Screening of cowpea, bambara groundnut and AMARANTHUS germplasm for drought tolerance and testing of the selected plant material in participatation with targeted communities
Report No 944/1/04
Water scarcity in South Africa has emphasized the need to improve water use efficiency. The challenge for farmers and researchers is to find ways to increase the crop output per unit of water.
Mechanisms used by plants to cope with changes in the environment are widely reported. Amongst them are root architecture, leaf morphology, physiological characteristics and others associated with the developmental biology. These components include cellular, developmental as well as biochemical traits. By selecting for these traits, it is possible to improve complex traits such as yield under stress conditions. Usually a combination of these attributes is present in crops that produce good yields under drought conditions. In environments where water is a limiting factor, water use efficiency is of the utmost importance.
Drought is a very common abiotic stress condition, thus economically important crops with high levels of drought tolerance are of great value. Under field conditions, drought severity, timing and duration vary from year to year and a cultivar, which is successful in one year, might fail in another year. The unpredictable and variable forms in which drought stress manifests itself, complicates the selection of superior plant material as well as the breeding programmes. Significant potential exists for the improvement of crop productivity by selecting plants that are better equipped to cope with unfavourable environmental conditions, such as drought.
One approach to improve crop performance is to select for genotypes that have improved yield during water deficit conditions. The ability of some plants to maintain a higher yield under drought than others is of great value. Average losses of some major crop plants due to environmental stresses may amount to 50-80% of their genetically determined productivity. The highest proportion of yield losses can be directly attributed to drought. The drought related responses in plants are of a complex nature and result from genomic re-organisation and alterations in gene expression. Drought tolerance has shown to be a highly complex trait, influenced by multiple genes.
The main objective of this study was to evaluate the drought tolerance of vegetable crops grown in environments where the crop yields are influenced by limited water supply.
Capacity buildingformed an important part of this project.
The distribution of the desired genotype(s) to the farmers will improve in the end crop productivity and quality and will thus be of value for millions of people that are dependent on indigenous crops as source vitamins and proteins. It will also have a positive impact on subsistence farmers in areas with low precipitation or variable rainfall patterns.
1. Collection of germplasm
Germplasm of cowpea, bambara groundnut and Amaranthus germplasm was collected by personnel of the University of Zululand and the Sustainable Rural Livelihood (SRL) unit of ARC-Roodeplaat. The SRL unit collected seed in Gauteng (Soshanguve), Northern Province (Polokwane and Bushbuckridge), Mpumalanga and KwaZulu-Natal (Ladysmith). Some Amaranthus seeds were also collected in Venda. The personnel of the University of Zululand collected seeds from various street markets and communities around the University, Empangeni, Mahlabatini, Pietermaritzburg, Richardsbay, Durban and Komatipoort. Some of the seeds that were collected originated from countries like Mozambique, Nigeria, Zimbabwe, Portugal and Ghana.
2. Optimisation of tissue culture and multiplication of selected lines
The tissue culture techniques for Amaranthus, bambara groundnut and cowpea were investigated. It was possible to establish a multiplication system for Amaranthus plants. The possibility for using in vitro plant material for screening amaranth for drought tolerance was also investigated, with the aim to reduce large and expensive glasshouse and field trials. PEG was used to induce drought stress in vitro and this was used as an alternative to dry land field trials to measure anti-oxidative stress activity, TTC reduction assays and proline production for amaranth. Due to the high variation within treatments and difficulty to relate to in vivo results with the in vitro results, it is suggested that in vitro screening results should always be first correlated to the results of the field trials. For these crops it is suggested that in vitro screening should not be used, seeing that the procedure itself causes the plants to stress and this resulted in very high variation within the treatments, rendering the results insignificant.
3. Physiological and anatomical evaluation of drought tolerance
A multidisciplinary approach was followed to measure the effect of drought stress on the physiology, biochemical and morphology of these plants, and to identify mechanisms that allow the plants to survive severe drought stress. Selected lines/ species as well as plant material collected from the communities were cultivated under optimum greenhouse conditions until the plants were subjected to drought stress by withholding water after which various screening methods were used to determine the levels of drought tolerance.
