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Open Access
Cryopreservation of chethikoduveli (Plumbago rosea L.) and assessment of genetic fidelity of regenerated plantlets using molecular markers
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Anand, Vishnu Prakash; KAU
Investigations on “Cryopreservation of Chethikoduveli (Plumbago rosea L.) and assessment of genetic fidelity of regenerated plantlets using molecular markers” were carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2011-2013. Plumbago rosea var. Agni plants were collected from AMPRS, Odakkali, Ernakulam and maintained at the Department of Plant Biotechnology, College of Agriculture, Vellayani as source of explant during the course of the study. The objectives of the present study was to standardise cryopreservation protocol by encapsulation dehydration technique for long term conservation of P. rosea and genetic fidelity assessment of plantlets recovered and regenerated from cryostorage using molecular markers. The project was carried out in two phases viz., in vitro regeneration and in vitro conservation by cryopreservation of P. rosea. In vitro regeneration protocol was optimised for P. rosea var. Agni. Various steps of in vitro regeneration viz., surface sterilization, axillary shoot proliferation, in vitro rooting and acclimatization and planting out has been standardised. For surface sterilizing, single nodal explants (3-4 cm long) were subjected to fungicide treatment with 0.1 per cent carbendazim 50 per cent W. P. (for 30 min) followed by aseptic sterilisation dip with absolute alcohol. Further, the explants were surface sterilised with 0.2 per cent mercuric chloride (for 5 min) which gave 100 per cent survival without any contamination. Enhanced release of axillary buds from single nodal explants, with maximum shoot proliferation (5.28 shoots/culture) was obtained in the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The best response (10.67 roots/culture) of in vitro rooting of plantlets was obtained in the medium, MS + NAA 1.0 mg l-1. In vitro rooted plants gave a maximum survival rate of 76 per cent and 72 per cent, when planted out in potting media consisting of red soil and coir pith (3:1) and red soil and coir pith (2:1) supplemented with VAM respectively at 25 per cent shade. In cryopreservation studies, preconditioning treatment (sucrose 0.5 M for 7 days) recorded maximum shoot proliferation (2.67 shoots/culture) when nodal segments with single axillary bud were cultured on MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1 medium. Among different encapsulation treatments, maximum shoot proliferation of (2.31 shoots/culture) was obtained in beads formed with sodium alginate 2.5 per cent and calcium chloride 100 mM, when cultured on the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. Pre-culture medium supplemented with sucrose 0.5 M for 3days gave maximum shoot proliferation (3.44 shoots/culture) when cultured on the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. A desiccation duration of 5 h at 18.13 per cent moisture level was found to be most effective giving 66.67 per cent survival and 62.50 per cent regeneration on thawing and culturing on the recovery medium MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The beads when stored in liquid nitrogen for different duration and cultured on recovery medium did not show any significant variation with respect to survival per cent. RAPD markers were tried to study the genetic fidelity of the regenerated plantlets from encapsulated and cryopreserved axillary buds. Six primers were screened and RAPD banding patterns of the cryoregenerated plantlets and control plants were compared. Polymorphism was not found with any of the primers tested. RAPD profiles of cryoregenerated plantlets were identical to those of the control. The in vitro regeneration protocol optimized included surface sterilization of single node cuttings with 0.2 per cent HgCl2 for 5 min, axillary shoot proliferation in MS medium supplemented with BA 1.5 mg l-1 and IAA 1.0 mg l-1, in vitro rooting in MS medium supplemented with NAA 1.0 mg l-1 and planting out in potting medium, red soil and coir pith (3:1). The protocol for encapsulation dehydration technique of cryopreservation was standardised for the axillary buds of P. rosea with preconditioning in semi solid MS medium supplemented with sucrose 0.5 M for 7 days, encapsulation using sodium alginate 2.5 per cent and calcium chloride 100 mM followed by pre-culture in liquid MS supplemented with sucrose 0.5 M for 3 days and 5 h dehydration (MC 18.13 %), rapid freezing in LN for at least 2 h and recovery in the medium MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The cryopreservation protocol using encapsulation-dehydration technique standardised could be utilised for long-term conservation of P. rosea.
