Vanilla

Minoo Divakaran, K. Nirmal Babu, and Michel Grisoni

Introduction

Vanilla planifolia G. Jackson (syn. V. fragrans Andrews), is a tropical climbing orchid (Figure 5.1) known for producing the delicate popular flavor, vanilla (Purseglove et al., 1981) and is the second most expensive spice traded in the global market after saffron (Ferrão, 1992). The major vanilla-producing countries are Madagascar, Indonesia, Uganda, India, and the Comoros with Madagascar ranking first. Following the discovery of the New World by Columbus in 1492, the earliest vanilla dissemination record from Mexico is the one by Father Labat who imported three V. planifolia vines into Martinique in 1697. The lack of natural pollinators in the areas of introduction prevented sexual reproduction and pod production until the discovery of artificial pollination in the first half of the nineteenth century (Bory et al., 2008d). Continuous vegetative propagation, lack of natural seed set, and insufficient variations in the gene pool all hamper crop improvement programs.

FIGURE 5.1 V. planifolia vine—in full bearing.

In a short span of time, biotechnology has had a significant impact on the pattern of development and quality of life globally (Bhatia, 1996). The later part of the twentieth century saw the rise of new industries based on discoveries made in the field of biological sciences and the progress made over recent years in molecular biology, genetic engineering, and plant tissue culture have provided a new dimension to crop improvement.

In vitro culture is one of the key tools of plant biotechnology, which makes use of the totipotent nature of the plant cells, a concept proposed by Haberlandt (1902) and unequivocally demonstrated for the first time by Steward et al. (1958). It can be employed for the production of disease-free clones, mass cloning of selected genotypes, gene pool conservation, selection of mutants, raising of hybrids between sexually incompatible taxa through somatic hybridization, incorporating the desired traits by genetic engineering, and in the production of secondary metabolites in cultured cells or tissues (Thorpe, 1990). However, the realization of these objectives necessitates prior standardization and optimization of tissue culture procedures.

Problems to be Targeted

As a cash crop, vanilla plays a major role in the economy of countries such as Madagascar, the Comoros, Indonesia, and Uganda. Continuous clonal propagation of V. planifolia leads to monoculture, exposing the crop to severe damage (Gopinath, 1994) as vanilla is affected by a large number of pests and diseases (see Chapters 7, 8, 9, and 20). The introduction of new genetic material is greatly constrained by factors such as its asexual propagation, the fact that the flowers are mostly self-pollinated, and the threat posed to wild populations of vanilla by land pressures (Lubinsky, 2003). The lack of sufficient variability in the gene pool, the threat of destructive diseases that wipe out vanilla plantations, as well as the destruction of its natural habitats, make the search for alternative methods to introduce variability into the gene pool vital. The narrow gene pool can be broadened by using interspecific hybridization to combine the available primary gene pool of the genus Vanilla, with the secondary gene pool, that is, the close relatives of V. planifolia, which is an important source of desirable traits such as self-pollination, a lower dependence of flower induction on the photoperiod, a higher fruit set, indehiscence of the fruits, and disease resistance (Lubinsky, 2003).

Different species of vanilla are found in various geographical regions and their flowering seasons are not synchronized, bringing about difficulties in the movement of pollen to the receptor species to enable pollination between species. It is in such instances that the development of methods for storing viable pollen for longer periods becomes significant.

Improvements in quality characteristics, such as higher vanillin content, larger bean size, improved aroma and taste, and so on would benefit vanilla processors and consumers. To perfect the germination of immature embryos into a complete plant, embryo rescue techniques can be used for retrieval and regeneration of nonviable hybrid seeds. Cell culture or protoplast culture is useful for creating somatic hybrids for the transfer of characters from alien sources. Protoplasts can be used as target organs for transformation, provided they are made to regenerate into a complete plantlet. Clonal propagation of elite lines, in vitro conservation, and international germplasm exchange are possible using micropropagation techniques. Molecular markers such as DNA markers [random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), amplified fragment-length polymorphism (AFLPs)] and biochemical markers (isozyme, protein) can be used for the characterization of germplasm and somaclonal variants.

