Manoj Mishra

Coffee is a high value agricultural commodity grown in about 80 countries. Sustainable coffee cultivation is hampered by multiple biotic and abiotic stress conditions predominantly driven by climate change. The NAC proteins are plants specific transcription factors associated with various physiological functions in plants which include cell division, secondary wall formation, formation of shoot apical meristem, leaf senescence, flowering embryo and seed development. Besides, they are also involved in biotic and abiotic stress regulation. Due to their ubiquitous influence, studies on NAC transcription factors have gained momentum in different crop plant species. In the present study, NAC25 like transcription factor was isolated and characterized from two cultivated coffee species, Coffea arabica and Coffea canephora and five Indian wild coffee species for the first time. The full-length NAC25 gene varied from 2,456 bp in Coffea jenkinsii to 2,493 bp in C. arabica. In all the seven co...

Isolation of high-quality RNA from coffee is challenging because of high level of polysaccharides, polyphenols and other secondary metabolites. In the present study, a rapid and efficient RNA extraction protocol from different tissues of coffee was optimized. Sufficiently high quality and quantity (225.6-454.8 µg/g) of RNA was obtained by using the optimized protocol. The presence of two distinct bands of 28S rRNA and 18S rRNA in agarose gel proved the intactness of the RNA samples. The average spectrophotometric values of the isolated RNA ranged from 1.96 to 2.02 () and 1.95 to 2.14 (), indicating the high quality of RNA devoid of polyphenols, polysaccharides and protein contamination. In the optimized protocol, addition of PVPP to the extraction buffer and a brief incubation of samples at 65 °C and subsequent purification with potassium acetate resulted in good-quality RNA isolation. The suitability of RNA for downstream processing was confirmed by PCR amplification with cytochrom...

Start codon targeted polymorphism (SCoT), a novel and gene-targeted marker, has recently become the marker of choice in genetic diversity studies. In the present study, 31 SCoT primers were tested for their suitability in the genetic analysis of 21 coffee genotypes representing 18 species. A total of 647 distinct PCR amplified fragments were produced with a mean of 20.9 fragments per primer and 80.80% of which were polymorphic. The polymorphic information content of SCoT primers ranged from 0.16 to 0.86, with a mean value of 0.63. Resolving power ranged from 6.19 to 28.29, with a mean value of 20.2. Species-specific unique PCR amplified fragments were identified for 16 species, which could be used as genetic fingerprints. The genetic similarity among various coffee species calculated using the Dice similarity coefficient ranged between 0.60 and 0.89. The dendrogram constructed using the unweighted pair group of arithmetic means (UPGMA) clustered the 21 coffee genotypes into two majo...

In Coffea arabica (arabica coffee), the phenotypic as well as genetic variability has been found low because of the narrow genetic basis and self fertile nature of the species. Because of high similarity in phenotypic appearance among the majority of arabica collections, selection of parental lines for inter-varietals hybridization and identification of resultant hybrids at an early stage of plant growth is difficult. DNA markers are known to be reliable in identifying closely related cultivars and hybrids. Sequence Related Amplified Polymorphism (SRAP) is a new molecular marker technology developed based on PCR. In this paper, sixty arabica-hybrid progenies belonging to six crosses were analyzed using 31 highly polymorphic SRAP markers. The analysis revealed seven types of SRAP marker profiles which are useful in discriminating the parents and hybrids. The number of bands amplified per primer pair ranges from 6.13 to 8.58 with average number of seven bands. Among six hybrid combina...

Several stomatal characteristics viz. stomatal frequency, epidermal cell frequency, stomatal index, leaf area served per stoma, stomatal plastid number and stomatal guard cell length and leaf architecture including major and minor venation pattern was studied in ten Indian arabica (Coff ea arabica L.) cultivars. Significant variation was observed for all stomatal characteristics as well as the leaf venation pattern such as leaf size, areole size, number of vein endings entering the areole and vein islets termination number in different cultivars and is attributed to their origin involving different parents and selection pressure. The coefficient of variability calculated for all the stomatal features indicated that both stomatal guard cell length and stomatal plastid number were least variable whereas leaf area served per stoma was the most variable character among cultivars. Among all the stomatal features, high heritability (h2) was observed for epidermal cell frequency. In all the cultivars, leaves were simple opposite with moderate mid-vein and entire margins. The major venation pattern was camptodromous type with festooned brochidodromous secondaries. Intersecondary veins were noticed in all cultivars. The marginal ultimate venation was either incomplete or incompletely looped. The functional significance of stomatal features and leaf vein architecture is discussed.

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 pl...

Coffee is one of the most important plantation crops, grown in about 80 countries across the world. The genus Coffea comprises approximately 100 species of which only two species, that is, Coffea arabica (commonly known as arabica coffee) and Coffea canephora (known as robusta coffee), are commercially cultivated. Genetic improvement of coffee through traditional breeding is slow due to the perennial nature of the plant. Genetic transformation has tremendous potential in developing improved coffee varieties with desired agronomic traits, which are otherwise difficult to achieve through traditional breeding. During the last twenty years, significant progress has been made in coffee biotechnology, particularly in the area of transgenic technology. This paper provides a detailed account of the advances made in the genetic transformation of coffee and their potential applications.

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of...

The South-Western highlands of Ethiopia are considered to be the centre of origin and diversity of the arabica coffee, Coffea arabica. More than 80 accessions of arabica coffee collected from Ethiopia are available in Indian gene bank. However, the genetic diversity of these accessions is not studied in detail. In the present study, genetic diversity analysis of 48 accessions collected from eight provinces of Ethiopia was carried out using Sequence-related amplified Polymorphism (SRAP) marker. Among the thirty two SRAP primer combinations tested, 14 primer pairs were polymorphic and generated 203 distinct fragments. The number of fragments ranged from 7 to 21 with a mean of 14.5 fragments per primer combination. Of the total 203 amplified fragments, 182 (89.65%) were polymorphic and the percent of polymorphism ranged from 53.84% to a maximum of 100% using different primers. The average resolving power (Rp) and average polymorphism information content (PIC) of the 14 SRAP primer comb...

Three molecular marker systems, RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat) and SRAP (sequence related amplified polymorphism), were employed for identification and genetic relationship between five indige- nous coffee species from India and cultivated species Coffea canephora. A total of 304, 140 and 234 bands were detected by 22 RAPD, 12 ISSR and 20 SRAP primer combinations, among which 95%, 91.3% and 96.1% bands were polymorphic respectively. The average PIC of SRAP primers (0.82) was higher than RAPD primers (0.78) but lesser than that of ISSR primers (0.83) where as the average RP of SRAP primers (8.3) is higher than ISSR primers (8.2) but less than RAPD primers (10.3). Some of the RAPD and SRAP primers were able to distinguish all the indigenous coffee species independently. The genetic similarity among the species ranged from 0.34 to 0.75 using RAPD, 0.30 to 0.69 using ISSR and 0.23 to 0.63 using SRAP marker systems. Based on the marker analys...

The identification and genetic relationships of 23 coffee species and one coffee-related species Canthium diccocum were studied using ISSR and SRAP markers. The average polymorphism information content of SRAP primers (0.81) was lower than ISSR primers (0.86), whereas the average resolving power of the SRAP primers (9.74) is higher than the ISSR primers (8.64). The genetic similarity among the species ranged from 0.30 to 0.89 using ISSR and 0.11 to 0.90 using SRAP marker systems. Based on marker analysis, all twenty three coffee species were clustered into two major groups. Both the markers amplified species-specific fragments and are useful in genetic diversity analysis of coffee.