Intraspecies Diversity of Ricinus Communis L. in Bangladesh and Their in Vitro Conservation

Abstract

The castor oil plant, Ricinus communis, is a species of flowering plant in the spurge family, Euphorbiaceae and it belongs to a monotypic genus, Ricinus. Castor seed is the source of castor oil, which has a wide variety of uses. The seeds contain between 40% and 60% oil that is rich in triglycerides, mainly ricinolein. The seed contains ricin, a toxin, which is also present in lower concentrations throughout the plant. The castor oil plant can vary greatly in its growth habit and appearance. In Bangladesh, castor plant growing all over the country without any special care. In the present aspect of climate change castor plant holds a great promise for producing environmentally friendly biofuel as a source of green energy . In respect of research, castor provides a virgin field in Bangladesh as it is not a mandatory crop plant of any national research institute in the country. In the beginning of our research work we have made a survey on the existing germplasms of castor plant over the country in order to preserve the land races with their particular gene pools for breeding purposes. A number of varieties of castor plant found to grow here and there over the country with distinct morphological variation and peculiarities. In the present investigation, a thorough and extensive survey was made and different castor cultivars were collected from different corners of the country and established a germplasm bank in the Institute of Biological Sciences, Rajshahi University, Bangladesh. Under present study the diversity of castor was studied both on phenotypic and genotypic perspectives. Under the purview of morphological study a number of 28 characters of castor plant were analyzed during its growth period. An attempt was taken to assess the genotypic influences on the nature and extent of variation in different morphological characters of different castor germplasms collected from different corners of the country. The characters were categorized under this study depending on their morphological nature and they were (1) plant height (cm), (2) stem diameter (cm), (3) branch number, (4) branch length (cm), (5) node number, (6) internodal distance (cm), (7) leaf length (cm), (8) leaf breadth (cm), (9) leaf area (cm²), (10) lobes number, (11) petiole length (cm), (12) petiole diameter (cm), (13) root length (cm), (14) lateral root number, (15) lateral root length (cm),(16) inflorescence number, (17) male flower’s region (cm), (18) female flower’s region (cm), (19) number of male flower, (20) number of female flower, (21) fruit number, (22) thorn number, (23) flowering time (days). (24) Seed number, (25) seed length (cm), (26) seed breadth (cm), (27) seed weight (gm), and (28) hundred seeds weight (gm). The results indicated the presence of substantial amount of genetic variability in the collected gerplasms and there exists ample scope for selection. The result also showed that phenotypic coefficient of variation (PCV) in general was higher than genotypic coefficient of variation (GCV) and very high estimate of broad sense heritability (more than 90%) were observed for all characters except number of seeds per fruit. Distinct variation was very obvious even in visual observation on stems, leaves, petioles, inflorescence and flowers. The present study confirmed that the castor cultivars were significantly different in regards of vegetative growth and yield component parameters. The characters plant height, branch number, stem diameter, node number, internodal distance, length and breadth of leaves, leaf area and length of root were recognized as the most important characters in castor plant for evaluating their morphological characterization and they hold the merit of receiving greater importance and priority in selection under any breeding programme for the improvement of castor genotypes Following the study, six distinct cultivars were recognized as cultivar shabje, cultivar shadatae, cultivar roktima, cultivar lalchay, cultivar badami and cultivar dhusar based on local identity particulars. An attempt was taken to confirm the molecular diversity and phylogenic relationship among six cultivars of castor by Random Amplified Polymorphic DNA (RAPD) markers. The RAPD provides a quick and efficient technique for DNA sequence-based polymorphisms at a very large number of loci. DNA fingerprinting of six cultivars of castor were performed using the seven RAPD markers in the present investigation. The seven primers used in our RAPD experiments were: OPA-8, OPA-9, OPA-10, OPB-17, OPC-17, OPD-3, OPE-6. RAPD experiment which showed promising result for the proper identification of the varieties using genetic marker. Among the seven primers used for DNA fingerprinting in six cultivars of castor 3 RAPD primers successfully generated distinct banding pattern. Three RAPD primers generated 50 bands and average 16.66 from the six cultivars of castor and the primers OPA-9, OPA-10 and OPB-17 produced 26, 17 and 7 bands respectively. Differential banding pattern was observed among four cultivars of castor. The