Available online a t www.pelagiaresearchlibrary.com Pelagia Research Library Asian Journal of Plant Science and Research, 2015, 5(2):52-62 ISSN : 2249-7412 CODEN (USA): AJPSKY Biotechnology for micropropagation and enhancing variations in Vanilla Minoo Divakaran 1, K. Nirmal Babu* 2, P.N. Ravindran 3 and K.V. Peter 4 1 Department of Botany, Providence Women’s College, Calicut - 673009, Kerala, India 2 Project Coordinator, All India Coordinated Research Project on Spices, Indian Institute of Spices Research, Calicut – 673 012, Kerala, India. 3 Consultant, Tata Tea Research Foundation, Bangalore, India. 4 Director, World Noni Research Foundation, Chennai. _____________________________________________________________________________________________ ABSTRACT Vanilla (Vanilla planifolia), a tropical orchid cultivated for its pleasant flavour, is a native of Mexico, and an introduced crop to other countries. Continuous vegetative propagation and lack of sufficient variations in the gene pool hampers crop improvement programmes. Introduction of somaclonal variation through callus cultures has been attempted to broaden the narrow genetic base. A callus induction and in vitro plant regeneration system has been optimized from both vegetative and reproductive tissues. The best results were obtained using vegetative tissues and over 80% callusing was achieved in Murashige and Skoog’s medium supplemented with 4.44µM BA and 2.68µM NAA. Callus differentiated into shoots which could be multiplied successfully in 1: 12 ratio in a combination of 4.44µM BA and 2.46µM IBA, when supplemented to MS medium. In vitro rooting was induced with an efficiency of 100% in basal MS media devoid of any growth regulators. This ability of dedifferentiated tissue to regenerate is a crucial prerequisite for future genetic transformation experiments. The protocol was successfully extended to the endangered wild species, V. aphylla, offering the potential of applying the protocol for mass multiplication as well as induction of variations in Vanilla species, in a limited time. Preliminary studies on the callus regenerants indicated variations in morphology and RAPD profiles. Key Words : Callus, callus regeneration, Vanilla planifolia , V.aphylla , micropropagation, RAPD. Abbreviations : MS : Murashige and Skoog’s medium (1962) BA : 6- benzyl adenine IBA : Indole-3-butyric acid NAA : α-naphthalene – acetic-acid RAPD : Randomly amplified polymorphic DNA _____________________________________________________________________________________________ INTRODUCTION Vanilla, Vanilla planifolia Andrews, (Syn. Vanilla fragrans Salisb .) Ames is cultivated for its pods which, when processed, yield vanilla extract making it the most economically important orchid. Three species of Vanilla are of commercial importance: V. planifolia Andrews [ V. fragrans (Salisb.) Ames], V. pompona Schiede, and V. tahitensis J. W. Moore. About 5 million pounds of beans are produced in a year and half of the world’s production is consumed by the US [1]. Vanilla is native to Mexico and Central America, but is now cultivated in other parts of the 52 Pelagia Research Library K. Nirmal Babu et al Asian J. Plant Sci. Res., 2015, 5(2):52-62 _____________________________________________________________________________ tropics mainly in Madagascar and Indonesia [2]. The history of cultivated vanilla suggests that almost the entire stock of cultivated vanilla outside Mexico is a single genetic individual (clone) propagated by vegetative cuttings with practically no variability available for crop improvement. Continuous clonal propagation leads to monoculture making vanilla susceptible to diseases and pests [3]. The introduction of new genetic material is greatly constrained by factors like its asexual propagation, the fact that the flowers are mostly ‘selfed’ and the threatened wild populations of vanilla by land pressures [4]. Lack of sufficient variability in the gene pool, threat of destructive diseases that erase vanilla plantations as well as destruction of its natural habitats, makes the search for alternate methods to introduce variability in to the gene pool, vital. Vanilla is suspected to be highly heterozygous with extensive inherent genetic variations because of its cross- pollinated nature and mitotic associations [5]. Seedling progenies of V. planifolia were developed earlier and isozyme analysis indicated the variability among them [6,7]. In the above context, utilization of biotechnological tools to introduce variability has been attempted. Reports on micropropagation concentrate on production of true-to-type plants, and few reports of callus regeneration [8, 9] are available. However protocols for induction and characterization of variations among callus regenerated plants