Genetic Diversity in Vanilla

V. planifolia is a crop that differs a little from its wild progenitors. This can be attributed to limited breeding and to recent domestication (Bory et al., 2008c; Lubinsky et al., 2008a). Several types have been recognized within the cultivated vanilla of Mexico differing in vegetative appearance or reproduction mode (Soto Arenas, 2003). The analysis of isoenzyme data of specimens from the vanilla plantations of northern Veracruz, Oaxaca, and elsewhere in Mexico showed little genetic variation in general (Soto Arenas, 1999), although plants originating from two main areas could be differentiated. Nucleotide sequence variation within introns of two specific protein-coding genes—namely, the calmodulin and the glyceraldehyde 3-phosphate dehydrogenase—were detected but were not able to differentiate among the Mexican types of vanilla. Outside the countries of origin, vanilla is likely to be of clonal origin and very little variation can be expected. The vanilla plantations of Réunion, Madagascar, Mauritius, and Seychelles have derived from a single cutting (Lionnet, 1958) and as per the present information, few differences in cultivated types of V. planifolia have been observed. However, recent studies revealed that self-progenies as well as polyploidization events have generated phenotypic diversity in cultivated vanilla in Réunion (Bory et al., 2008a, 2008b, 2008c).

At the genus level, molecular markers such as RAPD, AFLP, and sequence single repeats (SSR) have been developed over the last decade for studying genetic diversity.

RAPD was used to estimate the level of genetic diversity and interrelationships among different clones of V. planifolia and related species. The data confirmed the very limited variation within accessions of V. planifolia, indicative of its narrow genetic base and its close relationship with V. tahitensis J.W. Moore (Besse et al., 2004; Minoo et al., 2008; Schlüter et al., 2007). In a study including both leafy and leafless types, such as V. planifolia, V. tahitensis, V. andamanica Rolfe, V. pilifera Holtt., and V. aphylla Blume (Figure 5.2), there was reasonable variability indicating the possibility of natural seed set in the wild species. In spite of superficial morphological similarity, V. andamanica is not closely related to V. planifolia or V. tahitensis and its accessions are the most divergent from all other species studied, forming a separate and unique cluster (Minoo et al., 2008). There was considerable variability among the eight different accessions of V. andamanica, supporting the probability that this species did originate in the Andaman Islands, where sexual reproduction is likely (Minoo et al., 2008). Earlier, Rao et al. (2000) have reported the occurrence of natural seed set in India for V. wightiana.

FIGURE 5.2 Flowers of Indian species of Vanilla: (a) and (b) V. andamanica with varying label colors, (c) V. pilifera showing indication of insect visits, and (d) V. aphylla.

AFLP profiles were developed to analyze Vanilla species, interspecific hybrids, and selfed progenies (Bory et al., 2008c; Lubinsky et al., 2008a; Minoo et al., 2006b). All these analyses converged in showing that most of the V. planifolia accessions cultivated outside of Mesoamerica exhibit very low levels of genetic diversity, as they derived from a single accession, possibly the Mexican cultivar Mansa from Papantla.

The patterns of diversification of the cultivated species were also studied and compared with other cultivated (V. tahitensis) and wild (V. aphylla, V. bahiana, V. insignis, V. odorata, and V. pompona) species. Clear polymorphism was detected in these related species, interspecific hybrids, and selfed progenies.

The development of SSR markers (microsatellites) have been reported by Bory et al. (2008b). The isolation and characterization of 14 microsatellite loci from V. planifolia have been described. These were monomorphic within cultivated accessions, as expected based on the probable single clonal origin of this crop and previous genetic studies. These markers were transferable to V. tahitensis and 11 loci were polymorphic between these two closely related species. Furthermore, some of these markers were transferable and polymorphic across 15 other wild American, African, and Asian species and revealed consistent relationships between species, together with a strong pattern of Old World versus New World differentiation in the genus. Furthermore, the use of microsatellites allowed the first molecular-based estimation of heterozygosity levels in this species, which was not possible when dominant markers such as AFLP or RAPD was used.

