Introduction

Indigofera tinctoria, commonly known as Indian indigo or Indigofera, is a medicinally and commercially valuable leguminous plant (Simon et al. 1984; Morris 1999; Takawira-Nyenya and Cardon 2005). The plant is largely utilized in cosmetic, pharmaceutical, and dyeing industries. Its leaf extract is reported to have remarkable effect on hair growth and in preventing juvenile graying of hair (Nair et al. 1991). Within the indigenous system of medicine in India, Indigofera is used in preparations to treat hydrophobia, epilepsy, nervous disorders, bronchitis, sores, ulcers, and hemorrhoids (Saraswathy et al. 1998). The aerial parts of the plant have been shown to possess antihepatoxic (Sreepriya et al. 2001; Singh et al. 2004), antidyslipidemic (Narender et al. 2006), and anticancerous (Kameswaran and Ramanibai 2008) activities.

The plant bears seeds profusely, which are utilized for propagation and conservation. Accessions of Indigofera are usually preserved in seed gene banks. Several germplasm collections of Indigofera are maintained, the largest being at the Commonwealth Scientific and Industrial Research Organization (CSIRO), St. Lucia, Queensland, Australia (365 accessions) and the International Center for Tropical Agriculture (CIAT), Cali, Colombia (250 accessions). In Africa, collections are maintained in Ethiopia at the International Livestock Research Institute, Addis Ababa (60 accessions), and in Kenya at the National Genebank of Kenya Crop Plant Genetic Resources, Kikuyu (40 accessions) (Takawira-Nyenya and Cardon 2005). However, the seeds lose their viability in a short span of time under ambient storage conditions (Roos 1980; Morris and Hopkins 2000). Reports from the seed bank Centre for Medicinal Plant Research, Aryavaidyasala, Kottakkal (Kerala State, India) indicate that Indigofera seeds lose up to 60% viability after 1 yr under ambient storage conditions.

The success of preservation in seed gene banks depends on continuous monitoring of viability and plant regeneration capacity. Periodical renewal for germplasm maintenance is required if the viability goes below 85% (Genebank standards-FAO/IPGRI 1994). Germplasm conservation calls for germplasm exchange programs in which seeds are not a preferred entity as there is risk of seed-borne pests and pathogens. To overcome such quarantine problems, in vitro grown plantlets/cultures are preferred for germplasm exchange (Malaurie et al. 1998a; Reed et al. 2004). Propagation of plants through axillary meristem culture allows for recovery of genetically stable and true-to-type progenies (Begum et al. 2004; Barik et al. 2007; Sul and Korban 2007). Standardization of in vitro propagation systems is a prerequisite for in vitro conservation.

Cryopreservation is the process of freezing living plant material (shoot tips, meristems, cells, somatic embryos, zygotic embryos, etc.) at or near the temperature of liquid nitrogen, −196°C. At this temperature, physical and metabolic cellular processes are effectively stopped, and the living tissue is in a state of suspended animation. The transfer of cells from room temperature to −196°C is done in such a way that the viability of the stored material is retained, so that their biological functions and growth can be reactivated after thawing and transfer to regrowth medium (Towill 1991). Cryopreservation offers the only safe and cost-effective option for the long-term conservation of plant genetic resources (Dixit et al. 2004; Engelmann 2004).

The encapsulation–dehydration technique of cryopreservation, developed by Fabre and Dereuddre (1990), is based on the technology for the production of synthetic seeds. It involves the inclusion of meristems in alginate beads, the subsequent culture of the meristems in a highly concentrated (0.7–1.5 M) sucrose solution, followed by physical dehydration and direct immersion in liquid nitrogen. Culture of meristems on sucrose-enriched medium (0.3–0.7 M), prior to encapsulation, usually improves survival after desiccation and freezing (González-Benito et al. 2004, Shatnawi et al. 2004). Physical desiccation is carried out either with silica gel or in the air flow of a laminar flow cabinet (Paulet et al. 1993). Water contents of around 20% (fresh weight basis) have proven appropriate for high survival (50% to 75%) after freezing of vegetative explants in several species (Engelmann 1997; González-Benito et al. 1998; Malaurie et al. 1998b; Cho et al. 2002). By using the encapsulation–dehydration protocol, Clavero-Ramirez et al. (2005) obtained recovery rates between 23% and 63%, depending on genotype, in strawberry. The plantlets are regenerated from the cryopreserved meristems without intermediary callus formation in this cryogenic procedure (Wang et al. 2005).

