3.1. Callus induction
For androgenic callus induction, anthers, bearing early-to-late uni-nucleate stages of microspores from 4.0 mm size flower buds of TV 19 cultivar of tea (Fig. 1A) were utilized. Surface sterilization of flower buds with 0.8% sodium hypochlorite for 7 minutes yielded more than 93% aseptic cultures. MS medium in combination with various growth regulator combinations and the carbon sources were tested, callus induction was observed in the four media combinations listed below.
MS (60 g/l sucrose) + 2,4-D (1 µM) + NAA (1 µM) + BAP (5 µM).
MS (40 g/l sucrose) + 2,4-D (3 µM) + TDZ (18 µM).
MS (30 g/l sucrose) + 2, 4-D (5 µM) + kinetin (5 µM) + L-glutamine (800 mg/l) + L-serine (200 mg/l).
MS (60 g/l glucose) + 2, 4-D (5 µM) + kinetin (5 µM) + L-glutamine (800 mg/) + L-serine (200 mg/l).
Among the four media combinations tried, the highest percentage callusing was observed on MS1 medium (Table 1). Percentage response on callus induction medium was higher for the cultures incubated continuously in dark, compared to those incubated in light, at 25±2ºC (Table 1).
The anthers were exposed to two different temperature pre-treatments before being transferred to 25±2ºC light conditions. Maximum callusing was observed at 25ºC under dark incubation (Control), followed by cold pre-treatments at 5ºC for 5 days in the dark. Pre-treatment of anther cultures at 33ºC for 5 days in the dark was not effective for callus induction, rather it showed adverse effect (Table 1). Two-way ANOVA was done to study the effect of responding media and temperature pre-treatments on anther cultures. Both the factors, media and temperature pre-treatments were found to be significant factors with p-value less than 0.05. Cold or heat treatment beyond 5 days was found to be inhibitory for callus induction.
Although incubation of anthers in the dark at 25ºC (control) showed highest percentage of callusing, a better response in terms of induction of calluses from inside the anther locules was recorded when anthers were subjected to cold pre-treatment at 5ºC for 5 days in the dark (Table 1). Maximum callusing 41% was induced from bursting of anthers at 5ºC for 5 days in the dark on MS1 medium while cultures incubated in the dark at 25ºC temperature on same medium exhibited only 35 % of callusing (Fig. 2).
Further, the effect of addition of two carbon sources, at varying concentrations, on anther culture was studied. It was observed that sucrose was significantly better (p<0.05) than glucose (Fig. 3). But, the percentage of anthers that showed callusing and the extent of callusing differed with varying concentration of sucrose (30 - 120 g/l). Distinctly better callus induction was found with (60 g/l) sucrose, followed by 30, 90 and 120 g/l. The concentrations of sucrose above 9%, however inhibited callusing (Fig. 3).
The anthers enlarged to almost double of their original size on best responding medium within two weeks of culture initiation and a complete longitudinal furrow appeared on the anthers after 6 weeks from culture initiation time (Fig. 1 B). After seven weeks, anther sacs burst open, oozing off small calluses from inside the locules, which later showed shiny, white, transparent appearance after 8 weeks (Fig.1 C). After 10 weeks, distinct white, shiny, profuse callus was seen from each anther locule (Fig.1D) and, entire anthers were covered with cream and brown callus later. Therefore, after 10 weeks, the anthers were subcultured into a new medium with similar composition and maintained in the dark. Some callusing was also observed from walls of anthers, however, only the calluses that originated from inside anther locules were considered for further experiments on regeneration.
3.2. Callus multiplication and regeneration
Callus was subcultured twice at six weeks intervals on callus induction medium, with dark incubation. Though rate of callus multiplication was slow on callus induction medium, the calluses grew into white hard callus without any sign of regeneration (Fig. 1E) until after the first two subcultures. During the third subculture, the calluses were shifted to diffused light conditions at 25 ± 2ºC, where it turned green, hard and compact nodular (Fig. 1F) on the induction medium. The rate of callus multiplication was low, thus, to obtain profusely growing calluses, a range of growth regulator combinations were tested. Best callus proliferation in terms of fresh cell biomass increase was observed on MS + NAA (5 µM) + BAP (10 µM) medium in single growth cycle of 8 weeks. Initially the calluses were soft and fast growing. However, at the end of 8 weeks, texture of the calluses turned into hard, compact and nodular callus (Fig. 1G). Later histological examination of the nodular callus confirmed the presence of meristemoids within the calluses (Fig. 1H).
3.3. Embryo regeneration, maturation and germination
Regeneration in the calluses was observed via embryogenesis when the nodular calluses from callus multiplication medium were transferred to the regeneration medium that consisted of MS + BAP (10 µM) + GA3 (3 µM) + L-glutamine (800 mg/l) + L-serine (200 mg/l). Asynchronous embryogenesis was observed on regeneration medium after two subcultures of 8 weeks each. More than 18 embryos per culture were developed in clusters. All the stages of embryos development, globular, heart, torpedo and dicotyledonous, were seen within a single cluster in single passage and the embryos were attached loosely to the surface of calluses (Fig. 4A, B). The SEM images also confirmed asynchronous embryogenesis (Fig. 4 C, D).