Sequencing of neutral genes has been used for reconstructing the evolutionary history of Vanilloid orchids, including a few Vanilla species (Cameron, 2000, 2004, 2009; Cameron and Molina, 2006; Cameron et al., 1999). Nuclear (internal transcribed spacer, ITS) and plastid (rbcL gene) DNA sequences were also used for unraveling the origin of the Tahitian vanilla (Lubinsky et al., 2008b). Recently, the length polymorphism of the nonneutral caffeic acid O-methyl transferase gene was also used to analyze 20 Vanilla species, and confirmed the strong differentiation of Old World versus New World species in the genus (Besse et al., 2009). On the basis of sequencing data for nuclear and plasmidic DNA, Cameron (2005) suggested in setting up a bar code system (Lahaye et al., 2008) for vanilla using the ITS region and the psbA-trnH intergenic spacer. This system may allow routine identification of vanilla specimens to the species level, and perhaps even to the accession level. To build a robust phylogeny for the Vanilla genus, reference herbarium specimens will need to be included. For this purpose, the development of plastid mononucle-otide microsatellites should be considered for vanilla (particularly when using degraded DNA samples extracted from herbarium material), as have already been successfully used for biogeographical studies of orchids (Fay and Krauss, 2003; Micheneau, 2002).

Given the difficulty in using classical phenotypic markers for perennial crops such as vanilla, molecular markers are powerful tools for studying the variability in cultivated vanilla, unraveling species interrelationships, identifying interspecific hybrids, and fingerprinting important genotypes (Minoo et al., 2006a). They are, therefore very helpful for monitoring and evaluating the achievements resulting from biotechnologies.

Propagation and Breeding Methods

Commercial vanilla is always propagated by stem cuttings of healthy vigorous plants and may be cut from any part of the vine. The length of the cutting is usually determined by the amount of planting material available. Short cuttings, 20 cm in length, will take 3–4 years to flower and fruit. Cuttings of 90–100 cm in length are usually preferable as they tend to flower earlier. When available, with their free ends hanging over supports, these will flower and fruit in 1–2 years. Cuttings are usually planted in situ, but they may be started in nursery beds when necessary. Because of their succulent nature, cuttings can be stored or transported for a period of up to two weeks, if required.

Traditionally, vanilla germplasm is conserved in clonal repositories belonging to botanical gardens and scientific institutions. The high costs of traditional conservation systems limit the number of accessions that can be preserved. In order to reduce the losses of biodiversity, attempts to conserve Vanilla species, in vitro, were made (Jarret and Fernandez, 1984; Minoo et al., 2006b) and have been extended to conserve the endangered species.

For breeding purposes, vanilla can be grown from seeds. Hybridization and the production of plants from seeds have been carried out in Puerto Rico and Madagascar. The seeds should be disinfected, washed in sterile distilled water, and cultured in nutrient medium (Knudson, 1950). The germination of vanilla seeds is better if the cultures are maintained in a dark incubator at 32°C. Seeds of interspecific crosses between V. planifolia and V. pompona required a higher temperature of 34°C for germination.

In Vitro Seed Germination

Vanilla produces numerous minute seeds that do not germinate under natural conditions. Tissue culture technique can be used to successfully germinate the seeds. Protocols for seed and embryo culture of vanilla have been standardized (Gu et al., 1987; Knudson, 1950; Minoo et al., 1997; Withner, 1955).

Seed culture in different basal media indicated that vanilla seeds had no stringent nutritional requirements for the initiation of germination unlike some terrestrial orchids of temperate climate (Minoo et al., 1997). The germination of seeds began within four weeks of culture and the initial stages of germination were typical of most orchids, such as swelling of the embryo followed by rupturing of the seed testa, and the subsequent emergence of protocorms (Figure 5.3). Seeds germinated directly into plantlets in the medium supplemented with benzyladenine (BA) (0.5 mg L⁻¹) alone, without any intervening callus phase, and could thus be utilized for the production of selfed progenies/seedlings. The addition of tryptone had a growth-promoting effect on the size and development of protocorm, irrespective of the basal medium to which it was added. In treatments with BA, most of the protocorms remained the same with the scale-like leaf primordial and developing into shoots, whereas treatment with auxin supplements showed the gradual disorganization of the protocorms into callus. Murashige and Skoog’s (MS) medium gave a better response than Knudson’s medium, for in vitro cultures of vanilla. The minimum germination (26%) was observed in MS medium at half strength and the maximum (85%) was recorded in full strength MS medium supplemented with 2 g L⁻¹ tryptone (Minoo, 2002).