have not been reported in vanilla. This study reports protocols to regenerate plants via callus from both vegetative and reproductive tissues, which was required to identify the most suitable physiological state for callus proliferation and regeneration and presence of variations among the regenerants. This can be utilized as a tool for increasing the variability available in vanilla germplasm since the variations were observed among few somaclones in morphology, tolerance to disease causing organisms and Randomly Amplified Polymorphic DNA (RAPD) profiles. An efficient micropropagation protocol optimized for Vanilla species, will not only be a powerful tool for commercial multiplication but will also open new avenues for future breeding programmes which were hitherto hampered. MATERIALS AND METHODS Explant source Shoots tips and nodal segments, collected from field grown vines were established in vitro , after surface sterilization with 0.1% mercuric chloride solution for 5-7 minutes and subsequent washing off with 3-4 changes of sterile distilled water. Shoot tips and subsequent nodes from such established cultures of V. planifolia were used as explants for micropropagation and callus regeneration. Pods were dipped in alcohol and flamed thrice before splitting them open longitudinally and transferring the numerous minute ex-albuminous seeds on to the culture medium for direct as well as indirect germination via callus phase. Culture medium MS medium [10] fortified with 87.64mM sucrose and gelled with 0.65% agar was used as basal medium. Growth regulators viz ., cytokinins - Benzyl adenine (BA) at 2.22 – 4.44 µM, kinetin at 2.32 - 4.64 µM and auxins - Indole- 3-butyric acid (IBA) at 2.46 - 4.92 µM, α-naphthalene acetic acid (NAA) at 2.68 - 5.37 µM were supplemented to MS medium singly or in combinations to induce multiple shoots, rooting, callus and regeneration of plants from callus. Culture conditions The pH of the medium was adjusted to 5.8 in all cases prior to autoclaving at 15 lbs pressure and 120 0C temperature for 20 min. All cultures were incubated at 25 ± 20C with a photoperiod of 14 hrs and a light intensity of 2500 lux. Hardening In vitro regenerated plantlets were transferred to polybags with sterilized sand, garden soil and vermiculite in equal proportions and kept in humid chamber for 30 days for hardening. Characterization of variability Observations on the variations in plant characters at the nursery stage among a few callus-regenerated progenies in comparison to the cultivated parent were made. Anatomical and cytological studies were made to study the regenerative pathway. 53 Pelagia Research Library K. Nirmal Babu et al Asian J. Plant Sci. Res., 2015, 5(2):52-62 _____________________________________________________________________________ Virulent strains of Phytophthora meadii and Fusarium oxysporum isolates from the Division of Crop Protection, Indian Institute of Spices Research were used for screening the somaclones for resistance to these major diseases affecting vanilla. Infection was affected by placing discs of these isolates in the axils and development of disease symptoms was recorded. DNA amplification, band separation and RAPD profiles were also developed [11. 12].Genomic DNA was isolated from shoots at the beginning of the experiment and after retrieval from callus to compare the differences. The dNTPs, Taq polymerases and other chemicals were procured from Amersham Pharmacia Biotech, Sweden. Sixteen arbitrary primers with 60% -70% GC content and no self-complementary ends, from Operon Technologies Inc. Alameda, California, were used for screening. Primers that exhibited high polymorphism and showed best readability were chosen for the study. The amplicons, named by primer and molecular mass in base pairs (bp), were scored as presence (1) or absence (0) of homologous bands and a binary matrix of the different phenotypes were put together. Paired Affinity Indices (PAI) was calculated to estimate the extent of variation between each of these somaclones. Statistical analysis All experiments were set up in a completely randomized design. Differences between means were scored with Duncan’s Multiple Range Test. Analysis of samples from each treatment was statistically evaluated by analysis of variance (ANOVA, p ≤ 0.05%) using MSTAT-C software. RESULTS Callus induction The vegetative and reproductive tissues viz ., shoot and seed explants showed varied responses in different combinations of media (Table 1). Among the auxins tried, NAA supplemented at 2.68µM and 5.37µM induced callus from shoot explants whereas no callus was produced in IBA supplemented media, which induced rooting. The callus was hard, organogenic and white in colour initially which proliferated and turned green in colour