Browsing by Author "Snyman, Sandra Jane."

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Analysis of historical flowering data, investigations into aspects of pollen biology and selected biotechniques to complement sugarcane breeding in South Africa.
(2016) Mhlongo, Nonsikelelo Yvonne.; Snyman, Sandra Jane.
Abstract available in PDF file.
Development of in vitro culture and gene transfer techniques in sugarcane (Saccharum species hybrids).
(1992) Snyman, Sandra Jane.; Huckett, Barbara Isobel.; Watt, Maria Paula Mousaco Deoliveira.
In vitro cell and tissue culture systems were developed for sugarcane in order to utilise current transformation techniques to introduce genes to South African sugarcane varieties, which would be difficult, if not impossible to achieve in conventional breeding programmes. Embryogenic calli were initiated in the dark from stem explants of sugarcane varieties NCo376 and N13, on a MS medium containing sucrose (20-50 g/l), 2,4-D (2-4 mg/l), casein (1 g/l), inositol (100 mg/l) and agar (9g/l). After 2 months the somatic embryos were cultured in a light/dark photoperiod for a further 2 months. The best combination of sucrose and 2,4-D for callus initiation, and subsequent plant regeneration, was 20 g/l and 2 mg/l, respectively. Plant yields ranged from 16 to 36 plants per gram fresh weight callus, and the yields were not significantly increased by the addition of activated charcoal to the regeneration medium. When plantlets reached a height of 10 cm, they were transferred to autoclaved soil in pots, hardened-off and placed in the glasshouse. Suspension cultures were initiated from friable NCo376 calli in liquid MS medium shaken at 100 rev/min in the dark at 27°C, and were subcultured every 3-7 days. Protoplasts from various sources (leaf, calli and suspension cultures) were obtained after enzymatic digestion in cellulase (20-30 g/l), macerozyme (0,2 g/l), hemicellulase (5 g/l), and sorbitol (0,55 M) in a calcium and magnesium salt solution. Protoplasts cultured for 48 h resulted in a loss in viability of 84%. The potential of the seed as a recipient for direct gene uptake was investigated, as this eliminated the need for in vitro culture and plant regeneration. Uptake of [3H] pBR322 DNA by seeds was demonstrated, and seeds with the testa removed exhibited higher initial uptake rates than those with intact seed coats. However, transient expression, using the GUS reporter gene (coding for bacterial B-glucuronidase) carried on plasmid pBI221, could not be conclusively shown using the histochemical GUS assay, due to GUS activity generated by either microbial contamination or endogenous plant GUS activity. Neither microwaving to eradicate contaminants nor the addition of methanol (20%) to the GUS incubation buffer were successful in overcoming positive results observed in control seeds. An alternative approach to sugarcane transformation, using PEG-mediated DNA uptake and subsequent transient expression of GUS by protoplasts was investigated, but microbial contamination was a persistant problem and no positive results were observed. Further examination and elimination of endogenous contamination is required before transformation studies can be continued.
The establishment of in vitro screening methods for evaluating the susceptibility of sugarcane (Saccharum spp. hybrids) to the fungal disease, smut (causal agent : Ustilago scitaminea H. and P. Sydow) and the stalk borer, Eldana saccharina Walker (Lepidoptera : Pyralidae).
(2010) Devnarain, Natrisha.; Snyman, Sandra Jane.; Hunter, Charles Haig.