Successful cryopreservation involves submitting selected explants to a series of successive, potentially stressing events during pregrowth, cryoprotection, dehydration, rapid immersion in liquid nitrogen, thawing, recovery, and plant regeneration (Reed 2001). The freezing process may result in the formation of intracellular ice crystals, leading to damage of cellular components with the potential to disrupt the nucleus, chloroplast, and/or mitochondrion (Coger and Toner 1995). Dehydration injury causes cell shrinkage, the formation of membrane invaginations, and the possible loss of plasma membrane to the cytoplasm (Dowgert and Steponkus 1984). Moreover, chances of somaclonal variation in regenerated plants cannot be ruled out. Hence, the long-term genetic consequences of dehydration and freezing injury for in vitro conservation need to be assessed (Harding 1996; Castillo et al. 2007; Gagliardi et al. 2007).

The random amplification of polymorphic DNA (RAPD) technique is considered to be a fast, simple, and efficient method for evaluating the genetic stability of cryopreserved material and can be used after the completion of a freezing experiment as a quick complement to other genetic stability evaluation methods (Hirai and Sakai 2000). This technique has been successfully used to study the genetic stability of cryopreserved materials of Arachis species (Gagliardi et al. 2003), Diascorea bulbifera (Dixit et al. 2003), and Vitis vinefera (Zhai et al. 2003).

This paper reports the effect of plant growth regulators on in vitro axillary shoot proliferation; the impact of desiccation duration on survival and regeneration of encapsulated, dehydrated, and cryopreserved axillary shoot meristem of I. tinctoria; and the verification of genetic stability of the regenerated shoots using RAPD techniques.

Materials and Methods

Preparation of explant for axillary shoot proliferation.

Mature ripe pods were harvested from I. tinctoria (accession no. IT 100) maintained at the Instructional Farm, College of Agriculture, Vellayani, India. Seeds were extracted and washed in running tap water. Seeds were then immersed in water with a few drops of wetting agent (Labolene) for 30 min, followed by washing several times with distilled water. They were then transferred to a sterile beaker and treated with 0.08% v/v mercuric chloride for 15 min, followed by washing five times with sterile distilled water. Seeds were germinated in vitro in 150 × 25 mm test tubes containing 15 ml of Murashige and Skoog (MS) medium (Murashige and Skoog 1962), supplemented with 0.09 M sucrose, adjusted to pH 5.7 and gelled with 0.8% w/v agar (Merck) and autoclaved at 121°C and 1.06 kg cm−2 pressure for 20 min.

Single nodal cuttings, 0.5–0.8 cm in length and possessing a single axillary bud, were excised from 14-d-old in vitro-raised seedlings and were used as explants for axillary shoot proliferation. Five single-node cuttings were obtained per seedling. The explants were cultured on MS medium supplemented with different combinations of the cytokinins N 6-benzyl adenine (BA) and 6-furfurylaminopurine (Kn) at concentrations of 0.1 to 1.0 mg l−1, the auxin indole-3-acetic acid (IAA) at 0.05 to 0.1 mg l−1, and sucrose at 0.09 M. Media were adjusted to pH 5.7, gelled with 0.8% w/v agar, and autoclaved at 121°C and 1.06 kg cm−2 pressure for 20 min. The explants were implanted horizontally on the culture medium in 150 × 25 mm test tubes containing 15 ml culture medium. The cultures were incubated in a culture room maintained at light intensity of 40 µE m−2 s−1 using white fluorescent bulbs for a photoperiod of 16 h and dark period of 8 h. The temperature of the room was regulated using an air conditioner at 20 ± 2°C with a relative humidity of 60%.

Experiments were conducted in a completely randomized design (CRD), each treatment with six replications and repeated thrice. The number of days for bud initiation, number of shoots produced, length of the longest shoot, and number of leaves were recorded and the mean value calculated.

Preparation of explant for cryoconservation.

