Biotechnology research is carried out at CCRI with the short term and long term objectives of micro propagation, germplasm preservation and genetic improvement. Research efforts are focused on stem, leaf integument, apical bud and node cultures for micro propagation, anther culture, embryo culture, endosperm culture, protoplast culture, genetic transformation and molecular markers for genetic improvement and in vitro preservation techniques including cryopreservation for conserving the germplasm.
In coffee, micro propagation is possible by micro-cutting production and somatic embryogenesis from stem and leaf tissue in more than 20 genotypes of Arabica, Robusta and hybrids. Genotypic differences were found in respect to callus induction, somatic embryogenesis and plant regeneration among the different genotypes. Embryo encapsulation techniques were standardized. Hardening protocols for tissue culture trial plots established in different agro-climatic zones for evaluation of tissue cultured plants. Three improved selections viz., Cauvery, Sln, 9 and CxR are used for large scale field evaluation of micro-propagated plants against seedling progeny. The growth and yield of tissue culture plants was found to be on par with their seedling progenies. Apart from these, plant regeneration was achieved from the apical bud and nodal explants, but further refinement of the technology is required. Plant regeneration was also achieved from integument tissues of CxR cultivator of Robusta coffee.
With the objective of using the tissue culture technology for genetic improvement, research was done on embryo culture, anther culture and endosperm culture. Plant regeneration was achieved through embryo culture by direct germination, as well as somatic embryogenesis. Plantlets have been regenerated through embryogenesis from the anther culture of C x R and endosperm calli of S.2803 genotype. The inter-specific hybrids of C. racemosa X Sln. 3R (C. congensis X C. canephora) were evolved at RCRS, Chundale in view of developing varieties with low caffeine content and early ripening. However, the F1 hybrids have shown very poor fruit set and sterility. For overcoming these, fruits were collected from these F1 plants and in vitro plantlets were successfully regenerated through embryo rescue method.
In vitro preservation and protoplast culture
In vitro preservation of zygotic embryos up to 2 years under slow growth condition was achieved in C. Arabica. Successful cryopreservation of zygotic embryos was achieved in three coffee species. Protocols have been optimized for isolation of protoplasts from embryogenic calli.
Molecular marker assisted selection
DNA marker technology has practical application in coffee breading. Several molecular markers such as RAPD, ISSR, AFL and SSR were used for DNA finger printing of coffee species, cultivars, hybrids and germplasm. In coffea Arabica. Coffee leaf rust is a major disease caused by fungus hemileia vastatrix. Resistance of this coffee leaf rust is determined by nine resistance genes (SH1 to Sh9), either singly or in combination. Availability of molecular markers linked to SH genes could be very useful for marker assisted breeding for coffee leaf rust resistance. In this regard, studies were conducted and two SCAR markers viz., sat 244 and BA-124-12 k linked to SH3 gene for leaf rust resistance were validated in collaboration with plant breeding and plant pathology division in S.795 plants carrying SH3 gene were identified and grafted for developing the pure seed plot. The mapping population of S.795 raised from homozygous and heterozygous plants with and without SH3 markers were maintained and evaluated.
Agrobacterium tumefaciens mediated genetic transformation protocol has been developed for both Arabica and Robusta cultivars. Transformated plants of both Arabic and Robusta carrying the reporter gene (gusA) and selectable marker gene (hpt) were regenerated and integration of transgenes was confirmed through molecular analysis. In addition, Cauvery cultivar carrying rice endochitinase gene was regenerated following Agrobacterium mediated transformation and T1 progeny was derived for studying transgene integration and inheritance
Understanding molecular mechanism of stem borer resistance
Molecular biology research was carried out to understand the profile of gene expression during white stem borer infestation in CXR cultivar. A DNA library was constructed during the early stages of infection. A suppression subtractive hybridization protocol was employed to gain insight of the genes associated with defence mechanism during WSB infestation.
Identification of Bt isolates active against WSB
Research efforts are also being directed to identify the Bt genes effective against the WSB. In this regard , an inter- institutional collaborative project funded by 3000 natives Bacillus thuringiensis isolates, including those coffee growing areas, were collected. These isolates were evaluated to identify isolates active against coffee white stem borer. PCR and bioassay techniques were used for the purpose. Primers were designed targeting 14 Cry gene reported to br active against coleopteran insects and used to screen the Bt isolates to identify coleopteran-active cry genes. It was observed that, Cry11 was present frequently. Cry3, Cry11 and 35 genes were also present rarely. Bioassay methods for producing spore-crystal mixtures from the Bt isolates were developed and the artificial diet-based bioassay protocol was optimized. In bioassay analysis, most of the native Bt isolates were either inactive or very weakly active against WSB larvae. It was found that one native Bt isolate containing a Cry3 gene was active against WAB.
SOIL AND CLIMATIC REQUIREMENTS
The coffee soils in Indian belong to the red and laterite soil groups. They differ in texture from sandy loam to clayey loam with colour varying from light grey to deep red. The soils are usually rich inorganic matter and acidic to neutral in reaction (pH). The total soluble salts are well below the sensitivity limits. They are well supplied with potassium but are generally low in available phosphorus. They are also poor in calcium and magnesium. They respond well to liming, manuring and other soil management pratices. For good growth of coffee, the soil should be deep(75cm), well drained, slightly acidic in reaction and rich in organic matter content.
Geographic parameters like elevation, aspect and environmental factors like rainfall, temperature and atmospheric humidity can influence economic production of coffee much more than soil factors. Under South Indian conditions, high summer temperature combined with poor sub soil moisture can be a severe limiting factor where as at Northern latitudes cold winter temperature can be usually limiting.