Browsing by Author "Bairu, Michael Wolday."

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Characterisation of Taro (Colocasia Esculenta (L) Scott) germplasm collections in South Africa : towards breeding an orphan crop.
(2017) Jansen van Rensburg, Willem Sternberg.; Modi, Albert Thembinkosi.; Shanahan, Paul Edward.; Bairu, Michael Wolday.; Shimelis, Hussein Ali.
Amadumbe (Colocasia esculenta), better known as taro, is a traditional root crop widely cultivated in the coastal areas of South Africa. Taro is showing potential for commercialisation. However, very little is known about the genetic diversity, potential and its introduction and movement in South Africa. This study was undertaken to (1) determine the genetic diversity in the ARC taro germplasm collection using agromorphological characteristics and microsatellite markers, (2) to determine if it is possible to breed with local taro germplasm and (3) to determine the effect of four different environments (Roodeplaat, Umbumbulu, Owen Sithole College of Agriculture and Nelspruit) on ten agro-morphological characteristics of 29 taro landraces Taro germplasm was collected in South Africa in order to build up a representative collection. Germplasm was also imported from Nigeria and Vanuatu. The South African taro germplasm, and selected introduced germplasm, were characterised using agromorphological descriptors and simple sequence repeat (SSR) markers. Limited variation was observed between the South African accessions when agro-morphological descriptors were used. Non-significant variations were observed for eight of the 30 agromorphological characteristics. The 86 accessions were grouped into three clusters each containing 39, 20 and 27 accessions, respectively. The tested SSR primers revealed polymorphisms for the South African germplasm collections. Primer Uq 84 was highly polymorphic. The SSR markers grouped the accessions into five clusters with 33, 6, 5, 41 and 7 accessions in each of the clusters. All the dasheen type taro accessions were clustered together. When grown under uniform conditions, a higher level of genetic diversity in the South African germplasm was observed when molecular (SSR) analysis was performed than with morphological characterisation. No correlation was detected between the different clusters and geographic distribution, since accessions from the same locality did not always cluster together. Conversely, accessions collected at different sites were grouped together. There was also no clear correlation between the clustering pattern based on agro-morphology and SSRs. Thus, in order to obtain a more complete characterisation, both molecular and morphological data should be used. Although the results indicated that there is more diversity present in the local germplasm than expected, the genetic base is still rather narrow, as reported in other African countries. Fourteen distinct taro genotypes were planted as breeding parents and grown in a glasshouse. Flowering were induced with gibberellic acid (GA3). Crosses were performed in various combinations; however, no offspring were obtained. This might be due to the triploid nature of the South African germplasm. It might be useful to pollinate diploid female parents with triploid male parents or use advanced breeding techniques, like embryo rescue or polyploidization, to obtain offspring with the South African triploid germplasm as one parent. The triploid male parents might produce balanced gametes at low percentages, which can fertilize the diploid female parents. Twenty-nine taro accessions were planted at three localities, representing different agroecological zones. These localities were Umbumbulu (South of Durban - KZN), Owen Sithole College of Agricultural (OSCA, Empangeni, KZN) and ARC - Vegetable and Ornamental Plants (Roodeplaat, Pretoria). Different growth and yield related parameters were measured. The data were subjected to analysis of variance (ANOVA) and additive main effects and multiplicative interaction (AMMI) analyses. Significant GxE was observed between locality and specific lines for mean leaf length, leaf width, leaf number, plant height, number of suckers per plant, number of cormels harvested per plant, total weight of the cormels harvested per plant and corm length. No significant interaction between the genotype and the environment was observed for the canopy diameter and corm breadth. From the AMMI model, it is clear that all the interactions are significant for leaf length, leaf width, number of leaves on a single plant, plant height, number of suckers, number of cormels harvested from a single plant and weight of cormels harvested from a single plant. The AMMI model indicated that the main effects were significant but not the interactions for canopy diameter. The AMMI model for the length and width of the corms showed that the effect of environment was highly significant. There is a strong positive correlation between the number of suckers and the number of leaves (0.908), number of cormels (0.809) and canopy diameter (0.863) as well as between the number of leaves and the canopy diameter (0.939) and between leaf width and plant height (0.816). There is not a single genotype that can be identified as “the best” genotype. This is due to the interaction between the environments and the genotypes. Amzam174 and Thandizwe43 seem to be genotypes that are often regarded as being in the top four. For the farmer, the total weight of the cormels harvested from a plant will be the most important. Thandizwe43, Mabhida and Amzam174 seem to be some of the better genotypes for the total weight and number of cormels harvested from a single plant and can be promoted under South African taro producers. The local accessions also perform better than introduced accessions. It is clear that some of the introduced accessions do have the potential to be commercialised in South Africa. The study indicate that there are genetic diversity that can be tapped into for breeding of taro in South Africa. However, hand pollination techniques should be optimized. Superior genotypes within each cluster in the dendrograms as well as Thandizwe43, Mabhida and Amzam174 (identified by the AMMI analysis as high yielding) can be identified and used as parents in a clonal selection and breeding programme. Additionally, more diploid germplasm can be imported to widen the genetic base. The choice of germplasm must be done with caution to obtain germplasm adapted to South African climate and for acceptable for the South African consumers.
