Browsing by Author "Minimol, J S"

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Breeding for drought tolerance in cocoa (Theobroma cacao L.)
(Department of Plant Breeding and Genetics College of Horticulture, Vellanikkara, 2019) Juby, Baby; Minimol, J S
Candidate gene analysis on self incompatibility in cocoa (Theobroma cacao L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Sharath Prabhakaran; Minimol, J S
Cocoa is a perennial tree with typical plant habit and specific fruit characteristics. It is highly influenced by climate changes and growing environment, which makes long term and dynamic breeding programme necessary (Malhotra and Hubali, 2016). Physiological and genetic investigations have unveiled that the yield potential of cocoa is not yet fully exploited (Bertus, 2004). Demand for chocolate is increasing at a rate of 15-20 per cent every year. To meet this demand, more area has to be brought under cocoa cultivation using improved genetic stock. Development of superior hybrids have significantly contributed to improve the cocoa productivity in many countries (Kennedy et al., 1987; Dias et al., 2003). Cocoa hybrids showed wide adaptability, low environmental interaction and improved yield, when compared to traditional cultivars (Dias et al., 2003). Self-incompatibility is a pollination control mechanism which prevents self-fertilization. Hence, this can be exploited in hybrid production by avoiding emasculation, which is a cumbersome process (Minimol and Amma, 2013). Moreover, emasculation will damage the flowers leading to reduced success rate. Conventionally, the self-incompatibility is measured by selfing 100 flowers per tree. If no fruit set is observed, then the plant is classified as self-incompatible (Mallika et al., 2006). This is a tedious process which will reduce the pace of breeding programme. In various other crops, many candidate genes have been reported for self-incompatibility (McCormick, 1998). However, the actual sequence variations in candidate genes are yet to be studied in cocoa. Identification of appropriate genes involved in self-incompatibility will help to identify its mechanism at an early stage and quicken the breeding programme. In this study, 10 candidate genes viz. Serine Receptor Kinase (SRK), S Locus Glycoprotein (SLG), Barely Any Meristem 1 (BAM1), Barely Any Meristem 2 (BAM2), COMPASS-like H3K4 histone methylase component (WDR5a), Voltage-dependent L-type calcium channel subunit (alpha-1F), Gamete Expressed Protein (GEX1), Zinc finger AN1 domain-containing stress-associated protein 12 (PMZ), ARM repeat-containing protein (ARC1) and Hapless 2 (McCormic, 1998; Lanaud et al., 2017) were characterized. These genes were reported to have involved in self-incompatibility in other crops. Genomic nucleotide sequences from reported host plant species were retrieved from the NCBI GenBank database. Using this information, homologous gene sequences of the candidate genes in cocoa were retrieved. Primer sets targeting major exonic regions for each of the candidate genes were designed. Genomic DNA was isolated from self-compatible genotype (GVI-167 x GIV-18.5) and self-incompatible genotype (IMC20) and the candidate genes were PCR amplified. Amplified products were sequenced and the variations in the sequences between the self-incompatible and self-compatible genotypes were analyzed, in comparison with a self-compatible reference genome (Argout et al., 2010). Between the self-incompatible and self-compatible genotypes, a total of 31 different SNPs were discovered among the genes studied. All of them were found to be heterozygous at the locus either in self-compatible or self-incompatible genotype. The maximum number of SNPs, a total of 12, were found in GEX1 gene. Four SNPs each were found in genes SRK, BAM2, WDR5a and Alpha1F whereas three SNPs were found in PMZ. No variation was seen in BAM1 and ARC1. SNP locus homozygous in self-compatible and heterozygous in self-incompatible, with the corresponding locus of self-compatible reference genome can be used as potential candidate for developing markers to distinguish them. Such SNPs are identified and recommended for further validation.
Characterization and taxonomic evaluation of landraces of capsicum spp. in Kerala
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2018) Asish I Edakkalathur; Minimol, J S
DNA fingerprinting of promising cocoa (Theobroma cacao L.) varities of KAU
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Sujith, S S; Minimol, J S
Cocoa (Theobroma cacao L.) also known as ‘chocolate tree’ is a major cash crop of tropical countries and belongs to the family Malvaceae. The plant is a native of Andes, South America and was introduced to India during 1970s. In India, cocoa is cultivated as an important intercrop with in coconut, arecanut, rubber, oil palm etc. The statistics shows that, 80 per cent of cocoa plantations in India is established with planting materials distributed from Kerala Agricultural University (KAU). In India, the genetic base of cocoa is widened by the systematic introduction of germplasm from University of Reading, UK. Cocoa Research Centre (CRC), KAU holds Asia’s largest germplasm with 640 accessions. Exploitation of these germplasm has resulted in the release of 15 cocoa varieties from KAU. Central sub-committee on crop standards, notification and release of varieties for agricultural crops has made it mandatory to provide DNA finger printing data along with the varieties where ever the proposal for national release/ notification is submitted. DNA markers, that are not subjected to environmental influences act as an efficient tool to identify and differentiate accessions and cultivars which are similar in morphological characteristics and with indistinct traits. DNA finger printing is successfully applied for cultivar identification, controlling seed purity of hybrids and checking the genetic similarity between cultivars. Hence, the technique act as a powerful tool to protect Plant Breeder’s Right (PBR). In the present investigation, eight promising cocoa varieties CCRP 1, CCRP 2, CCRP 4, CCRP 5, CCRP 6, CCRP 7 (selections), and CCRP 8 and CCRP 9 (hybrids) released from KAU were characterized using morphological and molecular markers. For morphological characterization, six qualitative and nine quantitative characters were recorded. And it was observed that, CCRP 6 and CCRP 8 were superior based on the performance of their yield contributing characters. Molecular characterization was performed with genomic DNA isolated using modified Chandrakant’s (2014) protocol. Based on the polymorphism, ten ISSR (inter simple sequence repeats) and eleven SSR (simple sequence repeats) primers were selected. These selected primers were used for developing DNA fingerprints of varieties under study. In the further analysis, amplicon generation pattern were carefully scored to locate polymorphism. IndividualISSR and SSR amplification pattern further converted into variety wise fingerprints and thus consolidated DNA fingerprints on each marker system were developed. ISSR primers UBC 810 and UBC 826 were found to differentiate CCRP 6 from other genotypes. Primers UBC 827, UBC 846 and UBC 866 were generated unique amplicons in CCRP 9. UBC 841 and UBC 846 were capable of distinguishing CCRP 5 from other genotypes. Primers UBC 835 and UBC 866 were generated amplicons in hybrids CCRP 8 and CCRP 9 alone and the markers can be used for differentiating these hybrids. SSR marker analysis was performed using selected eleven primers. Selected primers generated polymorphic amplicons and were capable of distinguishing between varieties. Primer mTcCIR 8 generated unique amplicon at 220 and 420 base pairs (bp) in CCRP 1 and CCRP 4 respectively which was a fingerprint for those varieties. The unique amplicon generated by mTcCIR 33 at 320 bp was a fingerprint of CCRP 2. Polymorphic amplicons generated by mTcCIR 42 at 200 bp (CCRP 4) and at 220 bp (CCRP 9) were fingerprints of respective varieties. In future, more morphological characters have to be screened and correlated with the markers shared by two or three varieties. This will help to know whether the shared bands are responsible for expression of some distinct traits. DNA fingerprints for remaining seven released cocoa varieties also have to be designed.
DNA fingerprinting of selected cocoa (Theobroma cacao L.) varities of Kerala agricultural university
(Department of Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Megha Totaganti; Minimol, J S
Cocoa (Theobroma cacao L.), is an important tropical tree-crop belonging to the family Malvaceae. It has originated in South America (Wood and Lass, 1985) and was introduced to India, in 1798 at Courtallam in Tirunelveli district of the old Madras state (Ratnam, 1961). Now, cocoa is extensively cultivated in South Indian states and its cultivation is slowly extending to North Eastern states. Central sub-committee on crop standards has made the DNA fingerprinting data mandatory for the national release and notification of varieties. DNA markers, act as active tool to categorize and distinguish accessions and cultivars which have similar morphological characteristics. DNA fingerprinting is effectively used for cultivar identification, assessing seed purity of hybrids and to compare genetic similarities between the cultivars. DNA fingerprints act as a powerful tool to protect Plant Breeder’s Rights (PBR). In the present study, seven cocoa varieties, CCRP 3 (selection), CCRP 10, CCRP 11, CCRP 12, CCRP 13, CCRP 14 and CCRP 15 (hybrids) were fingerprinted. Molecular characterization was performed with genomic DNA isolated using modified Delloporta method (Ileana, 2005). Thirty five ISSR (inter simple sequence repeats) primers and 30 SSR (simple sequence repeats) primer combinations were screened for marker polymorphism, of which 23 ISSR and 17 SSR primer combinations were selected for further study. ISSR and SSR amplification patterns differed among the varieties and thus the DNA fingerprints from each primer combination were developed. ISSR primer UBC810 was found to distinguish CCRP 3 from other genotypes. Whereas, UBC835 and UBC857 produced unique amplicons in variety CCRP 10. Primer UBC810, UBC826, UBC841 and UBC854 generated unique amplicons and formed specific DNA fingerprints of the hybrid CCRP 11. Primer ISSR3, UBC815, UBC827, UBC854 and Oligo 05 gave highest (6) unique bands in CCRP12, whereas UBC 854 produced specific band in CCRP 13. Hybrid CCRP 14 generated unique amplicons with primers UBC855 and Oligo 07 which formed specific fingerprint of the hybrid. Similarly, CCRP15 generated three unique fingerprints with the primer UBC844 and Oligo05. In SSR marker analysis, all the seven genotypes have at least one unique band. mTcCIR40 generated unique amplicons at 250bp length in CCRP 3 and SHRSTc53 at 230bp in CCRP 10. mTcCIR10, mTcCIR8, mTcCIR11 and mTcCIR121 generated distinct bands in CCRP 11. In CCRP 12 genotype, primer mTcCIR18 (345bp), mTcCIR40 (200bp) and mTcCIR42 (210bp) produced specific band. Unique bands were generated by primers mTcCIR10(340bp), mTcCIR11 (310bp), mTcCIR12(270bp), mTcCIR22(200bp), mTcCIR24 (150bp), mTcCIR33 (300bp) and SHRSTc53 for CCRP12. DNA fingerprint generated for CCRP 14 revealed that unique bands were developed at 250bp (mTcCIR37) and 200bp (SHRSTc64). Similarly, CCRP15 developed specific fingerprint at 355 bp (mTcCIR18). ISSR amplicons shared by maximum of three varieties and SSR shared in four varieties were considered for developing final DNA fingerprint profile. The present study had facilitated to characterize the selected cocoa varieties of KAU and the data generated will be useful for varietal notification and in case of any third party litigations.
Genetic analysis of cocoa (Theobroma cacao L.) hybrids and screening superior hybrids for major biotic stress
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2019) Shillpa, K S; Minimol, J S
Cocoa is a crop highly influenced by climate change and growing environment, which make it necessary to have long term and dynamic breeding programme. Yield improvement was the prime objective of most of the earlier breeding programmes. However, with the emergence and spread of many diseases and pests, more emphasis is given for evolving disease and pest tolerant cocoa varieties, without sacrificing yield. At present, one of the main challenges faced by cocoa growers is Phytophthora pod rot caused by Phytophthora palmivora. Since this disease is prevalent during rainy season it is very difficult to control using fungicides. Cultivation of resistant varieties is the most effective and ecofriendly method of control. Tea mosquito bug (TMB) (Helopeltis theivora) is a major sucking pest of cocoa causing damage to young shoots, cherelles and pods. The development and use of mirid resistant cocoa varieties is the only effective alternatives to chemical control against TMB. Twenty cocoa hybrids evaluated in the comparative yield trail (CYT) were considered for the present study. Morphological characterization of the hybrids were carried out based on quantitative and qualitative characters. Thirteen pod characters, twelve floral characters, six bean characters and flush colour of leaves were studied. Except colour of petal and number of ridges and furrows, all other characters expressed high variability among the hybrids. Hybrids were also evaluated based on biochemical properties of beans including fat content, total polyphenol content and total antioxidant activity. Hybrids exhibited significant difference for biochemical characters. More over phenol content showed significant correlation with antioxidant activity. Based on biochemical and economic characters hybrids were scored and ranked. Six hybrids having top rank were selected as superior genotypes and they are PII 12.11, PIV 19.9, VSDI 33.4, VSDI 11.11, VSDI 29.9 and PIV 59.8. II The twenty cocoa hybrids included in the study were screened for Phytophthora resistance by artificially inoculating Phytophthora culture on detached cocoa pods. Based on the disease resistance reaction, the hybrids were classified using the score chart. Hybrids exhibited differential response towards Phytophthora pod rot resistance screening. Hybrids PIV 59.8, VSDI 10.13, VSDI 11.11 and PIV 31.9 were found to be highly resistant whereas, hybrids PII 12.11, SIV 5.15, SIV 1.6, PIV 26.8 and VSDI 29.9 exhibited resistance towards Phytophthora. Correlation studies between Phytophthora pod rot resistance and pod husk biochemical properties revealed that pod husk phenol and pod wax content are positively correlated to Phytophthora resistance. Artificial screening for TMB resistance was carried out on budded plant and detached pods to study the reaction of different hybrids against Helopeltis theivora. Budded plants of all the hybrids and medium matured freshly collected pods of selected six hybrids were screened. Based on the number of feeding punctures, hybrids were grouped into different classes following score chart. Hybrids PIV 59.8, PIV 60.9, PII 12.11, VSDI 33.4 and PIV 56.9 with average number of feeding punctures less than three on plants were included in highly resistant class. When cocoa pods of six hybrids were screened for TMB resistance, PII 12.11 and VSDI 33.4 had the least number of feeding punctures after 72 hours of feeding by TMB. Among the six hybrids selected based on economic and biochemical performance, five hybrids i.e. PII 12.11, VSDI 33.4, VSDI 29.9, VSDI 11.11 and PIV 59.8 exhibited highly resistant to slightly resistant reaction towards both the biotic stress considered.
Genetic stock development for phytophthora pod rot disease resistance in cocoa (Theobroma cacao L.)
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2017) Veeresh S Akki; Minimol, J S
Cocoa production is ruthlessly affected by pod rot disease caused by many Phytophthora species. Among these, the pod rot caused by Phytophthora palmivora is of prime importance. Since, the disease infection period is rainy season, the application of fungicides has not evidenced as a successful control measure. Hence, the effective and eco-friendly way to tackle this disease is by developing resistant varieties. The success of any breeding programme depends upon the availability of well characterised genetic resources. In this context, the current study was formulated for characterisation of exotic germplasm and identifying genetic stock of cocoa for Phytophthora pod rot resistance. Morphological characterization of 30 genotypes were carried out by recording ten qualitative and 23 quantitative characters governing leaf, flower, pod and bean. High variability was observed for most of the characters except petal colour and number of ridges and furrows. The germplasm when characterized based on biochemical parameters such as fat, polyphenols and mineral content (Na, K and Ca) also expressed wide variability. It is essential to quantify the diversity available among genotypes in order to design an effective breeding programme. Hence, cluster analysis was carried out by D2 statistics and principal component analysis. Among all the qualitative clusters, cluster III was the biggest with eight members. Cluster analysis of quantitative characters showed that most of the genotypes were placed separately in distinct cluster due to wide variability available in the germplasm. Cluster analysis of biochemical characters also exhibited wide variability which is evident from the fact that it formed 15 clusters even at 25 per cent similarity. Correlation studies and path analysis were employed to know the nature and relationships among the yield attributing characters. Here, it was found that wet bean weight (g) showed positive correlation with pod weight (g), furrow thickness (cm), pod length (cm), pod breadth (cm), weight of the bean (g) and number of beans per pod. Results of path analysis revealed that total wet bean weight (g) was directly influenced by pod thickness (cm), number of beans per pod, single dry bean weight (g) and wet bean weight (g). Since, quantitative and qualitative descriptors serve as an effective tool to discriminate among the genotypes, a evaluation data was constructed for all the genotypes considering distinct characters governing them. The non-pricking and pricking methods of pod inoculation with pure culture of pathogen were employed to know the disease resistance reaction exhibited by different genotypes. In the non-pricking method, five genotypes (ICS 41, SIAL 339, PNG 250, PNG 336 and IMC 20) with zero per cent infection and 14 genotypes (CRU 12, MO 109, GDL 7, GU 261/P, NA 149, PA 156, LX 43, POUND 4/B, JA 10/12, DOM 14, ICS 75, DOM 25, POUND 18 and POUND 16/A) with infection less than 15 per cent were grouped under highly resistant category. However, these genotypes did not show same disease resistance reaction in pricking method which indicated that the resistance was influenced by certain morphological characters apart from the internal resistance and the significance of those morphological characters were lost when pods were pricked. Binomial logistic regression revealed that different phenes like ridge thickness, polyphenol content and calcium content were positively contributing to disease resistance. Whereas, phenes like pod rugosity, pod basal constriction and pod length were negatively correlated with disease resistance. If these phenes are considered for selection, ample increase in the level of resistance will be noticed in the resultant population. Genetic stock was developed considering disease resistance and yield. As opined by many scientists “Disease resistance is a double-edged sword”. The phytotoxin developed in plants against pathogen is not only toxic to the pathogen but also to the plants resulting in yield reduction. Here also same trend was noticed and majority of the genotypes which expressed high resistance were low yielders. The accessions manifested high resistance can be used for further breeding programme.
Inheritance of molecular markers linked to vascular streak dieback disease resistance in hybrid progenies of cocoa (theobroma cacao L.)
(Department of Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Midhuna, M R; Minimol, J S
Theobroma cacao L. (also known as the chocolate tree) is a major cash crop and the costliest beverage crop. Andhra Pradesh is the leading cocoa producing state in India but Tamil Nadu ranks first with an area of 26,969 ha. Vascular Streak Dieback (VSD) caused by the fungus Ceratobasidium theobromae is a serious disease in cocoa. Since it is a vascular pathogen, chemicals have little effect on disease control. The most tenable and economic technique to tackle this disease is by evolving resistant materials. Kerala Agricultural University had initiated VSD resistant breeding since 1995. Seedlings from hybridization, exhibiting high levels of resistance were selected and field established. Nineteen hybrids, exhibiting resistance to VSD (after screening for a period of thirteen years), were selected for the present study. The progeny obtained from these hybrids by crossing it among themselves were used as plant materials for the study. Two thousand two hundred and thirty seven flowers were pollinated and seven pods were obtained. About two hundred and sixty nine seedlings were grown from the seven hybridized pods in which nursery screening for disease resistance was done. Inoculum was dispensed by keeping already infected seedlings around the experimental materials. High humidity was ensured by providing over head sprinkler system. Visual screening recorded one hundred and eighty seedlings as disease resistant, fourteen seedlings as partially resistant and seventy five seedlings as disease susceptible. Three ISSR markers (UBC 811, UBC 815 and UBC 857) and one SSR marker (mTcCIR42) linked with VSD resistance gene, identified and validated from the previous studies were used for screening of the one hundred and twenty seedlings out of which one hundred and six were resistant and fourteen were partially resistant. The polymorphic band of 950 bp, which was found to be linked with the gene conferring VSD resistance was recorded in seventy seven resistant seedlings and three partially resistant seedlings, when screened with the primer UBC 811. When screened with the primer UBC 815 (750 bp) and UBC 857 (450 bp), the polymorphic marker band which was found to be linked with VSD resistant gene from the previous studies, was present in only twenty five resistant and one partially resistant seedling and twenty one resistant and one partially resistant seedling respectively. When screened with SSR marker, the 200 bp marker band, which was tagged with the VSD resistant gene was detected in fourty six resistant and six partially resistant seedlings. The ISSR marker UBC 811 and SSR marker mTcCIR42 were found to be having comparatively good percentage of inheritance among the segregating progeny screened with a mean inheritance percentage of 71.70 per cent and 48.78 per cent respectively. Flanking sequences of the ISSR markers UBC 811, UBC 857 and SSR marker mTcCIR42 were extracted from the whole genome database of cocoa. The ORFs from the flanking sequences of UBC 811 were identified to be uncharacterized proteins by using BLASTp tool. One ORF from the upstream sequence of the UBC 857 had shown identity with beta tubulin chain. Analyzing the distance between the marker and the flanking region, it was deduced that UBC 857 is a part of beta tubulin gene. Two ORFs were identified from both the upstream and downstream flanking sequences of the SSR marker. Using BLASTp tool, it was analyzed that both the ORFs showed more than 97 per cent identity to beta tubulin gene. Analysing the spacing between the marker and the flanking sequences, it was deduced that both the ORFs are part of the same gene and the SSR marker mTcCIR42 lies within the beta tubulin gene. Tubulin beta chain belongs to the microtubular component of cytoskeletal elements which provides resistance by not allowing the fungi to penetrate the outer epidermal wall of the plants, hence protecting the plants from infection. The ISSR marker UBC 857 and the SSR marker mTcCIR42 are linked to the beta tubulin gene, which provides VSD resistance by giving resistance against penetration of the plant cell by the fungus. Nineteen seedlings were identified to be having three or more markers expressed. They can be planted in the field and can be further evaluated for yield and yield contributing characters. Sequence of the beta tubulin gene can be used for primer designing, which can be used for confirmation by screening in resistant genotypes of cocoa.
Metallophytes, a unique biological resource : Its molecular mechanisms and applications
(Centre for Plant Biotechnology and Molecular biology, College of Horticulture, Keala Agricultural University, 2020) Sharat, Prabhakaran; Minimol, J S
Morphogenesis and reproductive biology of sacred lotus (Nelumbo nucifera gaertn.)
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2004) Minimol, J S; Prasanna kumari, K T
Sacred lotus (Nelumbo nucifera) belonging to Nelumbonaceae is the only genera in that family. This legendary flower has very long and close association with history, culture, religion, literature and arts. Hence it is chosen as our national flower. Despite its immense potentialities as medicinal, ornamental and vegetable crop, this plant has received only very little attention of crop improvement workers. It was in this background the present investigation entitled ‘Morphogenesis and Reproductive Biology of Sacred Lotus’ was under taken with the objectives of evaluating the growth and development pattern of leaf, flower and seed and elucidating the reproductive biology. Six different genotypes collected from diverse ecological conditions were evaluated under ex situ conditions during December 1999 to March 2003 at College of Horticulture, Vellanikkara. Variability was observed in different biometric characters like size of lamina, longevity of leaves, petiole length etc. among the ecotypes evaluated. Study of the seasonal effect on these characters revealed that rainy season favoured growth in size of leaves and spring season favoured longevity. The growing tip of the rhizome was found to be the best propagule with respect to rhizome yield. Significant variability was observed in various floral characters among the ecotypes. Flower production started with the onset of Monsoon and reached the peak in Spring season and then declined with no flower production at all in Summer season. Flowers were found to be solitary, ebracteate, actinomorphic and complete with the different whorls arranged in a spiral fashion on the floral axis. Stigma became receptive 32 hours before flower opening and the receptivity was retained for 52 hours even after flower opening. Pollen dehiscence occurred only after complete opening of the flower bud. Pollen grains of lotus were found to be fertile, round and triporate. However, no seed set was obtained in protected buds indicating cross pollination. The temperature inside the flower bud remained between 30 to 35oC till the fourth day during the period of anthesis despite the changes in environmental temperature between 27 to 33oC. The period of this thermoregulation corresponded to receptivity of stigma. This is considered as a floral adaptation favouring cross pollination (Seymour and Schultze-Motel, 1998). Unlike other dicot, in sacred lotus, plumule emerged first and radicle was aborted. Adventitious roots were found anchoring the plant in mud. The seeds matured in 30 days after fertlization. Lotus seeds are reported to have the longest period of dormancy. The germination trials conducted after giving different pretreatments revealed that embryo as such is nondormant. Mechanical scarification followed by leaching improved germinability indicating that hard fruit wall along with thick waxy coating and water soluble inhibitors are responsible for dormancy.
Preliminary evaluation of double cross hybrids for yield and vascular streak dieback (VSD) disease resistance in cocoa (Theobroma cacao L.)
(Deapartment of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2020) Alfiya, A R; Minimol, J S
Cocoa is highly influenced by the climate change and growing environment, necessitating a long term and dynamic breeding programme. Even though the breeding programmes primarily focus on the development of high yielding varieties, outbreak of new pests and pathogens shift the priority to the development of resistant varieties. Vascular Streak Dieback disease (VSD) caused by Ceratobasidium theobromae (Samuels et al., 2012), pose a great threat to cocoa crop, causing complete defoliation and eventual death (Abraham et al., 2002). Even the high volume spray of chemicals was ineffective in disease control (Prior, 2007), and the only way to tackle it is to breed resistant varieties. Resistance breeding may result in yield reduction (Xu et al., 2017) however, breeding for double cross hybrids can overcome this situation (Gallais and Guy, 1971). Average yield superiority of the double cross hybrids over the F1 hybrids has been shown by many scientists (Sriani et al., 2003; Ghanwat et al., 2016). Twenty double cross hybrids, bred for vascular streak dieback disease and planted during 2017, were used for the present study. Morphological characterization of the hybrids was carried out based on the quantitative and qualitative characters. Thirteen pod characters, twelve floral characters, six bean characters and flush colour of the leaves were studied. Except colour of the petal and number of the ridges and furrows, all other characters have expressed high variability among the double cross hybrids. The double cross hybrids have exhibited significant difference for fat and polyphenol content. All the twenty double cross hybrids were screened and scored for the VSD resistance in the field condition, using the score chart (Abraham et al., 2000). Based on the disease intensity, they were classified into eleven resistant and nine partially resistant.
Screening for drought tolerance in cocoa hybrids (Theobroma cacao L.) and expression analysis of identified gene
(Department of Plant Breeding and Genetics, College of Agriculture, Vellanikkara, 2025) Santhra Mohanan.; Minimol, J S
Cocoa (Theobroma cacao L.) is a high-value, export-oriented beverage crop, empowering small-scale farmers through global value chain participation. Originating from the humid rainforests of South America, cocoa requires annual rainfall between 1500–3000 mm, and summer irrigation of about 24 liters per plant every four days. However, climate change-induced rainfall unpredictability, particularly in non-traditional cocoa growing regions of India like Tamil Nadu, Andhra Pradesh, and North Eastern states, severely limits water availability, increasing the vulnerability of crop to drought. Shallow root system further exacerbates its sensitivity to water stress, restricting its ability to access deeper soil moisture and significantly affecting yield and supply. Despite the identification of drought resistant genotypes in controlled conditions, their field level efficacy remains largely unexplored. Plants exhibit complex morpho-physiological and biochemical mechanisms in response to drought, notably through signaling molecules such as reactive oxygen species (ROS) and osmolytes that mediate stress responses. However, translating these findings into field resilience in cocoa remains a major challenge. This study aims to identify drought resistant cocoa hybrids under field conditions by evaluating key biophysiological parameters along with gene expression analysis, thereby contributing to the sustainable enhancement of cocoa production amidst growing climate variability. Twenty cocoa hybrids planted during 2018 at Cocoa Research Centre, which are at yielding stage served as the base population for the present study. Morphological characterization based on qualitative and quantitative characters were carried out. Thirteen pod characters and ten bean characters were studied among the hybrids, of which all the characters except for number of furrows and ridges showed high variability. The hybrids were evaluated for the biochemical properties with a focus on determining fat and total polyphenol content of beans. A significant difference in biochemical characters was observed among the hybrids with the highest fat and polyphenol content recorded in H102 (55 %) and H103 (8.56 %), respectively. Correlation study between the weight of beans per pod and other quantitative traits showed a significant positive association with pod weight, total beans, single bean dry and fresh weight, bean length, and width. Path analysis further indicated that five of these traits exerted a direct positive influence on bean weight per pod, whereas the contribution of bean length was minimal, with a negligible path coefficient value of 0.063. Based on scoring and ranking in terms of yield parameters, H101 and H153 were selected as top performing hybrids. Field screening for drought resistance was conducted on these twenty hybrids by initially flood irrigating until the relative water content reached 80 per cent, followed by drought induction through the suspension of irrigation until the onset of incipient wilting symptoms, and subsequently alleviating the stress by reapplying flood irrigation. Based on the days taken to wilt, hybrids were classified using the score chart. Hybrids exhibited differential responses to drought resistance screening. H152, H153, H101, and H102 were found to be highly resistant where as H141, H169, and H103 exhibited moderate resistance towards drought. Highly resistant hybrids retained relatively high values for physio-biochemical parameters under drought stress than the susceptible ones. Correlation studies between days taken to wilt and physio-biochemical parameters revealed significant positive associations with all parameters except transpiration rate (r = -0.652**) and stomatal conductance (r = -0.524*). According to path analysis, eight variables including Relative Water Content (RWC), chlorophyll content, catalase, membrane stability, glycine betaine, proline, transpiration rate, and stomatal conductance exerted a direct effect on days taken to wilt, with RWC exhibiting a very high positive direct effect (1.33). Principal component analysis delineated three components (PC1, PC2, and PC3) with eigen values exceeding the Kaiser criterion threshold (>1), accounting for 75.78 per cent of total variation, with PC1 contributing maximum variability (56.94 %). RWC and superoxide dismutase exhibited maximum contribution to PC1 with a strong positive correlation of 0.901 and 0.890, respectively with PC1. Scoring and ranking of hybrids based on significant physio-biochemical characters regarded H103 and H169 as drought resistant. According to the prior transcriptomic study, laccases (LACs) were found to be upregulated in drought tolerant genotypes and were selected for the present gene expression profiling. The expression study was undertaken at different soil moisture regimes (100% and 40%), using two contrasting hybrids identified as drought resistant (H103, H169) and susceptible (H8, H55). H103 exhibited a pronounced upregulation with a 9.81-fold increase of the targeted gene under 40 per cent field capacity (FC) relative to its 100 per cent FC. In contrast, the susceptible hybrid (H65) manifested a comparatively attenuated response, with only a 1.89-fold increase, indicating a limited capacity for stress-inducible gene activation. Although H103 and H169 exhibit strong drought resistance, their yield performance under ambient conditions remains suboptimal. Hence, H101 and H141 were identified as top-performing hybrids due to their excellent yield and strong drought resistance.
