Browsing by Author "Rajmohan, K"

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Agrobacterium mediated genetic transformation in dendrobium
(Department of Pomology & Floriculture, College of Agriculture, Vellayani, 2005) Swarnapiria, R; Rajmohan, K
Agrobactrium tumefaciens mediated genetic transformation in dendrobium variety sonia 17 with 1- aminocyclopropane- 1 carboxylic acid (acc) synthase antisense gene
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Karthika Karunakaran; Rajmohan, K
Amplification and sequencing of spacer region between two tRNA genes and its flanking region in the chloroplast genome of Centella asiatica L.
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Manju Elizabeth, P; Rajmohan, K
The study entitled “Amplification and sequencing of spacer region between two tRNA genes and its flanking region in the chloroplast genome of Centella asiatica L.” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2005-2006 with the objective of isolating a spacer region and its flanking regions from the chloroplast genome of Centella asiatica to develop a species specific vector for the chloroplast transformation. Heterologous primers were designed based on the chloroplast genome sequences of Arabidopsis thaliana, Nicotiana tabacum and Panax ginseng using Pimer3 software for the spacer regions trnG-trnfMet, trnE-trnT, trnT-trnL and rps16-trnQ and were amplified on genomic DNA of Centella asiatica. The entire isolated regions were sequenced except trnT-trnL spacer region. All sequenced regions were subjected to BLASTN and BLASTX similarity search. The trnG-trnfMet spacer region (270bp) showed maximum similarity to the same region in Panax ginseng (GI: 51235292, AY582139.1) chloroplast genome. The spacer region between genes rps16 and trnQ (1749bp) showed maximum similarity to rps16 gene and its intron in Centella asiatica (GI: 6692894, AF110603.1) chloroplast genome and to rps16 gene, spacer region after rps16 and starting region of trnQ gene in Panax ginseng (GI: 51235292, AY582139.1) chloroplast genome. Primers for flanking regions of rps16-trnQ spacer were designed manually based on the primers of this spacer and the chloroplast genome of Panax ginseng. The right flanking region amplified (1582bp) with primers Hf and Hr showed maximum similarity to trnQ gene, spacer region after trnQ, psbK gene, spacer region after psbK and psbI gene in Panax ginseng chloroplast genome. The left flanking region of rps16-trnQ spacer sequence amplified (1227bp) with primer combination Ff and Fr showed similarity to rps16 gene, spacer region after trnK gene, trnK gene and spacer region after matK gene of Panax ginseng chloroplast genome. The sequence amplified (1089bp) with primers Gf and Fr showed similarity to rps16 gene, spacer region after trnK of Panax ginseng and to matK gene of Centella erecta (GI: 2281236, US8599.1). The spacer region between rps16 and trnQ gene and its flanking region isolated can be used in developing a Centella asiaticaL. specific vector for chloroplast transformation.
Characterisation of traditional mango (mangifera indica L.) varieties of southern Kerala
(Department of Pomology and Floriculture, College of Agriculture, Vellayani, 2006) Simi, S; Rajmohan, K
Attempts were made at the Department of Pomology and Floriculture and the Department of Plant Biotechnology, College of Agriculture, Vellayani during June 2003 to December 2005 for characterizing the traditional mango (Mangifera indica L.) varieties of southern Kerala. Field visits were made in order to locate the varieties, to conduct survey and to collect the research materials. Fifty varieties were located in the four districts of southern Kerala, Trivandrum (17), Kollam (10), Pathanamthitta (6) and Alappuzha (17). Wide variability could be observed in the vegetative characters. The varieties / accessions varied remarkably in the vegetative characters like tree height, tree habit, leaf shape, leaf margin and leaf tip. Leaf length, leaf width and petiole length varied significantly between the varieties. The varieties / accessions varied widely with respect to