Relationships of Selected Endogenous Factors Associated with Direct Somatic Embryogenesis of Coffee (Coffea arabica L.) Mayoli,

Coffee is one of the most important cash crops produced in the world with great economic returns to growers and national gross domestic product. Somatic embryogenesis is a morphogenetic processes leading to plantlet regeneration and these processes are coupled with changes in the levels of primary metabolites. The present experiment established relationships of endogenous substances with direct somatic embryogenesis of coffee ‘Ruiru 11’. Laboratory experiments were set up at Coffee Research Institute, Ruiru-Kenya between 2014 and 2017. The set up was in a completely randomised design, replicated three times and repeated once. Third leaf pair explants were excised from 8-month-old greenhouse-grown mother plants and cultured in half strength Murashige and Skoog basal salts augmented with Thidiazuron. Once embryos had developed, the cultures were analysed for endogenous substances using HPLC and GCMS. Sucrose, phenolics, alkaloids, amino acids, fatty acids and their derivatives correlated positively, whereas fructose and glucose correlated negatively with the other biochemical components. Endogenous sucrose, chlorogenic acid, caffeine amino acid, fatty acids and their derivatives are potential biomarkers for coffee somatic embryogenesis, whereas endogenous fructose and glucose are inhibitors of the same. Further studies regarding the status of the biochemical components, especially in particular stages of embryo development should be conducted to establish treatments that can improve coffee direct somatic embryo development.

and 3 g/L gelrite added before autoclaving for 15 minutes at 121 o C and 100 kPa. Culture medium (25 ml) was poured into Magenta vessels (Sigma Chemical Co.) and 5 leaf discs (1 cm 2 ) cultured in each vessel maintained for about 8 months in darkness, 25 ± 2 o C and 70% relative humidity. In total, 627 culture vessels were prepared, but 183 (29%) were discarded due to fungal contamination.

Treatments
Treatments for this experiment were selected from the remaining 444 clean cultures. Culture vessels with green and brown leaf discs with and without embryos as shown in Plate 1 were used to characterise caffeine, chlorogenic acid, amino acids, sucrose, glucose, fructose and phyto-components in the leaf discs, embryos and medium. Fresh culture media and leaf explants excised from greenhouse-grown mother plants were used as the controls. The experimental layout was a completely randomized design, with three replications and six culture vessels per treatment. The experiment was repeated once.
Plate 1: Treatments lay out A: Brown leaf discs with embryos. B: brown leaf discs without embryos. C: green leaf discs with embryos. D: Green leaf discs without embryos, E: Fresh leaves (control). F: Fresh media (control)

Extraction and evaluation of sugars
The sugars sucrose, fructose and glucose were extracted as described by Osborne and Voogt (1978) with modifications. The leaf discs, somatic embryos and culture medium from culture vessels with green and brown leaf discs, with and without embryos were separately weighed and placed into round bottomed flasks. About 100 ml of 96% ethanol was added into each flask. The extraction of the sugars was done for one hour under reflux by boiling the leaf discs, somatic embryos, or media with the ethanol, while continually cooling the vapour to liquid and returning it back to the flasks for 25 minutes and the contents left to cool. The extract was filtered and evaporated to dry. The extract was reconstituted to 2 ml for the leaf and 5 ml for the embryo and media using mobile phase acetonitrile: distilled water at a ratio of 80:20. The extract sample was filtered through a 0.45 µm micro-filter (Chromafil) and analyzed with High Performance Liquid Chromatography (HPLC).

Extraction and evaluation of chlorogenic acid
Extraction of chlorogenic acid was done as described by Kathurima and Njoroge (2012). Leaf discs (1 cm 2 ), somatic embryos and culture medium from culture vessels with green and brown leaf discs, with and without embryos were weighed into 100 ml conical flasks and the weights recorded. About 50 ml of 96% ethanol (AR) and 10 ml acetone were added to each sample. The samples were first homogenized at 4 o C and then transonicated using an ultrasonic bath for 10 minutes. Filtering was done using Whatman No. 42 (12.5 cm) paper. The filtrate was recovered in a 100 ml round bottomed flasks and evaporated at 40 o C. The samples were reconstituted with 2 ml of 50% methanol, filtered through 0.45 µm micro-filters and analyzed with HPLC.