Stay green genes A potential tool in plant breeding
(College of Horticulture, Kerala Agricultural University, 2020) Alfiya, A R; Minimol, J S
Tagging of phytophthor pod rot disease resistance gene in cocoa (Theobroma cacao L.) using ISSR markers
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Jeughale Kishor, Pundlik; Minimol, J S
Cocoa (Theobroma cacao L.) known as ‘Chocolate tree’, is a major cash crop in tropical countries. Cocoa production is seriously affected by pod rot diseases caused by many Phytophthora species. Among these, the pod rot caused by Phytophthora palmivora has been reported in India. Yearly losses to the cocoa growers around the world from Phytophthora diseases were assessed at 30 per cent of the total yield loss. Disease resistance can be scored using a number of morphological and physiological characters. However, the morpho-physiological characters greatly depend upon the environment which ultimately affect the experimental data. Hence, confirmation of transfer of genes by tagging with the help of a strong tool is of utmost importance in crop breeding. Molecular markers such as Inter simple sequence repeats (ISSRs) have already proven to be a good tool to detect and tag the genes of interest and will help to reduce the breeding cycle. In this context, the present study was taken up with an objective to develop a strategy to tag gene(s) for Phytophthora pod rot (PPR) resistance in cocoa using ISSR markers. Morphological characterization of 28 hybrid progenies of SVI 1.26 × PII 12.11 was carried out by recording five pod and bean characters. High variability was observed for characters viz., pod weight, pod length and breadth, wet bean weight per pod and single dry bean weight among the progeny of the same cross. Detached pod inoculation technique was adopted to classify the hybrids into resistant and susceptible ones. The wide variability was also recorded for disease reaction among the progenies. Based on the resistance score, three resistant and three susceptible hybrids were selected from the segregating progeny. The eight accessions were screened with fifty ISSR and 15 SSR primers to observe polymorphism between resistance and susceptible genotypes. Polymorphism was observed in 11 ISSR primers and from these, six primers viz., UBC 810, UBC 826, UBC 827, UBC 857, Oligo ISSR 04 and Oligo ISSR 08 were eluted and cloned. Plasmid DNA was isolated from clones and sequenced. Though various SSR primer sets screened were found to yield polymorphism, none of them was successful to give a clear distinction among the resistant and susceptible hybrids. This may be due to the fact that, Quantitative trait loci (QTLs) associated with these reported SSR primers may be absent in the genotypes considered for the study. BLASTn analysis specific to plants was done for all six sequences. Upon analysis, Oligo ISSR 04561 had shown 98 per cent identity with Predicted: T. cacao histidine-containing phosphotransfer protein 1 (HPt). HPts play an important role in propagating cytokinin signal transduction. Cytokinins are instrumental in mediating disease resistance by generating a green island around the infection zones, exhibiting delayed leaf senescence and upregulating the expression of the pathogenesis related (PR) gene/s. In addition to this, the auxin-cytokinin antagonism that occurs as part of a complex hormonal interplay, exerts a critical influence on the core SA-JA/ET plant immunity pathways. The BLASTn analysis of marker UBC 810877 resulted in 99 per cent sequence identity with Predicted: T. cacao phospholipid: diacylglycerol acyltransferase (PDAT) 1 mRNA. This protein regulates the synthesis of triacylglycerol, which is a building component of oils in the plant. Accumulation of oil content in plant cells could impart resistance against the pathogen. UBC 827571 had shown 73 per cent sequence identity with T. cacao clone TCC_BA049P20 complete sequence and it is reported to be QTL rich region associated with different traits of T. cacao. Moreover, ISSR markers UBC 810877, UBC 826535 and UBC 857839 are located on chromosome nine, six and four respectively as inferred from NCBI Genome Data Viewer tool through BLASTn annotations. These markers are found to be located in PPR resistance regions rich in defense associated genes. Further validation and exploitation of polymorphic amplicons or markers in response to PPR would be required. The linkage of Oligo ISSR 04561 and UBC 810877 with HPts and PDAT correspondingly have to be validated to elucidate the association and role of cytokinin and triacylglycerol with PPR disease resistance. If validated, UBC 810877, UBC 826535 and UBC 857839 and Oligo ISSR 04561 could be employed as a marker in PPR resistance breeding programmes in cocoa.