This study was conducted to compare the growth and yield of one of the commercial hybrid coffee cultivars (Coffea congensis x Coffea canephora) of robusta coffee established from somatic embryogenesis as well as conventional seedlings. Results indicated no statistically significant differences in the growth pattern or the cumulative yield between the somatic embryogenesis derived plants and the seedlings. The genetic fidelity of somatic embryogenesis derived plants and the mother plant was tested using sequence related amplified polymorphism (SRAP) markers. A total of 24 SRAP primers were employed for DNA analysis which produced a total of 153 clear, distinct and reproducible amplicons of variable size. Out of 24 SRAP primers, 9 primers produced amplification patterns which are identical between the mother plants and plants derived from somatic embryogenesis. Cluster analysis revealed more than 95% genetic similarity between the somatic embryogenesis derived plants and the mother plants indicating a high degree of genetic fidelity. The present study clearly demonstrates the usefulness of SRAP markers in genetic fidelity analysis of coffee.
Coffee is one of the most important plantation crops and agricultural export commodities in the world. It is grown in about 80 countries of the tropical and subtropical regions of the world, especially in Africa, Asia and Latin America. The total global production of green coffee is about 143.25 million bags (60 kg capacity) with an export earning of over US$31.8 billion during 2014. Although more than 100 coffee species are known, only two species, i.e. Coffea arabica (known as arabica coffee) and Coffea canephora (known as robusta coffee), are commercially cultivated and consumed. Arabica coffee is favored over robusta coffee because of its quality and aroma and constitutes about 60% of total global coffee production. However, in recent years, there has been a shift towards robusta coffee cultivation because of the rising production costs, labor scarcity and other difficulties in providing timely inputs especially for disease and pest management associated with arabica cultivation. This challenging scenario has impelled focused research on robusta coffee in India.
Traditionally coffee is propagated through seeds and new plantations are established by planting the seedlings. However, in C. canephora, propagation through seeds is not desirable and leads to the appearance of off type plants in the progeny due to cross pollination and segregation. Thus, in vitro propagation is the method of choice to palliate this problem and propagate elite mother plants in large numbers. There are several reports on micropropagation and in vitro multiplication of plants via somatic embryogenesis in coffee. However, successful reports on field establishment and performance of micropropagated plants of coffee are very much limited. The progress on micropropagation in coffee has been reviewed [1-3]. In Brazil, an experimental field was established to evaluate the tissue culture plants of Bourbon obtained from somatic embryos produced in both solid and liquid media and compare their performance with the seedlings of the same line . In another study, five robusta clones are being field tested in five coffee producing countries (4000 plants/location): Philippines, Thailand, Mexico, Nigeria and Brazil. The visual inspection of 8000 plants under field conditions in Philippines revealed that all the micropropagated robusta plants have normal vegetative and reproductive growth with normal flowering and fruit set after 2 years of field planting . Similarly, Etienne et al.  and Etienne & Bertrand  evaluated 20,000 field grown arabica tissue culture plants in four Central American countries and reported the trueness-to- type and agronomic characteristics of tissue cultured plants produced by embryogenic cell suspensions. Muniswamy et al.  reported the field performance of tissue cultured plants of Coffea arabica. However, micropropagation through somatic embryogenesis involving a prolonged callus phase is considered to be unreliable due to the genomic changes of in vitro raised plantlets [9,10]. Thus the genetic fidelity of the invitro derived plantlets should be tested as early as possible especially in woody perennials which have a long life cycle.
Several strategies have been employed to assess the genetic fidelity of regenerated plants of which DNA based molecular markers have gained paramount importance during recent years. Various molecular markers such as RAPD [11-14], AFLP [15,16], SSR  and ISSR  are used for determining the genetic fidelity of micropropagated plants. In coffee (C. arabica), Landey et al.  analyzed genetic fidelity of in vitro propagated plants of C. arabica using AFLP and SSAP markers and confirmed that genetic variation observed in micropropagated plants are caused by abnormal chromosome numbers. Interestingly, these authors could not detect genetic variability among micropropagated plants using AFLP markers. Recently, a new class of molecular marker known as sequence related amplified polymorphism (SRAP) has been increasingly used for genetic diversity and gene mapping . Recently we have reported the use of SRAP markers in coffee [21-23]. The SRAP is a PCR based marker system that preferentially amplifies coding sequences randomly distributed throughout the genome. Moreover SRAP markers possessed multi loci and multi allelic features which made them more suitable than other marker systems in genetic analysis compared to other marker systems, the multi allelic features of SRAP markers make them more suitable for genetic analysis . More recently, SRAP markers were used in assessing the genetic fidelity of some plants obtained through tissue culture [25-27]. Here we describe the field performance and genetic stability of in vitro regenerated plants of Coffea canephora (CxR) hybrids using SRAP marker system. To our knowledge, SRAP has been used for the first time in assessing the genetic fidelity of coffee obtained through somatic embryogenesis.