FIGURE 5.3 In vitro seed germination.

The requirement of cytokinin for germination is considered to be related to the utilization of lipids that constitute the primary storage material in most orchid seeds and it has been observed that unless storage lipid is utilized, germination does not continue (De Pauw et al., 1995).

Vanilla, a cross-pollinated crop, is known to have many meiotic and postmei-otic chromosomal abnormalities (Ravindran, 1979). As a result, it is possible to get various cytotypes in the seed progenies. Culturing of seeds can thus give many genetically varied types. Studies on in vitro germination of vanilla seeds and the resultant progeny showed morphological and biochemical variations. Isozyme profiles of superoxide dismutase (SOD) and peroxidase (PRX) were studied in selfed progenies of V. planifolia. The profiles clearly indicated differences among prog-enies as expressed by the presence or absence of specific bands. The maximum similarity that these progenies exhibited was 47.37%, indicating high segregation and level of heterozygosity existing in V. planifolia (Minoo et al., 1997). This heterozygosity was further confirmed by AFLP analyses (Bory et al., 2008c). Thus, in vitro culture can be used for the germination of seeds and the selection of useful genotypes from segregating progenies that might be mass propagated for obtaining disease-free planting material.

Micropropagation

In vitro propagation of vanilla is essential to generate uniform, disease-free plantlets and for conserving the genetic resources. In vitro propagation using apical meristem has been standardized for the large-scale multiplication of disease-free and genetically stable plants (Cervera and Madrigal, 1981; George and Ravishankar, 1997; Kononowicz and Janick, 1984; Minoo, 2002; Minoo et al., 1997; Philip and Nainar, 1986; Rao et al., 1993b). In vitro propagation of V. tahitensis (Mathew et al., 2000) and endangered species of vanilla, such as V. wightiana, V. andamanica, V. aphylla, and V. pilifera (Minoo et al., 2006b) have been standardized to protect these species from extinction.

Clonal propagation methods for the efficient multiplication of V. planifolia by induction of multiple shoots from axillary bud explants (Figure 5.4) using semi-solid MS medium supplemented with BA (2 mg L⁻¹) and α-naphthale neacetic acid (NAA, 1 mg L⁻¹) have been reported (George and Ravishankar, 1997). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg L⁻¹ and NAA at 0.5 mg L⁻¹ for 2–3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots was obtained from a single axillary bud explant over a period of 134 days. The use of an intervening liquid medium was found to enhance the multiplication of shoots.

In another study (Minoo et al., 1997), the subculture of the explants onto proliferation MS media containing various levels of cytokinin (BA) and auxin (indole butyric acid, IBA) was evaluated (Table 5.1). The initiation of preexisting buds to grow in vitro could be induced in MS medium with low cytokinin. However, a combination of cytokinins and auxin promoted multiple shoot formation. The ideal medium for multiplication was MS supplemented with BA (1 mg L⁻¹) and IBA (0.5 mg L⁻¹). In this medium, an average of 15 multiple shoots were induced in 90 days of culture (Figure 5.4). Nodal segments gave a better response, with a mean of 15 shoots per culture compared to the shoot tips, which gave a mean of seven shoots per culture (Minoo, 2002). The culture media and conditions favorable to micro-propagation of V. planifolia were suitable for other related species, such as V. anda-manica, V. aphylla (Figure 5.5), and V. pilifera. The number of shoots induced in different species varied (Table 5.2). About 12–15 shoots/culture could be induced in V. planifolia, followed by V. aphylla (8–10 shoots). Among the species studied, the lowest multiplication rate was observed in V. pilifera. Elongated shoots from proliferation medium were rooted on MS growth regulator free medium containing 30 g L⁻¹ sucrose (Figure 5.6). In vitro plantlets with well-developed roots were acclimated with a survival percentage of more than 70%. The root initiation on microcuttings started between four and six days after culture, reaching 100% of the cultures after two weeks, indicating that the optimal endogenous levels of plant growth regulators required for rooting were already present in the tissue/explants.

FIGURE 5.4 Micropropagation of V. planifolia.

