PhD

Anthurium is the largest genus in the family Araceae, encompassing more than 800 species, native to tropical America, from Mexico, Costa Rica, Cuba to Brazil and Argentina. The spadix is composed of a multitude of flowers, which are perfect having two-carpelled ovary and four anthers. A few commercially grown plants are classified as "rat tail" anthuriums as their inflorescences have a long spadix and a small non-descript spathe. Anthuriums with colourful inflorescences have been grown for cut flowers. With the introduction of compact interspecific hybrids through breeding and the selection of somaclonal variants, the new commercially available types were developed.
Propagation is not easy for anthuriums and is considered a long-term crop which will take long time for the propagator to multiply. In light of afore said views, an attempt was made to standardize in vitro multiplication and DNA fingerprinting of selected hybrids and their parents in Anthurium andreanum Linden.
The explants after standardizing for the surface sterilization and survival were cultured on selected media with different hormone concentrations to get maximum callus induction. For callus induction the culture flasks were kept in dark at 25˚C and subcultured every third week. Calli were transferred to regeneration medium and embryogenic calli induction medium. Regenerants were selected and placed in rooting medium; further hardened and transferred to the field condition.

Preconditioned embryos were suspended in calcium free half strength NN medium supplemented with 1.5 per cent sodium alginate and 0.5 M sucrose. This mixture was dispensed with a micropipette into 0.1 M calcium chloride. Twenty minutes after encapsulation beads were pre cultured on modified half strength NN liquid medium supplemented with 0.75 M sucrose and three per cent DMSO into 100 ml Erlenmeyer flasks for one day without agitation. Beads were then transferred to fresh medium of same composition and incubated in darkness at 4ºC for three days. Beads were desiccated in a sterile laminar air flow chamber. Dehydrated beads were transferred to 4 ml cryo vials and stored at – 80˚C. On rewarming over a water bath at 25˚C, the beads were transferred to culture medium for germination.

Surface disinfection treatments were standardized for the different explants, irrespective of the explants and varieties and double sterilization was found to be effective. Among the explants, the highest number of sterile cultures was observed in double sterilization, followed by the treatment with 70 per cent Ethyl alcohol for 20 minutes. Majority of the contamination found in the cultures was due to the presence of systemic infection of Xanthomonas compestris pv dieffenbachiae. This directly influences the percentage of contamination occurred in the culturing condition and the size of explants which also play a major role in creating the bacterial contamination. Candle explants were found to exhibit more systemic infections than other explants and seed explants were found to be free from systemic infections. Leaf explants are highly vulnerable to exhibit systemic infections and are more sensitive; unable to recover even after treatments with antibiotics. The callus cultures exhibiting systemic infections can be recovered by kanamycin 50 mg per litre containing multiplication medium.

In most studies of in vitro culture of anthurium, MS medium has been used. In the present study also, it was observed that Nitsch and Nitsch medium was better than MS medium for multiple shoot induction. Nitsch and Nitsch medium is especially suitable for morphogenesis, meristem culture and regeneration.

As the genotype showed different nutrient requirements for their survival and growth, the present investigation was planned to standardize the media by screening with modified MS, half strength MS, modified NN and half strength NN medium. Modified NN with activated charcoal and coconut water showed better response. Half strength NN with coconut water and activated charcoal, modified MS with activated charcoal and coconut water and half strength MS activated charcoal and coconut water were also found to support the explants without hindering the survival. Addition of inositol and glycine along with folic acid was found to be essential but the presence of small amount was inefficient. In the present investigation no callus initiation was observed when inositol was reduced to half of the reported quantity.

Various treatments were tried for callus multiplication. The maximum fresh weight of callus 4.2458 g was observed in PR X DT inoculated in NN medium with major nutrients at normal strength followed by 4.1325 g in OG X DT for the same composition in NN medium. From the economic point of view NN medium can be recommended for callus multiplication.

Among the treatments, combination of 2, 4-D and zeatin was found to be the best. It stimulates callus formation and strongly antagonizes organized development. The low auxin requirement may be due to the high potency of the auxin which was used for callus initiation. The young developing leaf may be a rich source of endogenous auxins due to which lower exogenous application is required.