Characterization and control of micropropagation problems in aloe, devil's claw and banana.
(2008) Bairu, Michael Wolday.; Van Staden, Johannes.; Stirk, Wendy Ann.
The development of the science of micropropagation from the very initial concept of totipotency to the modern day advancement and sophistication has been affected by a wide range of problems such as hyperhydricity, shoot-tip necrosis and somaclonal variation. These problems are largely the result of the obvious fact of trying to grow plants in an environment that is different from the one plants are used to naturally. The extent of these problems ranges from minor technical inconvenience to significant economic loss. Characterization and control of micropropagation problems has been one of the priorities of plant tissue culture research due to the enormous contribution of this discipline for plant production, improvement and conservation. The prevalence and severity of these tissue culture problems varies widely among plant species. The rationale of this research project was therefore, to identify plant species most affected by the problems studied, characterize the problem and find mechanism(s) to control or minimize the damage caused by the problem. The literatures reviewed provide sufficient background information for the experimental chapters. Due to the different nature of the problems and variation in the plant species they affect, the model plant, the methodologies used and parameters analysed were also different. The findings of these investigations, in their own different way, addressed certain problems that individually and collectively pose difficulties to the micropropagation industry. The difference in the content of the experimental chapters is therefore the result of the broader objective of the research project to tackle such difficulties. The success and failure of tissue culture system greatly depends on the choice of PGR’s. This choice can be made based on comparative study of their biological activity. Some promising reports on the role of topolins in micropropagation led to the idea of testing these cytokinins for their potential in tissue culture. As a prerequisite to subsequent investigations, the biological activity of some selected topolins and BA derivatives was tested using the soybean callus bioassay. The activity of the cytokinins tested varied significantly. The results demonstrated that the structure of a cytokinin dictates its activity. Modifications of side-chain improved the activity of oT but had no effect on pT. The presence of the methyl group had an enhancing effect on cytokinin activity of topolins or at least it did not reduce it. BA derivatives BA9THP (conjugated at N9 position), 3FBA and 2Cl6(3OHBA)R (halogenated derivatives) also showed good cytokinin activity and hold good promise for future research. In an attempt to alleviate hyperhydricity in Aloe polyphylla and optimize the micropropagation protocol, meta-topolin and its derivatives were tested at various concentrations together with BA and zeatin. Of all the cytokinins tested mT produced the best results in terms of shoot and root growth. Five μM was found to be the optimum concentration at which complete control of hyperhydricity was achieved without compromising shoot and root growth. Plantlets rooted in a multiplication media. BA generally had a negative effect on growth and development both in vitro and ex vitro. Acclimatization of plantlets was achieved easily by initially transferring plantlets to a mist house (for three weeks) followed by transfer to the greenhouse. The type of cytokinin also had an effect on ex vitro growth with BA-treated plants producing the lowest shoot and root biomass. Various experiments were conducted to characterize and control factors affecting STN in Harpagophytum procumbens. Media type and strength, PGR, carbon sources, sub-culturing, calcium and boron were tested. Results indicated that all of the tissue culture components tested affected STN. From the different media types tested, half strength was MS found to be the preferred medium. Increasing cytokinin concentration increased the incidence of STN and the problem was aggravated by the addition of auxin to the multiplication medium. Optimum shoot multiplication was achieved by omitting auxin and using the cytokinin mTR. Plantlets produced basal callus which interfered with rooting. The quantity of this basal callus was minimum when mTR was used. Sub-culturing plantlets onto fresh medium every two weeks helped minimize STN. Off all the sugars tested 3% sucrose was optimum. Other sugars either aggravated STN or inhibited growth when compared at equi-molar concentration. Increasing the concentration of either Ca or B prevented the development of necrotic shoots. When the