Extraction and evaluation of caffeine
Caffeine was extracted as described by Kathurima and Njoroge (2012). Leaf discs (1 cm 2 ), somatic embryos and culture medium from culture vessels with green and brown leaf discs, with and without embryos were weighed into 250 ml flat-bottomed flasks with round necks. Thereafter, 0.5 g magnesium oxide (Merck) and 200 ml distilled water were added into the flasks. Refluxing was done by boiling while continually cooling the vapour to liquid and returning it back to the flasks for 25 minutes and the contents left to cool. After cooling, filtration was done under vacuum on celite and the filtrate recovered in 250 ml volumetric flasks. The volume was topped up with distilled water to the marks and 20 ml of the filtrates were drawn and put into 100 ml volumetric flasks. The volume was adjusted to the marks with the 20% acetonitrile. The eluates were filtered through a 0.45 µm micro-filters and analyzed with HPLC.

Extraction and evaluation of phyto-components
Phyto-components were extracted as described by Shettima et al. (2013) with modifications. Fifty (50) ml methanol was added to the culture vessels containing green and brown leaf discs, with and without embryos and immediately deep-frozen overnight at -20°C. The polarity of the samples was increased before partitioning against ethyl acetate, and about 1 ml of distilled water which had been adjusted to pH 8 was added. The pH of the samples was further adjusted higher than 9 with 1 M KOH to keep the phytocomponents ionized and then partitioned against 100% ethyl acetate. The aqueous and organic phases were separated using a funnel and the lower aqueous phase was transferred to a new 10 ml tube. The pH of the solution was lowered to below 3 using concentrated acetic acid to conserve the phyto-components in protonated form. The acidic sample was partitioned against 100% ethyl acetate and dried by passing it through anhydrous sodium sulphate. The sample were spiked with 50 ppm internal standard (Benzophenone) and analyzed with Gas Chromatograph-Mass Spectrophotometer.

Extraction and evaluation of amino acids
Amino acids were extracted as described by AOAC (1997). Leaf disc, embryo and media from culture vessels with green and brown leaf discs with and without embryos were weighed into screw-cap Pyrex borosilicate tubes. About 2 ml of Performic acid was added and the samples were incubated at 0°C for 16 hours. About 0.84 g sodium metabisulfite and 3 mL 6N hydrochloric acid were added to the mixture. The tubes were thoroughly flushed with nitrogen, quickly capped, and placed in an oven at 110°C for 24 hours. Approximately 250 µl of the hydrolysed extracts was evaporated under a stream of nitrogen. The residue extract was redissolved in 1 ml 0.1 M borate buffer and filtered through 0.2 µM syringe filters and analysed with HPLC.

Data collection on the sugars
The sugars glucose, fructose and sucrose were analyzed by injecting about 50 µl of the extract/sample into Knauer HPLC equipped with a Eurospher 100-5 NH2 column and a reflective index detector. The mobile phase was 75% acetonitrile HPLC grade (SCHARLAU) and 25% distilled water at a flow rate 1 ml/minute under ambient temperature. Glucose, fructose and sucrose were identified by comparing the retention time with that of sucrose standard (Fischer Scientific)and their concentration was calculated from peak areas using calibration equations, where: Concentration of analyte (C1) = Peak area of analyte/slope of the standards' calibration curve. Content (mg/g) of the analyte = [C1 x V x 1000 -3 ]/W, Where C1= concentration (mg/L) of the analyte in the test solution, V = the volume (mL) of the test solution, and W = the weight (g) of the sample used for the preparation of the test solution.