the floral characters like position of inflorescence, shape, length, colour and density of flowers and season and regularity of flowering. The percentage of hermaphrodite flowers ranged from 8.4 (Velutha Muvandan) to 96.0 (Eara Local). The fruit characters like shape, presence of basal cavity, beak type, sinus type, presence of groove, type and slope of shoulders and apex exhibited by the varieties / accessions showed remarkable variation. High variability in fruit length, breadth, thickness, weight and volume could also be observed among the varieties / accessions. Muthalamookan recorded the highest length, breadth, thickness, weight and volume of fruits and Puliyan manga recorded the lowest values. Fruit weight ranged from 37.5 g to 826.0 g. Skin characters like colour, thickness, texture and weight also showed high variability. Percentage contribution of skin weight to the fruit weight varied from 8.4 (Ambalathara Local) to 37.7 (Puliyan). The different varieties varied considerably in the various flesh characters like weight, texture, adherence of skin to pulp, fibre content and colour. Pulp weight ranged from 14.8 g to 676.0g. Stone characters showed wide variation among the accessions. Quality characters of fruits varied widely among the accessions. Titrable acidity ranged from 0.12 per cent (Nedungolan) to 4.03 per cent (Eara Local). Ascorbic acid content ranged from 3.08 (Neendakara manga) to 119.05 (Natumav Type-2) mg / 100g. The total carotenoid content varied from 0.21 (Natumav Type-4) to 7.97 mg / 100g (Karpoora Varikka). The TSS of ripe fruits ranged from 8.77 to 25.71 0 Brix. Total sugar content ranged from 2.0 (Natumav Type-4) to 22.2 per cent (Neenda Karpooram). The reducing sugar content ranged from 0.9 per cent (Kalluketty) to 6.1 per cent (Perakka manga). Non- reducing sugar content varied from 0.81 per cent (Natumav Type-4)to 16.8 (Neenda Karpooram ). The crude fibre content in fruit pulp ranged from 0.4 per cent (Nedungolan) to 2.92 per cent (Natumav Type-3). Perakka manga was rated as the best in organoleptic evaluation. The accessions, Pulichi, Natumav Type- 2, Natumav Type-5, Kalluketty, Vellari Type-1and Kalkanda Vellari can be recommended as outstanding in pickling qualities. Of the table types, Nedungolan, Perakka manga, Muthalamookkan, Vellari Type-2, Karpooram manga and Neenda Karpooram can be recommended as superior with respect to important economic characters like fruit weight, pulp content and eating quality. Among the dual types, Kotookonam Varikka, Velutha Muvandan, Karpoora Varikka, Ambalathara Local and Kizhakkan Thali can be recommended as excellent in overall acceptability. DNA was extracted from young leaves using CTAB method (Dellaporta et al., 1983) with slight modification (Anuj et al., 2004). A total of 157 RAPDs (average of 3.74 bands per primer) were generated on PCR amplification using 42 decamer primers, of which 96.18 per cent (151 bands) were polymorphic. This accounts to an average of 3.6 bands per primer. Of these, ten primers yielded 92 scorable bands with an average of 9.2 bands per primer. The number of bands resolved per amplification varied from six to fourteen. A genetic similarity matrix was constructed using the Jaccard’s coefficient method. The pair wise similarity coefficient values ranged from 0.217 to 0.833. In the dendrogram, the thirty accessions subjected to RAPD analysis, were observed to group into seven clusters. The largest cluster contained 20 accessions. Four table varieties, Kappa manga, Mylapore manga, Neenda Karpooram and Kandiyoor Local were grouped together. All these were soft fleshed. Muthalamookan and Kolambi manga clustered together. Puliyan, Perakka manga, Kalluketty and Champa Varikka formed four separate clusters. Similarity assessment based on morphological and quality characters suggested that there was very high diversity among the traditional mango varieties. The pair wise similarity coefficient values varied between 0.042 and 0.708. The clustering pattern based on RAPD analysis was not strictly in accordance with that based on morphological and quality characters.