Full and ½ MS basal medium (only major salts reduced to half) were used as controls for maturation and germination of embryos, but did not support growth. Maturation and germination of embryos occurred, after 8 weeks, only when cultures were transferred to ten times reduced concentration of growth regulators and nitrogen sources present in embryo differentiation medium. On MS + BAP (1 µM) + GA3 (0.3 µM) + L-glutamine (80 mg/l) + L-serine (20 mg/l), complete bipolar embryos were observed showing long radicular end and green plumular end (Fig.4 E). Fully developed bipolar embryos germinated into complete plantlets when transferred to ½ MS medium (major salts reduced to half strength) containing BAP (10 µM), GA3 (0.5 µM), IBA (1 µM), L-glutamine (80 mg/l) and L-serine (20 mg/l) (Fig.4 F) within 10 weeks.
3.4. Ploidy analysis
3.4.1. Cytological analysis
Cytological analysis of shoot-tips from field-grown plants (Control) showed the diploid number of chromosomes (2n=2x=30) (Fig. 5 A), while root-tips of in vitro developed plantlets revealed that the majority of the cells were in haploid state (2n=x=15) confirming androgenic haploid induction (Fig. 5 C).
3.4.2. Flow cytometry analysis
The linear fluorescence intensity histograms of relative nuclear DNA content of leaves from field-grown plant (control) and in vitro derived plantlets showed distinct G0/G1 peaks with coefficients of variation (CV) less than 5.0% for leaves from field-grown plant and in vitro derived plantlets. The representative histograms from leaves of field grown donor plants (control) is seen in (Fig. 5 B), where G1 peak was observed at channel position 699 and G2 phase at 1397. In contrast to control plants, the ploidy analysis of in vitro regenerated haploid plant leaves showing G1 peak at channel position 353 and G2 phase at 712 channel position (Fig. 5 D). The results of flow cytometry confirm that regenerated plantlets have half the DNA content of that found in control diploid plants. With this it can be concluded that in vitro grown cultures have maintained their haploidy status.
3.5. Total phenolic content of androgenic lines
The total phenolic contents from various extracts of TV19 cultivar of tea, i.e. hot water, methanol, ethyl acetate and hexane, was determined through a linear standard curve (y = 0.002x+0.074; r2 = 0.986). The total phenolic contents was mentioned in order of young leaves from donor plants > embryos > calluses. Hot water extract was found to be the most suitable solvent for extraction of phenolics (Fig. 6). Among the androgenic cultures, embryo extract contained the highest amount of phenolics as 43.12 ±2.21 mg GAE/g dry weight when compared to the content in the callus.
3.6. Antioxidant activity of androgenic cultures
3.6.1. DPPH radical scavenging activity of various extracts
The percent scavenging activity of various solvent extracts, obtained from control and androgenic lines (calluses and embryos) of TV19 cultivar (percentage inhibition i.e. I%) are represented in (Fig. 7). The slope of DPPH inhibition curve was greater in extracts prepared from leaves of donor plant (control) (Fig. 7A) followed by embryos (Fig. 7 B) and then calluses (Fig. 7C). Solvent wise DPPH inhibition pattern could also be seen in the order of hot water > methanol > ethyl acetate > hexane (Fig. 7 A, B, C). IC50 values of reference standards and each extract was calculated from the linear equations of the DPPH inhibition curves, respectively. Lower the IC50 value, higher is the antioxidant activity. IC50 values were in the order of young leaves from donor plant < embryos < calluses. Among solvents, maximum antioxidant activity was found in hot water extracts, followed by methanol, ethyl acetate and hexane (Fig. 7). The hot water extracts of TV 19 leaves (control), embryos and calluses exhibited DPPH inhibition with IC50 values of 29.98±1.34 μg/ml, 46.21±2.21 μg/ml and 53.37 ±2.15 μg/ml, respectively. In order to understand the free radical scavenging potential of sample extracts, their activities were compared with relative activities of standard antioxidant compounds (ascorbic acid, catechin (C), epicatechin (EC), epigallocatechin gallate (EGCG), gallic acid and vanillic acid). The IC50 values of these reference standards has been added as a supplementary data.
3.6.2. Ferric-reducing antioxidant power (FRAP) of androgenic cultures
Maximum antioxidant activity in above section was found in hot water extracts, therefore, ferric-reducing antioxidant power (FRAP) analysis was performed with the hot water extracts only. The FRAP measures the capability of compounds to act as an electron donor while DPPH measures their capability to act as hydrogen donor. The FRAP values obtained for hot water extracts from all three sources (leaf of control plant, embryos and calluses) of TV19 cultivar are 43.96 mg GAE/g, 26.19 mg GAE/g and 24.86 mg GAE/g respectively (Fig. 8).