Data collection on caffeine and chlorogenic acid
Caffeine and chlorogenic acid were analyzed by injecting about 50 µl of eluate/sample into Knauer HPLC equipped with a super Co Discovery C-18 column for caffeine and BDS HYPERSIL C-18 column for chlorogenic acid. The detector was diode array at wavelengths 278 nm and 324 nm for caffeine and chlorogenic acid, respectively. The mobile phase was methanol HPLC grade (PANCREAC) 35%, distilled water 65%, acetic acid (PROLABO) 0.1%, at a flow rate of 1 ml/minute under ambient temperature. Caffeine and chlorogenic acid were identified by comparing the retention time of caffeine standard (99%) (Fischer Scientific) and chlorogenic acid standard (Acros Organics) and the sample peaks. The concentration was calculated from peak areas using calibration equations, where: Concentration of analyte (C1) = Peak area of analyte/slope of the standards' calibration curve. Content (mg/g) of the analyte = [C1 x V x 1000 -3 ]/W, Where C1 = the concentration (mg/L) of the analyte in the test solution, V = the volume (mL), of the test solution, and W = the weight (g) of the sample used to prepare the test solution.

Data collection on phyto-components
Identification relied on matching the mass spectrometric fragmentation pattern corresponding to the various peaks in the samples total ion chromatogram with those present in the National Institute of Science and Technology mass spectral database. Integration was done automatically for the individual peaks. The sample was spiked with 50 ppm internal standard (Benzophenone) and injected into GC-MS (Model QP2010 SE, Shimadzu) and fitted with DB 5 column. Detector used was MS and Helium was used as the carrier gas at a flow rate of 1 ml/min. The phyto-components were estimated using the following equation: Concentration of analyte (C1) = Peak area of analyte/peak area of internal standard x concentration of internal standard.
Content (µg/g) of the analyte = [C1 x V]/W, Where C1= the concentration (ppm) of the analyte in the test solution, V = the volume (mL) of the test solution, and W = the weight (g) of the sample used for the preparation of the test solution.

Data collection on amino acids
Amino acids were analyzed by injecting about 50 µl of the eluate/sample into Knauer HPLC system equipped with a super Co Discovery C-18 column at a flow rate of 1 ml/minute under ambient temperature. A Knauer fluorescent detector was used at excitation wavelength of 350 nm and emission wavelength of 450 nm. Two mobile phases were used: Mobile Phase A: was 0.01M Na2HPO4 at pH 8.2 which was prepared by dissolving 1.42 g Disodium phosphate heptahydrate (Na2HPO4) and 3.81 g Sodium borate (Na2B4O7.10 H2O) in 950 ml milliQ reagent grade water then adding 32 mg sodium azide (NaN3). The pH was adjusted to 8.

Data Analysis
The XLSTAT computer software was used to perform statistical correlation analysis using Pearson correlation coefficients to establish the relationships among the endogenous factors identified. The data values were subjected to Principal Component Analysis (PCA) using XLSTAT 2018 software and a distance biplot was constructed.

RESULTS
A total of 71 endogenous biochemical components were identified and quantified (Table 1). To narrow down which of the endogenous biochemical components present during somatic embryogenesis of Ruiru 11 could be utilized as biochemical markers for somatic embryogenesis, they were analysed to identify a core set that were consistently detected in embryogenic and non-embryogenic cultures.

GE -Green leaf discs with embryos, BE -Brown leaf discs with embryos, GW -Green leaf discs without embryos, BW -Brown leaf discs without embryos
From the PCA, high sucrose content was associated with embryogenic cultures and was not present in brown non-embryogenic cultures, whereas disparlure was associated with brown non-embryogenic cultures only. The 5-Hydroxymethylfurfural and steroid derivative, Ethyl iso-allocholate clustered together and was present in brown embryogenic cultures.
Esters and ketone clustered together and were not present or present in minimal amounts respectively in green non-embryogenic cultures. Endogenous alcohol, Hydrocarbons, Fatty acids and caffeine were clustered closely together in the PCA and were least in green non-embryogenic cultures. Fructose and glucose clustered closely and were high in brown non-embryogenic cultures. Correlation analysis was performed to establish relationships of the endogenous biochemical components (Table 2). Glucose and fructose were positively  Bolded values are different from 0 at P = 0.05