Open Access
Isolation and Characterization of cDNA encoding chalcone synthase from flower buds of orchid Dendrobium variety sonia 17
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2007) Anjana, G R; KAU; Soni, K B
The study entitled “Isolation and characterization of cDNA encoding chalcone synthase gene from the flower buds of orchid Dendrobium variety Sonia 17” was conducted at the Department of Plant Biotechnology, Vellayani, Thiruvananthapuram during the period from 2005 to 2007 with an objective of studying the isolation and characterization of cDNA encoding chalcone synthase gene involved in anthocyanin pigmentation in orchid flower buds. . Heterologous forward and reverse primers were designed based on the gene sequences of Oryza sativa, Fragaria ananasa and Phalaneopsis orchid using primer3 software. Total RNA was isolated from immature floral tissues 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). Reverse transcription-polymerase chain reaction was carried out to study the expression of gene. The RT-PCR amplified products representing chalcone synthase (CHS) gene was eluted and purified. The product was sequenced and studied the similarity of the same using homology search. All sequenced regions were subjected to BLASTN and BLASTX similarity search. Rice chalcone synthase specific primer produced an amplified sequence of 460 bp long and showed maximum similarity to the cDNA clone 5', mRNA sequence of. flower bud of Phalaenopsis violacea and flower bud of Phalaenopsis equestris Lambda ZapII cDNA Library in BLASTN similarity search.BLASTX analysis of the sequence showed similarity to maturase K protein of Aerangis kirki. The cDNA amplified with strawberry chs specific primer showed maximum similarity to the cDNA clone 5’, mRNA sequence of Phalaenopsis violacea flower bud and flower bud of Phalaenopsis equestris in the BLASTN similarity search. BLASTX analysis of the sequence showed similarity to LFY-like protein of Serapias lingua. The results of the nucleotide to nucleotide search (BLASTN) of the cDNA of orchid, amplified using chalcone synthase specific primer from orchid showed similarity to cDNA 5', mRNA sequence of Ipomoea batatas in the BLASTN similarity search. BLASTX analysis of the sequence showed similarity to retrotransposon protein of Oryza sativa (japonica cultivar-group). The result of the sequences obtained from the study shows similarity with the genes involved in the biosynthetic pathway of Phalaenopsis orchid flower fragrance.
Open Access
Management of biodegradable plant tissue culture lab wastes through biomethanogenesis
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Abdulla Fayas, T; KAU; Rajendran, P C
Generating renewable source of energy from tissue culture laboratory waste by the process of biomethanogenesis is the focal theme of present investigation. Unlike developed countries, the developing countries are hesitant to establish more number of biotechnology/ tissue culture laboratories due to financial constraints. Easy and regular availability of biogas from TC wastes will be a boon to establish self-sustainable TC laboratory in view of present energy The biogas experimental units required for the study was designed and various treatments were employed for the biodegradation of tissue culture waste, using the methanogenic bacteria Methanobacterium ruminatium, Methanobacterium formicicum; Methanosarcina barkeri, Bactereoides ruminicola, Selenomonas ruminatium, Eubacterium tortuosum and Clostridium butyricum. Treatment involving TC waste and cow dung was also conducted for biomethanation in the present study. Quantity of gas production and its combustibility was noticed for various treatments. In bacterial treatments the quantity of gas generation was highest for Clostridium butyricum. Only treatments involving cow dung produced combustible gas. Molecular characterization, of methanogenic bacterial cultures was also done for finding the genetic similarity between them. RAPD followed by scoring . of the bands by UPGA routine showed maximum similarity between bacterial cultures of Methanobacterium ruminatium and Methanobacterium jormicicum with Methanosarcina barkeri. Physio-chemical characters like CIN ratio of the TC wastes, pH and temperature of medium and Hydraulic retention time was also observed for the various treatments. The CIN ratio of the TC wastes was found to be very low and nowhere near the optimum CIN ratio of 20-30 required for gas production. Other parameters like pH of the treatments and Hydraulic retention time was also. • noticed. The pH of the treatments involving bacterial cultures was very low, considering the normal pH of 6.8 to 7.5 required in biogas generation. The main constraints in the biogas generation were found out to be the low CIN ratio of the TC waste and the low pH of the medium. The present study indicated the possibility ofbio-gas generation from TC waste through fortification using various supplements like coconut water and coir pith which have higher CIN ratio.