concentration of both elements is increased simultaneously negative effects on both growth and STN were observed. Using 6 mM Ca in half strength MS medium was optimum. B was toxic at higher concentrations. Plantlets rooted readily in half strength cytokinin-free MS media supplemented with 2.5 μM IAA. Rooted plantlets produced using the optimized protocol were acclimatized successfully by transferring directly to a greenhouse in a 1:1 ratio of sand and soil mixture. The effect of meta-toplins on micropropagation and somaclonal variation of banana was investigated. Tissue cultured explants of cultivars ‘Williams’ and ‘Grand Naine’ were cultured in MS media containing the cytokinins BA, mT, MemT, MemTR and mTR at various concentrations. Results of the investigation revealed that superior multiplication and lower abnormality index was recorded from the mTR and mT treatments at 22.2 μM concentration. These treatments, however, had an inhibitory effect on rooting. The effect of these treatments (22.2 μM mT and mTR) in comparison with equi-molar concentration of BA on somaclonal variation of ‘Williams’ banana was tested using RAPD-PCR at the 7th multiplication cycle. No significant difference was found between the treatments. It should however be highlighted that cultures were initially maintained for three multiplication cycles in media containing BA. The inherent stability and initial effect of BA could have influenced the results.
Plant growth regulators and somaclonal variation in Cavendish banana (Musa AAA cv. Zelig)
(2004) Bairu, Michael Wolday.; Van Staden, Johannes.; Fennell, Catherine W.
Cavendish bananas are the most important sub-group of all bananas. They includes more than 30% of the global banana production and almost all bananas exported are of the Cavendish type. This sub-group is also an important food source. Most of the fruit is consumed locally, such that only 35% enters the international market. To meet the regular demand for domestic consumption and market supply there must be a reliable production strategy. The technique of tissue culture is a better option than conventional propagation techniques. However. the high incidence of somaclonal variation among plants derived from tissue culture is a problem for commercial producers. Several factors such as genotype, tissue source, duration in culture, and the tissue culture technique employed, cause somaclonal variation. The impact of plant growth regulators on somaclonal variation was studied on Cavendish banana cv. 'Zelig' obtained from African Biotechnologies Ltd., South Africa. In vitro grown plants at the 4th multiplication cycle were supplied for the investigation. The first component of the investigation dealt with the effect of types of plant growth regulators. Combinations of the auxins IAA, IBA and NAA with the cytokinins BA and TDZ were used to culture the plants for ten multiplication cycles. Plants were then randomly selected to collect leaf material for DNA extraction and RAPD analysis. The second aim of this study was to investigate the effect of the cytokinin BA on plantlets subcultured over 5-10 multiplication cycles. The auxin IAA at the concentration of 2 mgl-1 was combined with BA at concentrations of 2.5, 5.0 and 7.5 mgl-1. Plants were analyzed at each level of subculture from the 5th to the 10th cycle for respective cytokinin concentrations. Plants were then randomly selected for the collection of leaf material for DNA extraction and RAPD analysis. DNA was extracted from leaf tissue of in vitro grown plants using a modified CTAB extraction procedure. DNA amplification products were scored for the presence and absence of bands in a particular locus. Results were clustered according to their similarities. The relationship between multiplication rate and variation was assessed using correlation analysis. Results of the investigation showed that treatments with higher multiplication rates produced higher rates of variation. A variation rate of 55% was recorded for treatments containing IAA and BA. A higher rate of variation (72%) was identified in the treatment with IAA (2mgl-1) plus BA (7.5 mgl-1) over 10 cycles. In all cases the dwarf off-type was the most common type of variant obtained, contributing 87.7% of the total amount of variation. The dwarf specific marker (OPJ-041500) reported previously in Williams Cavendish was identified in cv. 'Zelig' in this study. Another band similar in size was amplified by primer OPC-15 and named OPC-151500 . This band consistently appeared in all the normal plants and was absent in all the dwarf types and hence could be used as a dwarf specific marker.
Propagation of Romulea species.
(2012) Swart, Pierre Andre.; Van Staden, Johannes.; Kulkarni, Manoj G.; Bairu, Michael Wolday.; Finnie, Jeffrey Franklin.