Differential expression of genes involved in anthocyanin pigmentation in red banana and green-red clones
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Rehna Augustine; Rajmohan, K
The study entitled “Differential expression of genes involved in anthocyanin pigmentation in Red banana and Green-red clones” was conducted at the Department of Plant Biotechnology, Vellayani, Thiruvananthapuram during the period from 2004 to 2006 with an objective of contrasting the expression of genes (chalcone synthase, dihydroflavonol 4-reductase and UDP: glucose 3-oxy-glucosyl transferase) involved in anthocyanin pigmentation in Red banana and Green-red clones. Experiments were also conducted to differentiate the clones based on total sugar concentration and protein profile. Reverse transcription-polymerase chain reaction was carried out to study the expression of genes. Heterologous primers designed based on the gene sequences of Oryza sativa and Vitis vinifera were used for RT-PCR. Total RNA from tissue culture shoot, leaves and fruit from three different stages of Red banana and Green-red clones were isolated using hot phenol method which gave an yield of 80 - 200 µg g -1 of the tissue and a A260/A280 ratio ranging between 1.6 –2.0. Messenger RNA was purified from the total RNA using the mRNA purification kit from GENEI (Bangalore). The RT-PCR amplified products of the two clones, representing chalcone synthase (CHS), dihydroflavonol 4- reductase (DFR), and UDP: glucose flavonoid 3-oxy-glucosyl transferase (UFGT) were eluted and purified. The cDNA fragments were cloned to pCRII vector (TA Cloning Kit, Invitrogen. Inc., USA) and sequenced. The nucleotide to nucleotide BLAST of cDNA clone of 183 bp from Red banana showed similarity with the chalcone synthase mRNA sequence of rice (Acc. No. X89859). cDNA from Green-red clone showed no significant similarity to any other chalcone synthase gene during homology search. The translated query vs. protein database (blastx) search exhibited similarity with shikimate 3- dehydrogenase, an enzyme in the biochemical pathway for aromatic amino acids. The cDNAs synthesized from Red banana and Green-red clone with gene specific primers for dihydroflavonol 4- reductase were having 354 bp and 325 bp length respectively. The homology search (blastn) revealed no similarities with any of the nucleotide sequences specific for dihydroflavonol 4- reductase. The cDNAs amplified from Red banana and Green-red clone with UDP: glucose 3-oxy-glucosyltransferase specific primers were of 361 bp and 345 bp, respectively. The homology search using blastn showed similarity with mRNA sequence for UDP: glucose 3-oxy-glucosyltransferase in grapes. In Red banana the blastx search revealed similarity with a glucosyltransferase from Synechocystis sp. There was no similarity for UDP: glucose 3-oxy-glucosyltransferase cDNA of Green-red clone in the NCBI database. SDS-PAGE analysis showed no difference in the protein profile of Red banana and Green-red clone. Total sugar content in the peel, pulp, peel together with the pulp of red banana and Green-red clone did not showed any significant difference. The expression analysis of the key genes of the general pathway showed no significant difference in both the clones. All the three genes selected for the study were present in both Red and Green-red clones. The genes isolated were not totally identical in the two banana clones. Further studies are needed to get a better insight into the cause of colour change.
Effect of anthelmintic treatment on milk production in subclinical nematode infections of cows
(Department of Parasitology, College of Veterinary and Animal Sciences, Mannuthy, 1994) Rajasekharan Nair, K G; Rajmohan, K
A study was conducted on the incidence of subclinical gastrointestinal namatodiasis in diary cows and heifers, and the effect of anthelmintic treatment on their milk production. It was found that all the animals examined in the two livestock farms of Kerala Agricultural University were harbouring the infection. Faecal culture was found to be the method of choice for detection of nematode infection when compared to microscopical examination of faeces.
Effect of green leaves used in traditional food preparations on DNA repair
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2009) Arpitha, Y R; Rajmohan, K
The thesis entitled “Effect of green leaves used in traditional food preparation on DNA repair” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thirvananthapuram during 2007-2009 with an objective to test the effect of green leaf extracts of plants (Azima tetracantha, Cissampelos pareira, Myxopyrum serratulum, Pongamia pinnata and Thespesia populneus) used in traditional food preparations on DNA repair by employing the Comet Assay. In the first set of treatments in the genotoxicity study, tomato seedlings, treated with two mutagens (Ethyl methanesulfonate and Methyl methanesulfonate) at different concentrations (0-4mM) over night under room temperature and dim light, showed significantly higher DNA damage, compared to control. In the second set of treatments in the genotoxicity study, tomato seedlings, treated with plant leaf extract, obtained by following two methods of extraction protocols, showed no genotoxicity effect. In all the treatments (pre-supplementation, post-supplementation and co-supplementation treatments) of plant leaf extracts obtained by following two methods of extraction protocol, before, after and simultaneous treatments with mutagens (EMS and MMS at 4mM), in antigenotoxicity studies, tomato seedlings recorded significantly lower tail moment value, compared to control, indicating effective protection of DNA from damages by mutagens. This may be due to phytochemicals and biologically active components (non nutrient) of the test plant leaf extracts. These results can be of use in testing the leaf extracts of test plants in animal system, which in turn can be of use as a prophylactic measure against cancer and other disorders.