Open Access
Characterisation of Pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Agatha Shiny, A; KAU; Deepu Mathew
Cowpea (Family: Fabaceae) is an important pulse cum vegetable crop of suitable for the tropical and sub-tropical regions of the world. The grain type cowpeas better tolerates the biotic and abiotic stresses against the vegetable types. Under humid conditions, vegetable types, especially the pole types are susceptible to many diseases and among them, anthracnose caused by Colletotrichum lindemuthianum (Sacc. & Magn.) Br. and Cav. is very severe. In Kerala, complete yield loss in vegetable cowpea is reported due to anthracnose during monsoons. The study entitled “Characterization of pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.” was carried out with objective to develop the protein profiles of resistant and susceptible bush and pole genotypes through SDS-PAGE analysis at different time intervals of infection and to characterize the differentially expressed proteins by MALDI-TOF followed by in-silico analyses. Two bush type varieties Pusa Komal and Kanakamony, the former reported to be highly susceptible and the latter immune to anthracnose and two pole type varieties Lola and Arimbra Local, of which the former susceptible and the latter resistant were used in the study. Pure culture of the pathogenic fungus was developed and maintained on selective medium (Neopeptone-Glucose-Agar) at the Dept. of Plant Pathology. The identity of Colletotrichum lindemuthianum has been established from the spore characteristics observed under phase contrast microscope and the pathogenicity was confirmed through artificial inoculation under controlled conditions. The pot culture experiment was conducted with 50 pots per variety. Artificial inoculation of pathogenic fungus was done and the leaf samples were collected at 0, 6, 12, 18, 24, 48, 72, 96, 120, 144,168 and 192 hours after artificial inoculation. The total protein was extracted using Tris-HCl buffer (pH-7.5), quantified using spectrophotometer and analyzed by SDS-PAGE method. The defense enzymes like peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were assayed. By artificial inoculation, disease responses for anthracnose were confirmed to be highly susceptible in Pusa Komal and Lola; highly resistant in Arimbra Local and immune in Kanakamony. Protein expression was found to be higher from the initial hours in resistant varieties whereas in susceptible varieties, the expression was reduced immediately after infection then peaked at 18hr and gradually decreased later on. Two prominent and differentially expressed protein bands at 56 kD and 14 kD were sequenced in MALDI-TOF to obtain the peptide mass fingerprint. Through in-silico analyses using Mascot server software, they were identified to be the large and small subunits of the chloroplastic enzyme RuBisCo. Thus the capability of a variety to maintain high levels of RuBisCo was found to be the deciding factor for anthracnose disease resistance. Further, protein profiles developed after purification of proteins by dialysis have clearly identified the differentially expressed band at 29 kD in the resistant varieties which is in the size range of already reported PR proteins. PO and PAL activities were proportionate to the resistance behavior, with the peak values at 18 and 24 hr after inoculation. With the results of this study, these defense enzymes are recommended as biochemical markers for identifying the resistance in the accessions. Capability to maintain higher levels of RuBisCo, PO and PAL enzymes is the characteristic of anthracnose resistant vegetable cowpeas and the future breeding programmes could be oriented in this direction
Open Access
DNA fingerprinting of selected black pepper (piper nigrum L.) varieties.
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Manjunath, Mogalayi; KAU; Nazeem, P A
Black pepper (Piper nigrum L.) famous as “Black Gold” and also known as “King of Spices” is one of the important agricultural commodities of commerce and trade in India since pre-historic period. The crop is the major source of income and employment for rural households in the predominantly pepper growing State of Kerala where more than 2.5 lakh farm families are involved in pepper cultivation. Karnataka, Tamil Nadu are the other major pepper producing States in the country. Kerala accounts for 80-90% of the total pepper production in the country. Idukki and Wynadu are the two major pepper producing districts in Kerala. Different cultivars/varieties are popular among the farmers and there phenotypic identity is not very distinct. The study entitled “DNA fingerprinting of selected black pepper (Piper nigrum L.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2009-2011. The objectives of the study were to characterize the released black pepper varieties of KAU using different molecular markers - RAPD, ISSR and SSR and to develop DNA fingerprint with which the variety could be identified and its fidelity detected. Seven black pepper varieties (Panniyur 1 to Panniyur 7) collected from Pepper Research Station, Panniyur and maintained at CPBMB, COH were used for the study. DNA extraction was done with CTAB (Rogers and Benedich, 1994) method with slight modification. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis. The PCR conditions were optimized for RAPD, ISSR and SSR assay. 30 RAPD, 34 ISSR and 29 SSR primers were screened with bulked DNA of black pepper varieties for amplification and those which gave reliable distinct banding pattern were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analysis were separated on 1.3 to 2 percent agarose gel and the amplification patterns recorded. The genomic DNA from each variety was amplified with 10 each of selected RAPD and ISSR primers and 8 SSR primer pairs. The amplification pattern was scored and depicted to develop fingerprint for each variety. The Resolving power (Rp) worked out for the different primers ranged between 7.42 to 9.42 in RAPD and 5.42 to 12.28 in ISSR analysis; indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.86 to 0.90 for RAPD analysis and it was between 0.80 and 0.89 in ISSR analysis indicating the variability among the genotypes. Distinct bands were used to develop DNA fingerprint of black pepper varieties Panniyur 1 to Panniyur 7 through RAPD, ISSR and SSR analysis. Sharing of amplicons developed for each primer with other varieties was also analyzed and demarcated with different colour codes in the fingerprints developed. Most of the amplicons were found shared among the varieties. However, the pattern of sharing was different and good enough to separate out the varieties. Combined DNA fingerprint of each variety with RAPD, ISSR and SSR data was also developed. The amplification pattern observed in RAPD, ISSR and SSR analysis was scored and analyzed for quantifying the variability among the varieties. The computer package NTSYS-Pc was used for cluster analysis. Maximum variability observed was 48 percent for the variety Panniyur 4. The varieties Panniyur 1 and Panniyur 3 having the same parentage indicated 76 percent similarity. The fingerprint developed was good enough to provide varietal identity and the analysis could reveal variability/relatedness among the seven varieties. Separate and combined fingerprints were developed for all the seven varieties through RAPD, ISSR and SSR analysis. The DNA fingerprints thus developed could be utilized for the variety registration and settling IPR issues.
Open Access
In vitro shoot regeneration and micrografting in nutmeg (Myristice fragrans houtt.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Liffey Zachariah, Antony; KAU; Valsala, P A
Nutmeg (Myristica fragrans Houtt.), is dioecious and dimorphic in branching habit with erect growing orthotropic and horizontally growing plagiotropic shoots. The long gestation period and dioecious nature of the crop causes difficulty in the production of quality planting materials of known sex. Vegetative propagation, budding and grafting with orthotropic scion material produces erect growing tree with upright tree architecture. In vegetative propagation, scarcity of orthotropic scion material is a limiting factor in large scale production of planting materials. So the programme “In vitro shoot regeneration and micrografting in nutmeg (Myristica fragrans Houtt.)” was taken up. The objectives of the study were: (1) To identify the culture conditions for multiple shoot production from orthotrops of gynoecious plants of nutmeg through enhanced release of axillary buds and shoot tip culture and (2) To standardize micrografting technique with in vitro and in vivo shoots as scion. The work was done at CPBMB, College of Horticulture, Vellanikkara. SH medium (Schenk and Hildebrandt, 1972) was found to be the best basal medium for in vitro culture establishment of nodal segments of nutmeg compared to ½ MS (Murashige and Skoog, 1962) and WPM (Lloyd and Mc Cown, 1980). Surface sterilization of nodal segments by soaking in (0.1%) carbendazim for 10 minutes followed by (0.1%) HgCl2 treatment for six minutes and sterile water wash, recorded 33% survival of cultures. The best explant for culture initiation was nodal segments. The best season for culture establishment was summer months (April-May) compared to rainy season (June- July). Loss of cultures was due to fungal contamination and necrosis of tissues. The media combination SH + Thidiazuron (TDZ) 0.03mg l-1 + Activated Charcoal (A.C.) 0.5% recorded bud expansion in 50% of the cultures within a period of nine days. Nodal segments are superior to shoot tips in culture establishment. Culture condition for culture establishment was 26 ± 20C at a light intensity of 1000 lux. The carbon source; 2% Sucrose + 1% glucose or 5% sucrose supported bud elongation and leaf expansion Refinement of culture establishment media was attempted with organic supplements; Coconut water (5, 10, 15 and 20% v/v), Casein hydrolysate (10, 25 and 50mg l-1) and Brassinolide (0.05, 0.1 and 0.2mg l-1) with nodal segments from juvenile seedlings, regenerants from coppiced trees and mature trees. In explants from juvenile seedlings