Browsing by Author "Sujatha, R"

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Alleilic difference in the putative gene ipk1 sequence and phytic acid (INSP6) content in Black Pepper
(Department of Plant Breeding & Genetics, College of Agriculture, Padannakkad, 2019) Gladish Mary Joy; Sujatha, R
Black pepper (Piper nigrum L.) is one of the world’s highly demanded and most traded spices with high medicinal and therapeutic values. A unigene pnc135 (995 bp) was developed by the Expressed sequence tags data obtained by next generation sequencing. This unigene was found to show similarity with ipk1 gene reported in other crop species which encodes for inositol pentakisphosphate-2 kinase enzyme (Unpublished data, Sujatha,R.). This enzyme is involved in the phosphorylation of inositol pentakisphosphate to inositol hexakisphosphate or phytic acid (InsP6), last step in the biosynthetic pathway of phytic acid. This unigene was later partially sequenced (1072 bp) towards the 3’ end by directional genome walking by Giridhari (2017). Phytic acid functions as the major storage form of phosphorus in seeds, cereals and legumes possessing significant benefits including signalling, plant communication, messenger RNA transport etc. However, phytic acid also acts as an anti-nutrient in animals as its chelating property will cause malnutrition in organisms and also leads to environmental pollution due to phosphorus excretion by non-ruminant animals. Therefore researchers are finding ways to create ipk1 mutants for either to decrease or increase the phytic acid content in organisms. However the genetic information about the black pepper crop remains very limited and the metabolic pathways and the genes related to it are also poorly understood. So in this study entitled “Allelic difference in the putative gene ipk1 sequence and phytic acid (InsP6) content in black pepper (Piper nigrum L.)”, the objective was to find out the flanking region towards the 5’ region of pnipk1 gene fragment (1072 bp) reported earlier by Giridhari (2017), to identify the allelic differences in pnipk1 gene in 10 black pepper genotypes and to estimate and quantify the phytic acid content in these 10 black pepper genotypes using polyacrylamide gel electrophoresis (PAGE). Genomic DNA was isolated from Panniyur 1 variety of black pepper and used it for the whole genome amplification by Rolling Circle Amplification method using Phi 29 DNA polymerase and walker adaptors (WA1, WA2, WA3 and WA4) reported by Reddy et al. (2008). After whole genome amplification, genome walking using primer combinations of walker primers, locus specific primers and nested locus specific primers were performed to find out the flanking region towards the 5’ region of pnipk1(1072 bp) gene fragment of black pepper. The walker primers (WP1 and WP2) used for genome walking were same as that of reported in Reddy et al. (2008) and the locus and nested locus specific primers were designed on the basis of pnipk1 gene fragment (1072 bp) sequenced by Giridhari (2017). From the nested PCR amplification four products, two amplicons, A1R3 and A4R3, each at two different temperatures viz., 51.6⁰Ϲ and 56.8⁰Ϲ were obtained and sequenced. On assembling the sequences a contig of length 523 bp was obtained towards the 5’ region of pnipk1 gene fragment and this showed similarity to ipk1 gene in other crops. This 523 bp contig was assembled with ipk1 gene fragment (pnipk1-1072 bp) to get a total length of 1535 bp. The newly assembled ipk1 gene sequence (pnipk1-1535 bp) was analysed in ORF finder for the coding region and found an Open Reading Frame (ORF) with 645 bp encoding for 214 amino acids. Phylogenetic analysis of the sequence and translated amino acid sequence showed closer evolutionary relationship with that of Dendrobium catenatum. Primers were designed based upon the pnipk1 gene sequence (1535 bp) to amplify the genomic DNA of Panniyur 1 and other 10 black pepper genotypes. The selected 10 genotypes were Panniyur 5, Panniyur 7, Chettanvally, Kottanadan, Karimunda 7, Thottamundy, Karimunda kuttyatur, Payyanganam 2, PRS 160 and Chumala. Amplification of pnipk1 gene (1535 bp) was obtained from genomic DNA of Panniyur 1, Panniyur 5, Panniyur 7 and Karimunda 7 with the expected of amplicon size indicating a similar sequence among these genotypes. Whereas amplification was not obtained in genomic DNA in rest of the genotypes showing allelic variation is present for ipk1 gene in these genotypes. To estimate and quantify the phytic acid content in Panniyur 1 and 10 black pepper genotypes, polyacrylamide gel electrophoresis (PAGE) was performed. Phytic acid was extracted from black pepper varieties same as that of selected for allelic difference analysis in ipk1 gene (pnipk1-1535 bp). The samples were loaded with phytate standards and band intensities of each concentration were determined with Gelquant.NET. The values of phytic acid in black pepper genotypes were estimated by the standard curve. The quantity of phytic acid in samples are: Panniyur 5 with 502.5nmoles/g, Panniyur 7 with 367.5nmoles/g, Chettanvally-511.5nmoles/g, Kottanadan-463.5nmoles/g, Karimunda7- 387nmoles/g, Chumala-201nmoles/g, Karimunda kuttyatur-637.5nmoles/g, Payyanganam 2-196.5nmoles/g and Panniyur 1- 275 nmoles/g, Thotamundy- 198nmoles/g, PRS 160-697.5nmoles/g. Based on the phytic acid content in the black pepper genotypes, they can be classified into low (<210 nmoles/g), medium (210-510 nmoles/g) and high (>510 nmoles/g) phytic acid content. The ipk1 gene fragment (pnipk1-1535 bp) was amplified in the genotypes Panniyur 1, Panniyur 5, Panniyur 7 and Karimunda 7. These genotypes all came under the category of medium phytic acid content group. The study resulted in sequencing a total of 1535 bp long segment of ipk1 gene black pepper variety Panniyur 1 and analysing the presence of allelic variation in ipk1 gene and phytic acid content in selected black pepper genotypes.