Effect of plant growth substances on the in vitro propagation of jack (Artocarpus Heterophyllus Lam.)
(Kerala Agricultural University, 1988) Rajmohan, K; Mohanakumaran, N
Explants from shoot apices of fresh stem sprouts of five-year old jack (Artocarpusheterophyllus Lam.) trees registered a multiplication rate of 4.5 x. when cultured for five weeks on MS proliferation medium containing BA (5 mg/l) and NAA (0.2 mg/l). The normal strength of inorganic salts and organic growth factors of the MS medium, with 30—40 g/l of sucrose or 20—30 g/l of glucose was found to support the multiplication and growth of the cultures. GAa did not influence the shoot proliferation or growth. Adenine sulphate at 20 mg/l was found to increase the multiplication rate by 27.3 per cent, without
Enhancing the in vitro response of explants from mature jack (artocarpus heterophyllus lam.) trees
(Department of Horticulture, College of Agriculture, Vellayani, 1993) Reena Philip; Rajmohan, K
The objective of the present study was to improve the propagation efficiency of mature phase jack trees by various pretreatments. The treatments tried involved stock plant treatments, explants treatments and in vitro treatments. Surface sterilization with HgCl2 for 13 minutes was found to be the most effective in reducing microbial contamination during culture establishment. Among the stock plant treatments tried, grafting on to juvenile rootstock was found to be the most effective in improving the in vitro response of explants from mature jack trees. Stress treatments were found to be the most effective among the explants treatments. Cold shock for 5 minutes at – 200C was found to produce the best results followed by heat treatment at 420 C for 2 minutes. Among the in vitro treatments, incorporation of phloroglucinol at the rate of 10mg/l was found to be the most effective. The results also showed significant influence of season on the response of mature explants, the highest response being observed during March – April.
Evolving techniques for in vitro somatic embryogenesis in polyembryonic mango varieties
(Department of Horticulture, College of Agriculture, Vellayani, 1998) Ramesh, P; Rajmohan, K
Studies were conducted for evolving techniques for in vitro somatic embryogenesis in polyembryonic mango varieties during 1994 - 1996 in the Plant Tissue Culture Laboratory of the Department of Horticulture, College of Agriculture, Vellayani. Attempts were made to standardise the various stages of somatic embryogenesis, namely, induction, initiation, maturation and germination using nucellus and embryo mass as explants. Among the SIX varieties attempted for induction of somatic embryogenesis from nucellus, Kalluvarikka recorded the highest percentage of response (85.0) and Vellari Manga the least (68.9). Induction of somatic embryogenesis from nucellus was found to occur at its maximum on MS medium having half strength major salts, supplemented with 2,4 - D 5.0 mg/l, GA3 5.0 mg/l, glutamine 400 mg/l, sucrose 60 g/l, coconut water 200 ml/l, agar 6.0 g/l and activated charcoal 2.5 g/l, in darkness. The highest callus index was recorded when 2,4 - D 5.0 mg/l was substituted with BA 16 mg/l in the same medium. Initation of .somatic embryo genesis from nucellus occurred at its best in darkness on MS medium having half strength major salts, supplemented with BA 1.0 mg/l, glutamine 400 mg/l, casein hydrolysate 500 mg/I, sucrose 60 g/l, coconut water 200 mIll agar 5.5 g/l and activated charcoal 2.5 g/l. Maturation of nucellus derived somatic embryoids was found at its best on a combination of basal media with B-5 major salts and MS minor salts supplemented with ABA 3.0 mg/l, casein hydrolysate 100 mg/l, sucrose 40 g/l, PVP 10 g/l, coconut water 200 ml/l and agar 5.0 g/l, in darkness. Germination of somatic embryoids derived from nucellus occurred only in presence of light, on a combination of basal media with B-5 major salts, MS minor salts, supplemented with BA 0.1 mg/l, sucrose 50 g/l, PVP 10 g/1 and agar 5.0 g/l. Somatic embryos were produced directly from embryo mass explants in darkness on solid MS medium having half strength of major salts supplemented with 2,4 - D 5.0 mg/l, GA3 5.0 mg/l, glutamine 400 mg/l, sucrose 60 g/l, coconut water 200 ml/l, agar 6.0 g/I and activated charcoal 2.5 g/l. The embryoids having sufficient size resulted from this treatment were able to attain near normal germination in presence of light on medium having a combination of B - 5 major salts and MS minor salts, supplemented with BA 0.1 mg/l or 0.2 mg/l, sucrose 50 g/l, PVP 10 g/l and agar 5.0 g/1.
