## 4 What Are Hyphenated Techniques?
However, the GC-MS targets small and volatile compounds. The sample to be analysed is vaporised and then pushed through a column by a carrier gas and then detected at the detection end the instrument. The LC-NMR is the coupling of LC with NMR. NMR lacks high sensitivity but remains useful in chemical structure elucidation. The NMR works by determining the nuclear properties of compounds exposed to an external magnetic field. A spectrum corresponding to unique emitted frequencies of compounds in the analyte is generated and its unique features are used for compound identification. These techniques have been used extensively in various plant science fields including the understanding of plant defence and signalling molecules, and the data generated supplement molecular data like gene expression profiling. The internal environment of the plant is composed of housekeeping compounds and compounds for adaptation to stresses. The production of plant hormones and the monitoring of hormone levels within the plant are conveniently done using hyphenated techniques. A multitude of studies have proven the usefulness of hyphenated analytical techniques in various plants including cassava. Figure 2: A workflow representation of the use of hyphenated techniques for understanding plant biotic stress. How Have Hyphenated Techniques Been Used to Study Cassava? Biotic Stress Response and Hormonal Response? To understand biotic stresses and hormone signalling in plants, the use of hyphenated analytical studies plays a major role. These techniques/methods involve the use of a combination of analytical techniques to understand specific responses of a plant to different treatments from the same set of data. These studies can be from understanding the physiology of the plant, co-expression relationships using metabolome data [82; 84; 142] biochemical diversity of crops using metabolite profiling [8] and metabolite profiling in response to pests/pathogen attack [9; 10; 11; 143] among others. Specifically, for the detection, quantification and understanding of hormonal signalling in plants, hyphenated techniques have been broadly used [10; 11; 144; 145]. [PERSON] et al. [145] reported the accumulation of aromatic and aliphatic amino acids along with phenylpropanol intermediates using GC-MS based metabolite profiling, which suggests the induction of secondary metabolism during pathogenesis. Alterations in carbon metabolism along with perturbation of hormonal signalling were highlighted. In addition, [PERSON] et al. [144] highlighted the mobilization of carbohydrates, disturbance of the amino acid pool and activation of isoflavonoid, a-linolenate and phenylpropanol biosynthetic pathways in soybean in response to _Rhizoctonia solani_ infection. Components of the pathways included phytoalgebras, coumarins, flavonoids, signalling molecules and hormones, many of which exhibit antioxidant properties and bioactivity, helping the plant to counterattack the pathogen's invasion. Other studies that reported hormone signalling in plants in response to insect pests and pathogens include tomato-_Botrytis cinerea_ interaction [7], rice and _R. solani_ interaction [146] and cassava-whitelfy feeding [11] among others. Cassava production is currently receiving attention in terms of research, since it was labelled an orphan crop due to the slow progress in research for decades. Because cassava can be consumed fresh as well as processed, its improvement as a crop is of global relevance. Breeding cassava genotypes for resistance/tolerance to biotic stresses has been ongoing for decades. Multidisciplinary approaches such as the use of hyphenated techniques are fundamental to understand and identify plant responses to these stresses within a given condition. Among the most recently used technologies and techniques, the use of hyphenated analytical techniques plays a role in the breeding progress. Several studies have investigated the plant biotic stress interactions in cassava using these techniques [8; 9; 11].