Investigation of chlorophyll and stomatal chloroplast content in diploid and tetraploid black wattle (Acacia mearnsii de Wild).
Black wattle (Acacia mearnsii) is one of South Africa's leading commercial exotic species comprising nearly seven percent of South African forestry plantations. The planting of black wattle has become increasingly popular, initially for its high quality tannin content and in more recent times, for its wood and wood products. The industry also provides jobs for more than 36 000 people. Despite the commercial value of black wattle, if left unmanaged, it is one of South Africa's top invader species that aggressively colonise and rapidly out-competes indigenous vegetation. Thus, both plant breeders and environmentalists alike are faced with an interesting paradox of balancing the commercial significance of black wattle on the one hand with increasing environmental concern on the other. At the Institute for Commercial Forestry Research (ICFR), black wattle breeding programmes are being designed and implemented in order to reduce invasiveness whilst still maintaining product quality. One way of minimising invasiveness is to decrease fertility through the introduction of semi-sterility; while at the same time leaving product yield and quality unaffected. A method of achieving semi-sterility is by the induction of autopolyploidy that results in unviable gametes. Autopolyploidy, tetraploidy, is induced chemically through doubling of the chromosomes of diploids. These induced tetraploids may then be crossed with diploids to produce triploids. Thus, an effective method to identify polyploids at the seedling stage would greatly facilitate the success of the abovementioned breeding programmes in the black wattle industry. Polyploidy in plants is often associated with physiological and biochemical changes that become apparent as gigantism of organs which include fruits, flowers and leaves. Polyploidy is also associated with an increase in the number of organelles such as the number of stomatal chloroplasts and nucleoli, as well as an increased production of some proteins and pigments such as chlorophyll. These ploidy-related manifestations are often utilised in breeding programmes to increase the size and quality of plant products as well as a tool to discriminate between polyploids and diploids. Two putative diagnostic procedures to differentiate between diploid and tetraploid black wattle were developed in this investigation. The study focused on the discriminating power of stomatal chloroplast numbers and arrangements as well as the chlorophyll content in the two different ploids. A number of associated experiments were initially conducted to establish the optimal conditions for chlorophyll content analyses such as the type of leaf material and storage conditions. Stomatal chloroplast frequencies were determined in diploid and tetraploid black wattle and comprised three lines per ploidy level with five plants per line. A thin epidermal layer from the abaxial surface of a pinnule was stripped, stained, mounted and 15 stomatal guard cells per plant were viewed at 40X magnification. The mean number of chloroplasts per cell in diploids (9.89 ± 0.222) was found to be statistically different (p < 0.001) to that of tetraploids (22.43 ± 0.222) with no overlapping of the mean chloroplast values between the two ploidy levels. The ratio of diploid and tetraploid stomatal chloroplast numbers was roughly 1:2. An analysis of the least significant difference (LSD) was performed and indicated significant differences between plants within lines, between lines of different ploids (LSD =0.6266), as well as between the different ploids (LSD =0.2802). Furthermore, stomatal chloroplasts spatial arrangements were distinctly different in diploids and tetraploids. In diploids, chloroplasts were clustered into two regions, each towards the extreme ends of the kidney shaped stomatal cells. In the tetraploids, no clustering of chloroplasts could be identified, with an even distribution around the convex curvature/perimeter of the cells. There are a number of factors that influence chlorophyll content and degradation, which are either environmental or genetic in nature. Environmental factors that were considered are sample age and sample storage conditions. Genetic factors include genetic composition and, specifically, the number of sets of chromosomes, that is, the ploidy. Chlorophyll content was investigated by chemically extracting chlorophyll from leaf material and obtaining absorbance spectra with a PerkinElmer UV/vis spectrometer for wavelengths from 400 nm to 700 nm. Chlorophyll absorbance spectra were generated in terms of leaves stored prior to chlorophyll extraction, leaves of different ages, trees of different ages and ploidy. The effects of storage of leaves on chlorophyll content were determined in five non-identical two year-old nursery diploid black wattle genotypes. Fifteen leaf samples from each genotype were