Rahul Sreekumar

Over-dependency on fossil fuel can result in depletion of natural resources and acceleration of global climate change. Thus, there is a need to find alternatives to fossil fuel to meet the growing energy demand. Establishment of biofuel crops on marginal lands could be a viable option. However, at present, little is known about such crops. There are many non-edible feedstocks that can grow well on marginal lands. Calophyllum inophyllum L. also known as beauty leaf tree (BLT) is one such tree crop that naturally occurs on marginal soils. Interestingly, its kernels contain up to 65% non-edible oil which can be converted into biofuel. At present, however, little information exists on its stress tolerance and high oil yielding genotypes.

The current study evaluated in pot trials, a wide range of BLT genotypes for seed germination, salinity tolerance and kernel oil content. The study also assessed the effectiveness of novel technologies such as fourier transform infrared (FTIR) and fourier transform near infrared (FTNIR) spectroscopy to predict kernel oil content and tissue chloride (Cl-) concentrations, respectively.

Seed germination studies showed significant variability between the three genotypes and the germination ranged from 82.8% to 98.6%. Based on the early seedling growth, superiority of three genotypes were ranked as RB2 (Rosslyn Bay)>Nash1 (Cairns)>CAS1 (Darwin). Screening of 12 genotypes in a glasshouse for salinity (75 mM NaCl) tolerance showed significant reduction in seedling height increment, leaf area, net CO2 assimilation rate (A), transpiration rate (E) and biomass, as compared to their controls. Principal component analysis of studied parameters ranked the genotypes BR35, 3A, BR23 and 29B as being the most tolerant and the genotypes 21A, 15A and 32A as the least salt tolerant. 

Effects of NaCl concentrations (0, 50, 150, 300, 500 mM) on three genotypes (RB2, Nash1 and CAS1) showed little change in biomass accumulation at 50 mM, and up to 57% reduction at 500 mM. At 300 mM NaCl, Na concentration in the mature leaves was 6.5 times higher than that in the controls, whereas the Cl- concentration was higher by 28 times. In an outdoor experimental study, 3-year-old saplings of BLT were assessed for long term (16 months) responses to NaCl (75 mM). In this study no significant differences were observed between the control and NaCl-treated plants for growth, biomass, chlorophyll fluorescence and photosynthetic parameters. There was a significant difference between younger and older leaves in Cl- accumulation. While the leaves of control plants contained ~495 mg kg-1 Cl, those exposed to 75 mM NaCl accumulated ~1151 mg kg-1 of Cl. Results of this experiment helped confirm that the BLT is a moderately salt tolerant species. 

FTIR-ATR spectroscopy was tested to predict leaf tissue Cl-. Clusters of wavenumbers were identified, with each wavelength displaying differing correlation with tissue Cl-. Application of exhaustive feature selection technique enabled selection of 806 spectra (out of 1746) that highly correlated (r=0.93) with the chemometrically determined Cl-. It was also found that a feature selection process can be a valuable exercise to predict tissue Cl- or to estimate the contribution of wave numbers to Cl-. Overall, this study demonstrated that the BLT tissue Cl- can be estimated using FTIR spectra.

Kernel oil, resin, press cake and residue content were determined using a screw press for 50 accessions of BLT that were collected from various parts of Australia. The same kernels were used to obtain FTNIR spectra. The resulting spectra were employed to develop calibration models using partial least squares regression. The model produced coefficient of determination in cross validation (R2) and standard error in cross validation (SECV) values of 0.60% and 4.15% for oil content, 0.78% and 2.6% for resin, 0.77% and 4.86% for cake and 0.76% and 2.8% for residue content, respectively. The results showed that the FTNIR technique could be successfully used as a faster and cheaper alternative technique to other laboratory methods to screen BLT genotypes for oil, resin and cake contents, as their residual predictive deviation (RPD) values ranged between 2 and 1.5.

In summary, this research has examined germination and salinity tolerance of various genotypes of BLT. Based on these results, the BLT has been declared as a moderately salt tolerant species as compared to other Australian tree species. The research has also demonstrated that the FTIR and FTNIR technologies can be employed to estimate leaf Cl- and kernel oil content non-destructively. These technologies will be invaluable in selecting salt tolerant genotypes that also contain high oil%, as the kernels having high oil% can be germinated and the resulting seedlings can be subsequently screened for salinity tolerance. The study helped identify superior genotypes and demonstrated that the infrared spectroscopy techniques could be used to rapidly screen BLT genotypes for low tissue Cl- (hence higher salt tolerance) and high kernel oil.

With the world being over-dependent on fossil fuels for energy generation, there is a great need for alternative renewable energy sources. The production of biofuel from the seeds of Beauty Leaf Tree (BLT) could potentially help to meet this need. As Beauty Leaf Trees thrive in coastal or saline conditions, the study aims to look at the viability of growing the trees in marginal soils by testing seedlings for stress tolerance and physiological responses. The research focused on elucidating the variability in morphology, growth rates, germination, oil content and the mechanism of salt tolerance in BLT. It also examined the suitability of modern novel technologies like fourier transform infrared (FTIR) and fourier transform near infrared (FTNIR) spectroscopy in assessing stress tolerance in plants, enabling identification of salt tolerant genotypes in a very efficient, convenient and non-destructive way.