Janarthanam et al. (2005) and Kalimuthu et al. (2006) have devised simple and rapid protocols for the mass multiplication of V. planifolia. A commercially viable protocol for the mass propagation of V. tahitensis, another cultivated species of Vanilla, was standardized with a multiplication ratio of 1:4.7 over a culture period of 60–70 days (Mathew et al., 2000). Rao et al. (2000) have reported the occurrence and micropropagation of V. wightiana Lindl., an endangered species. Giridhar et al. (2001) and Giridhar and Ravishankar (2004) have studied the effects of other additives, namely, silver nitrate, thidiazuron, zeatin, coconut milk, and so on, on in vitro shoot multiplication and root formation in V. planifolia.

BA = benzyladenine, IBA = indole-3-butyric acid, Kin = kinetin, NAA = α-naphthaleneacetic acid.

FIGURE 5.5 In vitro multiple shoot production in V. aphylla.

The conversion of root tips into shoots was observed in V. planifolia and V. aphylla when cultured on MS medium supplemented with BA (1.0 mg L⁻¹) and IBA (0.5 mg L⁻¹). These shoots developed into plantlets and were hardened and established in soil. The conversion of root meristem into shoots in in vitro cultures of vanilla was earlier reported (Philip and Nainar, 1988). These meristematic conversions without callus stage are assumed to minimize the chances of induced epige-netic changes. Earlier studies by Sreedhar et al. (2007) indicated no difference in the AFLP-banding patterns of any of the micropropagated samples for a particular primer, suggesting the absence of variation among the micropropagated plants.

BA = benzyladenine, IBA = indole butyric acid, Kin = kinetin, NAA = α-naphthaleneacetic acid.

^a All growth regulators were supplemented on MS basal medium at 0.5 to 1.0 mg L⁻¹.

FIGURE 5.6 In vitro rooting in V. planifolia cultures.

Plant Regeneration through Callus Cultures

Continuous vegetative propagation and lack of sufficient variations in the gene pool hamper crop improvement programs. 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 MS medium supplemented with 1 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA. Callus differentiated into shoots that could be multiplied successfully in 1:12 ratio in a combination of 1 mg L⁻¹ BA and 0.5 mg L⁻¹ IBA, when supplemented with MS medium (Table 5.3). In vitro rooting was induced with an efficiency of 100% in basal MS media devoid of any growth regulators. This ability of dedif-ferentiated tissue to regenerate is a crucial prerequisite for 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.

BA = benzyladenine, IBA = indole butyric acid, Kin = kinetin, NAA = α-naphthaleneacetic acid.

Reports on variability among callus-regenerated plants in vanilla are few. They concern successful plant regeneration from leaf- and seed-derived callus (Davidonis and Knorr, 1991; Davidonis et al., 1996; Janarthanam and Seshadri, 2008; Xju et al., 1987) and studies among indigenous collections of vanilla, through polyacrylamide electrophoretic (PAGE) studies (Rao et al., 1993a). A study comprising randomly selected callus-regenerated progenies showed variability in morphology and RAPD profiles (Figure 5.7) among the callus-regenerated plants in comparison with the control plant V. planifolia (Minoo, 2002). It showed that a significant amount of variability can be generated with this protocol and be utilized in vanilla improvement programs for developing variants with desirable agronomic characters.

FIGURE 5.7 RAPD profiles of callus-regenerated progenies of vanilla using OPERON primer OPA10. 1: 1 kb ladder, 2–23: callus-regenerated plants, 24: control (V. planifolia).

Callus cultures initiated from leaf explants of V. planifolia showed better callus initiation than those from nodal explants with callus biomass production maximal when cultured on MS basal medium containing 2,4-dichlorophenoxy acetic acid and BA. Callus transferred to MS basal medium supplemented with 3 mg L⁻¹ BA and 2.5 mg L⁻¹ μM NAA showed superior growth response. Davidonis et al. (1996) have patented the production of callus of V. planifolia, extraction of vanillin, and the use of ferulic acid to increase the content of vanillin.