Axillary buds obtained from seedlings germinated on MS-based culture medium supplemented with 1.0 mg l−1 BA and 0.1 mg l−1 IAA (Table 1), which gave maximum number of multiple shoots (Fig. 1 B), were used as the explant for cryoconservation experiments. Axillary shoot meristems approximately 4 mm long, obtained from shoot cultures after up to three subcultures on the same medium (MS medium supplemented with 1.0 mg l−1 BA and 0.1 mg l−1 IAA), were used for encapsulation. Using a scalpel with disposable blades, 4 mm long buds were excised from the shoots, leaving behind the stem portion. The buds were then subjected to a series of treatments before cryopreservation.

Table 1. Effect of cytokinins and auxin on release of axillary buds from nodal explants of I. tinctoria after 4 wk of culturea
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Figure 1.
figure 1

A, Bud break in nodal segment of I. tinctoria. B, Shoot proliferation in MS medium supplemented with 1.0 mg l−1 BA and 0.1 mg l−1 IAA. C, Regeneration of shoot meristems of I. tinctoria in recovery medium, MS medium supplemented with 0.5 mg l−1 GA and 0.2 mgl−1 BA.

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Preconditioning of axillary buds.

Axillary buds were inoculated onto half-strength (half the macro and micro nutrients and full-strength vitamins) MS medium (devoid of glycine) supplemented with 0.5 M sucrose and 0.65% w/v agar (Merck) in Petri plates. Cultures were incubated at 25 ± 2°C under 12 h photoperiod at a photon flux intensity of 30–50 µE m−2 s−1 for 3 wk.

Encapsulation of preconditioned axillary meristems.

Preconditioned axillary buds were suspended in calcium-free, half-strength MS medium that was devoid of glycine and supplemented with 0.5 M sucrose and 2.5% w/v sodium alginate (medium viscosity from Sigma, St. Louis, MO). This mixture was dispensed with a 5 ml micropipette into encapsulation medium, which was half-strength liquid MS medium supplemented with 0.1 M calcium chloride and 0.29 M sucrose, in a 100 ml Erlenmeyer flask.

Preculture of encapsulated axillary meristems.

After 30 min in the encapsulation medium, 50 encapsulated axillary shoot meristems were precultured in 25 ml of half-strength MS liquid medium, devoid of glycine and supplemented with 0.75 M sucrose and 3% v/v DMSO, in a 100 ml Erlenmeyer flasks for 24 h without agitation. Beads were then transferred to fresh medium of same composition in a new 100 ml Erlenmeyer flask and incubated in darkness at 4°C for 3 d.

Dehydration of precultured encapsulated meristems.

To determine the optimum drying time, encapsulated, precultured axillary shoot meristems were desiccated for 0 to 5 h in a sterile, laminar flow cabinet. Liquid nitrogen tolerance was tested for each drying time at 1-h intervals. Moisture content of the beads after every 1 h was determined on a fresh weight basis from three replicates of beads dehydrated prior to oven drying at 103°C for 2 h.

Cryopreservation and recovery.

Dehydrated beads were transferred to 4 ml cryovials and directly immersed in liquid nitrogen, where they were stored for at least 1 h. Cryotubes were removed from liquid nitrogen and transferred to water under constant circulation in a water bath maintained at 40°C for 30–60 s to achieve rewarming. The rewarmed encapsulated meristems were transferred to recovery medium, consisting of half-strength MS supplemented with 0.5 mg l−1 gibberellic acid (GA), 0.2 mg l−1 BA, and 0.09 M sucrose, gelled with 0.6% w/v agar, and incubated in a culture room under diffused light intensity for 1 wk (30 µE m−2 s−1) and then under bright light (60 µE m−2 s−1) for the remaining recovery period. Each treatment consisted of ten beads and was repeated three times.

Results were expressed as survival percent and/or regeneration into shoots. Survival was estimated as percent of treated shoots remaining green and showing early symptoms of in vitro response (shoot initiation). Regeneration was estimated as percentage of the surviving meristems that differentiated into shoots in 4 to 6 wk of culture. The regenerated shoots were again subcultured in the same medium for a further 4 wk. CRD was used to analyze the data.

Isolation of genomic DNA for RAPD technique.