Assessment of variability and compatibility in Tagetes spp.
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2007) Kishore Boddu; Sujatha, R
The present investigation on “Assessment of variability and compatibility in Tagetes spp.” was carried out in the Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, during 2006 – 2007. The experimental material consisted of 14 accessions of African marigold and seven varieties of French marigold, collected from Andhra Pradesh, Karnataka and Kerala. The major objectives include assessment and interpretation of genetic variability and the inter relationship between different traits, investigation of the reproductive biology and compatibility behaviour between different genotypes, formulation of an efficient selection method for identifying superior genotypes, estimation of the heterotic potential as well as characterization of superior hybrids at biochemical and molecular level along with their parents. A local cultivar from Kapugal (Ka) in Andhra Pradesh had maximum flower yield of 114.69g per plant. On an average, it produced 24.66 flowers per plant with maximum fresh weight of flowers (4.64 g) and flower diameter (4.11 cm). It was closely followed by the accession collected from Ananthagiri (A) (Andhra Pradesh) with an average flower yield of 113.54g per plant. For this accession the number of flowers (31.16) and flower diameter (4.51 cm) were higher than Ka. In French types variety French vanilla had the highest flower yield (29.76 g per plant), followed by Boy O Boy (27.13 g per plant). Highest genotypic and phenotypic coefficients of variation were observed for the character number of whorls of ray florets followed by yield per plant. Heritability and genetic gain were maximum for the characters such as number of whorls of ray florets, fresh weight of flowers, yield and plant height. Correlation studies revealed the traits number of flowers per plant and fresh weight of flowers had strong correlation with yield. Comparison of different genotypes based on the selection index value revealed the superiority of genotypes Kapugal (Ka) followed by Ananthagiri (A) and Thammara (T). The 14 genotypes were grouped into five clusters based on genetic distance. Reproductive biology and compatibility studies showed that there was no self or cross incompatibility in African marigold but in inter specific hybridizations with French types, seed set and germination was lower. Six superior hybrids Th-1 X C-6, Tr-5 X Pil.O-3, Th-6 X Ka-3, Th-1 X A-3, Vk1-5 X Pil.O-4 and Tr-3 X Vk2-3 for the characters plant height, number of whorls of ray florets, number of flowers, fresh weight of flowers and flower yield were selected based on heterobeltiosis and relative heterosis values. There was no much difference between direct and reciprocal crosses except for the character seed set percentage. Biochemical analysis of the selected hybrids and parents for total carotenoid content showed that overall mean of parents was 5.56 mg and that for the hybrids it was 5.54 mg per one gram of fully opened flowers. Molecular characterization of the selected hybrids and parents produced a total of 20 bands with three primers, The bands of approximate size 1375 bp and 1584 bp were present in all the parents whereas 1904 bp band was found in all except Th-6 and A-3. Similarly the bands of approximate size 1375 bp and 1584 bp were shared by all the hybrids except two and 1904 bp was present in all the hybrids except three.