2.1 Selection of elite plants and somatic embryogenesis
Superior mother plants of C. canephora (cv. CxR) were identified in some of the estates in Karnataka and used as the source material for in vitro multiplication. CxR is an interspecific hybrid between C. congensis and C. canephora which was developed at Central Coffee Research Institute (CCRI) and widely cultivated in India. Leaf explants were incubated in Murashige and Skoog (1962) medium  supplemented with 2, 4-D (1 mg L−1) and kinetin (5 mg L−1) for callus induction. After four weeks of incubation in callus induction medium, the developed calli were transferred to MS medium supplemented with 2, 4-D (0.1 mg L−1), IAA (1 mg L−1) and kinetin (4 mg L−1) along with proline (20 mg L1) for somatic embryo induction. The somatic embryos were transferred to MS medium with kinetin (0.1 mg L−1) for germination and development. The well grown in vitro plantlets were planted in net pots/ polybags containing soilrite mixture and hardened in a partially shaded area under polytunnels. After hardening, these somatic embryo derived plants and the seedlings of the corresponding mother plants were field planted and established in different locations. In each location, an average of 600 tissue culture derived plants and seedlings raised from the identified mother plant were planted in the field for agronomic evaluation. One hundred randomly selected tissue culture plants and equal number of seedlings were selected and growth and agronomic features like stem diameter, bush spread, number of primaries, length of primaries, number of nodes per primary, leaf area and total fruit yield were recorded for five consecutive years and data was subjected to statistical analysis. The projected mean yield of somatic embryo derived plants and seedlings were compared statistically The seed samples collected from the somatic embryo derived plants and seedlings were graded and analyzed for both bean parameters as well as organoleptic evaluation at the Coffee Board’s Quality Evaluation Centre.
2.2 DNA isolation
Genomic DNA was isolated from fresh young leaves using a modified CTAB method as described earlier by Mishra et al. . About 200 mg of freshly collected leaf tissue was ground to fine powder in liquid nitrogen, transferred to a 30 ml tube containing 5 ml preheated extraction buffer (2% CTAB (w/v), 100 mM Tris-HCL (pH 8.0), 25 mM EDTA, 2M NaCl, and 0.1% beta-mercaptoethanol). The tubes were incubated at 60°C for one hour with occasional shaking for thorough mixing. After incubation, the tubes were cooled to room temperature and centrifuged at 6000 rpm for 20 min following which the supernatant was transferred into a fresh tube and extracted twice with chloroform-isoamyl alcohol (24:1). The supernatant was transferred to 2 ml tubes, precipitated with 0.7 volume of ice cold isopropanol at room temperature for 30 min, and then centrifuged at 8000 rpm for 20 min at 4°C. The resultant pellet after centrifugation was washed with 75% (v/v) ethanol for 10 min and dissolved in 60 µl of Tris-EDTA (1-10mM). The concentration of DNA was measured using a 0.8% agarose gel stained with ethidium bromide as well as via a UV spectrophotometer at 260 nm. The ratio of the absorbance at 260 and 280 nm (A 260/280) was used to assess the purity of DNA. The re-suspended DNA was then diluted to 10 mg/l concentration in sterile distilled water and kept at -20°C for further use in amplification reactions.