Heritable somaclonal variations with respect to various resistance traits have been reported, namely, resistance to methionine sulfoxime (Carlson, 1973) and Pseudomonas syringae (Thanutong et al., 1983), in tobacco, resistance to Fusarium oxysporum in tomato (Evans et al., 1984), and resistance to Helminthosporium sati-vum (Chawla and Wenzel, 1987) in wheat. In future attempts to genetically transform vanilla, the ability of transformed tissue to regenerate is a crucial prerequisite. The regeneration protocol optimized (Minoo, 2002) could shorten the length of genetic transformation experiments while inducing a high frequency of regeneration.

Ex Vitro Establishment of Seedlings

Most plant species grown in vitro require a gradual acclimatization and hardening for survival and growth in the natural environment. The survival of in vitro plants depends upon their ability to withstand water loss and carry out photosynthesis. However in vanilla, the survival rate of transferred plants is currently over 80% during hardening process (Minoo, 2002). Plantlets should be removed from culture vessels (Figure 5.8), washed, treated with fungicide, transferred to polybags containing potting mixture (sand, soil, and vermiculite) and hardened for 30 days under controlled conditions (26–28°C, 80–90% RH). Initiation of new growth occur through development of the axillary branch. These plants are successfully transferred to soil after initial hardening period of three weeks (Figure 5.9) and can be. They were later field planted with proper shade and support.

FIGURE 5.8 Hardening of in vitro developed plantlets.

FIGURE 5.9 Tissue-cultured plants growing in pots.

Interspecific Hybridization

Interspecific hybridization is an age-old mechanism by which useful genes from wild progenitors and species can be brought into cultivated species. The cultivated types of many crop species were improved through interspecific hybridization and backcrossing. Interspecific hybridization is very common in orchids to produce new and novel varieties of flowering plants.

Natural occurrences of interspecific hybrids have been reported in vanilla by Nielsen and Siegsmund (1999) between V. claviculata and V. barbellata in localities in Puerto Rico where they coexist. Progenies were discovered having morphological characters intermediate between the two parents.

The cultivated species of V. planifolia has been crossed with other American species including V. pompona and V. phaeantha, which are resistant to Fusarium (Purseglove et al., 1981). Interspecific hybridization was also conducted in Java between cultivated and wild vanilla to develop lines resistant to stem rot caused by Fusarium oxysporum (Mariska et al., 1997).

In India, V. aphylla and V. pilifera flower synchronously but V. aphylla occurs naturally in South India and V. pilifera in Assam, North East India. When cultivated in Kerala, flowers of both species opened sequentially and lasted for one day in V. pilifera, whereas it lasted for two days in V. aphylla. In the former, signs of fruit set were observed even without manual pollination whereas V. aphylla flowers did not set fruit. Since rostellum is present in both species, natural pollination without an aid is ruled out. It can be suspected, that the fragrance of the V. pilifera flowers attracts insects (which were found to frequent the flowers often) to visit them and bring about effective pollination (D. Minoo, unpublished data). Self and interspe-cific hybridizations between the two species were done manually and fruits set was observed.

Successful attempts were made to increase the spectrum of variation of V. plani-folia by interspecific hybridization with V. aphylla which is tolerant to Fusarium (Minoo, 2002). Morphological characters and molecular profiles revealed the true hybridity of the interspecific hybrid progenies. Seedling progenies of V. planifolia, and interspecific hybrids obtained from crosses between V. planifolia (female) and V. aphylla (male) were evaluated using a number of different loci as markers by using AFLPs and RAPDs loci. The profiles indicated similarity between the parents, selfed progenies, and interspecific hybrids and that all the progenies tested were variable when compared to each other, which can be exploited for crop improvement in vanilla (Minoo et al., 2006a).

Thus, these successful introgressions of male and female characters into the hybrids (Minoo et al., 2006a) by interspecific hybridization, confirmed by molecular profiles are promising to help solve the major bottlenecks in vanilla breeding.