Irrespective of the source of explant all the callus cultures were able to be converted into plantlets by redifferentiation. The number of days taken for regeneration ranges from 55.5 to 82. This variation is due to the varietal difference and difference in hormonal effect. Modified NN with activated charcoal and coconut water along with 22.2 μM BA, 11.42 μM IAA and 4.09 μM biotin was found to produce regenerants.

Each genotype is varying with the response to change in media composition in producing somatic embryogenesis. The treatments with MS and modified MS media were found to be insignificant when compared with NN and modified NN media. Among these modified NN was found to be the best one.

Within two weeks on embryo development medium, the globular embryos developed a bipolar shape. Embryos at this stage were comprised of cells larger than those at the globular stage. Bipolar embryos had an extended upper region that formed the cotyledon and the epicotyl, and a lower region that formed the radicle.The main difference between the mature embryos of monocotyledons in vitro and in vivo is the absence or presence of suspensor. The presence of single cotyledon which is the terminal structure and the shoot initials present at the sides or hidden creating a heart shape. When the cotyledon starts growing the embryo will have a single cotyledon at the terminal end which is some what cylindrical in shape.

In anthurium tissue culture, no special rooting treatments were needed and the shoots developed in vitro were found to develop roots spontaneously even in the absence of additional growth hormones in the supporting medium. The spontaneous root formation was not due to the carry over effect of the hormones supplied in the previous cultures for shoot formation. Irrespective of the supporting medium the shoots were able to form roots even in sterile sand supplied with sterilized compost materials. For in vitro studies Completely Randomized Block Design (CRD) was followed for statistical analysis wherever necessary.

Molecular characterization of twelve hybrids and their parents were carried out with RAPD using AP-PCR. Young leaf samples from each genotype were collected for DNA isolation.Young copper coloured leaf tissues were used immediately after collection for DNA extraction. Leaf samples were pre-chilled at -80ºC for half an hour with pestle and mortar and then pulverized in liquid nitrogen by rapid grinding to a fine powder.The frozen powder was used to extract the total genomic DNA using CTAB extraction buffer. The purity of the DNA was analysed by running in 0.8 per cent agarose gel with 1 X TAE buffer.

The optimized PCR mixture with 50ng of template DNA for a final volume of 20μl was used in thermal cycling in a PCR machine. The amplified products were run in 1.6 per cent agarose gel with 1X TAE (Tris buffer, Glacial acetic acid and EDTA pH 8.0) buffer.

A total of 114 AP-PCR bands were generated by the 25 primers, of which 74.56 per cent were polymorphic (88 bands) and 26 were monomorphic. Ten primers showed high level of polymorphism out of which seven were selected.Seven promising primers were identified for AP-PCR analysis based on performance in DNA amplification, production of highest number of polymorphic bands as well as intense bands and reproducibility viz. OPA 10, OPB15, OPA13, OPB20, OPB6, OPB8 and OPB18 primers were found to produce polymorphism in Anthurium andreanum Linden.A total of 50 scorable bands (average of 7.143 bands per primer were generated by the selected seven primers of which only 8 were monomorphic and the rest were polymorphic. The number of bands ranged form 4 to 11 with an average of 7.143 per primer. The reproducible bands were scored for their presence (1) or absence (0) for all the hybrids and parents. A genetic similarity matrix was constructed using Jaccards’s similarity co-efficient methods.

From the cluster analysis based on the dendrogram, TR X MW was found to be extreamly different from the other accessions and its own parents showing the significance of hybridization. The hybrids like OO X KR, PR X DT, OG X DT, FK X LR and PR X MW are not closely related to either of their parents and hybrids were distinguished from others. Some hybrids like LJ X W and PR X MW; PR X LR and FK X DT shows 30 to 39 per cent similarity. This shows there is considerable variability among the genotypes selected and can be further utilized for crop improvement. Confirming that, they were quite different from the other hybrids and varieties.

Pair wise genetic distances based on RAPD [(AP-PCR) (Nei and Li Genetic Distance GDNL)] genetic distance co-efficient values for twelve varieties and twelve hybrids ranged from 0.1935 to 0.7037 indicating the wider diversity. The AP-PCR profiles show the relatedness and diversity of the hybrids and varieties. The bands were found within 1.5kb from 100bp. Most of the bands were concentrated between 300bp and 1200bp.