Young leaves from the cryopreserved and regenerated plants as well as control plants were used for DNA extraction. A cetyltrimethylammoniumbromide (CTAB) protocol (Murray and Thompson 1980) was adopted with slight modification for DNA extraction. Leaf material (0.5 g) was first washed in running tap water and then in distilled water two to three times after chopping the leaves to a size of 1 cm2 using a sterilized scalpel with disposable blades. They were then dried by spreading on tissue paper. Dried leaves were then pulverized in liquid nitrogen in a precooled mortar and pestle by rapid grinding to a fine powder. The dry powder was transferred to a 2-ml centrifuge tube, mixed thoroughly with 1 ml extraction buffer (0.7 N NaCl, 1% CTAB, 50 mM Tris–HCl (pH 8.0), and 10 mM EDTA), 100 µl (% or conc) PVP, 50 µl (% or conc) SDS, and 20 µl β mercaptoethanol and incubated in a water bath at 65°C for 1 h. The mixture was then centrifuged at 15,000 rpm for 10 min at 4°C. The upper phase was collected and subjected to repeated chloroform/isoamyl alcohol (24:1) extraction, until the interphase disappeared. The final supernatant was collected and added to one-tenth volume of 3.0 M sodium acetate and double volume of chilled absolute isopropyl alcohol. Extracts were placed in a refrigerator at 4°C for 30 min and then centrifuged at 10,000 rpm for 10 min at 4°C to pellet the DNA. The supernatant was discarded and the pellet washed in 70% alcohol and centrifuged at 10,000 rpm for 5 min at 4°C. The supernatant was again discarded and the pellet air dried for 20 min. The pellet was then dissolved in 0.5 ml of 1× Tris–EDTA buffer (10 mM Tris HCl, 1 mM EDTA—pH 8.0) and stored at −20°C.

Agarose gel electrophoresis.

Agarose gel electrophoresis was carried out in a horizontal gel electrophoresis unit (Bangalore Genei, Bangalore, Karnataka, India). The TAE-buffered gels (0.04 Tris acetate, 0.001 M EDTA at pH 8.0) were either 0.7% w/v agarose for visualizing genomic DNA and 1% w/v agarose for amplified products. After cooling to about 50°C, ethidium bromide was added to a final concentration of 0.5 μg ml−1. The DNA sample was mixed with the required volume of gel loading buffer (6.0× loading dye 1.17 M (40%) sucrose, 0.25% bromophenol blue). Each well was loaded with 20 μl of sample. One of the wells was loaded with 5 μl of molecular weight marker along with required volume of gel loading buffer. Electrophoresis was performed at 75 V until the loading dye reached three fourths of the length of the gel. The gel was visualized using an ultraviolet–visible (UV–Vis) transilluminator (Appligene Oncor, France).

Random amplified polymorphic DNA analysis.

DNA amplification was done using arbitrarily designed primers (Operon Inc., California, USA) adopting the procedure of William et al. (1990) with required modifications.

Polymerase chain reactions (PCR) of genomic DNA were performed in 12.5 µl reaction mixtures containing 1.25 µl 10× PCR buffer, 5 pM primer, 200 µM each of dNTPs, 0.75 units of Taq, and 20 ng genomic DNA. Amplification was performed in a programmable thermal controller (MJ Research, Inc., Waltham, Massachusetts, USA) for an initial denaturation at 95°C for 3 min followed by 45 cycles of denaturation at 95°C for 1 min, annealing at 40°C for 1 min, and extension at 72°C for 2 min. The synthesis step of the final cycle was extended further by 5 min. Finally, the products of amplification were cooled to 4°C. The DNA fragments produced and the PCR molecular weight markers were visualized by agarose gel electrophoresis, stained with ethidium bromide, and photographed with the help of gel doc system.

The genetic fidelity of cryopreserved axillary shoot meristem was verified by comparing the RAPD banding pattern with that of the noncryopreserved (control) plantlets.

Results

Axillary shoot proliferation.

Explants cultured on MS medium containing growth regulators (BA, Kn, IAA) singly as well as in combination showed various type of responses with respect to number of days for bud initiation, number of shoots regenerated per culture, average shoot length, and number of leaves per shoot. Irrespective of the presence or absence of growth regulators in the culture medium, all the cultures recorded 100% survival.