Ex vitro establishment of jack (Artocarpus Heterophyllus Lam.) plantlets
(Department of Horticulture, College of Agriculture, Vellayani, 1990) Ramesh, B; Rajmohan, K
The problem of poor ex vitro establishment of jack plantlets was addressed in a study conducted during 1988-90 at the Plant Tissue Culture Laboratory of the Department of Horticulture, College of Agriculture, Vellayani. A reliable protocol could be standardized based on the analysis of the factors influencing plantlet production and their ex vitro establishment and characterization of the morphological, histological and physiological peculiarities of the in vitro raised plantlets. A rooting medium (MS) containing half strength inorganic nutrients (particularly inorganic nitrogen and calcium salt), sucrose 30.0 g/l and agar 6.0 g/l was identified as ideal for the in vitro rooting and ex vitro establishment of jack plantlets. Activated charcoal was not useful for the purpose. Shoots of 3.0 cm length, with three to four leaves, recorded 100.0 percent rooting, 5.5 roots per shoot and good root branching (2.75). Erlenmeyer flasks of 100 or 150ml capacity were the superior culture vessels for in vitro rooting. A light intensity of 50 µE m-2S-2 for 21 days was required during the prior to planting out stage for successful ex vitro establishment. Plantlets of 18 days and above old recorded the maximum survival ex vitro. Sand was identified as the best potting medium. However the moisture level of sand has to be maintained at an optimum of 13.4g/100g of soil. Plastic pot (5.0 x 5.0 x 7.5 cm size with small holes) was found to be superior to the other containers tried. Mist chamber was found to be convenient and successful as a humidity maintenance device for the hardening of the plantlets. Use of antitranspirants was not advantageous for the establishment of the plantlets. Inoculation of the potting medium with the vesicular arbuscular mycorrhiza Glomus fasciculatum and G etunicatum favoured 80.0 to 100.0 percent ex vitro establishment of the plantlets; the plant growth was significantly increased in such cases. Nutrient starter solutions cannot be recommended during the initial period of acclimatization as they reduced the survival of the plantlets. Leaves of the in vitro raised plantlets had improper deposit of epicuticular wax and underdeveloped palisade parenchyma, Spongy parenchyma, mechanical tissues and vascular bundles. The stomatal aperture was comparatively large. The stomata did not close when exposed to stress conditions. The morphological and histological peculiarities caused high rate of water loss (16.0 mg/cm2 in 105 minutes) from the plantlets when planted out and hence necessitated an initial humidity acclimatization. The mean number of stomata per unit area, total chlorophyll chlorophyll a and chlorophyll b contents were less in the in vitro grown leaves. The morphological, histological and physiological characteristics of the plantlets were normalized during the ex vitro establishment, especially as the new leaves were produced.
In vitro germination of hybrid seeds of banana
(Kerala Agricultural University, 1992) Lekshmy, M L; Valsalakumari, P K; Rajmohan, K
In vitro rooting of jack shoot cultures
(Kerala Agricultural University, 1983) Rajmohan, K; Mohanakumaran, N
Influence of explant source on the in vitro propagation of jack (Artocarpus heterophyllus Lam.)
(Kerala Agricultural University, 1985) Rajmohan, K; Mohanakumaran, N
Physiological ago of the explants exhibited significant influence on the in vitro propagation of jack. Shoot apices from the seedlings registered a multiplication rateof17.4x, with 100 percent rooting and 6.0 roots formed in 20.75 days. Explarits from fresh stem-sprouts of five, ten and thirty-year old trees recorded shoot multiplication rates of 4.50x, 2.80x and 2.09x, respectively in five weeks. The corresponding rooting percentages were 70 (with 5.43 roots formed in 13.43 days), 40 (with 2.59 roots formed in 24 days) and 15 (with 1.0 root formed in 46.7 days) after two to three subcultures. Explants from six-month old jack grafts failed to produce multiple shoots; but exhibited 50 per cent rooting with 2.0 roots formed in 20.5 days.