The study included responses with regard to:
Two pathways are evident in the maintenance of turgor in amaranth: the modulation of LWP and RWC. Restoration and repair upon rehydration are critical components of desiccation tolerance, and recovery of leaf area. The lower RWC and LWP values coincide with lower photosynthetic activity and rate of decrease in leaf area during drought stress. Furthermore, the ability of the membrane to retain and selectively transport cellular solutes gives an indication of the cellular membrane function. CMS assay indicates excessive leakage of cellular electrolytes and is usable as an indication of cellular membrane injury. TTC reduction assay measures the dehydrogenase activity of the intact cell. Proline accumulation screening seems to be highly useful for determining genotype variation in drought tolerance in amaranth. This is possible since differences in the onset of proline accumulation, as well as amount of proline accumulated as a result of severe water deficit, were observed in the amaranth species tested. Proline accumulation also serves as a good indicator of the water status of the plant as there is an inverse relationship between proline content and water potential. The amaranth species tested at seedling stage showed strong drought avoidance characteristics during early drought screening. Although the leaves of the species were discarded during severe moisture stress (drought avoidance), the plants recovered very well after rewatering by sprouting again. Amaranth seedling roots exposed to extended periods of moisture stress recovered quickly to re-establish water uptake again upon rewatering.
The local collections that out performed some of the species under the drought conditions were: Community 3 and 4, Krugersdorp, Callaloo, Imbuya and Indigenous I. The best species were: A. tricolor, A. hybridus, A. hypochondriacus and A. candatus.
The screening methods, which showed the highest correlation with each other in cowpeas, were the JIP-test (chlorophyll fluorescence measurements), chlorophyll a and b levels, RWC, woodenbox technique, yield of the greenhouse plants and root architecture. The determination of the free proline levels and LWP showed a relatively good correlation with the above mentioned screening methods. The screening tests of the enzymes of the antioxidative system (SOD and GR) and the CMS were unable to reveal differences in the drought resistance levels of the cowpea lines.
The drought tolerant control line IT96D-602 was able to maintain a higher relative water content and water potential than the other lines tested over most of the seasons. This improved the viability of the plants, which was evident from the TTC reduction assay and the chlorophyll fluorescence values. The fact that IT96D-602 was experiencing lower stress levels than the other lines was also evident from the lower free proline levels accumulated and the higher Chl a and b levels maintained under drought stress than the other plants. Some qualities that were detected in IT96D-602, which might have contributed to the water retention, were smaller leaves and a more upright growth form. IT96D-602 also possesses a more extensive root system than the other lines, which helps with water uptake.
The community collected lines: Ghana black eyed bean, Manguzi 1 and 2, Okhaluleni and Phelandaba, performed exceptional well against the drought selected line IT96D-602. This illustrates the potential of the indigenous germplasm.
c. Bambara groundnut
Fifteen bambara groundnut lines were evaluated over four seasons. The proline levels changed little over the stress period, as bambara groundnuts are generally very drought tolerant. The SOD enzyme activity decreases in all lines but SB1-1. The AP activity levels show little change over the drought stress period, except SB1-1 that experienced higher AP levels with increased stress. The RWC of all the lines were 100% before stress was induced and declined to between 50 and 70% after 2-3 weeks without water. After rewatering the RWC recovered to 90% within 4 days. The differences in the LWP and CMS were not significant between the different lines, and could not be used for distinguishing between different lines. The woodenbox experiments were however able to distinguished between the different lines during the drought stress as well as after recovery. The root architecture boxes were used to show differences between the unstressed lines. Some lines had a good distribution of long roots and some had more roots at the surface. The yield of the plants tested differed significantly between the lines, as well as between the treatments.
It was experienced that the bambara groundnut yield was very low in most lines under stress conditions. The lines SB1-1, SB7-1, SB20-1, AS18 and SB9-1 have the characteristic to yield even under dry conditions. The lines that performed best during the test period were: SB7-1, SB1-1, MAD1, SB9-1, SB20-2A, NB and KwaHgnase.