A total of 40 SRAP primer pairs involving ten forward and thirteen reverse primers were initially screened of which 24 primer pairs (Table 1) have given consistent banding pattern and therefore were used in the genetic fidelity analysis. SRAP analysis was performed by adapting the procedure described by Li & Quiros  with minor modifications as described earlier by Mishra et al. . Each 20 µl PCR reaction mixture consisted of 30 ng template DNA, 2 µL of 10x reaction buffer (75 mM Tris-HCl, pH 8.8, 20 mM (NH4)2 SO4, 0.01% Tween 20), 200 µM dNTP mixture, 2.5 mM MgCl2, 3 µM each of forward and reverse primer, and 1.0 U Taq DNA polymerase. The PCR amplification program followed was 4 min initial denaturation at 96°C; 5 cycles consisting of 1 min denaturation at 94°C, 1.15 min primer annealing at 35°C; and 2 min extension at 72°C followed by 30 cycles consisting of 1 min denaturation at 94°C, 1.15 min primer annealing at 50°C; and 2 min extension at 72°C; and a final extension of 15 min at 72°C. PCR products of SRAP were run on 2.0% (w/w) agarose gels containing 0.5 µg ethidium bromide/ml in 1X TAE buffer and then visualized and photographed using the UV-transilluminator (Biorad).
|Name||Forward primer (5’ – 3’)||Name||Reverse primer (5’ – 3’)||Primer combinations used|
2.3 Data analysis
SRAP amplified bands were scored for presence (1) or absence (0). The total number of bands, distribution of bands across all the collections, polymorphic bands, and average number bands per primer were calculated. A pairwise similarity matrix was constructed using the Dice similarity coefficient . The relationship between various germplasm collections was displayed as a dendrogram constructed using NTSYS –PC 2.1 software  based on the unweighted pair group method using arithmetic averages (UPGMA). Statistical support of the clusters was assessed by means of 1000-bootstrap replicates.
3.1 Somatic embryogenesis and plant establishment
More than 90% of the leaf explants produced calli following incubation in the callus induction medium. Initially the calli were thin, greenish and developed all along the cut surface of the explant. After one month, the calli developed profusely and enlarged and subsequently turned slightly brownish but still attached to the leaf explants. Forty five days after incubation, globular somatic embryos started developing as tiny structures in the callus mass (Figure 1A). The number of these globular somatic embryos increased and after 90 days of incubation these somatic embryos were prominent. When the somatic embryos were transferred to auxin free MS medium with kinetin (0.1 mg L−1,) germination and development of somatic embryo took place with the rapid development of cotyledonary leaves (Figure 1B & 1C). However, all the somatic embryos did not give rise to plantlets but continued to produce secondary somatic embryos. The well grown in vitro plantlets with 3-4 pairs of leaves and well grown root system (Figure 1D) were planted in netpots/polybags containing soilrite mixture and hardened in a partially shaded area under polytunnels (Figure 1E). About 30% of the plants survived the hardening process. After hardening, these somatic embryo derived plants and their seedlings were field planted and established in different locations (Figure 1F & 1G).
3.2 Comparison of growth and yield
The vegetative growth and fruit yield data were recorded for 5 consecutive years and data was analyzed statistically. After the initial plant establishment, height of both the somatic embryo derived plants and their seedlings were restricted to 3 feet by topping. Morphologically, somatic embryo derived plants of C. canephora cv. CxR were more uniform and vigorous than seedlings in all the locations established. The average stem girth obtained in seedlings (6.24 cm) is slightly higher than somatic embryo derived plants (5.92 cm) (Table 2). Similarly, the average bush spread of seedlings (226 cm) was slightly higher than that obtained for somatic embryo derived plants (232 cm). The average number of nodes per primary was found to be similar in both somatic embryo derived plants (7.2) and the seedlings (7.06). The leaf area ranged between 240-266 cm2 in somatic embryo derived plants as compared to seedlings with 240-250 cm2.
