MemTTHP-treated and lowest in 10 μM MemTR-treated plantlets. Prolonging the culture duration as well as increasing CK concentrations reduced the pigment content. However, the drastic breakdown in chlorophyll pigments beyond 50 days was slightly inhibited by the presence of mT, mTR, MemTTHP and BA compared to the control. Current findings indicate the potential anti-senescence activity of the topolins such as mT, mTR and MemTTHP under in vitro conditions. This study articulates that the right choice and concentration of CKs applied during in vitro propagation may alleviate photomixotrophic-induced physiological stress that usually accompanies the transfer of plantlets to ex vitro conditions. Findings indicate that the effect of subculturing contributed significantly to the higher rate of variation in 'Williams' bananas in vitro. The presence of CK in the culture media apparently aggravated the stress on the explants as indicated in the relatively higher percentage polymorphic bands compared to the controls. Among the tested CKs, the use of mTR and MemTTHP caused the least detrimental effect on the regenerants while mT-treated plantlets had the most polymorphic bands. Hence, it is recommended that subculturing cycles from the initial explant establishment should be limited to a maximum of five. The use of SW and KAR1 improved the level of photosynthetic pigment and phenolic compounds in the micropropagated bananas. However, they had a negative effect on shoot proliferation; hence their inclusion is more desired when used at the rooting phase of micropropagation. Perhaps, these compounds could be used in conjunction with auxin to increase the number of roots prior to the acclimatization stage. The enhanced photosynthetic pigment level resulting from addition of SW and KAR1 would also play a vital role during acclimatization of the micropropagated plants. The present finding serves as an alternative approach, available to researchers for improving the quantity of secondary metabolites in micropropagated plants. The highest regeneration rate (93%) was observed in BA + roscovitine treatment while mT + INCYDE-treated plantlets produced most shoots. Treatment with BA + roscovitine had the highest shoot length and biomass. Although not significant, there was more proanthocyanidins in BA + roscovitine treatments compared to the treatment with BA alone. On the contrary, total phenolics were significantly higher in mT + roscovitine treatment than in the mT-treated regenerants. The presence of roscovitine and/or INCYDE had no significant effect on the photosynthetic pigments of the banana plantlets. Forty-seven aromatic and isoprenoid CKs categorized into nine CK-types were detected at varying concentrations. The presence of mT + roscovitine and/or INCYDE increased the levels of O-glucosides, while 9-glucosides remained the major derivative in the presence of BA. Generally, the underground parts had higher CK levels than the aerial parts; however the presence of INCYDE increased the level of CK quantified in the aerial parts of both CK treated plantlets. Apparently, the presence of INCYDE serves to enhance transportation of the CK towards the aerial regions. From a practical perspective, the use of roscovitine and INCYDE in PTC could be crucial in the alleviation of commonly observed in vitro-induced physiological abnormalities. Soil drenching with SW significantly increased the root length (1:1000 and 1:500 dilutions) as well as fresh and dry weight (1:1000; 1:500 and 1:250 dilutions) when compared to foliar application. Vermicompost leachate (1:10 and 1:5 dilutions) significantly enhanced the shoot length, root length, leaf area and dry weights. Vermicompost leachate (1:20; 1:10 and 1:5 dilutions) also significantly increased the number of off-shoots. The positive effect on rooting is beneficial for acclimatization and establishment of tissue-cultured banana plantlets in nurseries and subsequent transfer to the field. However, field trials will be necessary to substantiate the effects demonstrated by these compounds. In an attempt to contribute to improving banana micropropagation, the current findings provide additional evidence on the increasing advantage of topolins over BA. Nevertheless, some detrimental physiological effects observed with some of the topolins (for example, MemT and MemTR) are clear indication that they should not be taken as a panacea in PTC. Besides optimizing efficient PTC protocols through stringent choice of CKs, other associated physiological and metabolic events taking place in culture during the optimization process need more in-depth investigation. In addition to contributing towards the better understanding of the mode of action of these CKs, such an approach will help solve associated physiological and developmental problems in vitro.