Thesis (MSc. (Botany)) -- University of Limpopo, 2015 / Soybean is an important source of high quality protein and oil for both humans and animals, especially in protein formulations for pharmaceutical and nutriceutical use. This crop is adversely affected by both biotic and abiotic stresses impacting on its productivity. Soybean productivity can be improved via techniques such Agrobacterium-mediated genetic transformation. Soybean is recalcitrant and depends on suitable explants from which new shoots can be regenerated and be amenable for transformation. The goal of this study was to produce transgenic soybean plants that are tolerant to drought stress through Agrobacterium tumefaciens-mediated transformation. Multiple shoot induction on double and single coty-node explants, obtained from soybean seedlings derived from seeds germinated in vitro on Murashige and Skoog culture medium supplemented with cytokinins was studied. The effect of different concentrations of benzyladenine (1.57, 2.00 and 4.00 mg/l), and benzyladenine (2.00 mg/l) in combination with kinetin (1.00 mg/l) was tested. The results show that the double coty-node explants produce the highest number of shoots per explant, an average of 7.93 shoots on Murashige and Skoog medium supplemented with 2.00 mg/l benzyladenine. The lowest number being 1.87 shoots obtained from single coty-node explants cultured on Murashige and Skoog medium containing 4.00 mg/l benzyladenine. The single coty-node explants showed lower frequency (10–57%) of shoot induction when compared to the double coty-node explants (50–83%). The suitability of aminoglycoside antibiotics (hygromycin, tetracycline and rifampicin) for efficient elimination of Agrobacterium tumefaciens after co-cultivation was tested using a well agar diffusion assay. Co-culturing double coty-node explants with Agrobacterium containing pTF 101 vector carrying the Oryza cystatin 1 gene resulted in 76.6, 63.3 and 60.0% shoot regeneration on Murashige and Skoog shoot induction media (shoot induction medium 1, shoot induction medium 2 and shoot induction medium 3) containing hygromycin, tetracycline and rifampicin at 500 mg/l respectively. These antibiotics showed the highest zones of inhibition against pTF 101 using the well agar diffusion assay. On the other hand, 85% plant regeneration was obtained during in vivo transformation following Agrobacterium injection into seedlings. These results imply that vi both in vitro and in vivo protocols were suitable for transgenic shoot regeneration and plant establishment since all the plants continued surviving in the presence of 6.00 mg/l glufosinate-ammonium. Future work will focus on screening of transgenic plants using beta-glucuronidase and isolating the protein encoded by the Oryza cystatin 1 gene to further confirm the generation of transformed plants carrying the gene of interest.

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M. Tech. Agriculture / This study evaluated plant growth and symbiotic performance of two soybean genotypes inoculated with five inoculant formulations (i.e. EMDPeat, EMDLiquid, SoyLiquid, SoyPeat and Granular) and a Control of the Bradyrhizobium japonicum strain WB74 at three field sites in Mozambique and a pot experiment in South Africa. The sole aim was to assess whether the inoculant formulations can increase nitrogen fixation and plant growth on the two soybean genotypes.

Thesis (MTech. degree in Agriculture.)--Tshwane University of Technology, 2011. / This study evaluated plant growth and symbiotic performance of four promiscuous-nodulating soybean genotypes and three commercial varieties supplied with a peat-based inoculant of Bradyrhizobium japonicum strain WB74 at three field sites in Mozambique and a pot experiment in South Africa. The sole aim was to assess whether these promiscuous-nodulating soybean genotypes can benefit from inoculation.