and mature trees, 5 to 15%coconut water supported culture establishment. In juvenile explants, shoot elongation was also observed at 10% coconut water. Casein hydrolysate supported bud expansion in juvenile and mature tree explants at 10 to 50 mg l-1. Bud elongation and leaf expansion was observed at 25 mg l-1 concentration. Brassinolide (0.2 mg l-1) supported bud expansion in juvenile explants. The suggested media for explants from juvenile as well as coppiced trees for culture establishment of nodal segments of nutmeg is SH + 0.03 mg l-1 TDZ + 25 mg l-1 Casein hydrolysate + 2% sucrose + 1% glucose + 0.5% A.C. Casein hydrolysate concentration for explants from mature trees could be 50 mg l-1. In vitro seed germination was observed in mature seeds in presterilized bottles with water soaked cotton/ little water. Somatic embryos were formed at the cut portion of six month old seeds in the medium of ½ MS + 2% Sucrose + A.C. 0.5%. Proliferation of callus and somatic embryos was observed with the medium B5 + 0.1 mg l-1 Kin + 0.01 mg l-1 NAA + 0.01 mg l-1 GA3 + 10.0 mg l-1 Casein hydrolysate + . 0.5% A.C. The response was obtained two and a half months after inoculation. Three days old in vivo germinated seedlings did not established under in vitro condition even though surface sterilization treatment with 0.1% Emissan for thirty minutes followed by 0.1% HgCl2 for six minutes was given. Feasibility of grafting in juvenile plants was studied with epicotyl grafting and got 80% success. Grafting was done on twenty days old seedling with scion material from different seedling. In vitro epicotyl micrografting was done with in vitro raised scion and root stocks. It was also done on in vivo germinated seedlings after surface sterilization. Scion shoot of 2.5 cm length was grafted on twenty days old root stock. Graft was cultured in liquid nutrient medium and survived for two weeks. Later fungal contamination destroyed the cultures. Grafting with in vitro shoots on in vivo raised root stocks did not succeed
Open Access
Exploitation of invitro cultures of Indian Madder(Rubia cordifolia.Linn) for anticancerous compounds
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2009) Labade, Dinesh Sitaram; KAU; Asha Sankar, M
The present investigation on “Exploitation of in vitro cultures of Indian Madder (Rubia cordifolia L.) for anticancerous compounds” was carried out at the Plant Tissue Culture Laboratory of the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara and Amala Cancer Research Centre, Thrissur during the period 2006-2008. The study was undertaken with the objective to standardize the in vitro techniques for initiation and proliferation of static and suspension cultures of Rubia cordifolia and to screen the in vitro cultures for synthesis of naphthoquinone and quantify it. It was also envisaged to enhance the level of product synthesis in in vitro cultures and to assess the anticancerous activity of in vitro and in vivo extracts in terms of cytotoxicity, antioxidant and prooxidant activities in vitro. Leaf, nodal and root derived callus cultures of Rubia cordifolia were established in vitro. Explants were pre treated with the fungicide, Bavistin 2.5 per cent for 15 minutes. Surface sterilization with mercuric chloride (HgCl2) at 0.1 per cent for 1 min and 30 sec was effective for yielding healthy, contamination free cultures from nodal segments and leaves, respectively. MS medium at full strength, supplemented with NAA at 2 mg l-1 along with BA at 0.5 mg l-1 was observed ideal for initiation and proliferation of calli. The auxin synergist phloroglucinol, when supplemented to the medium, did not not yield encouraging results, with respect to callusing in the experimental species. Root derived cultures were inferior with respect to callus initiation and proliferation, registering low values for all the parameters studied. Incubating in vitro cultures under illuminated condition at 26 ± 2 C was superior to dark incubation, with respect to callus initiation and proliferation. Chloroform – methanol at 8.5 :1.5 ratio was indentified as the appropriate solvent system for detection of naphthoquinone on thin layer chromatograms in the test extracts of the experimental species, with alcoholic KOH (10 per cent) as the spray reagent. Ms medium at full strength, fortified with NAA and BA at 2.0 mg l-1 and 0.5 mg l-1 respectively, which recorded maximum naphthoquinone synthesis, was standardized as the production medium. Enhancing concentration of sucrose to 5 per cent in the production medium, did not elicit a positive response on naphthoquinone production in vitro. Reducing nitrate concentration of the production medium, to half and one fourth the original concentration, resulted in enhanced in vitro synthesis of the target compound. Supplementing the production medium with yeast extract (1 per cent and 2 per cent) as well as precursor feeding with phenyl alanine and tyrosine each at levels of 50 mg l-1, 100 mg l-1 and 150 mg l-1 exerted a favourable influence on synthesis of naphthoquonines, in vitro. Incubation in dark resulted