In Vitro Conservation

Effective procedures for in vitro conservation by slow growth in selected species of vanilla have been standardized (Minoo et al., 2006b). The addition of mannitol (10–15 g L⁻¹) and reduction of sucrose to lower levels (15–10 g L⁻¹) induced slow growth and subsequently 80–90% of the cultures could be maintained for a period of 360 days, when the culture vessels were closed with aluminum foil. Supplementing mannitol and sucrose in equal proportions at 10 or 15 g L⁻¹, could help to maintain the cultures for one year and thus were maintained in vitro for more than seven years with yearly subculture. The plantlets maintained in this medium showed reduced growth rate and maximum survival. The conserved material was transferred to MS medium fortified with 30 g L⁻¹ sucrose and supplemented with 1 mg L⁻¹ BA and 0.5 mg L⁻¹ IBA, for retrieval of normal shoots and their multiplication. The conserved material was transferred to the multiplication medium (MS + 30 g L⁻¹ sucrose and 1 mg L⁻¹ NAA) for normal growth. The small-sized plantlets kept in the conservation medium for over one year showed good growth and developed into normal-sized plants with good multiplication rate (1:5). These plantlets were transferred to soil (garden soil:sand:perlite in equal proportions) and established easily with 80% success when kept in a humid chamber for 20–30 days after transfer. They developed into normal plants without any deformities and defi-ciency symptoms and exhibited apparent morphological similarities to the mother plants. After more than seven years of slow growth storage, involving over five subculture cycles, the genotypic stability of few species was assessed using molecular markers. No changes were observed in DNA fingerprinting vis-à-vis nonconserved controls in the authors’ laboratory.

Jarret and Fernandez (1984) have reported storage of V. planifolia shoot tips as tissue cultures for 10 months and Philip (1989) has discussed the possibility of using root cultures for conservation of vanilla germplasm for assured genetic stability. In vitro conservation of V. planifolia (Jarret and Fernandez, 1984) and V. walkeriae using slow growth method (Agrawal et al., 1964) has been reported and the effects of polyamines on in vitro conservation of V. planifolia have been studied by Thyagi et al. (2001).

Conventional and in vitro genebanks are complementary as the active and base collections of genetic resources. Although in vitro conservation cannot be viewed as a method to replace in situ conservation, the advantages of in vitro conservation as a component that can be incorporated into an overall vanilla long-term conservation strategy for a safe and economical storage of the germplasm were demonstrated.

Cryopreservation

Protocols for conservation of gene pools have been developed for slow growth as well as cryopreservation of vanilla accessions as encapsulated shoot tips, pollen, and DNA (Minoo, 2002). Combining the available gene pool in the genus will help in broadening the genetic base and in converging the useful genes into cultivated vanilla from wild species. Interspecific hybridization requires synchronized flowering between the species and availability of viable pollen. Pollen from two asynchronously flowering species of Vanilla, namely, cultivated V. planifolia and its wild relative V. aphylla, were cryopreserved after desiccation, pretreated with cryoprotectant dimethyl sulfox-ide (5%) and cryopreserved at −196°C in liquid nitrogen (LN). This cryopreserved pollen was later thawed and tested for their viability both in vitro and in vivo. A germination percentage of 82.1% and 75.4% in V. planifolia and V. aphylla pollen, respectively, were observed indicating their viability. These cryopreserved pollens of V. planifolia were used successfully to pollinate V. aphylla flowers resulting in fruit set. The seeds thus obtained were successfully cultured to develop hybrid plantlets (Minoo, 2002). Viability and fertility assessment of cryopreserved pollen (Figure 5.10) from Vanilla species thus showed that it is possible to use cryogenic methods for conservation and management of the haploid gene pool in this species. This is of great importance for the facilitating crosses in breeding programs, for distribution and exchange of germplasm, and for preserving nuclear genes of the germplasm.

FIGURE 5.10 Germination of cryopreserved pollen.

A procedure for storage of vanilla germplasm by cryopreservation of shoot tips using encapsulation/dehydration method has been standardized (Figure 5.11). The in vitro-grown shoot tips were encapsulated in 4% sodium alginate. The encapsulated beads were subjected to pretreatment by progressive increase of sucrose concentration from 0.1 to 1.0 M, followed by dehydration for 8 h to a moisture content of 22%. This was followed by rapid freezing by plugging into LN. The cryopreserved shoot tips were thawed after 12 h in LN by keeping them in water bath at 40°C for 3 min. The thawed propagules were allowed to recover on MS with 3% sucrose, 1 mg L⁻¹ BAP, and 0.5 mg L⁻¹ IBA in dark for one week and then transferred to light for regrowth and multiplication. Seventy percent of the propagules have for recovered, grown, and multiplied into full-fledged plants (Ravindran et al., 2004).