In the N$ sb2$ fixing legume symbiosis, suboptimal root zone temperatures (RZTs) not only decrease N$ sb2$ fixation. but reduce the formation and development of nodules. The purpose of this thesis was to elucidate the mechanism by which suboptimal RZTs affect nodulation and nodule development in legumes, such as soybean (Glycine max (L.) Merr.) and to attempt to find ways to overcome this inhibition. Initial studies characterized the RZT response in soybean plants inoculated with Bradyrhizobium japonicum. In plants grown at RZTs from 25 to 17$ sp circ$C, the time between soybean inoculation with B. japonicum and the beginning of N$ sb2$ fixation increased by 2.5 days for every $ sp circ$C decrease, whereas below 17$ sp circ$C RZT each $ sp circ$C appeared to delay the onset of N$ sb2$ fixation by 7 days. RZTs less than 17$ sp circ$C strongly inhibited the nodulation process and, as a result also sharply decreased N$ sb2$ fixation per plant. The greater sensitivity below 17$ sp circ$C is due to events related to, or occurring before infection initiation. Coinoculation of soybean with B. japonicum and other microorganisms beneficial to legumes, either vesicular-arbuscular (VA) mycorrhizae or plant growth promoting rhizobacteria (PGPR), increased soybean nodulation and N$ sb2$ fixation, but these increases were temperature dependent. Vesicular-arbuscular mycorrhizal colonization had a negative effect on nodule establishment below 18.5$ sp circ$C RZT, but a positive one above this RZT. At each temperature tested some PGPR increased the amount of fixed N and number of nodules formed, whereas some decreased the level of these variables. The most stimulatory strain at each temperature was: 15$ sp circ$C-Serratia proteamaculans 1-102, 17.5$ sp circ$C - S. proteamaculans 1-102 and Aeromonas hydrophila P73, and 25$ sp circ$C - S. liquefaciens 2-68. / Because our research indicated that an event before infection thread initiation was most sensitive, and because the first known step in establishment of the symbiosis is production of a plant-to-bacterial signal molecules. I tested whether the poor nodulation at suboptimal RZTs was related to disruption of plant-to-bacterium signalling. Inocula bacteria were preincubated with genistein, a major isoflavonoid signal molecule in soybean. This shortened the period between inoculation and root hair curling, and hastened the onset of N$ sb2$ fixation under both controlled environment and field conditions. At 15 and 17.5$ sp circ$C RZTs, 20 and 15 $ mu$M genistein was found to reduce the inhibition of suboptimal RZTs, increase nodulation, and accelerate the onset of ${ rm N} sb2$ fixation. When applied to the plant rhizosphere in the field, genistein also reduced the inhibitory effects of cold spring soils on nodulation and N$ sb2$ fixation. Direct measurements of genistein accumulation in soybean roots indicated that, with decreasing RZTs, genistein accumulation decreased. B. japonicum USDA110 containing plasmid ZB977 with nodY-lacZ fusion genes incubated with genistein under different temperatures indicated that higher genistein concentrations and longer incubation times were required to activate the lacZ gene to a maximum level under low incubation temperature. Overall, these findings suggested that plant-to-bacteria signal molecules such as genistein may be an important limiting factor in the nodulation of legume plants at low RZT.