Molecular analysis of spike branching observed in black pepper (Piper nigrum L.) type from Idukki
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2009) Vimarsha, H S; Rajmohan, K
The study entitled “Molecular analysis of spike branching observed in black pepper (Piper nigrum L.) type from Idukki” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2006-2008. The objective of the study was to analyse the sequence homology for the inflorescence architecture gene (TFL1) and flowering gene (FT) in spike branching black pepper type from Idukki, using polymerase chain reaction technique. Spike branching in black pepper is very rare phenomenon. Profuse spike branching was observed in an uncharacterized black pepper type from Idukki. Single spike had three to four times more berry yield, compared to improved varieties. This type needed to be characterized at morphological and molecular levels to know its parental descendent and possible involvement of gene regulation responsible for spike branching. As an initial approach, molecular analysis was done to find out the presence of TFL1 and FT genes, which have been reported to be involved in inflorescence branching in modal plants. TFL1 primer pair (TFL-F1 and TFL-R1), designed for the fourth exonic sequence of TFL1 gene in Arabidopsis thaliana, could amplify a 700 bp DNA sequence in the spike branching type, as well as in Karimunda and Vellamundi, indicating homology for the gene sequence and possible conserved exonic sequence in black pepper. Presence of sequence homology for the gene TFL1 indicated the possible involvement of TFL1 gene, which had been reported to be associated with inflorescence branching in Arabidopsis thaliana, in the spike branching trait of the black pepper type. This result is significant, as five out of the eight cultivars tested did not give any positive response for the primer pair designed based on TFL1 gene. However, two non-spike branching varieties including Karimunda and Vellamundi have also shown amplification for the TFL1 primer pair. As an initial step towards characterisation of the spike branching type, RAPD analysis of it along with seven other cultivars/varieties was done. This analysis revealed variability, accounting for 83 per cent polymorphism. In the dendrogram, at a similarity index of 0.43 the plants grouped into two big clusters, indicating 67 per cent dissimilarity. All the eight plants under study formed individual clusters at similarity index 0.74, except the spike branching type and Vellamundi. Results of gene specific PCR which yielded single amplicon can be hypothecated for the presence of sequence homology for the gene TFL1 and has conserved fourth exon in black pepper cultivars and varieties. Further transcript level and expression level studies are essential to find the specific role of the TFL1 gene in spike branching black pepper.
Molecular characterization of banana (Musa AAB plantain subgroup) clones
(Department of Pomology and Floriculture, College of Agriculture, Vellayani, 2001) Simi, S; Rajmohan, K
Attempts were made for characterizing eleven banana (Musa AAB Plantain subgroup) clones at molecular level during January 2000 to October 2001 at the Department of Pomology and Floriculture and the Plant Molecular Biology and Biotechnology Centre, College of Agriculture, Vellayani. Tissues from fully unfurled leaves of the clones, were used for isolating DNA, using modified Walbot's method. Storage of leaves at -SYC did not affect either the DNA yield or purity ratio. Gel elctrophoresis using agarose concentrations of 0.09 per cent and 1.4 per cent were the best for visualizing the genomic DNA and RAPD pattern, respectively. The best voltage level was 75 V. TBE buffer could produce better separation of bands compared to TAE buffer. Twenty nanogram of DNA, 200 IlM each of dNTPs, 0.6 units Taq DNA polymerase and 5 pM primer in presence of the assay buffer gave good PCR amplification results. The programme consisted of an initial denaturation at 95°C for 3.0 minutes, followed by 45 cycles of denaturation at 95°C for 1.0 minute, annealing at 36°C for 1.0 minute 30 seconds and extension at 72°C for 2.0 minutes. The synthesis step of the final cycle was extended further by 6.0 minutes. The products of .ampl ification were kept at 4.0°C until