|Materials||Stem diameter (cm)||Bush spread (cm)||No. of primaries||Length of the primary (cm)||No. nodes/ primary||Range of fruits /node||Fruit yield/ plant||Growth Index Score[*]|
|Somatic embryoderived plants||5.92||232||4.56||107.16||7.18||20-56||4.54||40.21|
In somatic embryo derived plants 20-56 fruits were produced per node whereas only 12-30 fruits were produced per node in seedlings. Fruit yield per plant was higher in somatic embryo derived plants (4.54 kg/plant) as compared to seedlings (3.80 kg/plant) (Table 2). The weight of 100 fruits was similar in both the plant types with 195.8 g in somatic embryo derived plants and 190 g in seedlings. The percentage of floats is more or less same in somatic embryo derived plants (5.1%) and seedlings (4.98%). Values of growth index was computed based on four morphological characters, viz., number of primaries, length of the primary, nodes on primary and stem girth, which are presented in Table 2. Somatic embryo derived plants of C. canephora cv. CxR registered highest growth index (40.21) compared to seedlings (31.15) (Table 2)
Yield data was recorded for five consequent years after four years of field planting in both somatic embryo derived plants and seedlings of C. canephora cv. CxR plants. Highest fruit yield were recorded in the fifth year in somatic embryo derived plants (8091 kg/ha) followed by seedlings (7096 kg/ha) in one of the trial plot in Coorg, Karnataka (Figure 2). Highest average clean coffee of 1348 kg/ha was recorded in somatic embryo derived plants followed by 1182.7 kg/ha in seedlings.
The coefficient of variation (CV) was slightly lower for stem diameter in somatic embryo derived plants, bush spread and length of primaries and highest for number of primaries in somatic embryo derived plants of C. canephora cv. CxR plants (Table 3). Though somatic embryo derived plants showed higher CV for fruit yield and number of nodes per primary in different locations, no significant variation in yield was obtained in a paired t-test (Table 3).
|Materials||Stem diameter (cm)||Bush spread (cm)||No. of primaries||Length of the primary (cm)||No. nodes/ primary||Fruit yield/plant||Mean yield (kg ha-1) (CV)|
|Somatic embryoderived plants||10.89||14.56||19.89||13.87||5.34||41.30||5441 (41.30)|
|Paired ‘t’ test for mean yield||Non-Significant|
3.3 Bean grade percentage and cup quality
Bean parameters and organoleptic quality analysis was carried out and presented in Table 4. In somatic embryo derived plants percentage of pea berries, AA and A grade seeds recorded was higher than that obtained in the seedlings. Higher percent of B and C grade beans was obtained in seedlings. The average to good cup quality obtained in somatic embryo derived plants of C. canephora cv. CxR in all the locations was fair average quality (FAQ) whereas the cup quality of seedlings was below fair average quality (below FAQ).
|Materials||PB||AA||A||B||C||cuts & bits||Cup quality rating|
|TC plants||9.68||52.24||10.23||2.91||0.28||24.66||AVERAGE FAQ|
|Seedlings||5.66||--||2.41||56.06||33.62||2.28||BELLOW AVERAGE FAQ|
3.4 Assessment of genetic uniformity of in vitro regenerated plants
Forty SRAP primer combinations were initially tested of which 24 primer combinations produced unambiguous and clear amplification patterns. These 24 primers could produce 153 distinct scorable bands among the 11 regenerated as well as the mother plant. The number of amplified fragments ranged from 1 (Me4-Em1) to 16 (Me3-Em12) with an average of 6.37 bands per primer combination (Table 5). The size of the amplified fragments ranged from 50 bp to 2000bp (Figure 3). Of the total 153 amplified fragments, 49 were polymorphic with an average 2.04 polymorphic fragments per primer combination. Nine out of 24 primer combinations showed 0% polymorphism (Figure 3) whereas primer combination Me1-Em12 showed 100% polymorphism with mean polymorphism of 29.42%. The resolving power (Rp) of 24 primer combinations ranged from 2.0 (Me4-Em1) to 26.77 (Me3-Em12) with a mean of 11.16. Similarly, the average PIC or the genetic diversity of the 24 SRAP primers ranged from 0 to 0.408 (Me1-Em12) with a mean of 0.148.