4. Propagation of selected lines
Selected amaranth, cowpea and bambara groundnut species, lines or selections that recorded high levels of drought tolerance were propagated for distribution to communities for testing of general performance and acceptability.
5. Evaluation of the crop for yield and acceptability through farmer participation
Demonstration trials of Amaranthus, bambara groundnut and cowpeas were established in Soshanguve and Bronkhorstspruit in co-operation with farmers in order to introduce the crops to community members and to encourage them to be willing to evaluate it for acceptability and potential income generation. Eleven families from KwaZulu-Natal and Gauteng were involved in the first community trials. The program in Gauteng was later extended to 20 farmers. The farmers had to give feedback on any problems experienced with the cultivation of the three crops, how well the crops were accepted by the communities and also how the proposed crops fitted in with the other crops that they were already cultivating. They were used to harvest Amaranthus in the field and therefore the cultivation of this crop was new to them. They felt that amaranthus could be an economically viable crop. The farmers preferred the dual-purpose runner types of cowpea, as they could consume the seeds as well as the leaves. The bambara groundnut is a very popular crop in especially Gauteng, but the unavailability of seed, especially of improved varieties, and lack of space poses problems. The farmers that participated in this study sold their crops to restaurants, hawkers and urban communities. Acceptability of the new selections was evaluated by palatability tests performed by taste panels consisting of local people who are familiar with the taste of the locally grown cultivars.
6. Capacity building
The adaptation of different physiological and anatomic techniques to screen large numbers of amaranth, bambara groundnut and cowpea plants for drought tolerance were mastered by the researchers during the course of the research project. These screening techniques were again passed on to various participants from Africa through training courses funded by AFRA and ICRO. The farmers through farmer participation in demonstration and community trials also learned a lot about the crops and are much more positive about the cultivation of these crops.
The information gathered through this study was of high scientific value seeing that two Ph.D. theses and two BSc honours degrees were completed. Fourty five papers and posters were also delivered at conferences and other scientific meetings. One scientific paper has been published, two are in press and seven to be submitted shortly.
Germplasm of cowpea, bambara groundnut and amaranth were selected in various places in South Africa. The aim was to evaluate the collected germplasm for tolerance to drought stress. In order to do this, different physiological and phenotypic techniques were used. The information obtained through this study contributes towards a better understanding of the physiological and morphological basis of drought tolerance in neglected crops. The techniques selected were able to distinguish between drought tolerance in the different genotypes tested. It was also noticed that the different species reacted differently to a drought stress. Some of the selections out performed the control drought tolerant lines, indicating the successful identification of increased drought tolerance in the germplasm screened in this study.
It was found that amaranth appears to tolerate water stress by means of mechanisms of osmotic, metabolic and photosynthetic adjustment. From these results it can be concluded that some of the most important physiological factors which effect drought tolerance in amaranth seems to be: (1) limitation of water loss by reduction of leaf area, (2) ability to maintain a high water potential during water deficit, (3) efficient rooting ability and root/shoot ratio to exploit all available soil moisture, (4) maintenance of water status through osmotic adjustment, (5) scavenging of toxic O3 species, (6) possibility of adapting photosynthesis to comply with the changing demands of the e- transport system.
The most suitable methods for screening large numbers of cowpea plants for drought resistance are: (1) chlorophyll fluorescence (JIP-test), (2) free proline levels and (3) woodenbox screening for drought resistance at the seedling stage and (4) relative water content. One of the strategies used by cowpeas to survive unfavourable conditions is very sensitive stomatal control to minimise water loss.
The most suitable screening methods for bambara groundnut selection are: (1) chlorophyll fluorescence, (2) free proline, (3) woodenbox screening and (4) yield, which all form an important part of the plants survival.
The communities targeted to participate in this project, were generally very enthusiastic about the new crops. The cultivation of the crops was well accepted by the eleven farmers in the test trial and they were all able to find suitable markets for the crops. The initial farmer participation was extended to 20 other farmers, who were generally very enthusiastic about the new crops and willing to grow them. They indicated that they learned a lot during the trials regarding cultivation, utilisation and general nutritional value. Valuable information was gathered on community preferences regarding these crops.
Recommendation for future studies: