Biotechnology for micropropagation and enhancing variations in Vanilla

Transcription

1 Available online at Asian Journal of Plant Siene and Researh, 5, 5():56 ISSN : 97 CODEN (USA): AJPSKY Biotehnology for miropropagation and enhaning variations in Vanilla Minoo Divakaran, K. Nirmal Babu*, P.N. Ravindran 3 and K.V. Peter Department of Botany, Providene Women s College, Caliut 6739, Kerala, India Projet Coordinator, All India Coordinated Researh Projet on Spies, Indian Institute of Spies Researh, Caliut 673, Kerala, India. 3 Consultant, Tata Tea Researh Foundation, Bangalore, India. Diretor, World Noni Researh Foundation, Chennai. ABSTRACT Vanilla (Vanilla planifolia), a tropial orhid ultivated for its pleasant flavour, is a native of Mexio, and an introdued rop to other ountries. Continuous vegetative propagation and lak of suffiient variations in the gene pool hampers rop improvement programmes. Introdution of somalonal variation through allus ultures has been attempted to broaden the narrow geneti base. A allus indution and in vitro plant regeneration system has been optimized from both vegetative and reprodutive tissues. The best results were obtained using vegetative tissues and over 8% allusing was ahieved in Murashige and Skoog s medium supplemented with.µm BA and.68µm NAA. Callus differentiated into shoots whih ould be multiplied suessfully in : ratio in a ombination of.µm BA and.6µm IBA, when supplemented to MS medium. In vitro rooting was indued with an effiieny of % in basal MS media devoid of any growth regulators. This ability of dedifferentiated tissue to regenerate is a ruial prerequisite for future geneti transformation experiments. The protool was suessfully extended to the endangered wild speies, V. aphylla, offering the potential of applying the protool for mass multipliation as well as indution of variations in Vanilla speies, in a limited time. Preliminary studies on the allus regenerants indiated variations in morphology and RAPD profiles. Key Words: Callus, allus regeneration, Vanilla planifolia, V.aphylla, miropropagation, RAPD. Abbreviations : MS : Murashige and Skoog s medium (96) BA : 6 benzyl adenine IBA : Indole3butyri aid NAA : αnaphthalene aetiaid RAPD : Randomly amplified polymorphi DNA INTRODUCTION Vanilla, Vanilla planifolia Andrews, (Syn. Vanilla fragrans Salisb.) Ames is ultivated for its pods whih, when proessed, yield vanilla extrat making it the most eonomially important orhid. Three speies of Vanilla are of ommerial importane: V. planifolia Andrews [V. fragrans (Salisb.) Ames], V. pompona Shiede, and V. tahitensis J. W. Moore. About 5 million pounds of beans are produed in a year and half of the world s prodution is onsumed by the US []. Vanilla is native to Mexio and Central Ameria, but is now ultivated in other parts of the 5

2 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5():56 tropis mainly in Madagasar and Indonesia []. The history of ultivated vanilla suggests that almost the entire stok of ultivated vanilla outside Mexio is a single geneti individual (lone) propagated by vegetative uttings with pratially no variability available for rop improvement. Continuous lonal propagation leads to monoulture making vanilla suseptible to diseases and pests [3]. The introdution of new geneti material is greatly onstrained by fators like its asexual propagation, the fat that the flowers are mostly selfed and the threatened wild populations of vanilla by land pressures []. Lak of suffiient variability in the gene pool, threat of destrutive diseases that erase vanilla plantations as well as destrution of its natural habitats, makes the searh for alternate methods to introdue variability in to the gene pool, vital. Vanilla is suspeted to be highly heterozygous with extensive inherent geneti variations beause of its rosspollinated nature and mitoti assoiations [5]. Seedling progenies of V. planifolia were developed earlier and isozyme analysis indiated the variability among them [6,7]. In the above ontext, utilization of biotehnologial tools to introdue variability has been attempted. Reports on miropropagation onentrate on prodution of truetotype plants, and few reports of allus regeneration [8, 9] are available. However protools for indution and haraterization of variations among allus regenerated plants have not been reported in vanilla. This study reports protools to regenerate plants via allus from both vegetative and reprodutive tissues, whih was required to identify the most suitable physiologial state for allus proliferation and regeneration and presene of variations among the regenerants. This an be utilized as a tool for inreasing the variability available in vanilla germplasm sine the variations were observed among few somalones in morphology, tolerane to disease ausing organisms and Randomly Amplified Polymorphi DNA (RAPD) profiles. An effiient miropropagation protool optimized for Vanilla speies, will not only be a powerful tool for ommerial multipliation but will also open new avenues for future breeding programmes whih were hitherto hampered. MATERIALS AND METHODS Explant soure Shoots tips and nodal segments, olleted from field grown vines were established in vitro, after surfae sterilization with.% meruri hloride solution for 57 minutes and subsequent washing off with 3 hanges of sterile distilled water. Shoot tips and subsequent nodes from suh established ultures of V. planifolia were used as explants for miropropagation and allus regeneration. Pods were dipped in alohol and flamed thrie before splitting them open longitudinally and transferring the numerous minute exalbuminous seeds on to the ulture medium for diret as well as indiret germination via allus phase. Culture medium MS medium [] fortified with 87.6mM surose and gelled with.65% agar was used as basal medium. Growth regulators viz., ytokinins Benzyl adenine (BA) at.. µm, kinetin at.3.6 µm and auxins Indole 3butyri aid (IBA) at.6.9 µm, αnaphthalene aeti aid (NAA) at µm were supplemented to MS medium singly or in ombinations to indue multiple shoots, rooting, allus and regeneration of plants from allus. Culture onditions The ph of the medium was adjusted to 5.8 in all ases prior to autolaving at 5 lbs pressure and C temperature for min. All ultures were inubated at 5± C with a photoperiod of hrs and a light intensity of 5 lux. Hardening In vitro regenerated plantlets were transferred to polybags with sterilized sand, garden soil and vermiulite in equal proportions and kept in humid hamber for 3 days for hardening. Charaterization of variability Observations on the variations in plant haraters at the nursery stage among a few allusregenerated progenies in omparison to the ultivated parent were made. Anatomial and ytologial studies were made to study the regenerative pathway. 53

3 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5():56 Virulent strains of Phytophthora meadii and Fusarium oxysporum isolates from the Division of Crop Protetion, Indian Institute of Spies Researh were used for sreening the somalones for resistane to these major diseases affeting vanilla. Infetion was affeted by plaing diss of these isolates in the axils and development of disease symptoms was reorded. DNA amplifiation, band separation and RAPD profiles were also developed [. ].Genomi DNA was isolated from shoots at the beginning of the experiment and after retrieval from allus to ompare the differenes. The dntps, Taq polymerases and other hemials were proured from Amersham Pharmaia Bioteh, Sweden. Sixteen arbitrary primers with 6% 7% GC ontent and no selfomplementary ends, from Operon Tehnologies In. Alameda, California, were used for sreening. Primers that exhibited high polymorphism and showed best readability were hosen for the study. The amplions, named by primer and moleular mass in base pairs (bp), were sored as presene () or absene () of homologous bands and a binary matrix of the different phenotypes were put together. Paired Affinity Indies (PAI) was alulated to estimate the extent of variation between eah of these somalones. Statistial analysis All experiments were set up in a ompletely randomized design. Differenes between means were sored with Dunan s Multiple Range Test. Analysis of samples from eah treatment was statistially evaluated by analysis of variane (ANOVA, p 5%) using MSTATC software. RESULTS Callus indution The vegetative and reprodutive tissues viz., shoot and seed explants showed varied responses in different ombinations of media (Table ). Among the auxins tried, NAA supplemented at.68µm and 5.37µM indued allus from shoot explants whereas no allus was produed in IBA supplemented media, whih indued rooting. The allus was hard, organogeni and white in olour initially whih proliferated and turned green in olour on transfer to the same medium and small shoot buds initiated after 5 to 6 days in ulture. In media supplemented with ytokinins, BA and kinetin, at.µm and.µm, singly, only multiple shoots were produed and no allus indution was notied. When auxins and ytokinins were tried in ombination, Interation of.68µm NAA and.µm BA gave the most favourable response. In this medium allus was indued at the base of the shoot explants and in root tip explants (Fig. a) after 3days of inubation. Further subulture into MS media with.µm BA and.68µm NAA, regeneration of shoot buds ourred from these alli (Fig. b). About 5 shoot primordia were formed from eah mass and eah of them was apable of regenerating into individual shoot and plantlet, whih were studied in omparison with parent (ontrol), Vanilla planifolia. Indution of allus from protoorms The initial stages of seed germination were typial of most orhids, involving swelling of the embryo followed by rupturing of the seed testa and emergene of protoorm. In treatments with BA, most of the protoorms remained the same with the sale like leaf primordial developing into shoots whereas treatments with auxin supplements showed gradual disorganization of the protoorms into allus (Fig. ). In medium supplemented with NAA, it was found that sudden disorganization of the protoorms ourred to form a allus tissue, whih was of yellowish olour initially. On ulture in medium supplemented with BA and NAA, regeneration of protoorm like bodies (PLBs) ourred from these alli (Fig. d). About 5 PLBs were formed from eah mass and eah of them were apable of regenerating into individual shoots and plantlets. Media supplemented with BA alone, prevented formation of allus and seeds germinated diretly into plantlets (Table ). Plant regeneration from allus Among the various media ombinations tried for allus indution and regeneration, the most suitable medium was MS medium fortified with.68µm NAA and.µm BA (Fig. 3) in whih 75% of the ultures developed allus and an average of plants ould be regenerated from them (Table ). The regenerated plants indued from allus tissue produed roots in growth regulator free medium, in ontrast to the BA supplemented media. NAA supplemented medium was found to indue thik fleshy velamen like roots, whih were of a disadvantage in the hardening stages [7, 3]. 5