either oven dried and then stored for one week or one month at room temperature, or frozen for one week or one month at -4 °C, before chlorophyll was extracted and absorbance spectra determined. Chlorophyll absorbance values of chlorophyll extracted from leaf material on the day of collection (day-0) was used as the control. An analysis of variance (ANOVA) revealed that the chlorophyll absorbance values of the different storage treatments were all significantly lower than the chlorophyll absorbance values of the control (p < 0.001). Assessment of the mean chlorophyll absorbance (TĀ), sum of the three peak absorbance values at three wavelengths, namely, 433 nm, 456 nm and 663 nm, revealed significant differences (p < 0.001) from the control (TĀ = 1.275) for all treatments. Dried leaves that were stored for seven days (TĀ = 1.132) resulted in the least amount of chlorophyll degradation followed by 28 day ice storage (TĀ = 1.114), seven day ice storage (TĀ = 1.103) and lastly 28 day dried storage (TĀ = 1.093). An analysis of least significant differences (LSD) revealed that chlorophyll absorbance values within lines and between wavelengths were significantly different (LSD = 0.005). Furthermore, LSD analysis revealed significant differences between all treatments (LSD =0.003) which also supported the ANOVA findings. Chlorophyll absorbance values within dried and frozen treatments were compared with respect to storage time periods of one week and one month. It was noted that whilst all treatments decreased from the control (day-0), dried samples responded differently to storage periods as compared to frozen samples. Chlorophyll absorbance values of dried material decreased steadily over time from control to seven-day storage to one-month storage, whereas, in the case of frozen material, a similar trend could not be identified. A greater decrease from the control to seven day ice storage was recorded than for the decrease from the control to 28 day ice storage. The effects of tree and leaf ages of diploid black wattle on chlorophyll content were determined. Two types of leaf flushes namely, old and new flush, were examined in relation to different tree ages; two, four, six, eight and nine year-old; in order to assess whether the choice of material impacts on chlorophyll absorbance values. Five leaf samples from each tree were collected, bagged and chlorophyll extracted within two hours of collection. These chlorophyll absorbance values were compared to young diploid seedling material as a base-value and as a control value. An analysis of variance (ANOVA), revealed significant differences between tree ages and between leaf ages (p < 0.001). An analysis of least significant differences (LSD) revealed that new flush of all tree age groups were significantly different from the control (LSD = 0.006). This was mostly true for old flush, except that of six year-old old flush which was not significantly different from the control (LSD =0.006). The chlorophyll absorbance values of both old and new flush of different age groups produced spectral graphs for which no specific trends could be ascertained. Therefore, the data from the two flush types were pooled and revealed a marked increase in chlorophyll absorbance as trees became older. Moreover, this increase was more apparent in new flush than in old flush. Interestingly, juvenile characteristics were identified in two year-old black wattle trees, where a marked increase in chlorophyll content was noted. The effects of the number of chromosome sets on chlorophyll content were assessed for diploid and tetraploid black wattle. Seedlings, bagged juveniles as well as two year-old field trees were analysed. Three genetic lines per ploidy level comprising of ten plants per line were used in the analysis. An analysis of variance (ANOVA) revealed significant increases of chlorophyll absorbance values (p < 0.001) for diploid seedlings (TĀ = 1.1086) to bagged trees (TĀ = 1.149) to field trees (TĀ = 1.224). Similar significant increases were recorded for the tetraploid seedlings (TĀ = 1.886) to bagged trees (TĀ = 1.931) to field trees (TĀ = 2.059). There were distinct differences in chlorophyll absorbance between the two levels of ploidy (LSD =0.002). Furthermore, chlorophyll absorbance within lines, between wavelengths were found not to be significant (p = 0.984), which was supported by an analysis of least significant differences (LSD = 0.004). Moreover, the ratio of diploid to tetraploid chlorophyll absorbance was roughly 2:3. Additionally, the increase of chlorophyll content from seedlings to bagged juveniles to field material of both diploid and tetraploid black wattle further supported the findings in the previous age study that there was an increase in chlorophyll content as the tree matures. Stomatal chloroplast frequencies and chlorophyll content have been identified as two methods that are able to effectively, and with ease, discern between diploid and tetraploid black wattle.