in marginal increase in in vitro production of naphthoquinones. Incorporation of autoclaved mycelia of Pythium aphanidermeatum at levels of 2.0 per cent and 5.0 per cent resulted in enhanced in vitro production of naphthoquinone. The abiotic elicitor, salicylic acid at concentration of 10 μM and 100 μM resulted in maximum synthesis of naphthoquinones in in vitro root cultures (8.76 units g -1 calli) of Rubia cordifolia. Immobilization of test calli with sodium alginate – calcium chloride complex as well as subjecting the in vitro cultures to stress conditions, as imposed by sorbitol failed to bring about an enhancement in the in vitro production of naphthoquinones. None of the explants employed in the study induced hairy roots, when co- cultured with the Agrobacterium rhizogenes strains, MTCC 2364 and MTCC 532. Based on cell count, subculturing intervals of leafs, nodal and root derived suspension were fixed as 24, 27 and 27 days respectively with the respective packed cell volume as 0.93 per cent, 0.83 per cent and 0.80 per cent. Naphthoquinone was detected, in ex vitro and in vitro test extracts at all levels of maturity tested. Both ex vitro and in vitro root extracts exihibited maximum cytotoxicity, as revealed by the percentage of cell death on DLA and EAC cell lines as well as their IC50 values. As compared to whole plant extract, in vitro systems of the experimental species exhibited least antioxidant action. Extent of pro-oxidant activity was higher in in vitro root extract of the experimental species.
Open Access
Genetic transformation of Amorphophallus paeoniifolius (Dennst) Nicolson
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Leen Abraham, N; KAU; Makeshkumar, T
A study on Agrobacterium-mediated genetic transformation of Amorphophallus paeoniifolius (Dennst.) Nicolson was conducted at the Central Tuber Crop Research Institute, Sreekariyam, Thiruvananthapuram during 2013- 2014. Calli were initiated using petiole and leaves of in vitro plantlets of elephant foot yam cv. Gajendra in callus induction media. After four weeks of incubation, actively dividing globular, hard and creamy white calli were developed. Subculture of developed calli was repeated periodically (20 days) in CIM with an approximate size of one cm2. 15 days old calli was found to be suitable for transformation study. Calli sufficient for the transformation study was obtained after 3 months of subculture. Experiments were conducted to evaluate the sensitivity of elephant foot yam calli to different doses of antibiotics viz. geneticin, hygromycin, ticarcillin. It was observed that complete death and discoloration of the calli obtained with 20 mgl-1 geneticin and 10 mgl-1 hygromycin from sixth week treatment. Statistical analysis of sensitivity response of calli indicated that LD100 was 20 mgl-1 and 5 mgl-1 with geneticin and hygromycin respectively. Sensitivity of the calli to ticarcillin was studied and the responses are analysed with ANOVA. The lowest lethal concentration of ticarcillin was found to be 650 mgl-1. So, concentration below 650 mgl-1 can be used for the successful elimination of Agrobacterium without affecting the regeneration potential of explant. 500 mgl-1 ticarcillin used in this study was observed sufficient for the successful elimination of Agrobacterium without affecting the regeneration potential of calli. For the optimization of parameters affecting transformation, experiments were conducted for the standardisation of optimum concentration of acetosyringone, time of co cultivation, temperature of co cultivation, and suitable Agrobacterium strain. In a study conducted for standardisation of optimum concentration of acetosyringone, increasing number of transformants was obtained with increase in acetosyringone. Significantly higher GUS staining of calli (21.5896) was achieved with the addition of 400μM acetosyringone in the co cultivation media. The effect of number of days of co cultivation on transformation was compared on the GUS expression of 14-day old selected calli. Two-three days of co-cultivation was determined to be the suitable for elephant foot yam because prolonged co-cultivation period (more than three days) was found to promote overgrowth of bacteria and subsequent death of the calli. Correspondingly the transformation percentage was found to decrease with the decrease (less than two days) of co-cultivation period. Investigation of the effect of temperature during co cultivation in elephant foot yam calli revealed that temperature plays an important role in transformation efficiency. Higher temperature, 28°C was found to be optimal to support the highest transient transformation frequency in elephant foot yam and dramatic transient expression reduction occurred when temperature decreased from 22 °C to 20°C. Transformation efficiency with respect to the different strain of Agrobacterium was investigated and the results showed that maximum percent of GUS stained tissue (24.5 percent) of transformants was obtained with the strain LBA4404 with pOYE153 vector followed by AGL0/pOYE153 (14 percent) and