Characterisation and conservation of promising genotypes of orchids from central western ghats
(Department of Plant Breeding and Genetics,College of Agriculture, Padannakad, 2016) Yusuf Abbas; Sujatha, R
Characterisation of field established tissue culture derived black pepper (piper nigrum L.) plants using morphological, cytological and molecular markets
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2001) Sujatha, R; Mallika, V K
Universally acclaimed as the ' King of Spices', black pepper (Piper nigrum L.) enjoys a unique position as a commercial crop of historical importance and assumes great economic importance to several nations of the world. Efforts to evolve high yielding types of black pepper through selection as well as hybridisation started as early as in 1953 (Nambiar et al., 1978) and by now, about ten improved varieties were evolved and released. In order to meet the insatiable demand from farmers for the planting materials of high yielding pepper varieties, mass multiplication protocol through micro-propagation has been standardised (Joseph et al., 1996). Babu (2000) tested the fidelity of TC plantlets of black pepper during the hardening stage using RAPD assay. A thorough evaluation and characterisation of these 'I'C plants in relation to their performance in the field is a prerequisite to establish their heritage and usefulness. In this background, the present study was taken up with the main objectives of establishing the genetic identity of TC derived black pepper plants in field; testing the intra-group, inter-clonal and inter- varietal polymorphism and obtaining a thorough and fool proof finger print of the TC plants using a combination of molecular marker techniques and conventional markers. Each of the experimental vines was characterised using morphological traits (biometric as well as qualitative) based on the descriptor formulated by NBPGR. The cytological studies ruled out any variation caused by a change in the chromosome number, which is possible in in vitro cultured plants. The somatic chromosome number was found uniform in all the vines with 2n = 52, which is characteristic of the species. The molecular markers provided a more specific identity for each of the experimental vines. The , zymogram based on peroxidase as well as RAPD banding pattern has given a very clear fingerprint characteristic of each vine. The biometric observations and visual assessment of the vines based on qualitative traits brought out the intra group (within conventional clones or within TC clones), inter- clonal (CC vs. TC clones) and inter varietal variation of the different characters. Among the 22 biometric observations, 17 were found homogenous within clonal groups and also within the TC groups irrespective of varieties. The remaining five traits were discussed with respect to each variety. Most of the qualitative observations also showed uniformity in. the clones as well as TC plants. The estimation of inter-clonal variablity proved the uniformity and better vigour of TC plants compared to the conventional clones. Another interesting observation was the great variablity in qualitative and quantitative traits between TC Subhakara and clonal Subhakara indicating a possible error in labelling. The molecular markers brought out the variablity in the different vines more specifically. In both isozyme and RAPD analysis, the clones under each variety were found to be monomorphic whereas certain variants could be detected within TC plants with respect to a few bands. The vines TC P2-7 and TC P1-1 were separated out as variants by both isozyme and RAPD markers. Other TC vines, which showed polymorphism with respect to either of these markers were Pl-I, PI-2, P2-10, P4-9, Su-9, Su-lO, P4-4 and P4-8. The assessment of inter clonal variability using molecular markers clearly pointed out the distinctly different traits of TC clones and conventional clones Subhakara. Morphological observations too supported this finding. Using RAPD, it was conclusively proved that the vines labelled as TC Su were in fact TC P 4. This confirmed the possibility of an error in labelling while the TC plantlets were transported from Vellanikkara to Panniyur. The inter-varietal polymorphism brought out by morphology and molecular markers was useful in discriminating the four black pepper varieties and to asses the genetic distance between them, which is an important criterion in the selection of parents for hybridisation. The results exposed the genetic proximity between PI and P2 and between P 4 and Suo The standardisations of various protocols for cytological and molecular marker analysis in black pepper as well as the relative efficiency of the different marker systems were also discussed. The RAPD technique was found most effective in assessing the genetic constitution of the individual vine.
Detection and quantification of piperine from in vitro cultures of black pepper (Piper nigrum L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Achintya Kumar Dolui; Sujatha, R
Developing dwarf specific scar markers from WCT inbreds of coconut (Cocos nucifera L.)