4 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5():56 The somalones regenerated also exhibited signifiant variations in vitro (Fig. ), in the rate of multiple shoots formation and also leaf size and internodal distane (Table 3). Miropropagation In order to generate suffiient material for further studies, shoot tips and nodal explants of V. planifolia, and the regenerants were initiated for bud break in MS medium with.µm BA and the sprouting shoots. Was used for miropropagation experiments. Of the ombinations tested, BA when used alone or in ombination with IBA indued multiple shoots. Interation of.µm BA and.6 µm IBA enhaned the proliferation of shoots (Table ). In this medium, an average of 5 multiple shoots were indued in 6 days after establishment, in more than 9% of the ultures (Fig. b) and was onduive for proliferation as well as elongation of shoots. Nodal segments gave better response with a mean of 5 shoots per ulture ompared to the shoot tips (mean of 7 shoots per ulture), due to absene of apial dominane. In the present study kinetin had no effet of the indution of multiple shoots and gave rise to single shoots whereas NAA supplemented media led to indution of allusy roots or velamen roots whih were unfavorable during transplantation and hardening. When BA was added singly, it indued multiple shoots whih had very short internodes (i.e., no elongation) and total absene of roots. These problems were overome by supplementing a ombination of auxin and ytokinin. On transfer to growth regulator free MS medium, the shoots elongated, developed good root system, and ould be harvested for further experimental studies, proliferation or establishment in soil. This method was effetively used throughout the study to multiply different genotypes / morphotypes generated. Hardening and planting out The rooted shoots were arefully removed from ulture vessels, washed thoroughly to remove any traes of nutrient medium, treated with.% Indofil and transferred to polybags ontaining potting mixture (sand, soil and vermiulite). They were hardened for 3 days under ontrolled onditions, before transferring them to pots. The plantlets hardened with over 8% suess and the plants were field planted (Fig. g) with Glyridia standards for proper shade and support. Charaterization of allus regenerated plants Morphologial haraters: The allus regenerated progenies exhibited high variations among themselves with plant and leaf haraters, shapes varying from ovate to laneolate and internodal length. S showed highest plant growth of 9.6 m after six months of planting out (Table 3). In general, S was haraterized by good vegetative growth in terms of plant height (8. m), leaf size (length of 3. m and breadth of.5 m) and internodal length (.55 m), when ompared to V. planifolia (plant height of.3 m). S3 was haraterized by omparatively shorter internodes (Fig. g), whih an be of immense agronomi importane sine the number of nodes per meter of the vine in inreased thereby inreasing the number of infloresenes obtained at every node, making a notable yield impat per meter of the vine. A few attempts to index these progenies ytologially were made and one of the allus regenerated progenies, V 8. did not show any ell with the normal somati ompliment of n = 3. It instead had variations in hromosome numbers viz., n=, 6, 8 and aberrations like lagging and lumping of hromosomes were observed, indiative of the high degree of variations existing in these plants (Fig. i). Reports of mitoti assoiations in vanilla and its potential in developing ytotypes have been disussed [5]. Anatomial hanges observed were similar for all genotypes. BA indued proliferation of vasular tissue and allus parenhyma ells within the allus. The differentiating meristemati tissue was haraterized by ells filled with dense ytoplasm and prominent nulei. Sreening of somalones for disease resistane The somalones were sreened against the infetion of Phytophthora meadii and Fusarium oxysporum, the ausal agents of foot rot and wilt diseases in vanilla. The disease progression was manifested as browning and water soaked pathes at the axil spreading out to either side of the internode. It was observed that the somalones exhibited high 55