result confirmed molecular diversity existed among the castor cultivars and considering the observed phenotypic and genotypic differences the collected germplasms of castor can be denoted as six distinct genotypes or cultivars. Another potential avenue of present investigation was to establish protocols for in vitro propagation of six cultivars of castor. Cotyledonary node and shoot tip segments were used as explants for proliferation of axillary shoots in MS, MMS1 and MMS2 medium with different concentrations (0.5-5.0 mg/L) of cytokinins viz. BAP, Kn and 2ip. Cotyledonary node explant showed better performance while MMS1 was proved as the best medium and 2.0 mg/L BAP proved as the best hormonal supplement giving 100% direct shoot regeneration in this media formulation. Callus induction was studied in six cultivars of castor from hypocotyl, internode and shoot tip explants in different concentrations of BAP (0.5-5.0 mg/L) with different concentrations (0.1-1.0 mg/L) of NAA, 2,4-D and IAA. Hypocotyl was proved as the best explant for callus induction producing 100% callus formation in suitable media formulation. On the other hand BAP 2.0 mg/L with 0.5 mg/L NAA proved the best formulation for callus induction. Nature of callus also varied in shape and colour, a very wide range of diversity existed in castor cultivars in producing whitish green, green compact, pinkish brown, pinkish nodular callus in their respective suitable media compositions. MMS1 medium supplemented with 1.0 mg/L BAP + 0.2 mg/L NAA + 0.8 mg/L TDZ was recognized as the best media formulation for indirect organogenesis of cultivar shabje, cultivar roktima and cultivar lalchay among the six cultivars of castor. MMS2 medium was identified as the best media formulation for root induction for castor cultivars. Particularly for root induction three auxins IBA, NAA and IAA were used in different concentrations (0.1 - 1.0 mg/L) and among them, 0.2 mg/L IBA was proved to be best for root formation. Rooting performance in castor cultivars were successfully enhanced by the application of additives to the artificial medium. Different concentrations of AgNO3 and activated charcoal were used with 0.2 mg/L IBA but 0.2 mg/L IBA + 0.6 mg/L AgNO3 showed best performance for root elongation of six cultivars of castor. Investigation was carried out for cell suspension culture of six cultivars of castor. Free cells isolated from friable embryogenic calli drived from hypocotyl explant of six cultivars of castor were subjected to grow under different concentrations and combinations of auxins and cytokinin (NAA, 2,4-D and BAP) on cell suspension culture. All the cultivars of castor produced successfully cell suspension culture in liquid medium from their respective friable callus under continuous agitation in dark. The peak period of cell growth was observed within 4th - 6th days of suspension culture and the highest weight of the cells and cell aggregates of all cultivars were obtained on the 14th days of suspension culture in modified liquid MS medium supplemented with 2.0 mg/L BAP + 0.3 mg/L NAA accompanied with 1.0 mg/L biotin and 1.0 mg/L glutamine. The cultivar shabje showed the maximum cell weight which was 0.143±0.13 gm while the maximum cell weight of other cultivars viz. shadatae, roktima, lalchay, badami and dhusar were 0.133±0.12 gm, 0.138±0.13 gm, 0.135±0.13 gm, 0.126±0.12 gm and 0.129±0.12 gm respectively. For callus induction from the isolated single cells modified MS medium was used supplemented with 2.0 mg/L BAP with five different concentrations of NAA (0.1, 0.2, 0.5, 0.8 and 1.0 mg/L). Callus induction was observed in all BAP and NAA formulations. Growth regulator played very important role in callus induction from single cell culture in semisolid artificial medium and 2.0 mg/L BAP with 0.5 mg/L NAA showed the best performance in callus induction from isolated single cell culture. Cell extracts of six cultivars of castor were taken after different periods of cell suspension culture and they were tested against five gram positive and six gram negative bacteria at the concentration of 25 µl/disc and they were compared with standard antibiotic ciprofloxacin 5 µg/disc. The cell extracts prepared for antibacterial test were taken from cell suspension cultures growing under different time regimes viz, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days and 16 days. All the castor cultivars showed antibacterial activity performing in different grades under different time periods. Over all the castor cultivars, cell extracts taken from 6 days to 16 days culture were found 100% effective against all the bacteria tested but the extracts taken from 4 days culture failed to show antibacterial activity against all the bacteria tested. But, the extract taken after 14 days from cell suspension culture showed the best antibacterial activities. Among the six cultivars of castor, comparatively cultivar roktima showed the highest level of lethality against all bacteria tested. The experimental results indicated that the castor cultivars also holds the diversity in the levels of their toxicity and mode of action in antibacterial activities.