3.7. Standardization of a methodology for separation of acid (+)-catechin, (-)-epicatechin, (-)-epigallocatechin gallate, caffeine and theophylline
Absorption maxima of the C, EC, EGCG and caffeine (CAF) standards, dissolved in water, was recorded using UV-Visible spectrophotometer (Cary, USA) which was later utilized as optimized wavelength for detection of these compounds through HPLC. For theophylline (T) standard compound, 273 nm was found as the absorption maximum. Initially, methanol was used as the mobile phase for HPLC, as it has higher absorption and gives higher interference at 280 nm. But, separation of catechins (C & EC) which are structural isomers, requires a solvent with low absorbance which would not obstruct process of peak separation. Hence, separation of all catechins, was carried out using acidic water along with acetonitrile and ethyl acetate i.e. acetonitrile: ethyl acetate: 0.05 % H3PO4 (12: 2: 86 v/v) at a flow rate of 0.5 ml/min, which provided clear separation of all four catechins. The chromatograms of standards C, EC, EGCG and CAF obtained by the same method is shown in Fig. 9 A-D. Acquisition time of all these compounds was less than 25 min. Separation of T standard was carried out using acetonitrile and water in ratio of (10:90) (v/v) as mobile phase with flow rate of 1 ml/min. The chromatogram of T is shown in (Fig. 9 E). An acquisition time of 20 min was required for its separation. The retention times (in minute) for all five compounds are mentioned in Fig. 9.
3.8.1 Linearity and precision
Calibration curves obtained for each of the five standard compounds exhibited clear linearity at tested concentrations in range of (0.025 mg/ml to 1 mg/ml) having the correlation coefficients (R2) of 0.992, 0.996, 0.991, 0.999 and 0.987 for C, EC, EGCG, CAF and T, respectively. The equation achieved from the calibration curve using the external standard was utilized for the estimation of the quantity of compounds that exist in the crude extract. Existence of similar compounds in the crude extracts was assured by co-injecting internal standards of all concerned compounds along with the crude extract in HPLC (Fig. 10 A, B). The accuracy of the method adopted during analysis, as described in materials and methods section, by running the uniform concentration of the standard samples, thrice on same day, RSD values obtained were 1.3 %, 0.9 %, 0.5 %, 1.2 % and 0.3 % for C, EC, EGCG, CAF and T, respectively. The variability in values obtained for inter-day run using similar concentration of all five standards were 1.1 %, 1.2 %, 0.8 %, 1.4 %, and 0.2 % for C, EC, EGCG, CAF and T, respectively (Fig. 9).
3.8.2. Quantification of compounds in crude extract
The amount of C, EC, EGCG, CAF and T in samples (callus and embryo) was calculated using the standard equations and are listed in (Table 2). The pattern of peaks was similar for both standards and sample compounds, where almost all the peaks present in the standard were present in the in vitro haploid callus and embryo samples as well (Fig.10 A-H). Hot water extract from leaves of field-grown donor plant (control) contained highest amount of metabolites. However, among in vitro cultures, embryogenic cultures possessed highest content of all metabolites tested in this study as compared to that in the dedifferentiated (callus) cultures.
3.8.3. Analysis of mass spectra
The HPLC eluted fractions of crude sample were collected at respective retention time, analyzed by mass spectrometry and the fragmentation pattern achieved was compared with that of HPLC eluted standards of respective compounds, procured from Sigma, Aldrich. All the five compounds were analyzed using, positive mode electrospray ionization (+ESI) conditions. Spectra was obtained in full scan mode. Base peak for caffeine was obtained at m/z 195 due to the addition of hydrogen ion in positive mode electrospray ionization [MH+], m/z 217 formed due to formation of sodium adduct [MH++Na+] in HPLC eluted crude sample as well as in standard (Fig. 11 A, B). For (+)-catechin m/z fragments in standard compound and HPLC eluted crude fraction are shown in (Fig. 11 C , D), where the base peak of m/z 291 was obtained due to the addition of hydrogen ion in positive mode electrospray ionization [MH+] and fragments peaks are m/z 139 and m/z 183 due to fragmentation of compound. Similarly in (-)-epicatechin, base peak of m/z 291 was seen due to the addition of hydrogen ion [MH+] and it’s fragment peak was seen as m/z 139 (Fig. 11 E, F). Mass spectra of HPLC eluted (-)-epigallocatechin gallate standard and crude samples is shown in (Fig. 11 G, H), base peak is observed at m/z 459 of [MH+] and two characteristic fragment peaks at m/z 139 and m/z 289, thus, confirming presence of EGCG; m/z 497 formed due to formation of potassium adduct [MH++K+] in HPLC eluted crude samples as well as in standards (Fig. 8 G, H). Mass spectra of HPLC eluted standard and crude samples of theophylline are presented in (Fig. 11 I , J) where base peak is m/z 181 due to addition of hydrogen ion in positive mode electrospray ionization [MH+] and m/z 200 is due to adduct with water [MH++ H2O].