(Department of Plant Biotechnology, Vellanikkara, 2018) Anto Varghese; Sujatha, R
Coconut is an important subsistence and cash crop in the humid tropical regions of the world. The coconut palm is often called “the tree of life” because of its numerous uses as food, drink, fuel, building materials and so on. Coconut cultivars are mainly classified into two: tall and dwarf. West Coast Tall (WCT) is the most common and superior tall cultivar extensively cultivated all over the west coast region, especially in Kerala. One of the main methods of crop improvement in coconut is hybridization between tall and dwarf types. However the heterozygous nature of tall types is a hurdle in selection of mother palms and seedlings. Efforts to develop homozygous inbreds of WCT were started as early as 1924. Chethana (2016) studied the extent of inbreeding depression in second generation (S2) inbred palms and recorded morphological observations of one year old third generation (S3) inbred seedlings. Based on height, she classified the S3 seedlings into Low, Medium and High, before planting in the field. She also carried out the characterization of selected third generation (S3) inbreds using RAPD marker system along with tall (WCT) and dwarf palms (COD, CGD). The results revealed two dwarf specific RAPD bands in two of the S3 seedlings belonging to IAS3-1 and IBS3-1 family, when amplified with primers OPAU03 and OPAW15. However, as RAPD markers have low specificity and sometimes lack repeatability, converting them into Sequence Characterized Amplified Region (SCAR) markers is more advantageous. A tall specific SCAR marker was reported in coconut using primer OPA 09 (Rajesh et al., 2013). The present investigation was carried out in the departments of Plant Biotechnology, College of Agriculture, Padannakkad and College of Horticulture, Vellanikkara during the period 2016-2018 was undertaken with an objective of developing SCAR markers from dwarf specific RAPD marker for screening superior inbreds with dwarfing nature. DNA samples were extracted from total 54 genotypes, 22 seedlings belonging to the inbred families IAS3-1and IBS3-1, 10 seedlings from other three families viz, IIS3, IIIS3 and VS3, 14 dwarf seedlings (including COD, CGD and MYD) and 8 tall seedlings (WCT). These were subjected to RAPD analysis using the 3 reported primers viz, OPAU 03, OPAW 15 and OPA 09. Though OPAU03 showed polymorphism between tall and dwarfs, the bands lacked repeatability and hence this was not included in further analysis. OPA 09, which was reported to produce a tall specific band (Rajesh et al., 2013), confirmed the result in the present study also. Primer OPAW15 was successful in amplifying a polymorphic band of size around 320 base pair specifically in dwarf seedlings and it was not present in the tall cultivar (WCT). The polymorphic dwarf specific band produced by primer OPAW 15 was eluted and cloned in pTZ57R/T vector and transformed into E. coli DH5 alpha cells. Cloned cells were subjected to blue white screening and transformed ones were selected, the plasmid was isolated and sequenced. The sequence after vector screening was subjected to homology search using BLASTn. The DNA sequence of polymorphic band showed similarity with chromosome 2 of Solanum lycopersicum. Based on the sequence, one pair of SCAR primer was designed and tested for dwarf specific band. Dwarf specific amplicon was found in all the dwarfs but absent in the tall WCT seedlings. Hence, this was used to screen the S3 inbred seedlings which were classified as Low, Medium, and High based on their height at the time of planting, to confirm the effectiveness of this classification based on morphology. The result showed that the dwarf specific marker is present in S3 inbreds belonging to Low and Medium types but absent in all those seedlings classified as ‘High’ based on height. This shows that the classification based on height in the one year old seedlings is effective to some extent. However, some of the seedlings in ‘Low’ and ‘Medium’ category lack the marker, which indicate that a classification based on morphological and molecular marker is more effective than classification based on morphology alone. The dwarf specific SCAR marker developed in the present study after validation using other tall and dwarf cultivars, along with the earlier reported tall specific SCAR marker can be successfully employed in breeding experiments for marker assisted selection. The SCAR markers which distinguish tall and dwarf coconut cultivars can be used in screening seedlings at an early stage which would be of immense importance to nurseries and growers while establishing coconut plantations.
Development and validation of novel EST-SSR markers in black pepper
(Department of Plant Breeding and Genetics, College of Agriculture, Padannakkad, 2017) Sherin Jose; Sujatha, R
Diversity and population structure analysis in coconut (Cocos nucifera L) using molcular markers
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2012) Renju, S; Sujatha, R
Genome walking for putative phytic acid (InsP6)unigene in black pepper (Piper nigrum L.)
(Centre for Plant Biotechnology and Molecurar Biology, College of Horticulture, Vellanikkara, 2017) Ananduchandra Giridhari; Sujatha, R
Heterosis in black pepper (Piper nigrum L.)
(Kerala Agricultural University, 1995) Sujatha, R; Namboodiri, K M N
A study was conducted to estimate heterosis in F, hybrids of black pepper with respect to yield and three reproductive characters using 45 hybrids belonging to eight different intervarietaJ combinations of crosses planted at the Pepper Research Station, Panniyur. One of the hybrids, viz. Culture 4968 belonging to the cross Uthirenkotta x Balankotta was found to exhibit high heterosis with respect to all the characters. Promising parental combinations were, Uthirenkotta x Cheriyakaniakkadan and Karivilanchy x Cheriyakaniakkadan. The combinations Uthirenkotta x Kottanadan, Uthirenkotta x Kuthiravaly and Panniyur 1 x Karimunda were found least promising.