Plants have evolved numerous strategies for the adaptation to drought. Although many investigations reported on the potential value of proline accumulation during environmental stress, it is still unknown whether or not a constitutive higher level of proline accumulation enhances plant tolerance. Thus, it was investigated if underproduction and overproduction of proline will influence the susceptibility to drought stress in soybean plants. This was made possible with the transformation of soybean plants with an L-Δ¹-pyrroline-5-carboxylate reductase (P5CR) gene. First, an Agrobacterium-mediated vacuum infiltration transformation system, using partially germinating Carnia 2233 soybean seed, was established through the assessment of several conditions that can affect transformation efficiency with the use of β-glucuronidase reporter genes. Transformation was confirmed with PCR and Southern blot analysis and results indicated that stable transgenic soybean plants were obtained within one generation with a transformation rate of± 30%. This technique was used in the transformation of Carnia 2233 soybean seed with the P5CR gene in the antisense orientation under the control of an inducible heat shock gene promoter (IHSP). It was confirmed that the P5CR-IHSP gene construct was integrated into the soybean cells and was conserved over three generations. Physiological screening of the antisense P5CR transgenic plants in the greenhouse proved that, with activation of the promoter, an under-expression of the P5CR gene and subsequent inhibition of the accumulation of proline were experienced during drought and osmotic stress. The decline of the viability of the transgenics with prolonged drought stress, as monitored with a woodenbox screening test, is an indication that proline is needed for survival of soybean plants under drought stress conditions. The transgenic plants demonstrated a sensitive reaction in contrast to the control plants that displayed a tolerant reaction to osmotic stress in a TTC assay. The underexpression of the P5CR gene resulted in a decline protein synthesis due to proline shortage as was observed with the evaluation of the efficiency of protein synthesis. All these results suggest that a decrease in the proline level due to the antisense P5CR gene, yielded plants that are more osmotic and drought stress sensitive. Subsequently, the soybean cultivar Ibis was successfully transformed with the P5CR-IHSP construct in the sense and antisense directions in order to test the reproducibility of the transformation process and to assessed the link between the biochemical traits involved in the drought stress mechanism. Three different experiments were conducted: a mild heat and drought stress on "To" transgenic plants exploring changes in chlorophyll fluorescence transients, a mild heat stress on "T1" transgenic plants comparing proline accumulation and chlorophyll fluorescence transients and a severe drought and heat stress on the "T1" transgenic plants comparing proline accumulation NADP⁺synthesis and chlorophyll fluorescence transients. Chlorophyll fluorescence transients were successfully used as a screening method for transgenic soybean plants during this study. The sense transgenics responded to the mild stresses with a significant decrease in their electron transport, trapping and absorption compared to the antisense plants that displayed significant increases in electron transport and trapping. During the severe stress, the antisense transgenics experienced total photoinhibition indicated by the enormous loss of electron transport but the sense plants had the ability to overcome the stress as is revealed in the increase in the electron transport. It was demonstrated that although proline accumulation yielded no significant differences during the mild heat stress, the sense plants accumulated substantially more proline than the control and antisense plants during the severe heat and drought stress. It was demonstrated that proline plays an important role in the plant's response to a drought stress as well as in the recovery phase after drought, as the sense plants also had the ability to reduce the accumulated proline during the recovery period in contrast to the antisense transgenics that experienced protein degradation. The transgenics responded to a period of heat and drought stress with a reduction in NADP⁺ levels in the antisense plants and increasing levels in the sense plants. The sense plants were able to fully recover after the stress period, thus adaptation to drought may depend on different mechanisms, including the capacity to maintain high levels of proline and to regenerate them through the "reduction" of NADP⁺. It was possible to alter the drought tolerance of Ibis by transformation with antisense and sense P5CR gene constructs, which resulted in respectively more sensitive and more tolerant Ibis plants. It can be concluded that over-expression of P5CR during a drought stress resulted in higher proline levels, better photosynthetic efficiency, higher NADP⁺ production and thus a more drought tolerant plant. This study gave additional proof that a constitutively higher level of proline accumulation enhances drought tolerance in soybean. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

The development of a genetic engineering system for soya bean (Glycine max L.) is described in this thesis. Routine tissue culture regeneration systems were developed for South African cultivars of soya bean despite the recalcitrant nature of this plant to in vitro manipulation. Regeneration of shoots was obtained when cotyledons were excised from seeds germinated for two days and cultured on B5 BA 20 medium containing 2 mg/I BA. The important problems of in vitro shoot elongation and rooting were overcome by culturing cotyledons in the dark for four weeks to produce shoots with unusually long stems. This was followed by one week of culture under conditions of high light intensity to obtain healthy green shoots which could be rooted , either in sorbarods or on solid Y2MS 30 medium. The use of a mist bed for the hardening off of rooted soya bean regenerants was essential for the recovery of fertile soya bean plants. Molecular techniques for the cloning of foreign genes into binary vectors suitable for plant genetic engineering were also studied and are described in the thesis. The Basta herbicide resistance gene, pat, was successfully cloned into the binary vector pBI121 which contains the [beta]-glucuronidase (GUS) reporter gene, uidA. The new construct, pB1121/Ac, was conjugated into various disarmed Agrobacterium tumefaciens strains and these strains, along with other binary vector-containing strains, were used to transform soya bean plant material. Although a protocol for the routine transformation of soya bean was not developed, transgenic soya bean material resistant to kanamycin and showing GUS activity was obtained. Transformation of wound sites on cotyledons was obtained in several experiments and transgenic shoots were regenerated from inoculated cotyledons. Only the A. tumefaciens strain C58C1 (pGV2260)(pJIT119) was able to transform cotyledonary cells of soya bean and, therefore, only kanamycin resistant soya bean shoots were produced. Transgenic soya bean plants resistant to the herbicide Basta were not produced due to the recalcitrant nature of the crop to genetic engineering. Transformation of the non-recalcitrant plant, tobacco, which is a model system for plant genetic engineering was achieved. The binary pat gene containing vector constructed in th is study, as well as vectors obtained from AgrEvo, were tested. The transgenic Basta resistant tobacco plants obtained were used to optimize assay systems for the analysis of transformed plant material containing the pat gene. These assay systems included the use of the polymerase chain reaction as well as digoxigenin-Iabelling of a DNA probe suitable for detection of the pat gene. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