The fungal disease smut (causal agent: Ustilago scitaminea H. & P. Sydow) and stalk borer Eldana saccharina Walker place major constraints on sugarcane agriculture in South Africa. The best approach for management is the introduction of resistant cultivars; however, conventional field-based screening for pest and disease resistance is a lengthy process. This study evaluated in vitro techniques combined with artificial inoculation of 12 week old in vitro plantlets and 8-10 week old embryogenic calli as rapid screening methods. Preliminary investigations were conducted on cultivars with known field ratings to smut and E. saccharina: NCo376, N26 and N39; and 5 „test‟ cultivars, whose identities were undisclosed until completion of experiments, were used to assess the accuracy of protocols. Infective U. scitaminea sporidia generated from teliospores, were used as inocula. Development of a callus protocol was unsuccessful due to sporidial and mycelial overgrowth, despite addition of a contact fungicide, Dithane M-45® (0.025 g/l) and a biocide/fungicide, PPMTM (5 ml/l), to media. Plantlet inoculation by injection, 1 cm above the apical meristem, resulted in 12% and 20% of smut susceptible NCo376 plantlets producing smut whips after 5 weeks when inoculated with 1 x 106 and 1 x 109 sporidia/ml, respectively. Smut whip production in 5 of the 8 (63%) cultivars inoculated with the lower sporidial concentration correlated with their field resistance ratings. In addition, whips harvested from in vitro plantlets were a valuable source of aseptic teliospores for future research. Ongoing work involves inoculation of NCo376 calli with such teliospores and maintenance on medium with PPMTM - emergence of whips from plantlets remains to be assessed. The E. saccharina screening protocol involved surface decontamination of eggs with 1% sodium hypochlorite (NaOCl) for 15 min. Feeding bioassays were conducted by placement of first instar larvae on in vitro plantlets and calli for 3 and 2 weeks, respectively. Larval mass, length and percentage infestation were recorded. Although greater larval size was expected in susceptible compared with resistant cultivars, the results did not support this. Significant differences in plantlet infestation were observed between susceptible (94-98%) and resistant (72-86%) lines. No significant differences were found in the callus feeding bioassay. However, a 24 h callus choice bioassay which investigated larval preference between callus genotypes compared with NCo376, showed significant differences and correctly discerned cultivar susceptibility according to field ratings.
Establishment of in vitro shoot multiplication and short-term embryo storage protocols for single genetic modification events in sugarcane.
(2013) Mupanehari, Edwin.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandra Jane.
Sugarcane (Saccharum spp. hybrids) is an important economic crop that accounts for more than 80% of world sugar production. Genetic improvement and selection to produce cultivars with traits of interest is difficult in sugarcane because of its complex polyploid and aneuploid genome. Genetic transformation and in vitro mutagenesis techniques are, therefore, being investigated for such purposes. However, it takes a long period to multiply plants regenerated from such events as: 1) the in vitro plants have to establish well in vitro; 2) then they need to acclimatize in the greenhouse and 3) then multiplied using setts, before they can be tested for traits of interest. Furthermore, once a plant is lost in culture, the whole genetic event is lost, which can occur due to lack of labour resources at the time of subculture. The aim of this study was, therefore, two-fold: 1) to develop a protocol to multiply single shoots regenerated from single transformation or mutation events in vitro; and 2) to establish a strategy for short term storage of somatic embryos developed from such events. Preliminary investigations were undertaken using sugarcane varieties NCo310 and NCo376. Two approaches were employed for shoot multiplication, viz. either multiply the shoots when they were well developed or multiply them immediately after embryo germination. In the former, shoots (2 cm in height), produced via indirect somatic embryogenesis, were cultured on six different media, each containing full strength MS salts and vitamins, 20 g l-1 sucrose and different combinations and concentrations of plant growth regulators (PGRs), viz. M1 (no PGRs), M2 (0.1 mg l-1 BAP and 0.015 mg l-1 kinetin), M3 (6 mg l-1 BAP and 1 mg l-1 kinetin), M4 (0.5 mg l-1 BAP and 0.25 mg l-1 kinetin), M5 (1 mg l-1 BAP, 0.1 mg l-1 kinetin and 1 mg l-1 NAA) and M6 (1 mg l-1 IBA, 1 mg l-1 kinetin and 0.5 mg l-1 GA3). The shoots were subcultured two or three times onto same medium. At 6 w, the total shoot yield (no. shoot/original shoot explant) was significantly highest on medium M5 for both NCo376 (12.7 ± 4.0) and NCo310 (7.2 ± 3.4). Similarly, this medium resulted in the highest total number of shoots per embryo at 6 w (11.0 ± 2.0) when applied to germinating embryos of NCo376. Modifications of this medium were investigated but none was found to be better than M5 (P < 0.05). After multiplication, the shoots were transferred to rooting liquid medium containing ½ strength MS salts and vitamins, 20 g l-1sucrose and 1 mg l-1 IBA. More than 80% of the shoots from each medium rooted within 3 w. The developed protocol was then applied to varieties N41, N50 and transgenic lines of NCo376. The results confirmed that M5 can be used for high yielding shoot multiplication for those varieties producing 6.6 ± 0.9 and 4.3 ± 1.3 shoots per shoot at 6 w for N41 and N50, respectively. Subculturing for a further 2 w increased the shoot yields to 18.6 ± 2.3 for N41 and 8.0 ± 0.3 for N50. Transgenic shoots multiplied using the developed protocol, were used to investigate the stability of the transgene in in vitro culture. This was done by testing for the presence of the gene in those shoots using end point PCR. The results showed the presence of the transgene in all the transgenic shoots indicating the protocol did not have a negative effect on the stability of the transgene. To establish a protocol for slow growth storage of somatic embryos, mature embryos of variety NCo376 were encapsulated in alginate beads and placed on semi-solid medium containing ½ strength MS salts and vitamins, 5 g l-1 sucrose and 9 g l-1 agar. The cultures were kept in the dark at room temperature for a month and at 18 oC for 1, 2 and 3 months. The embryos were assessed for germination capacity at the end of each period by transferring them to medium containing full strength MS salts and vitamins, 20 g l-1 sucrose, 0.5 g l-1 casein hydrolysate and 8 g l-1 agar. Embryos that were stored at 18 oC for 1 month had the highest survival percentage (66 ± 5.7% germination) compared with the other treatments and control (53.3 ± 6.7% germination).
Field assessment of agronomic traits and in vitro acetolactate synthase characterisation of imazapyr herbicide tolerant sugarcane.
(2013) Maphalala, Kwanele Zakhele.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandra Jane.; Rutherford, Richard Stuart.
Weed control is a major cost for growers in the sugarcane industry, especially for monocotyledonous species such as Cynodon and Rottboellia spp. The introduction of imazapyr-tolerant sugarcane would be advantageous as this herbicide has shown to be effective against the above-mentioned weeds but it also kills sugarcane. In a previous study in our laboratory, several sugarcane putative-mutant lines of variety N12 were generated by in vitro exposure of embryogenic callus to 16 mM ethyl methanesulfonate (EMS), followed by selection on imazapyr-containing medium. Tolerance to a low dose of imazapyr was confirmed in seven of those lines when the herbicide was applied (182 g a.i. ha-1) to 3 month-old plants in pots. The aim of the present study was to identify which of the seven herbicide mutant lines had agronomic characteristics at least equivalent to un-mutated N12. The objectives were to: 1) confirm tolerance to increased rate (312 and 625 g a.i. ha-1) of imazapyr in field plants; 2) measure the agronomic characteristics of these lines; 3) determine the effect of residual soil herbicide activity on germination of sugarcane setts. The seven mutant lines (Mut1-Mut7) and un-mutated N12 were clonally propagated in vitro by shoot multiplication followed by rooting and planted in three plots (untreated, sprayed with 312 or 625 g a.i. ha-1 imazapyr), in the field, in a randomized complete block design. In the untreated control plot there were no significant differences between the control and the mutant plants for agronomic traits (tiller number/plot, stalk height and stalk diameter) or estimated yield (kg/plot) after 10 months, indicating that the mutation process had no effect on general plant phenotype. In the sprayed (312 and 625 g a.i. ha-1) plots, Mut1, Mut4, Mut5, Mut6 and Mut7 plants showed tolerance to imazapyr as the leaves remained green compared with Mut2, Mut3 and N12 control plants, which displayed chlorotic leaves and eventually died in the plot sprayed with 625 g a.i. ha-1. Post-herbicide application, the yields of Mut5, Mut6 and Mut7 (52.33, 43.43 and 41.43 kg/plot, respectively) from the 312 g a.i. ha-1 plot were not significantly different from that of N12 control (53. 61 kg/plot) in the untreated plot. However, in the 312 g a.i. ha-1 plot, the yield and agronomic trait measurements of the untreated N12 control were significantly higher than those of the herbicide-susceptible plants Mut2 and Mut3. Similarly, in the 625 g a.i. ha-1 plot, the recorded yields for Mut4, Mut6 and Mut7 were 41.60, 43.44 and 36.30 kg/plot, respectively, indicating that their imazapyr tolerance and yield characteristics were comparable to the untreated N12 control. Imazapyr is conventionally applied to a fallow field 3-4 months prior to planting sugarcane as there is residual herbicide activity in the soil that suppresses sugarcane germination and growth. Therefore, in order to establish if the herbicide-tolerant mutants could germinate in iii an imazapyr-treated field, 3-budded setts of the mutant lines (Mut1-Mut7) and N12 control were planted in two plots, one unsprayed and one sprayed with 1254 g a.i. ha-1 imazapyr, 2 weeks previously. Germination was calculated after 3 weeks as the number of germinated setts in each plot/no. germinated setts in unsprayed plot x100. In the sprayed plot, the setts from Mut1, Mut4 and Mut6 displayed the highest germination percentages (60, 71 and 74%, respectively) compared with Mut2 (24%), Mut3 (46%), Mut5 (34%), Mut7 (40%) and the N12 control (12%). The in vitro acetolactate synthase (ALS) enzyme activity of 10 month-old plants from the untreated plot was assessed in the presence of 0-30 μM imazapyr to determine the herbicide concentration that inhibited ALS activity by 50% (IC50). The IC50 values for the mutated lines were between 3 and 30 μM, i.e. 1.5-8.8 times more tolerant to imazapyr than the N12 control plants, with Mut6 displaying the highest IC50 value (30 μM). On the basis of the results, it was concluded that Mut1, Mut6 and Mut7 lines were more tolerant to imazapyr than N12 and the other tested lines. Future work includes phenotypically assessing these lines for traits including sucrose content, fibre content, actual yield (tons cane ha-1) and altered pest and disease resistance. Once isolated and sequenced, the ALS gene conferring imazapyr tolerance can be used in genetic bombardment in the genetic modification approach as the gene of interest or as a selectable marker. In addition, the imazapyr-tolerant line can be used for commercial purposes in the field and as the parent plant in the breeding programme.
Field evaluation and characterisation of the mode of imazapyr tolerance in three mutant sugarcane genotypes.
(2016) Singh, Varnika.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandra Jane.; Rutherford, Richard Stuart.
Abstract available in PDF file.
In vitro culture and genetic transformation of selected ancestral and commercial sugarcane germplasm.
(2013) Pillay, Ellisha.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandra Jane.
Sugarcane is an economically important crop and its high demand has necessitated the use of biotechnology methods to produce and accelerate the production of desirable genotypes. One such method is genetic transformation. However, as sugarcane is a highly polyploid crop, which originated from interspecific crosses between Saccharum spontaneum and S. officinarum, efforts to transform it are inhibited by transgene promoter silencing. As ancestral lines have a simpler genetic makeup than modern varieties, they may be useful to test promoter function. Intrinsic to the generation of transgenic plants is the ability to produce plants from specific species and varieties, for which an indirect method of regeneration is needed. Consequently, the first objective of this study was to determine a high yielding protocol for somatic embryogenic calli. The second was to transform such calli and produce regenerated plants to assess transgene expression. A preliminary study was conducted using eight ancestral varieties to determine which were the most responsive in culture. Leaf roll disks were cultured on 5 mg.1¯¹ 2, 4-D and callus production was assessed. Based on these results and the availability of plant material, S. spontaneum Nigeria 1, S. spontaneum Nigeria 2, S. spontaneum Coimbatore, S. officinarum NG 77-69, and S. officinarum Black Cheribon and the commercial polyploid variety NCo376 were selected and tested on 11 different callus induction media. The S. spontaneum variety that generated the highest percentage of leaf disks that produced callus and plant yield was Nigeria 1 (61 % and 259 plants/10 disks, respectively), whilst the S. officinarum variety was Black Cheribon (75 % and 90 plants/10 disks, respectively). The best media for both comprised of MS salts and vitamins, 20 g.1¯¹ sucrose, 0.5 g.1¯¹ casein hydrolysate 5 mg.1¯¹ 2, 4-D and 8 g.1¯¹ agar. NCo376 produced the most amount of callus (93 %) when cultured on media containing 3 mg.1¯¹ 2, 4-D and gave a final yield of 450 plants/10 disks. Based on the yields obtained above and the availability of plant material, the varieties S. spontaneum Nigeria 1 and S. officinarum NG77-69 were selected for genetic transformation studies. Calli of these varieties as well as that of NCo376 were microprojectile bombarded with either pEmuKN + pAHC27 or pEmuKN + pR₁₁F¯. Following bombardment, the calli were cultured onto paromomycin-containing (1 ml.1¯¹) selection media and regenerated plants were obtained after 8-12 weeks. Transgene integration into the plant genome was assessed using PCR and qPCR techniques, and indicated that all NCo376 plantlets contained the GUS and npt II transgenes. However, only 4 out of 5 and 2 out of 3 S. officinarum NG77-69 plants transformed with pAHC27 and pR₁₁F¯- respectively, and 6 out of 10 S. spontaneum Nigeria 1 plants transformed with pR₁₁F¯- contained these transgenes. The transformation efficiencies achieved for NCo376, for the constructs pAHC27 and pR₁₁F¯- was 0.27 and 0.33 transgenic plants/blast, respectively. For NG77-69 it was 0.27 and 0.13 transgenic plants/blast, whilst that of Nigeria 1 was 0.20 and 0.40 transgenic plants/blast. Stable transgene expression in acclimatized plants was then assessed using a histochemical GUS assay and none of the plants expressed the GUS gene.
