Flowering, fruit growth and intracellular storage component formation in developing Moringa Oleifera Lam. seed as influenced by irrigation

Abstract

Moringa oleifera has become increasingly popular as an industrial crop in recent times due to its multitude of useful attributes as a water purifier, nutritional supplement and biofuel feedstock. Besides this, Moringa oleifera has also demonstrated high tolerance to sub-optimal growing conditions, particularly towards drought. As a result, the current and anticipated cultivation areas of this tree are in medium to low rainfall areas. To what extent this drought tolerance was at the expense of reproductive development remained unclear. This study therefore aimed to assess the effect of different irrigation rates on flowering, fruit growth and storage compound synthesis of Moringa oleifera. Established Moringa oleifera trees were subjected to three different irrigation treatments simulating total annual rainfall amounts of 900 mm/annum (900IT), 600 mm/annum (600IT) and 300 mm/annum (300IT). Irrigation was administered through surface drip irrigation, while semi-weekly soil water measurements using a neutron probe were performed at several depths to ascertain differences between treatments. After having exposed trees to the different irrigation rates for nine months, the treatment effects were assessed. Firstly, individual inflorescences from each treatment were tagged during floral initiation and monitored throughout until fruit set. Flower bud initiation was 65.3% higher at the 300IT and 4.6% higher at 600IT compared to the 900IT. Fruit set however, was 22.0% lower for the 300IT and 4.4% lower for 600IT, compared to the 900IT. Floral abortion, reduced pollen viability as well as moisture stress in the style were contributing factors to the reduction in fruiting/yield observed at the 300IT. Subsequently, microscopic studies of developing seed were performed to better comprehend storage compound biosynthesis and accumulation throughout seed growth. From these studies, the endosperm was found to be nuclear, becoming cellular at a fruit diameter of ±6 mm from the micropylar side towards the developing embryo. At a fruit diameter of ±8 mm the cellular endosperm had covered the entire inner integument, which coincides with the developing embryo reaching the globular stage. Cotyledon development commenced at a fruit diameter of ±12 mm and continued up until ±24 mm. At the end of this phase the cotyledons had filled the entire seed coat, while the unicellular epidermal layer of the inner integument remained distinctly visible between the cotyledons and the testa. Fruit growth measurements throughout the first 60 days after flowering (DAF), revealed a significant reduction in fruit growth rates and final fruit size with decrease in irrigation rate. The number of mature fruit, average seed count and time to maturity however, increased with the rate of irrigation. Average seed mass also increased from the 300IT to the 600IT, but decreased again between the 600IT and the 900IT. As photosynthesis is central to plant growth and development, the extent to which the irrigation treatments affected photosynthesis and ultimately tree performance had to be assessed. Photosynthesis measured during the vegetative, flowering and fruit development stages revealed not only a reduction in photosynthetic activity throughout the growing season, but also with reduction in irrigation rate. Lower photosynthetic rates were primarily as a result of stomatal (conductance and SI) and non-stomatal (possible RuBP regeneration, ATP synthesis and mesophyll conductance) limitations, while the decreases observed throughout the growing season were as a result of diminishing leaf chlorophyll concentrations. Harvested fruit were categorized according to their diameter (Ø0 mm - 28 mm) at 2 mm increments and the compositional changes monitored at each irrigation treatment. Starch was the first to accumulate during the initial histo-differentiation phase (Ø0 mm - 12 mm), while both oil and protein levels remained comparatively low. During the subsequent expansion phase (Ø12 mm - 24 mm) however, stored starch was remobilized and used in oil biosynthesis, thereby reducing the starch content percentage. Most of the oil and protein reserves were synthesized during this phase. As fruit reached their final maturation phase (Ø24 mm - 28 mm), the average oil content percentage was 24.8%, while the protein content percentage was 24.7% and the starch content percentage was 8.8%. The different irrigation treatments did not influence the final content percentage of the seed components as much as it did affect the time and rate of their synthesis throughout seed development. By using both light and electron microscopy in conjunction with histochemical staining techniques, the intracellular locality of storage compounds as well as the initiation of their synthesis could be determined. The storage compound detecting stains were; Sudan III (oil), Light Green SF (protein), Orange G (protein) and Periodic Acid-Schiff’s reagent (starch). During early seed development (fruits of 8 mm in diameter), starch was synthesized by the plastids and transiently stored in the cell periphery. Protein and oil bodies were synthesized in association with the endoplasmic reticulum and finally stored in the centre of cotyledonous cells. Protein and oil body formation only commenced in significant amounts inside cotyledons at a fruit diameter of ±14 mm. Intracellular protein bodies were largest at between ± 5 μm to 8 μm in diameter while oil bodies ranged from ± 0.2 μm to ± 1 μm in size. Identification of numerous intracellular compounds using histochemical staining proved very effective. This prompted further investigation into whether storage compound content could be quantified from stained sections using digital imaging software. The percentage stain coverage area calculated using Adobe® Photoshop® was compared to the content of the same compound determined analytically at the corresponding developmental stage. The best correlation between measured seed storage compound and stain coverage was observed for the protein and oil detecting stains, Orange G and Sudan III throughout seed growth. A stage specific factor based on seed mass was however necessary in order to estimate storage compound content (g) from image analysis results. Conclusively, results from this study suggest that moderate water stress prior to flower development encourages floral initiation. Irrigation should however be resumed once flowering has commenced to ensure good pollination, fruit set and yield. Between the fruit diameters of 12 - 24 mm, the majority of storage reserves were synthesized and as a result this stage of fruit development was found most susceptible to water stress. Increased irrigation rates not only shortened the time to storage compound synthesis initiation but also increased their synthesis rate. Reduced irrigation in contrast, delayed the onset of oil biosynthesis, and as a result starch levels continued to increase reaching much higher levels prior to its remobilization during oil biosynthesis. The highest oil content percentage and seed mass at maturity were measured at the intermediate irrigation treatment. Along with decent flowering and fruit set, 600 mm/annum appeared to be the most suitable irrigation/rainfall amount for Moringa oleifera. This irrigation amount was low enough to initiate floral initiation, but also sufficient to sustain the subsequent seed developmental processes. Digital image stain quantification proved reasonably effective for simple comparisons between storage compound contents at different developmental stages. Differences between storage compound contents of the same developmental stage from different irrigation treatments were however, not as clearly distinguishable using this method. Although the success of digital image analysis might be stain dependant, it certainly is a cost effective technique for compound content estimation. This study enabled the identification of sensitive stages throughout fruit/seed development, by providing detailed insight into oil, starch and protein biosynthesis. Ultimately these findings provide current and prospective Moringa growers with recommendations to ensure suitable site selection as well as irrigation management guidelines.