Histology of organogenesis from callus cultures of black pepper (Piper nigrum L.)
(Kerala Agricultural University, Vellanikara, 2003) Sujatha, R; Luckins C, Babu; Nazeem, P A
Hybridization using promising second generation inbred and molecular characterization of a the third generation inbreds of WCT coconut
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2018) Hassain, N; Sujatha, R
The research project entitled “Hybridization using promising second generation inbred and molecular characterization of the third generation inbreds of WCT coconut (Cocos nucifera L.)” was carried out in the Department of Plant Breeding & Genetics, College of Agriculture, Padannakkad during the year 2016-2018. The major objectives of this study include development of D x T hybrids using promising palms in second generation (S2) inbreds of West Coast Tall (WCT) as male parent and Malayan Yellow Dwarf (MYD) as female parent and characterization of third generation (S3) inbred seedlings of superior S2 palms of WCT using RAPD analysis. For hybridisation, the male parent was selected from the superior inbred family based on the reports on morphological evaluation and estimation of inbreeding depression during the period 2013-2015 (Chethana, 2016). The family IIIS2-1 showed lowest inbreeding depression for yield related traits among 12 families followed by VS2-2. The palm 313 (IIIS2-1) and 225 (VS2-2) were selected as male parents. Superior MYD palm to be used as the female parent was selected from among the MYD palms available at CoA, Padannakkad based on the general features of mother palm selection (Nair et al., 2008). Observations were recorded from these parental palms which included 24 morphological characters. The male parents showed inbreeding depression for several vegetative characters such as stem girth, functional leaves etc and reproductive characters such as number of female flowers per inflorescence, setting percent and nut yield per palm per year, compared to WCT. The results showed that inbreeding depression for these characters was more in palm 313 (developed by selfing) than palm 225 (developed by sibmating). Total five nuts were harvested from MYD (two nuts by using pollen from palm 313 and three nuts using pollen from palm 225) during May 2018 and were sown in the nursery. Molecular characterization of the 15nos of S3 inbred seedlings of IIIS2 family planted in the field in replicated plots were done using RAPD. Genomic DNA was isolated from 15 seedlings of IIIS3 family among which 10 were produced by selfing the S2 palms (IIIS3-1) and 5 were produced by sibmating the S2 palms (IIIS3-2) along with COD, CGD and WCT. After DNA isolation, samples were subjected to RAPD analysis using 10 decamer primers namely OPAU02, OPBA3, OPAW14, OPAW19, OPAW12, OPAW13, OPAW13, OPAW09, OPAU03, OPAW08 and OPAW15 selected based on screening 30 primers. Using primer OPAW08, a band of size of ~400bp was specifically found in inbreds alone while it was absent in dwarfs and WCT seedlings. In OPAU 03 primer, band size of ~1200bp was absent in WCT but present in all inbreds and CGD. The sibmated inbred NL1 showed a distict pattern when amplified with OPAW 09 compared to all other inbreds. The primer produced a specific band of size ~1050bp only in the sibmated inbred CL1 and was absent in all other samples. Amplification with OPAW19 primer produced a banding pattern in which it mainly distinguished CGD from all other seedlings where there were only two bands while others were having six bands. Primer OPAU 02 produced two unique amplicons: 1100bp present in selfed inbred I1M1 TH2 and MYD; 1000bp present in COD and MYD. Genetic similarity among the 15 IIIS3 seedlings, CGD, COD, MYD and WCT was estimated by using of software DARwin (Darwin 6.Ink). The dendrogram indicated three major clusters in which the grouping of several inbreds along with WCT, COD and CGD may be an indication supporting the hypothesis that the dwarfs might have been originated from the talls during the course of evolution followed by selection. The two dwarfs present in the group are COD and CGD which are from Chavakkad area in Kerala and WCT is the most common cultivar of Kerala. As a future line of study, further evaluation of these S3 inbreds regarding the flowering time and yield characters has to be carried out to find out the genetic potential of these inbreds as breeding lines for producing superior hybrids. The seedlings to be emerged from the hybrid nuts produced in the present study, though limited in number, needs to be evaluated for important traits and the hybridisation programme using superior palms in S2 generation needs to be continued for production of more number of hybrid nuts.
Influence of plant characters on yield in black pepper (Piper nigrum L.)