In vitro generation of somaclonal variant plants of sugarcane (Saccharum spp. hybrids) for tolerance to toxins produced by Fusarium sacchari.
(2012) Mahlanza, Tendekai.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandra Jane.; Rutherford, Richard Stuart.
The fungus Fusarium sacchari (Butler) Gams causes stem rot in sugarcane especially in association with the stem borer Eldana saccharina Walker. Sugarcane plants tolerant to F. sacchari PNG40 were obtained by chemical mutagenesis and in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF), followed by selection in the greenhouse. Somaclonal variants tolerant to F. sacchari PNG40 CF were established by treatment of calli with ethyl methanesulphonate (EMS) and various selection treatments. Investigations were conducted to test the effect of varying CF concentrations and the culture developmental stages (embryo maturation, embryo germination and plantlets) that were most effective in screening calli and plants. Incorporation of CF (0-100 ppm) in the media, at either embryo maturation or germination stages, resulted in significant callus necrosis, and consequent decreased plantlet yield. The highest callus necrosis of 95.55 ± 0.9 % and the lowest plant yield of 1.4 ± 0.45 plants/0.2 g were obtained after inclusion of 100 ppm CF in the germination medium compared with 61.5 ± 3.8 % and 43.8 ± 5.6 plants/0.2 g in the maturation medium, respectively. Exposure of whole plants with trimmed roots to 0-1500 ppm CF resulted in inhibition of root re-growth, with the 1500 ppm CF treatment having the greatest negative effect. Subsequent treatments involved immersing in vitro plantlets in varying concentrations of F. sacchari conidial suspensions. This resulted in 33.3 % and 100 % mortality with 103 and 105 conidia/ml treatments, respectively. Control and EMS-treated calli and potentially tolerant regenerated plants were selected using the established CF and inoculation treatments. Plants from EMS treatments displayed more varying root length. More plants with increased root growth, in the presence of CF, were produced from these treatments than from non-EMS treatments, indicating the ability of EMS to induce somaclonal variation. These putative tolerant plants were inoculated with PNG40 and those selected using CF in vitro were symptomless whilst the positive controls (plants unexposed to CF) were symptomatic. Re-isolation of Fusarium from the inoculated plants and identifying isolates as PNG40 using ISSR analysis confirmed tolerance of the asymptomatic plants and the fungus as the causal agent of the observed symptoms. This confirmed that tolerance to CF correlates to tolerance to F. sacchari PNG40. Future work includes testing stability of tolerance in the field and after sexual reproduction, and use of this protocol to produce plants that permit endophytic PNG40 colonisation towards biological control of E. saccharina.
The interaction between endophytic Fusarium species and Eldana saccharina (Lepidoptera) following in vitro mutagenesis for F. sacchari tolerance to control the borer in sugarcane.
(2015) Mahlanza, Tendekai.; Rutherford, Richard Stuart.; Snyman, Sandra Jane.