An infusion technique was used with an aqueous radiocalcium ( 45CaCl2) solution during the seed-filling stage of well-watered and moisture-stressed soybean in a greenhouse. The kinetics of infusion volume showed a quadratic reduction in absorption which approached zero on the sixth day for non-irrigated plants. The concentration of 45Ca increased quadratically from the point of injection towards the apex independent of both water status and plant parts. The difference in concentration of 45Ca between irrigated and non-irrigated plants was significant (P < 0.05) and concentrations attained the maximum values at the sixth node from the plant base. Seeds contained considerably less 45Ca than either stem or leaves. / The effects of a long (LD, 16h) and a short (SD, 12h) photoperiod with two water stress levels (SL) of stress (ST) and no stress (NS) on the distribution of 45Ca in plant organs (PO) of leaves, petioles, and stem at different node number (NN) of soybean were studied during the seed-filling stage. The univariate and Manova analyses showed the main effects of photoperiod (PP), SL, and PO to be highly significant (P < 0.001) on Ca distribution. The long PP increased Ca concentration in top leaves compared with the short PP regardless of SL. Water stress significantly (P < 0.001) modified the Ca distribution and reduced its concentration in PO within NN irrespective of the photoperiod (a measure of light stress). A possible mechanism for the regulation of Ca distribution is discussed in terms of nitrate reduction. / Ca uptake was also studied by immersing the central tip of a trifoliate leaf in various concentrations of 45CaCl2 solutions and drought conditions during the seed-filling period of soybean. The beta-ray gauging and the diurnal leaf temperature variation showed similar characteristics for leaf water status. The activities of 45Ca were significantly higher (P < 0.0001) at 5, 10, 20, and 30 mM concentrations for water-stressed and non-stressed leaves compared with the control. 45Ca activities at 5, 10, and 20 mM Ca concentrations between stressed and non-stressed leaves were not significant, but the difference in their mean values at 30 mM Ca concentration was significant (P = 0.0159). The relationship between 45Ca uptake and Ca concentration was parabolic for both stressed (R 2 = 0.77) and non-stressed (R2 = 0.81) leaves. Autoradiograms indicated Ca movement through the mid-rib and veins of the tip-immersed trifoliate leaf but showed no activity in other plant parts. An activity gradient developed between seeds when a pod-tip was immersed in the radioactive solution. Solutions of ruthenium red (RR, 0.01 mM), Ethylene Glycol-bis-(beta-aminoethyl ether)- N,N,N,N-Tetraacetic Acid (EGTA, 0.1 mM), calcium (Ca, 1 mM), and double distilled water (control) were fed through a bottom branch of soybean with (ST) and without (NS) water stress. The volume absorptions and transpiration rates were significantly higher for NS than ST plants and decreased almost linearly with time for all treatments. The transpiration rates of Ca-feeding ST plants and the control overlapped while the NS plants approached the same rate of transpiration by the third week. Ca was implicated in stomatal closure for the reduction in the transpiration rates. The relative amounts of chlorophyll decreased with time but chlorophyll was least affected for Ca-absorbing plants for both ST and NS plants. The use of RR (Ca transport blocker), and EGTA (Ca chelator) indicated the role of intracellular Ca conce

This study was conducted to improve knowledge of the impact of chronic soil water deficit and to test a novel technique of water management consisting of Lipo-chitooligosaccharides (LCO) spray application. It also aimed at evaluating changes in canopy reflectance due to water stress and LCO spray. Water stress during reproductive development resulted in an important decrease of plant physiological activity, vegetative growth, and productivity, and accelerated plant senescence at both water stress levels. Water deficit increased leaf reflectance in the visible and decreased it in the infrared ranges of the spectrum at both imposed stress levels. Foliar application of LCO affected overall plant physiological activity, increased flower and pod numbers. LCO treatment had the largest positive effect on the growth pattern of soybean at the medium stress level, which is the stress level most commonly observed in standard farm-field conditions. LCO treatment constitutes a potential technology for reducing water deficit effects. (Abstract shortened by UMI.)