5 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5():56 degree of variability in tolerane to the pathogens, the spread ranging from no infetion, superfiial infetion to omplete infetion, when V.planifolia was suseptible. In the absene of resistant lines in Vanilla planifolia, further studies for identifiation of tolerant line would be important for development of disease resistant varieties. RAPD profiles of allus regenerated progenies of Vanilla The few regenerated somalones were seleted based on preliminary morphologial differenes observed at nursery stage. Operon primers OPB, OPA and OPD3 gave good polymorphism (Fig. h) between the progenies. The somalones tested are variable (7% to 7.5%) when ompared to eah other, indiative of somalonal variation as the possible ause for this variation. They showed % dissimilarity with its parent, V.planifolia (Fig. 5). A few seedling progenies were analyzed with their respetive allus regenerated plants, a variability ranging from 5 3% was observed. V7. and its root allus regenerated progeny V7.R showed 85% similarity, 7% similarity in V79 and its allus regenerated progeny V79., 7.5% similarity among V and its allus regenerated progeny V. and 77.5% similarity among V and its allus regenerated progeny V.. RAPD profiles from shoots regenerated from allus were ompared to ontrol and few variations were deteted. The number of polymorphi bands ranged from.5 to % and the math perentage between mother miroutting and newly sprouted allus regenerating shoots were 76.5 %, sharing values of similarity between 6 to 87.5%. DISCUSSION Callus an be used as target tissues for geneti transformation experiments whih, is the next step to bring in desirable traits into this ommerially important speies. Sine the regeneration protool standardized in this system is simple, and of high frequeny, it an shorten the length of suh experiments. Though NAA alone indued allus, they ould not be direted to regenerate in to plants. Low ytokinins have been reported to enhane allusing []. Addition of low ytokinin espeially BA, was essential in dedifferentiation of the explant into regenerative allus in the present study. Regeneration potential of vanilla allus here is omparable to miropropagation indiating that hanges in auxin supplementation do not influene the regenerative potential of the tissue. Unfavorable ratio between total ell population and ells apable of stably expressing the foreign genes has been the most serious hindrane to monootyledonous geneti manipulation [5]. Fators affeting allus regeneration inluded explant and tissue type. Vegetative tissues produed longer shoots whih were harvestable and diretly utilized for further studies however shoots regenerated from reprodutive tissue needed to be exised and ultured for further elongation before they ould be multiplied (Fig. ). Using allus tissue, from embryos/ reprodutive tissues does put forth a onern sine Vanilla is basially a ross pollinated rop in its native ountry. Hene its use as target tissue for transformation may not be able to reveal whether the variations were inherent seed variability or transgeni effet. Embryogeni alli with regeneration potential were observed in ulture of early immature embryos (3 DAP) and 7 DAP and shoot base of young seedlings of Stenotaphrum seundatum (Li et al. 6). Suessful plant regeneration from allus has been reported in many orhids viz., Cymbidium [6], Phalaenopsis [7], Paphiopedilium [8] and these morphogenesis systems are being used in some to explore the ontrol proess on reduing juvenility and preoious flowering in vitro. Earlier studies in Vanilla planifolia [8] indiated that shoot setions from first nodes has best allus initiation and proliferation rates and allus from ytokinin ontaining Linsmaeir and Skoog medium and its subsequent regeneration from stem and leaf setions [9]. The potential of protoorm derived alli to regenerate PLBs in developing a allus assoiated miropropagation system in Geodorum densiflorum has been disussed by [3]. The present protool offers the possibility of its appliation to both ultivated and wild speies of Vanilla. Miropropagation of vanilla has been reported earlier [9,, 7]. In vitro transformation of root meristem from aerial roots into shoot and plantlets in V. planifolia as an effiient method for lonal propagation has been reported []. Miropropagation and in vitro onservation of five diverse speies of vanilla viz., V.planifolia, V.andamania, V. wightiana, V.aphylla and V. pilifera have been reported [] as an effetive alternative to onserve this important speies in laboratories or in vitro gene banks. In the present study, BA alone suppressed rooting while induing multiple shoots and IBA/NAA alone suppressed multiple shoot indution. BA is known to indue multiple shoots in a wide range of rops. However, its addition in 56