|Primer combinations||Total bands||Size range||Range and average number of bands||No. of polymorphic Percentage of||RP||PIC|
|Me-1- Em-12||5||100-1200||1-4 (3.53)||5||100||7.077||0.408|
|Me-2 – Em-3||6||50-1300||6-6 (6.0)||0||0.00||12.00||0.000|
|Me-2 – Em-4||12||50-2000||10-12 (11.53)||2||16.66||23.08||0.067|
|Me-2 – Em-6||6||150-1900||6-6 (6.0)||0||0.00||12.00||0.000|
|Me-2 – Em-7||5||250-850||3-5 (4.0)||2||40.0||8.00||0.228|
|Me-2 – Em-12||8||100-850||8-8 (8)||0||0.00||16.00||0.000|
|Me-3 – Em-3||5||150-1500||4-5 (4.15)||2||40.0||8.31||0.252|
|Me-3 – Em-4||2||100-1600||2-2 (2.0)||0||0.00||4.00||0.000|
|Me-3 – Em-11||11||150-1400||7-10 (7.23)||5||45.45||14.46||0.375|
|Me-3 – Em-12||16||50-1400||11-15 (13.38)||5||31.25||26.77||0.222|
|Me-4 – Em-1||1||800||1-1 (1)||0||0.00||2.00||0.000|
|Me-4 – Em-2||3||475-550||3-3 (3)||0||0.00||6.00||0.000|
|Me-4 – Em-11||4||250-600||1-4 (3.38)||3||75.00||6.77||0.263|
|Me-4 – Em-16||8||150-1300||6-8 (7.61)||2||25.00||15.23||0.087|
|Me-5 – Em-5||4||100-850||4-4 (4.0)||0||0.00||8.00||0.000|
|Me-6 – Em-5||6||150-1200||4-6 (5.0)||2||33.33||10.00||0.226|
|Me-6 – Em-15||5||100-1600||5-5 (5.0)||0||0.00||10.00||0.000|
|Me-10 – Em-13||8||250-1400||4-7 (6.76)||5||62.50||13.54||0.198|
|Me-10 – Em-16||7||100-900||2-7 (5.38)||5||71.42||10.77||0.319|
|Me-11– Em-16||8||150-900||5-8 (6.69)||3||37.50||13.38||0.211|
|Me-13 – Em-11||8||75-1900||6-8 (7.61)||3||37.50||15.23||0.088|
|Me-14 – Em-8||5||175-1250||5-5 (5)||0||0.00||10.00||0.000|
The genetic similarity between the tissue culture plants and the mother plants ranged from 0.84 (between the tissue culture variant and mother plant) to 0.96 (between mother plants and 7 tissue culture derived plants) (Table 6).The mean genetic similarity between various tissue culture plants and mother plants was found to be 0.95. The UPGMA clustering algorithm from SRAP analysis grouped the tissue culture derived plants and mother plants in to two major clusters. (Figure 4). Between the two major clusters one major cluster included tissue culture variant plant along with plant number 6 and the other major cluster divided into two minor clusters. The first minor cluster included tissue culture plant numbers 5 and 10 whereas the second minor cluster divided into two minor subclusters. The first minor subcluster included the mother plant whereas the second minor subcluster divided into two groups. The first group included plant numbers 1, 2 and 3 and the second group included plants 4, 7, 8, 9 and 11.
|Mother plant 1||TC Plant 1||TC Plant 2||TC Plant 3||TC Plant 4||TC Plant 5||TC Plant 6||TC Plant 7||TC Plant 8||TC Plant 9||TC variant||TC Plant10||TC Plant11|
|TC Plant 1||0.967||1|
|TC Plant 2||0.960||0.992||1|
|TC Plant 3||0.963||0.996||0.996||1|
|TC Plant 4||0.956||0.989||0.989||0.992||1|
|TC Plant 5||0.963||0.953||0.952||0.956||0.963||1|
|TC Plant 6||0.914||0.904||0.903||0.907||0.915||0.930||1|
|TC Plant 7||0.960||0.992||0.992||0.996||0.996||0.960||0.910||1|
|TC Plant 8||0.960||0.992||0.992||0.996||0.996||0.960||0.910||1||1|
|TC Plant 9||0.960||0.992||0.992||0.996||0.996||0.960||0.911||1||1||1|
|TC Plant 10||0.950||0.932||0.932||0.936||0.943||0.966||0.963||0.939||0.939||0.939||0.883||1|
|TC Plant 11||0.956||0.989||0.989||0.992||0.992||0.956||0.906||0.996||0.996||0.996||0.835||0.935||1|
Somatic embryogenesis is an important technology for rapid and mass multiplication of elite genotypes of many plant species . In coffee, the somatic embryogenesis system is well studied  and large scale propagation of improved cultivars of robusta and arabusta were taken up using bio-reactors [34,35]. In another study, agronomic performance of plantlets derived through somatic embryogenesis and microcuttings of axillary shoots were taken up and no significant differences in morphological and yield pattern were observed between somatic seedlings and microcutting derived plants . In the present study, no significant differences in morphological features were obtained between the seedlings and tissue culture plants. In Carcia papaya, no morphogenic differences were observed between the invitro raised plants and seedlings . Jain & Datta  compared various morphological characters such as leaf shape, petiole length, area of leaf lamina and internodal distance in both in vtro raised plants of Morus bombycis cultivar, Shimanochi and the vegetative propagated saplings and observed no significant quantitative variation in these characters between them and the results obtained in the present study supports their contention. In the present study, though minor differences in average yield was obtained between tissue culture plants and seedlings they were not statistically significant. In a previous study, Ducos et al.  did not observe any significant differences in yield pattern of somatic derived plants and plants derived from microcuttings of coffee and the present observation did not deviate from their findings. In Musa, micropropagated plants did not confer a yield advantage over conventionally propagated plants . Yield is controlled by many factors in the same environment and not always corresponds with the genotypic homogeneity.