Eldana saccharina is a major pest in the South African sugar industry. Stalk damage by this borer and infection of bored tissue by opportunistic fungi result in loss of biomass and sucrose content, respectively. Amongst integrated management approaches, the best is employing E. saccharina-resistant genotypes. Resistance is attributed to physical stalk traits that impede boring and biochemical defences via nitrogen-based antiherbivory compounds. Further, in vitro assays have shown that Fusarium strains may be beneficial (e.g. F. pseudonygamai SC17) or antagonistic (e.g. F. sacchari PNG40) to the insect. The first objective of this study was, therefore, to establish the effect of sugarcane stalk traits and infection by Fusarium spp. on resistance to E. saccharina. In the first of two glasshouse trials, mature and immature stalk internodes of seven cultivars of known E. saccharina resistance ratings were inoculated with 2nd instar larvae via nodal wounds. Stalk rind hardness was greatest in both mature (42.2 units) and immature internodes (25 units) of the resistant cultivar N33. The softest of both mature and immature stalk regions were from the very susceptible N11 (32 units) and susceptible NCo376 (17.7 units), respectively. Percent fibre content in mature internodes was highest in the resistant N33 and N17 (12.8 - 14.2%) and lowest in the susceptible N11 and NC0376 (10.9 - 11.2%) cultivars. In all but one cultivar, % nitrogen content/dry mass was higher in immature internodes (0.65 - 1.2 %) than mature ones (0.36 - 0.91%) and lower in stalks of the resistant N41, N29 and N33 (0.36 - 0.75%) than in those of the susceptible NCo376 and N41 (0.48 - 1.27%) cultivars. Damage and mass gain by larvae retrieved from stalks were not entirely consistent with the cultivar resistance ratings, probably because the inoculation method by-passed the rind; N29 and N33 were unaffected by lack of rind protection. Hence, the tested stalk traits may contribute to E. saccharina resistance to varying extents in different sugarcane cultivars. In another trial, immature and mature stalks of NCo376 and N41 were inoculated with SC17 and PNG40 and then with E. saccharina larvae. The stalk area discoloured by Fusarium infection was smaller in the immature (6.1 - 7.1 cm²) than the mature (12.3 – 17.8 cm²) internodes. The smallest stalk length bored was in PNG40-infected NCo376 (3.3 cm) and N41 (1.7 cm) mature internodes, whilst NCo376 stalks colonised by SC17 (8.2 cm) were the most damaged. Hence, the proposal that Fusarium strains affect E. saccharina differently thereby impacting cultivar resistance/susceptibility to the borer, is supported. The in vivo activity of F. sacchari PNG40 against E. saccharina was also established, corroborating its potential as a biological control agent against the borer. As this application of PNG40 is impeded by the fungus being the causal agent of Fusarium stem rot in sugarcane, F. sacchari-tolerant plants were then produced via induced mutagenesis. Embryogenic calli of NCo376 and N41 were exposed to 32 mM ethyl methanesulphonate (EMS) for 4h. They were then placed on 100 ppm F. sacchari PNG40 culture filtrate (CF) at embryo maturation, germination or both stages, where 30.7 - 86.9% of the calli became necrotic and plantlet yield decreased by 59.2 - 99.2%. Roots of the regenerated plants were trimmed and placed on 1500 ppm CF. Plantlets with roots that regrew on CF medium beyond the 10 mm established threshold were deemed putatively tolerant (26.6 – 47.6% for EMS treatments, 5-24% for controls). These plants were acclimated and inoculated with PNG40 in the glasshouse. After 8 weeks, absence of symptoms, low lesion severity, re-isolation of PNG40 from the lesion and molecular identity of the isolates, confirmed some as PNG40 resistant. Re-isolation of PNG40 from undamaged tissue above the lesion, in plants with low lesion severity and no symptoms, confirmed endophytic colonisation and tolerance to the fungus in the mutants. Polymorphisms were detected in some mutants, using 24 RAPD primers. The use of the tolerant mutants in F. sacchari PNG40-mediated control of E. saccharina was then investigated. Stalks of five tolerant mutants and parents of each NCo376 and N41 cultivars were inoculated with PNG40 and with E. saccharina larvae, 3 weeks later. The length bored was less (1.0 - 4.7 cm) in stalks of PNG40 infected-mutants and parents than in the controls (3.9 - 9.0 cm). However, the % stalk discoloured area due to PNG40 infection was less in the mutants (10.6 - 22.0%) than in the parents (N41 - 28.9% and NCo376 - 30.2%). Re-isolation of PNG40 from undamaged tissue, within the inoculated internode and that above it, confirmed endophytic colonisation and fungal spread across internodes. Amongst stalks inoculated with PNG40, one mutant of NCo376 and two of N41 displayed limited boring (1 - 2 cm) and % discoloured area (10.6 - 15.1%), and the highest % of endophytically colonised stalk sections (50 - 75%) in the internodes immediately above those inoculated. There were no differences between the mutants and their respective parents in stalk rind harness, fibre and nitrogen contents. This work, therefore, resulted in the production of F. sacchari-tolerant mutants, demonstrated the toxicity of F. sacchari PNG40 against E. saccharina in vivo, and the ability of the PNG40-tolerant mutants to support endophytic colonisation by the fungus. Demonstration of these Fusarium - E. saccharina interactions in the mutants under field conditions will lead to the application of biological control of E. saccharina using PNG40, as part of integrated management approaches for the pest.