(Kerala Agricultural University, 1995) Sujatha, R; Namboodiri, K M N
Data on 580 genotypes of black pepper were .utilised to estimate the correlations among yield and 20 quantitative characters. The results revealed that the reproductive characters viz., green spike yield per vine, green berry yield per vine, number of spikes per vine and number of under-developed berries per spike as well as the vegetative characters, thickness of node and intemode of orthotrope and angle of insertion of plageotrope are positively and significantly correlated with yield. The inter-correlations among these characters were also positive and significant. Path analysis revealed positive direct effect on yield by the characters green berry yield, spike number, spike length and angle of insertion of plageotrope. The effect of the remaining characters on yield were due to their indirect influence through other component characters.
Molecular characterization and testing hybridity of interspecific crosses in black pepper (Piper Nigrum L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2012) Ashok Babadev Jagtap; Sujatha, R
Molecular marker based linkage mapping in blake pepper (Piper nigrum L.)
(Centre for Plant Breeding and Molecular Biology, College of Horticulture, Vellanikkara, 2009) Rahul Kumar Varma; Sujatha, R
The study on the Development of molecular marker based linkagemapping in black pepper (Piper nigrum L.) was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture and Pepper Research Station (PRS), Panniyur during the period of 2006-2008. The research work included mainly three aspects; the development of mapping population, generation of molecular marker data for linkage mapping and the analysis of RAPD data for linkage map construction. A molecular linkage map covering a large region of the genome with informative DNA markers is very useful for effective and efficient plant selection in breeding programs. It is also essential for identifying and isolating the genes responsible for various quantitative traits. Genetic mapping is based on the principle that genes (markers or loci) segregate via chromosome recombination during meiosis, thus allowing their analysis in the progeny. It requires two genetically divergent parents and segregating plant populations. The recombination frequency of the identified polymorphic markers is an indication of the relative position of the markers in a linkage group. Developing a linkage map in black pepper (2n=4x=52) genome would be very useful because of its specific disadvantages in breeding programmes such as long juvenile period, inherent heterozygosity, perennial nature etc. Two genetically diverse and heterozygous cultivars viz., Cheriyakaniakkadan and Uthirenkotta were selected as the male and female parents of black pepper for the present investigation. The progenies developed by selfing of Panniyur-1(the F1 progeny of these parents) was taken as the F2 population; the progenies developed by back crossing Panniyur-1 with one of the parents gave the BC1 generation and a fresh hybridization of the parental cultivars produced the F1 generation. The seedlings were germinated through in vitro and ex vitro methods. The seed set of the F1 population and germination percentage of in vitro raised seedlings were not satisfactory hence only ex vitro raised F2 population (67 selfed seedlings) was used for linkage analysis. To have an assessment about the general variability in the mapping population morphological observations viz. number of leaves, height, length and breadth of leaf and the pigmentation at the growing tip were recorded and the statistical parameters such as Mean, Coefficient of variation (C.V) and Standard deviation (S.D) were worked out. The result showed that maximum variation was observed in the height of seedlings with the value of Coefficient of variation (C.V) 65.32 per cent. Random Amplified Polymorphic DNA (RAPD) marker was used to identify the polymorphic loci across the male and female parents. Twenty four loci were found polymorphic between the parents using 10 random primers. The identified polymorphic loci were used to genotype the entire mapping population. Linkage analysis was done using the software MAPMAKER/EXP Version 3.0. The results showed that the markers were mainly grouped into four linkage groups. First group consisted of 12 markers with a total genetic distance of 84.8 cM, second group consisted of four markers of 56.6 cM and the last two groups consisted of only two markers each with a total genetic distance of 6.3 cM and 3.3 cM respectively This is a preliminary study carried out with and objective to standardize the method for linkage analysis in black pepper so as to develop a frame work map using molecular markers. Further analysis with larger mapping population and varied markers will provide enough polymorphic markers to supplement this map. Once the seedlings have attained full growth, the morphological and yield related characters could also be subjected to linkage analysis in order to produce a more comprehensive genetic map of all the 13 linkage groups in black pepper
Morpho-molecular characterisation and evaluation of TxD, DxT and DxD hybrids of coconut cultivar ayiramkachi (Cocos nucifera L.)