Genetic variation has frequently been observed in plants regenerated from tissue cultures . The degree and type of variation is affected by several factors, including genotype and culture conditions . Jain  proposed that cultures which are exposed to 2,4-D for prolonged periods can accumulate mutations. According to Phillips et al. , the auxin 2,4-D,commonly used for the induction of callus in somatic embryogenesis systems, has been shown to cause genetic and/or epigenetic DNA variation. According to Kaeppler et al. , DNA methylation levels increased with increasing amounts of the auxin 2,4-D and may be an important factor affecting tissue culture-induced variation. In coffee, 10% somaclonal variation is highly genotypic dependent and varied from 3 to 30% in somatic embryogenesis derived plants .
Etienne & Bertrand  observed 3 to 10% off type plants in a population of 30,000 plants belonging to 20 clones of C. arabica F1 hybrids, derived through somatic embryogenesis. In our study, less than 1% of off type plants were observed in the somatic embryogenesis derived plants of Coffea canephora var. CxR which could be mainly attributed to the genotype since 2,4-D was also used by us for callusing and somatic embryogenesis.
In recent years, molecular markers have been increasingly used for capturing the morphologically indistinguishable somaclonal variants regenerated through various in vitro methods in different crops such as Solanum aculeatissimum , Olea europaea , Dendrocalamus hamiltonii , Capparis deciduas  and Camellia spp. . In the presen tstudy, we have used sequence related amplified polymorphism (SRAP) marker for unravelling the genetic variation between the mother plants and the plants regenerated through somatic embryogenesis. Molecular marker analysis demonstrated 95% genetic similarity between mother plants and the regenerated plants, indicating true-to-type progeny. In earlier studies, Sun et al.  and Li et al.  reported suitability of SRAP PCR for assessing the genetic fidelity of tissue culture plants and our result support their contention. In jojoba, Kumar et al.  used RAPD and ISSR markers and observed 100% genetic similarities between the micropropagated plants and mother plants. Bhatia et al.  evaluated genetic fidelity with 100% similarity of in vitro propagated plants and mother plant of gerbera using RAPD and ISSR markers. Similarly, Zarghami et al.  evaluated the genetic stability of cryopreserved and non-cryopreserved potato and obtained 97-100% similarity using AFLP markers. Recently, Landey et al.  observed high genetic and epigenetic stability of Coffea arabica plants derived from embryogenic suspensions as well as secondary embryogenesis using AFLP and MSAP markers. The present findings of high genetic similarities between mother plants and the plants derived from somatic embryogenesis are concurrent with the earlier findings. In conclusion, in vitro plant regeneration through somatic embryogenesis could offer a promising system for breeding program of Coffea canephora we have been pursuing for some years now.
The authors thank Dr. C.S. Srinivasan, former Joint Director of Research, CCRI, for helping with the statistical analysis of the data and Mr. Venkatesh, Coffee Quality Specialist, Coffee Board, Bangalore for analysis of cup quality.
MKM conceived the experiment. MB carried out the tissue culture experiments and field data collection. BK & MKM performed the SRAP analysis. MKM & RY prepared the manuscript. All authors have gone through the manuscript and approved.
Conflict of interest
Conflict of interest disclosure: The authors declare no conflict of interest.
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coefficient of variation
Murashige and Skoog
Cetyl trimethylammonium bromide
Indole Acetic Acid
Sodium Dodecyl Sulphate
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