DISCUSSION
There are different factors involved in the success or failure of the somatic embryogenesis response. An important "factor" that affects the somatic embryogenesis response is the release of organic molecules by the explants into the culture medium (Nic-Can et al., 2015); some of the secreted molecules have been demonstrated to induce or modulate the somatic embryogenesis response. Other compounds, mostly secondary metabolites, have been found to inhibit the embryogenic response of the cells. Among the first biochemical observations on somatic embryogenesis was the necessity of a ''factor'' required to coordinate cell division and morphogenesis. This "factor" includes polysaccharides, amino acids, growth regulators, vitamins, low molecular weight compounds and polypeptides (Chung et al., 1992). These molecules play an important role in the development of somatic embryogenesis as either inhibitors or inducers (Matthys-Rochon, 2005). Storage compounds are important markers of physiological quality of somatic embryos and a failure to produce them may affect the somatic embryo final developmental stages and conversion to plants (Cailloux et al., 1996).
Variations in the endogenous components indicated that embryogenic and non-embryogenic cultures utilize their nutrients differently. A positive correlation between fructose and glucose resulted and these sugars were highest in non-embryogenic cultures compared to embryogenic cultures. In addition, these two sugars were negatively correlated with all the other biochemical compounds and clustered separately from the rest. A negative effect of accumulation of 6-carbon sugars such as fructose and glucose in embryo cells has been suggested and being of reducing nature they cannot accumulate to high levels without harmful effects, but their great advantage is a more direct entry into metabolism (Lipavská and Konrádova, 2004).
Lopez-Molina (2001) and Rolland et al. (2002) reported that high levels of sugars during seedling growth may be an indication of untenable growth conditions culminating in arrested development as a protection measure. This may be the reason why cultures with high fructose and glucose did not develop somatic embryos. Sucrose on the other hand was highest in embryogenic cultures and this indicated that during embryogenesis, there is increased uptake, and/or synthesis of the same as reported by Mahmud et al. (2014). Positive correlations between sucrose and chlorogenic acid was attributed to the fact that these compounds are considered as signaling molecules for differentiation and embryo development (Guan et al., 2009;Khosroushahi et al., 2011). Phenolic compounds and intermediaries of phenylpropanoid metabolism may stimulate inactivation of indole acetic acid (IAA), thereby lowering the physiological levels of IAA in the plant tissue that is undergoing differentiation and create a situation that is more favourable for organogenesis (Jain et al., 2014).
Positive correlations that occurred among alcohols, aldehydes, hydrocarbons and esters may be related to the biosynthetic pathway of fatty acids. This is because the fatty acids undergo an acyl reduction pathway in which a reduction reaction occurs to form aldehyde intermediaries and can further reduce to primary fatty alcohols.
In an alkane pathway, a typical reduction reaction occurs to give the aldehyde intermediaries in the acyl reduction pathway followed by decarbonylation to form odd-numbered alkanes. Often, this is the end product of the pathway but in some cases, the alkanes can carry out further downstream conversions by either hydroxylation to secondary alcohols which can then be followed by oxidation into ketones (Sin, 2012).

CONCLUSIONS AND RECOMMENDATIONS
Sucrose, phenolics, alkaloids, amino acids, fatty acids and their derivatives correlated positively and this implied that they are promoters or signal molecules for somatic embryogenesis in coffee. Endogenous fructose and glucose were associated with non-embryogenic cultures. In addition, these sugars were negatively correlated with the other biochemical components. Thus endogenous fructose and glucose are inhibitors of coffee somatic embryogenesis. Endogenous sucrose, chlorogenic acids, caffeine amino acids, fatty acids and their derivatives are potential biomarkers for coffee somatic embryogenesis. Further studies regarding the status of the biochemical compounds identified, especially in particular stages of embryo development are needed in order to propose treatments to improve coffee somatic embryo development.