6 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5():56 onentration higher than. µm led to fasiated shoots. This may be a feedbak inhibition of multipliation at higher onentration of BA due to whih proliferation at vigorous rate is promoted at lower onentration of BA as has been observed in bamboo [3]. Inhibitory effets of many ytokinins on seedling development have been reported in angiospermi plants [, 5]. Other stimuli to hange the environmental onditions have been reported to improve shoot regenerative potential. Use of phenylaeti aid as primary fator to indue multiple shoots from axillary buds of V. planifolia and silver nitrate at µm for shoot and root formation. me genotypes performed better indiating that a proess of seletion and loning an identify and multiply lines of faster multiplying lines whih may suitably meet the demand for planting materials without hampering the natural resoures, have been reported [6, 7]. Reports on variability among allus regenerated plants in vanilla are not available, exept for studies among indigenous olletions of vanilla, through polyarylamide eletrophoreti (PAGE) studies [8]. The present study omprising of randomly seleted allus regenerated progenies, shows variability among the allus regenerants in general and also between a parent and its own allus regenerated plant. This is the first report of haraterization of plants regenerated through allus in vanilla, revealing important information on the amount of variability that an be generated. This study an be utilized for developing variants with desirable agronomi haraters like short internode and tolerane to disease for utilizing them in vanilla improvement programmes, and broadening the narrow geneti base. Earlier attempts have been made to inrease the spetrum of variability by effetive wide rossing between ultivated V. planifolia and V.aphylla, their moleular profiles indiating introgression of male and female haraters into the hybrids [] and isolation and fusion of protoplasts of V.planifolia and V.andamania, a speies indiating possibilities of natural seed set []. In vitro regeneration systems are being used for onservation of endangered orhid speies too [9]. In future attempts to genetially transform vanilla, the ability of transformed tissue to regenerate is a ruial prerequisite. The regeneration protool optimized here is very simple and not time onsuming, hene ould shorten the length of any geneti transformation experiments while induing a high frequeny of regeneration. In vitro roots ould be developed in growth regulator free medium, so that effet of exogenous hormones would not affet the hardening or planting out of plantlets. The rare natural seed germination hampers onventional propagation and harvesting natural variability through seed germination. This is overome by in vitro seed germination and the protool to multiply plants in large numbers. The media onduive to indue responses viz., multiple shoots, roots as well as plant regeneration from allus was similar irrespetive of the maturity / juvenility of the tissue, indiating the nonspeifiity of the optimized onditions to different genotypes, explants or speies. The protool thus optimized a highly oordinated pathway led by the interation of auxin and ytokinin (Fig. 3) into plant regeneration diretly and via allus phase in Vanilla while indiating the amount of variability that an be generated through this system. Thus it is the first report in Vanilla to establish an effiient indiret plant regeneration system via allus appliable to both ultivated V.planifolia and wild and endangered, V.aphylla. Table. Effet of auxins on allus indution and plant regeneration invanilla planifolia Explant type Media@ Callus indution rate (%)* No. of shoots regenerated Immature seeds Control + NAA (.68) + IBA (.6) + BA (.) + NAA (.68) + BA (.) + IBA (.6). ±.89 e 79. ±.88 ab e 76. ±.3 b.6 ±.3 e.8 ± 3.7 b Mature seeds Shoot base Control + NAA (.68) + IBA (.6) + BA (.) + NAA (.68) + BA (.) + IBA (.6) Control + NAA (.68) + IBA (.6) + BA (.) + NAA (.68) + BA (.) + IBA (.6) ±.73 e 8. ±.55 a. ±.5 e 8. ±.97 a.8 ±.77.6 ±.89 e 5.8 ± 6.6 d e 8.6 ± 5.6 a 6.6 ±.98 d. ±.5. ±.6 ±.7a ±.. ±.5.8 ±.77 a. Conentrations of growth regulators are in µm *Mean ± S.D. Means in the same olumn followed by d ifferent letters are signifiantly different at 5% probability level 57