FIGURE 5.11 Germination of cryopreserved shoots.

Cryopreservation, once fully implemented will provide an expeditious and cheaper means to duplicate the base collection for safety reasons, as well as for the distribution of germplasm sets to other countries/continents.

Production of Synthetic Seeds

Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, to produce good quality rigid beads ideal for withstanding low temperatures and cryopreservation. Higher concentrations of alginate were not suitable as they produced very hard matrix, which hindered the emergence of shoot buds and thereby affecting the rate of germination and recovery, while at lower concentrations of alginate, the beads were difficult to handle during cryopreservation and retrieval. The synthetic seeds were stored at 5°C, 15°C, and 22°C to study the effect of temperature on their storage and viability. Low temperatures (5°C and 15°C) were not suitable for synthetic seed. Shoot buds of 0.4–0.5 cm size were suitable for encapsulation as smaller buds failed to survive the storage and lost their viability within a month. However at 22 ± 2°C, synthetic seeds could be stored for 10 months (Figure 5.12). The plants derived from these encapsulated buds were apparently healthy and developed into normal plants.

FIGURE 5.12 Synthetic seeds.

Clonal propagation of V. planifolia using encapsulated shoot buds have been reported by George et al. (1995). Synthetic seeds are ideal for germplasm conservation and exchange, especially in vanilla, where there is no natural seed set.

Protoplast Isolation and Fusion

The techniques of protoplast isolation and fusion are important because of the far-reaching implications in studies of plant improvement by cell modification and somatic hybridization. The possibility of protoplast systems in spice crops such as cardamom, ginger, and vanilla was studied by Triggs et al. (1995) and Geetha et al. (2000).

Protoplasts were successfully isolated from V. planifolia and V. andamanica when incubated in an enzyme solution containing macerozyme R10 (0.5%) and cellulase Onozuka R10 (2%) for 8 h at 30°C in dark (Table 5.4). In vitro leaves were plasmolyzed in a solution containing cell protoplast washing salts with 9% mannitol before enzymatic digestion. Since it was difficult to peel off the lower epidermis in vanilla, the plasmolyzed leaf tissue was mechanically macerated by scraping the lower surface of the leaf with a sharp blade and incubating in different concentrations and combinations of enzyme solutions. Periodical microscopic observations showed the liberation of cell clusters and individual cells after 2 h of incubation in enzyme solution.

The isolation solution containing 9% mannitol was found necessary for the release and maintenance of viable protoplasts. The isolated protoplasts were round and filled with chloroplasts. Protoplasts of V. planifolia were bigger in size (0.031 mm) than those of V. andamanica (0.022 mm) and could be distinguished by the arrangement of chloroplasts—peripheral in V. planifolia and centrally scattered V. andamanica. The visually distinguishable nature of protoplast can be exploited for the purpose of identifying genetic transformation in these species. When subjected to polyethylene glycol (PEG)-mediated fusion, the protoplasts fused forming a heterokaryon. The fusion product was cultured on MS liquid medium with 0.5 mg L⁻¹ BA, 0.5 mg L⁻¹ IBA supplemented with 3% sucrose and 7% mannitol for 20 days. The cell wall development around the fusion product was observed after 36 h (Minoo et al., 2008). The fusion protoplast technology can be very useful in gene transfer of useful traits to V. planifolia, especially the natural seed set and disease tolerance observed in V. andamanica.

The Future of Vanilla Improvement

The landmarks that have been attained in the form of various technologies can be effectively used for the production of a spectrum of genetic variations in vanilla, thus overcoming a major bottleneck in vanilla breeding and crop improvement programs. The protoplast isolation and fusion technology developed can be used in transfer of useful traits through the production of somatic hybrids, thus making way for genetic manipulations in vanilla. The characterization of Vanilla species, accessions, seedlings, somaclones, and interspecific hybrids, proved the existence and extent of genetic variations that is available and brought by biotechnological tools. The in vitro conservation methods, through synthetic seed, slow growth, and cryopreservation will form an integral and important part of overall conservation strategy in genetic recourses management of vanilla germplasm. Furthermore, the synthetic seed technology forms an ideal means for exchanging disease-free planting material.

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