(Department of Plant Biotechnology, College of Agriculture, Padannakkad, 2021) Haritha, M R; Sujatha, R
Study on “Morpho-molecular characterisation and evaluation of TxD, DxT and DxD hybrids of coconut cultivar Ayiramkachi (Cocos nucifera L.)” was carried out at the Department of Plant Biotechnology, College of Agriculture, Padannakkad during 2018-2020. This investigation aimed at morphological and molecular characterisation of hybrids planted at RARS Pilicode during 1994, in the hybridization programme involving different tall and dwarf genotypes (WCT, Laccadive Ordinary, Philippines, Laccadive Micro, Andaman Ordinary and Malayan Yellow Dwarf) with Ayiramkachi, for important yield attributes and nut quality combined with dwarf stature. The evaluation based on morphological characters recorded during 2018-2020 revealed that the hybrids LMxAYK, MYDxAYK and LOxAYK were promising ones with high yield (155.50, 115.33 and 116.50 nuts/ palm/year respectively) and superior nut characters such as fruit weight, nut weight, shell and meat weight, kernel thickness and copra content. Hybrid PHIxAYK was superior for all the nut characters combined with a shorter stature but the yield was comparatively less (81.67 nuts/ palm/ year). The hybrid CCxAYK was high yielding (150.67 nuts/ palm/ year) but inferior in nut characters. Hybrid AOxAYK recorded good nut characters but was low yielding (61.50 nuts/palm/year). Hybrids of AYK with WCT recorded a moderate yield (95.00 nuts/ palm/ year) with inferior nut characters, and hybrid AYKxMYD was inferior for both yield (21 nuts/ palm/ year) and nut characters. On analysing the TxD, DxT and DxD groups with their tall (T) and dwarf (D) parents, all the hybrids recorded palm height statistically similar to the dwarf cultivars, which may be an indication of inheritance of shorter stature from the common parent AYK. Study on genetic parameters revealed that characters such as palm height, internode length, number of green leaves, rate of leaf production, petiole length, leaf length, number of female flowers per inflorescence, number of female flowers one month after pollination and shell and meat weight had shown high heritability (H2) coupled with high genetic advance as per cent means (GAM%). Hence choice of such characters are best suited for selective breeding. Yield and copra content had shownmedium heritability with high genetic advance. This might be due to the indirect effect of secondary characters contributing to them. Estimation of heterobeltiosis (superiority over better parent) and standard heterosis (superiority over standard check, Kerasree) for the nine hybrids of Ayiramkachi revealed that the hybrids LMxAYK and MYDxAYK were found to be better performers with respect to yield as well as nut characters, and can be exploited for developing commercially important hybrids suitable for culinary as well as processing purposes (copra and other value added products). The hybrid CCxAYK was high yielding but inferior in nut characters. As one of the parent in this cross, Cochin China, is suitable for tender nut purpose due to high nut water content, the hybrid nuts also may be further evaluated at tender nut stage for its suitability for tender nut purpose. Thirty four SSR markers were screened for polymorphism among the eight parental cultivars and the check palm (Kerasree). The markers produced an average of 52.94% polymorphism and 1 to 2 alleles/locus. Out of 34 SSR markers screened for polymorphism, 15 markers viz., CAC02, CAC11, CNZ40, CNZ44, CnCirA9, CnCirB12, CnCirE2, CnCirC7, CnCirH4, CnCirE12, CnCirC3, CnCirF2, CnCirH7, CnCirG11 and CnCirC5 were able to detect the polymorphism between the parental palms and hence can be utilized in future for fingerprinting the hybrid palms.
Morpho-molecular characterisation of D x D hybrids developed using 'annur', the dwarf ecotype of WCT coconut ( Cocos nucifera L.)
(Department of plant breeding and genetics, College of Agriculture, Padannakkad, 2021) Anuprasad, T E; Sujatha, R
Coconut is one of the most extensively grown crop in the world, playing a significant role in the economic, cultural, and social life of over 80 tropical countries. Coconut cultivars are classified into tall (variety typica) and dwarf (variety nana) types. Short statured palms are currently receiving more attention due to various socio-economic factors. However, the poor quality of nuts for various processing aspects and high susceptibility of dwarf palms to pest and diseases makes them unsuitable for large scale cultivation. The solution for this problem is development of suitable hybrids by combining the desirable features of tall and dwarf palms. West Coast Tall (WCT) is the most widely cultivated variety in Kerala and several ecotypes of WCT were reported. In a study initiated in 2005 at RARS Pilicode, an ecotype of WCT was detected in Annur, a place in Kannur district which showed dwarfing nature with nut qualities similar to WCT. The Annur ecotype was crossed in 2007-08 with the dwarf cultivars Malayan Yellow Dwarf (MYD) and Gangabondam (GB). The hybrid seed nuts along with interse seed nuts of Annur were planted at RARS Pilicode in 2009. The first flowering was recorded in 2014. The present study