Romulea is a genus with numerous attractive and endangered species with horticultural potential. This genus in the Iridaceae has its centre of diversity in the winter-rainfall zone of South Africa. This thesis uses ecophysiological and biotechnological techniques to investigate the physiology behind the propagation of some species in this genus. The ecophysiological techniques of soil sampling and analysis and germination physiology were used to determine the natural and ex vitro growth and development requirements of these plants, while biotechnological techniques are used to determine the in vitro growth and development requirements of these plants and to increase the rate of multiplication and development. Soil sampling and analysis revealed that R. monadelpha and R. sabulosa, two of the most attractive species in the genus, grow in nutrient poor 1:1 mixture of clay and sandy loam soil with an N:P:K ratio of 1.000:0.017:0.189 with abundant calcium. To investigate the physical properties of the seeds, imbibition rate, moisture content and viability of seeds were determined. The seed coat and micropylar regions were examined using scanning electron microscopy. To test for suitable stimuli for germination, the effect of temperature and light, cold and warm stratification, acid and sand paper scarification, plant growth promoting substances, deficiency of nitrogen, phosphorous and potassium, and different light spectra (phytochromes) on germination were examined. An initial germination experiment showed germination above 65% for R. diversiformis, R. leipoldtii, R. minutiflora and R. flava seeds placed at 15°C; while seeds of other species placed at 15°C all had germination percentages lower than 30%. More extensive germination experiments revealed that R. diversiformis and R. rosea seed germinate best at 10°C, R. flava seed germinates best when cold stratified (5°C) for 21 days and R. monadelpha germinates best at 15°C in the dark. Seeds of R. diversiformis, R. flava, R. leipoldtii, R. minutiflora, R. monadelpha and R. sabulosa seem to all exhibit non-deep endogenous morphophysiological dormancy while seeds of R. camerooniana and R. rosea appear to have deep endogenous morphophysiological dormancy. The suitability of various explant types and media supplementations for culture initiation was examined for various species of Romulea. Both embryos and seedling hypocotyls can be used for R. flava, R. leipoldtii and R. minutiflora in vitro shoot culture initiation. R. sabulosa shoot cultures can only be initiated by using embryos as explants, because of the lack of seed germination in this species. Shoot cultures of R. diversiformis, R. camerooniana and R. rosea could not be initiated due to the lack of an in vitro explant shooting response. Shoot cultures can be initiated on media supplemented with 2.3 to 23.2 M kinetin for all species that showed an in vitro response. The most suitable concentration depended on the species used. Some cultures appeared embryogenic, but this was shown not to be the case. A medium supplemented with 2.5 M mTR is most suitable for R. sabulosa shoot multiplication. BA caused vitrification of shoots in all the experiments in which it was included and is not a suitable cytokinin for the micropropagation of these species. The effect of various physical and chemical parameters on in vitro corm formation and ex vitro acclimatization and growth was examined. Low temperature significantly increased corm formation in R. minutiflora and R. sabulosa. A two step corm formation protocol involving placing corms at either 10 or 20°C for a few months and then transferring these cultures to 15°C should be used for R. sabulosa. When paclobutrazol and ABA were added to the medium on which R. minutiflora shoots were placed, the shoots developed corms at 25°C. This temperature totally inhibits corm formation when these growth retardants are not present. BA inhibited corm formation in R. leipoldtii. Corms can be commercialized as propagation units for winter-rainfall areas with minimum temperatures below 5°C during winter. Although an incident of in vitro flowering was observed during these experiments, these results could not be repeated. Although none of the corms or plantlets planted ex vitro in the greenhouse survived, a small viability and an ex vitro acclimatization experiment shows that the corms produced in vitro are viable. One embryo of the attractive R. sabulosa, produces 2.1 ± 0.7 SE shoots after 2 months; subsequently placing these shoots on a medium supplemented with 2.5 μM mTR for a further 2 months multiplies this value by 5.5 ± 1.3 SE. Each of these shoots can then be induced to produce a corm after 6 months. This means that 1 embryo can produce about 12 corms after 10 months or about 65 corms after 12 months (if shoots are subcultured to medium supplemented with 2.5 μM mTR for another 2 months). Embryo rescue can enable wider crosses within this genus. These results can be used for further horticultural development of species in this genus and their hybrids and variants.
The role of meta-topolins on the physiology of micropropagated 'Williams' bananas (Musa spp. AAA)
(2012) Aremu, Adeyemi Oladapo.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Bairu, Michael Wolday.