GV3103/pCAMBIA 1305.2 (6 percent). GUS assay of transformed callus showed blue colour and confirmation was done by PCR analysis with specific primers and southern blotting. PCR amplification of the DNA of the calli survived in selection medium yielded an expected band size of 280 bp for nptII primer, two bands of size 880bp and 700bp for GUS primer, 300 bp single band for hpt primer and GUSPlus primer. No amplification was obtained for untransformed calli DNA. Nucleic acid spot hybridisation of putative transformants of elephant foot yam further confirmation of the presence of transgene in the DNA. Hybridisation with nptII probe yield spots of varying intensity for all the transformants of AGL0/pOYE153 and LBA4404/pOYE153. Whereas only 5 out of the 8 transformants of GV3103/pCAMBIA1305.2 gave positive for hpt probe and the intensity of spot was low when compared to the spots obtained with nptII probe. Southern hybridisation with DIG labelled nptII probe yield a band for positive control (pOYE153 plasmid) whereas the bands in sample lane was not observed. It is possible that the concentration of DNA (10μl) used in the blot was too low for detection of T-DNA inserts. Hybridisation with hpt probe gave a single band corresponding to the putative transformants lane, which are visible after 30 min exposure indicated that successful hybridisation of the DIG-labelled hpt probe. But the absence of band for positive control was not expected.
Open Access
Development of biodegradable films from enzymatically modified cassava starch
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Edwin, K Wilson; KAU; Sajeev, M S
Cassava forms an important food crop in the tropical countries and are rich in starch (20-40%) having desirable physico-chemical and functional properties. Starch and starch derivatives form an important constituent in biodegradable film preparation due to its renewability, abundance availability, low cost, film forming properties, high oxygen barriers, odorless, tasteless, colourless, nontoxic, low solubility, biodegradability etc. But films from the native cassava starch often possess poor physico-mechanical and hydrophobic properties. Hence modified starches by chemical, physical and enzymatic methods offer better scope for the production of biodegradable films which has got wide applications in the food packaging industry. The objectives of the present study was to find out the film forming properties of enzymatically modified cassava starch viz., liquefaction by -amylase and debranching by pullulanase enzymes added with glycerol as plasticiser. Rheological properties were measured in terms of the dynamic mechanical spectra of the film forming solutions viz., storage modulus, loss modulus, phase angle and complex viscosity. Filmogenic solutions based on -amylase was prepared with starch 5%, glycerol 20%, amylase concentration: 100, 200 and 300 μl from the stock solution (0.l ml amylase in 100 ml distilled water), temperature: 80, 85 and 90°C and time: 20, 30 and 40 min for gelatinization. For developing the pullulanase modified starch based films, the starch (5%) was incubated with pullulanase at 2, 3 and 4 units concentrations at 45, 50, 55 0C for 8, 16 and 24 h and the filmogenic solutions added with 20% glycerol were gelatinised at 90 0C for 20 min. Both the experiments were designed using response surface methodology using Box- Behnken design. The physico-mechanical, functional hygroscopic, biodegradation and storage studies of the films were carried out. The dextrose equivalent of the filmogenic solutions varied between 1.6-8.4 with the amylase and 2.2-16.0 with the pullulanase treated starch. The higher values of storage modulus, complex viscosity and low phase angle of the solution containing 153pullulanase treated starch compared to -amylase treated solutions showed that the gel formed during gelatinization of the solution is having more solid nature in their visco-elastic character. The films containing -amylase treated cassava starch showed better whiteness properties. Thickness, moisture content, tensile force, elongation at break and swelling capacity of the films containing pullulanase treated starch was higher than that of the films with -amylase treated starch. The higher solubility of the -amylase based starch films helps easy degradation of the films in the soil whereas offers poor packing ability. Pullulanase film‘s packing ability is better owing to low permeability and solubility. Though the films with both the modified starch is easily biodegradable, pullulalanase took 4 weeks for completely degrade into the soil as evidenced from the soil burial test. The microbial analysis studies showed that the soil in which -amylase treated film buried for degradation had highest bacterial, fungal and actinomycetes population than that of the soils with pululanase treated films. Considering various physical, mechanical and functional properties, the pullulanase modified starch offers better scope for the production of biodegradable packing materials.

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