aims at evaluation of plant stature, yield, nut quality and other important morphological traits of Annur and the hybrids developed from it. The interse of Annur, Annur x GB and Annur x MYD were compared with corresponding characters of the respective parents as well as WCT, Keraganga (WCT x GB) and Kerasree (WCT x MYD). Significant reduction in height was observed for hybrids viz. Annur x MYD (2.251 m) and Annur x GB (2.065 m) which was on par with Annur (2.725 m) and considerably lower than the corresponding values of dwarf cultivars viz., MYD (5.2 m) and GB (8.885 m). Number of nuts per palm per year was on par in Annur (58.2) and Annur x MYD (55.4) while that was lower in hybrid Annur x GB (34.3). However, since the palms started flowering only in 2014, stabilization of yield is not attained. Nut weight and kernel thickness were significantly higher in Annur x MYD whereas these were on par in Annurand Annur x GB. Copra content in Annur and both hybrids were on par. In general, Annur x MYD showed superior performance for nut characters compared to Annur x GB. Annur palms were significantly similar to WCT for most of the characters except height, rate of leaf production, nuts per bunch, nut water content. When the yield of hybrids of Annur were compared with the standard check varieties viz., Keraganga (WCT x GB) and Kerasree (WCT x MYD), (101.5 and 104.25 nuts per palm per year) yield of Annur x GB and Annur x MYD were low. However, both hybrids showed nut characters similar to the respective released varieties ie, Annur x GB was similar to Keraganga and Annur x MYD showed similar nut characters with Kerasree except copra content (161.432 g and 193 g respectively). Better palms from each cross were analysed and palm numbers 1, 2, 15, 19, 20 and 28 were identified as better palms from interse of Annur. The better performing palms of hybrid, Annur x MYD were palm numbers 43, 44, 47 and 49. Three palms from Annur x GB (palm numbers 53, 55 and 56) were identified as better palms. Correlation analysis revealed significant positive genotypic correlation of nut yield per palm with all reproductive and yield characters and with most of vegetative and nut characters. Path coefficient analysis showed high positive direct effect on number of female flowers per inflorescence, number of bunches per palm per year, number of nuts per bunch, pole to pole circumference of nut, nut weight (with husk), shell and meat weight, quantity of liquid endosperm and copra content. High heritability coupled with high genetic advance was exhibited by the characters height of palm, leaf length, number of green leaves, number of inflorescences at the time of observation and number of female flowers per inflorescence. This indicates that these characters are governed by additive gene action and selection based on these characters will be effective. For both hybrids, height of the palm showed negative heterosis which indicates the dwarf nature of the hybrids. In hybrid Annur x MYD, most of nut charactersshowed heterosis over mid parent (relative heterosis) and better parent (heterobeltiosis). The standard heterosis was significant when compared with Keraganga but was not significant with Kerasree. Molecular characterization of the Annur (interse) and the hybrids from it was also attempted using Simple Sequence Repeat markers (SSR). Good quality genomic DNA is the prerequisite for any molecular work. DNA was isolated from all the parental palms as well as check cultivar Kerasree. The quality and quantity of genomic DNA obtained was verified using agarose gel electrophoresis and spectrophotometer. The quantity of DNA samples ranged from 350 to 1300 ng/μl with good quality. Screening of SSR markers for parents and check palms revealed polymorphism for the primers CAC02, CAC11, CNZ40, CnCirA9, CnCirB12, CnCirC3, CnCirC5, CnCirC7, CnCirE2, CnCirE12, CnCirF2, CnCirG11, CnCirH4 and CnCirH7. Out of 34 SSR markers, 27 were monomorphic between WCT and interse of Annur. Genetic Similarity analysis using the SSR markers revealed the high similarity between of interse palms of Annur and WCT. Dwarf palms viz. MYD and GB also showed high genetic similarity with each other. The present study revealed that the genotype Annur is a dwarf ecotype of WCT with similar nut quality and hence is a very promising genotype for breeding for dwarfness. The hybridization of ecotype Annur with MYD is found to be superior in yield characters compared to the hybridization with Gangabondam. However, both hybrids inherited the short stature from Annur and are promising. Since the yield characters needs to be stabilized, evaluation based on important vegetative and reproductive characters as well as molecular fingerprinting needs to be continued inorder to identify the full potential of these dwarf hybrids.
Somatic embryogenesis from immature inflorescence of coconut (Cocos nucifera L.)
(Department of Plant Biotechnology, College of Horticulture, Vellanikkara, 2019) Radhika, R; Sujatha, R
Variability in inter varietal hybrids and open pollinated seed progenies of black pepper ( Piper nigrum L .)
(Department of Agricultural Botany, College of Horticulture, Vellanikkara, 1991) Sujatha, R; Narayanan Namboodiri, K M