Technique establishment for in vitro selection of drought tolerant sugercane genotypes.
(2015) Mhlanga, Philisiwe Felicity.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandra Jane.
There is a need to have constant supply of sugarcane varieties adapted to different South African regions. However, the genetic improvement and selection of sugarcane cultivars with superior traits, e.g. drought tolerance, are difficult due to its complex polyploid and aneuploid genome. Biotechnology approaches are being investigated for the selection and/production of drought tolerant cultivars. Towards this end, the aim of this study was to establish: 1) the best source of meristematic explant for initiation and mass propagation of in vitro shoots; 2) in vitro conditions to screen and select for drought tolerance; and 3) physiological parameters as indicators of drought tolerance in vitro. Sugarcane stalks and shoots from single-budded setts of NCo376 were used. From the former, 1.3 cm-long meristems were isolated and used for shoot induction, shoot multiplication and rooting. The single-budded setts (approx. 50 mm) were first germinated in 20 ml sterile water or sterile moist paper, resulting in 100% and 60% sett contamination, respectively. With 1 mg lˉ¹ methylene blue (MB) there was 30% sett contamination, whilst 1 mg lˉ¹ MB in combination with 1 ml lˉ¹ Previcur® or 1 ml lˉ¹ BRAVO® resulted in 40% and 7% contamination, respectively. The uncontaminated germinated shoots (approx. 1 - 2 cm) were excised after 10 days in culture and used as the other source of meristems. Meristems from both sources were multiplied and rooted in vitro and their plantlet yield was 60 shoots/sugarcane stalk meristem and 10 shoots/meristem from in vitro-germinated sett. NCo376 and N41 varieties were used to determine the effect of mannitol (204, 326, 448 and 569 mM) on in vitro plantlet shoot and root re-growth. For both, increased mannitol in the media delayed shoot and root re-growth, with NCo376 being affected first. Stress was more significant on root than on shoot re-growth. For NCo376 plantlets, there were significant differences in root re-growth between 0 and 204 mM and the other tested treatments. For N41 plantlets, % root re-growth at day 10 on 569 mM mannitol was significantly higher than that at the other treatments. At 4 – 10 days, % shoot re-growth of NCo376 on 0 and 204 mM mannitol was greater than that at 326, 448 and 569 mM mannitol. Similar results were observed with N41 plantlets. The LD₅₀ and LD₉₀ for mannitol were 332 and 606 mM for NCo376, and 851 and 1493 mM for N41. There was no differences between the effects of polyethylene glycol-6000 (PEG-6000) and mannitol on root re-growth at the same osmotic potential. However, PEG-6000-containing cultures required to be aerated. As at 87 mM PEG-6000, NCo376 plantlets showed 50% root re-growth compared to 10% in non-aerated cultures, mannitol was used in subsequent investigations. Mannitol concentrations equivalent to LD₅₀ and LD₉₀ for NCo376 and N41 were used to screen N12, N36, N19 and N26 varieties. Based on the results obtained, the varieties were ranked on their tolerance to mannitol stress: N41 > N26 > N36 > N12 > N19 > NCo376. Leaf electrolyte leakage, leaf chlorophyll content measured with Soil Plant Analysis Development (SPAD) measurements, and histochemical detection of hydrogen peroxide (H₂O₂) (with nitroblue tetrazolium) and superoxide anion (O₂⁻˙) (with 3, 3’-diaminobenzidine) production were evaluated as indicators of stress using N41, N26, N19 and NCo376 on 332, 606 or 851 mM mannitol. N19 and NCo376 plantlets on 332 mM mannitol showed a higher % electrolyte leakage at day 5 (70%) than at day 10 (40 – 50%) of culture than N41 and N26 plantlets. A slight decrease in chlorophyll content was recorded at day 10 of culture in 332 and 851 mM mannitol, with no differences between NCo376 and N19, and N41 and N26. NCo376 and N19 accumulated more H₂O₂ than N41 and N26. O₂⁻˙ accumulation was also greater in NCo376 and N19 than in N41 and N26. All these parameters detected stress at lower levels of mannitol (332 and 606 mM), but not at 851 mM. It was concluded that mannitol stress in vitro (332 – 606 mM), in combination with the physiological assays allow for the discrimination of in vitro osmotic stress among sugarcane varieties. Further work is necessary before recommendations can be made regarding the use of the other stress biomarkers.