attended. One hundred and six RAPDs were generated when PCR amplification was carried out using forty decamer primers (Operon Inc., CA, USA) of kit A and kit B. Of these, 100 bands were polymorphic which accounted to an average of 2.5 polymorphic bands per primer. Eight primers (OPA-OI, OPA-03, OPA-13, OPB-OI, OPB-06, OPB-lO, OPB-12 and OPB-IS) produced reproducible banding patterns on at least two runs. These primers yielded 42 scorable bands with an average of 5.25 bands per primer. The amplification products ranged in size from 400 to 1500 bp. The number of bands resolved per amplification was primer dependent and varied from a minimum of three to a maximum of nine. Reproducible bands were scored for their presence (+) or absence (-) for all the plantain clones studied. A genetic similarity matrix was constructed u~ng the Jaccard's coeffecient method. The pairwise coefficient values varied between 0.3333 and 0.9355. The least similarity coefficient values were those of Zanzibar with Changazhikodan and Manjeri Nendran (0.3333). The highest value for similarity index was obtained for Koonoor Ethan - Quintal Banana pair (0.9355), followed by Manjeri Nendran - Myndoli pair (0.8889). The next value was for the Kaliethan - Koonoor Ethan pair (0.8529). Based on the similarity coefficients, distances between the clones were computed using SYSTA T software package. The distance was the least between Koonoor Ethan and Quintal Banana (0.042), followed by Manjeri Nendran and Myndoli (0.06). Zanzibar and Mysore Ethan showed the greatest distance (0.349), followed by Mysore Ethan and Padalamurian (0.167). In the dendrogram constructed by the nearest neighbour (single-link) method (Krzanowski, 1988), all the eleven plantain clones were found grouped under five clusters. Attu Nendran, Changanasseri Nendran, Changazhikodan, Kaliethan, Koonoor Ethan and Quintal Banana formed the largest cluster. Manjeri Nendran and Myndoli formed the second cluster. Padalamurian, Mysore Ethan and Zanzibar formed three separate clusters. Zanzibar, belonging to 'Horn Plantain', was different from the rest of the clones. Quintal Banana and Myndoli, which were considered to be identical, got grouped under two different clusters.
Molecular evaluation of genomic stability of banana plants developed by in vitro clonal propagation
(Department of Horticulture, College of Agriculture, Vellayani, 2001) Asha S Nayar; Rajmohan, K
Attempts were made for evaluating the genomic stability of in vitro propagated Peel banana plantlets at molecular level, during 1999- 2000, at the Plant Molecular Biology and Biotechnology Centre, College of Agriculture, Vellayani. Efforts were made to standardise the DNA isolation method and PCR amplification conditions, to identify the primer producing reproducible polymorphic bands and to compare the banding patterns characteristic to the subcultures and the mother plant. The emerging leaves of the Rxl banana plants before they fully unfurl, gave the highest DNA yield of2825 ng! III whereas, the in vitro leaves gave the highest optical density (OD) ratio of 1.76. The purity of DNA was the highest (0. D. ratio 1.81) while CTAB method (Scott et al., 1994) was adopted. The DNA quantity was the highest in the Walbot's method, viz. 3000 ng! Ill. The OD ratio increased from 1.33 to 1.46 on addition of proteinase k. Additional purification step increased the OD ratio from 0.93 to 1.18. One per cent polyvinyl pyrrolidone (PVP) and 1.5 per cent J3-mercaptoethanol, when added in the extraction buffer produced transparent DNA pellet. 0.9 per cent and 1.4 per cent of agarose concentration were found to be the best for the genomic DNA and RAPD banding patterns respectively. The optimum PCR programme Wdenaturation at 95° C for 1.0 minute, annealing at 36° C for 1.0 minute and 30 seconds, and extension at 72° C for 2.0 minutes. The synthesis step was extended further by 6.0 minutes. A total of 134 RAPDs were generated when PCR amplification was done of which 130 were polymorphic. OPA- 06, OPB-IO and OPB- 14 produced no amplification. No difference was found in the banding pattern of the three subcultures and the mother plant of Fed banana, when amplification reaction was carried out using OPA-20. A total of five intense bands and three faint bands were obtained with OPA-20.