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.
Screening acacia mearnsii (black wattle) seedlings for frost tolerance using an artificial frost technique, combined with proteomic analysis.
(2023) Jugmohan, Mayuri.; Laing, Mark Delmege.; Bairu, Michael Wolday.; Chan, Julian Moreno.
Acacia mearnsii de Wild. (commonly known as black wattle), was first introduced to South Africa in the 1800’s. Today, it is one of the leading commercially grown forestry crops in South Africa due to its usefulness and versatility. Black wattle is a source of high-quality tannin and raw material for wood pulp. It is also excellent for building and as a source of firewood. These uses have contributed to its popularity as a crop for both commercial farmers and small growers. Since its introduction to South Africa, abiotic stresses such as frost damage have affected the silviculture of black wattle, and this has resulted in major financial losses for the forestry industry. Over several decades, breeding research has been conducted with the aim of developing genetically improved seed for frost prone areas. However, this has yet to be achieved. Screening for frost tolerance in black wattle has mainly been conducted using field trials. While this provides the most realistic means of screening, it has several challenges. Field trials are time consuming, expensive and require much effort. Furthermore, frost events are unpredictable in timing and in magnitude, thus affecting frost damage screening trials, with levels of zero to extreme frost occurring in any one year at a selected site. This does not allow for a productive plant breeding program to consistently screen for frost tolerance. The current study focussed on the development of an artificial frost screening method for black wattle. A protocol comprising of eight days of moderately cold temperatures (adaptation) and one day of extremely cold temperature (frost tolerance) was established using a temperature-controlled chamber. The frost damage that resulted from the implementation of this protocol produced similar levels of damage as those experienced in previous field trials. This protocol was thereafter tested in two separate trials involving 100 families of wattle accessions that had previously been ranked in field trials that had been run in frost prone areas. A weak correlation was observed between the results of the artificial frost screening trials and the corresponding field results (r=0.24 to 0.28, rs=0.20 to 0.28). Kruskal-Wallis tests showed a statistical similarity between the medians of one of the artificial frost screening trials and the field frost damage evaluations, and a significant difference between the medians of the two artificial frost screening trials that were conducted. The understanding of the molecular aspects that contribute towards frost tolerance in black wattle is extremely limited. Several studies involving a molecular approach to understanding this trait in other woody species have shown promising results. Frost tolerance is a multigenic trait and therefore a proteomic approach was chosen as the best option to identify biomarkers associated with it. Protein extraction from black wattle has not been previously conducted and therefore a protocol that dealt with the interference of phenolics and that was compatible with downstream proteomic techniques was developed. During the protein extraction protocol development, three different protein extraction methods were compared. These methods differed in terms of their precipitation agent combinations. These combinations included acetone and methanol, phenol and ammonium acetate and ammonium acetate and methanol. The combination of phenol and ammonium acetate produced the highest protein yield, as well as the most distinct protein spots after separation by two-dimensional gel electrophoresis. This method was used to extract proteins from 40 black wattle families with varying levels of frost tolerance, before and after cold stress treatment. These extracted proteins were thereafter separated using two-dimensional gel electrophoresis so that changes in protein expression as a result of cold exposure could be analysed. Multivariate analyses of the proteomic data revealed that six proteins were upregulated in frost-tolerant black wattle families. The identified proteins were: two isoforms of oxygen-evolving enhancer protein 1, probable 1-acyl-sn-glycerol-3-phosphate acyltransferase 4, ribulose bisphosphate carboxylase/oxygenase activate, chaperonin 60 subunit alpha 1 and stromal 70 kDa heat shock-related protein. After identification by mass spectrometry, it was established that these proteins have previously been shown to contribute to the protection of cellular membranes, maintenance of photosynthetic processes and the prevention of protein misfolding and aggregation. These proteomic functions have been observed in previous studies to be associated with the process of cold acclimation in plants and thus seem to play a role in frost tolerance in black wattle. The establishment of an artificial frost screening protocol and the proteomic profiling of black wattle for frost tolerance are important tools for preliminary screening of families prior to field trials. By using these protocols fewer black wattle families will require field testing. This will be beneficial both in terms of cost, effort and time associated with field trials. The development of artificial methods for the induction of stresses and the proteomic changes that result from these are valuable tools for understanding the mechanisms that plants use to cope with abiotic and biotic threats.