7 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5():56 Table. Effet of growth regulators on multiple shoot and root indution from shoot explants of Vanilla planifolia on MS medium* Growth regulators (µm) Multiple shoots frequeny (%)* Average no. of shoots/ulture±sd* Roots development /ulture Kin BA NAA IBA No Type ± 3.5 ± ± Velamen Velamen.6.9 Long roots Long roots ± ±. 5.5 ± ± 3.5 velamen branhing velamen Healthy roots *Mean of repliates Table 3. Variations in plant haraters observed at the nursery stage among a few allus regenerated progenies of V. planifolia (six months after planting out) No Progenies S S S3 S39 S S3 S7 S3. Plant height (m) ±S.D.35± 8.±.3 9.8±.7.3±.8 9.6±.5.9±.6 7.3±.6±.6 Internodal length (m) ±S.D 5±.9.55±..86±.5.8±.5.±.39.7±.3.66±.37.57±.38 Leaf size (m) Length ±S.D Breadth ±S.D 3.86±.3.5±.3 3.±.7.5±..5±.3.±.35.±..7±..8±.36.7±..±.3.6±.3.±..3±.33.8±.7.75±.3 9 V.planifolia.3±..3± ±.3.±.9 values are an average of observations; S.D: Standard Deviation 58

8 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5():56 b a e d f h g Fig.. Plant regeneration from allus ultures in Vanilla a. Organogeni allus indued in shoot ultures of V.planifolia, b. Shoot regeneration from allus,. Callus indution from seeds, d. Plant regeneration from seed allus, e. Indution of allus from shoot bases of V.aphylla, f. Plant regeneration from allus ultures of V.aphylla, g. Somalone with small internode, h. RAPD profiles of allus regenerated plants of vanilla using OPERON primer OPA Lanes. kb ladder :V8.; 3 : V56.; : V9; 5 : V9.; 6 : V98.; 7 : V; 8 : V.; 9 : V; : V.; ; 5.; : V.; 3 : V.; : V56.; 5 ; V6.; 6 : V79; 7 : V79.; 8 : V58.; 9 : V.R; : VR; : V7.; : V7.R; 3 : V53R ; : V.planifolia (Control), i. Variations in hromosome numbers in adjaent ells in a allus regenerated variant 59

9 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5():56 % germination 8 6 Immature seed Mature seed Shoot bases Number of days 3.5 Number of regenerative shoots per allus ulture 8 6 V.planifolia V.aphylla Conentrations of growth regulators in µm Basal medium.µm BA.µM BA µM NAA.68µM NAA.µM BA.µM BA +.68µM NAA 5.36µM NAA Fig. 3. Callus regenerative potentials influened by auxinytokinin interation in V.planifolia and V.aphylla 6

10 K. Nirmal Babu et al Asian J. Plant Si. Res., 5, 5(): ts o s h b e r o f 8 m u6 N S S 3 9 S 3 S S Different regenerants 7 S. 3 S l n tro o Maximum no of shoots Polymorphi loi expressed by the varaints Fig. Variations in Regenerated regenerative variants apaity in Vanilla of different planifolia regenerated lines Fig. 5 Graphi representation of the polymorphism expressed by allus regenerated progenies of vanilla in pooled RAPD data Aknowledgement The authors are thankful to the sientists and staff of Indian Institute of Spies Researh, India for help during the ourse of the study. 6

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