Optimising in vitro somatic embryogenesis in polyembryonic mango (Mangifera indica L.) varieties
(Department of Horticulture, College of Agriculture, Vellayani, 1995) Bindu, C P; Rajmohan, K
Studies were conducted to optimise the in vitro propagation techniques via somatic embryogenesis in polyembryonic mango varieties (Vellari, Kalluvarikka, Thalimanga, Kilichundan, Pulichi and Varikka) of Kerala, during 1993-1994 at the Department of Horticulture, College of Agriculture, Vellayani. Culture media and conditions could be standardised for the first two stages of somatic embryogenesis, namely induction and initiation. However, attempts for inducing normal maturation and germination of the embryoids were not so successfu I . Five out of the six varieties of mango (except Ki I ichundan) responded to the induct ion treatments for somatic embryogenesis. Kalluvarikka recorded the highest per cent cultures (87.50) initiating somatic embryoids from the nucellar tissue. Puliehi was observed to initiate the highest per cent eultures (91.66) initiating somatic embryoids from embryo mass cultured. Somatic embryoids were induced and initiated from nucellus as well as embryo mass. From the nucellus, the embryoids were produced directly, without any intervening callus. The embryo mass gave rise to emb r y og e n i c ca I I us, multiple embryos or zygotic embryos. The somatic embryoids from nucellar tissue were best induced when cultured in darkness on half strength Murashige and Skooge basal medium supplemented with 2,4-D 5.0 mg/l, GA3 5.0 mg/l, glutamine 400.0 mgll, sucrose 60.0 g/l, coconut water 200.0 mIll, agar 6.0 g/l and activated charcoal 2.5 g/l. Somatic embryoids from nucellar tissue were found to be initiated in 55.50 per cent cultures on half strength Murashige and Skoog basal medium supplemented with 2,4-D 5.0 mg/l, BA 0.05 mg/l, glutamine 400.0 mg/l, casein hydrolysate 500.0 mg/l, sucrose 60.0 g/l, coconut water 200.0 mill, agar 6.0 g/l and activated charcoal 2.5 g/l. Darkness was essential for the initiation. Ambient temperature and in the culture room temperature (26°C) were equally effective for the initiation. Abscisic acid was tried, among other treatments, for inducing proper maturation of the somatic embryoids initiated from nucellar tissue. The maximum size of the embryoids was observed on half strength Murashige and Skoog basal medium supplemented with ABA 16.0 uM, casein hydrolysate 100.0 mgll, sucrose 40.0 gll, coconut water 200.0 mIll, agar 6.0 g/l and activated charcoal 2.5 g/l. the embryoids was not influenced by light. Size of Attempts for inducing normal germination of the somatic embryoids from the maturation medium were made using treatments involving plant growth substances (BA, 2iP, GA 3 and NAA), factors known to impart osmotic stress (Polyethelene glycol and high concentrations of sucrose and a g a r) , sodium butyrate, known to influence histone deacetylation, and activated charcoal, capable of absorbing inhibitors. However, the treatments were not very useful in inducing normal germination of the embryoids.
Plant biotechnology research at the kerala agriculture university
(Kerala Agricultural University, Vellanikkara, 2005) Rajmohan, K
Protoplast isolation, culture and regeneration in Mango (Mangifera indica L.)
(Department of Horticulture, College of Agriculture, Vellayani, 2000) Ullas Mony; Rajmohan, K
Attempts were made to standardise techniques for isolation, culture and regeneration of protoplasts in mango, during 1998-1999 at the Plant Molecular Biology and Biotechnology Centre, College of Agriculture, Vellayani. Efforts were made to standardise the source of explant, optimum concentration, temperature and duration of incubation and osmolarity of the enzyme mixture. Callus induced from nucellus of immature fruits was found to be the best plant material for protoplast isolation. The cell wall digesting enzymes Cellulase 'Onozuka' R-I0 and Macerozyme R-lO at l.0 and 0.5 per cent respectively at a pH of 5.8 yielded the highest number of protoplasts (101 protoplasts per " field). The optimum time of incubation was found to be eight hours. Pre- plasmolysis of the callus was found to be not beneficial. Treatment without pre-plasmolysis and with 9.0 per cent osmolarity recorded the highest yield. Incubation temperature of 28.0 "c was found to be optimum for the best yield of protoplasts. Cell wall formation and microcalli development from the protoplast was observed when Murashige and Skoog medium with half strength of major salts, supplemented with BAP 3.0 mg r', NAA 0.1 mg r ' along with sucrose or glucose 90.0 g r ' was used. Combinations of osmoticums like sucrose 70.0 g r', mannitol 10.0 g r' and glucose 10.0 g r' as !. well as sucrose 70.0 g r', mannitol 10.0 g r' and inositol 10.0 g r' were found to be ideal for cell wall formation and micro calli development.