Bud initiation is indicated by swelling of axillary buds, which later developed into shoots (Fig. 1 A). The earliest bud initiation took place at an average of 4.8 d when cultured on MS medium supplemented with 1.0 mg l−1 BA and 1.0 mg l−1 Kn. Treatments supplemented with either BA, Kn, or BA in combination with IAA gave the earliest bud initiation (Table 1). Bud break took 7.0 to 7.8 d for explants cultured on medium devoid of growth regulators as well as in treatments with Kn–IAA combinations (Table 1). Development of new shoots started within 15–20 d of culture. During the proliferation stage, axillary buds grew out from the original explant and, after 2 wk of incubation, several new buds could be seen developing from the base of the pre-existing ones. Shoots developed from the newly formed buds.

Explants inoculated on the cultures supplemented with growth regulators exhibited different levels of shoot proliferation (Table 1). The maximum response was observed from MS medium supplemented with 1.0 mg l−1 BA and 0.1 mgl−1 IAA (Fig. 1 B). Maximum number of shoots at 4.7, maximum length of the longest shoot at 5.18 cm, and maximum number of leaves at 4.03 were also obtained from explants cultured by this treatment. This clearly indicated that BA in combination with a low concentration of auxin gave maximum shoot proliferation. Shoot proliferation was very low (1.17 to 1.67 shoots per culture), in the treatments involving Kn alone or Kn in combination with IAA. Explants cultured in medium free of growth regulators invariably produced only one shoot per culture.

Single-node segments were dissected out from the axillary shoots developed after 4 wk of culture and inoculated on the same medium, MS medium supplemented with 1.0 mgl−1 BA and 0.1 mgl−1 IAA, for further multiplication. Shoot proliferation at the rate of 1.5 times per subculture at 4 wk interval was observed.

Regeneration of encapsulated, dehydrated, and cryopreserved axillary shoot mersitem.

Axillary shoot meristems obtained from the proliferated shoot cultures after up to three subcultures were used for encapsulation–dehydration experiments. Encapsulated axillary buds of I. tinctoria were subjected to desiccation for 0 to 5 h, during which the moisture content was reduced gradually from 66.0% to 9.4%. The survival percentage was determined after 2 wk of culture in the recovery medium.

With no desiccation (0 h), survival of nonfrozen control plants was 93.3%, and regeneration was 78.3%. However, explants that were not first dessicated resulted in 0% survival after freezing in liquid nitrogen. Maximum survival after freezing in liquid nitrogen was obtained from treatment in which the encapsulated explants were desiccated for 4 h. This reduced the moisture content to 16.0% and facilitated a survival rate of 56.7%. At the same desiccation duration, control plants, which were regenerated without exposure to liquid nitrogen, survived at 50.0%, showing that the 4-h desiccation is highly effective for protecting the explants from freezing injury (Table 2). Indeed, explants given a 5 h desiccation survived at 20.0% after freezing in liquid nitrogen, while 33.3% of nonfrozen controls survived. The meristems subjected to desiccation duration of 0–3 h, at a moisture content declining from 66.0% to 25.0%, did not survive at all when frozen in liquid nitrogen. Additionally, the control plants, which were not exposed to liquid nitrogen, recorded a declining trend in the survival rate, from 93.3% to 63.3%, due to increasing effects of desiccation stress.

Table 2. Effect of desiccation on encapsulated Indigofera meristems on moisture content (%), survival (%), and plant regeneration (%) with and without cryopreservationb
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The regeneration rate was estimated as percentage of surviving meristems that differentiated into shoots after a further 4 wk of culture in the same medium and conditions. At 0–3 h desiccation, control plants, not frozen in liquid nitrogen, exhibited a declining trend in regeneration, ranging from 78.3% to 63.6%. After 4 h desiccation, 62.2% of the meristems that survived cryopreservation differentiated into shoots (Fig. 1 C); while in nonfrozen controls, 58.9% of the surviving meristems differentiated into shoots. After 5 h desiccation, the regeneration was 47.2% and 33.3% for meristems cryopreserved in liquid nitrogen and noncryopreserved controls, respectively. After 4 wk of culture, regenerated shoots were subcultured onto fresh medium of same type for a further 4 wk.

Genetic stability of cryopreserved materials.

To verify the genetic fidelity of plantlets regenerated from cryopreserved meristems, the RAPD patterns were compared with those of control plantlets. All six primer pairs produced clear and reproducible bands. Primers and their nucleotide sequence are presented in Table 3.

Table 3. Nucleotide sequence of decamer primers employed for verification of genetic stability of plantlets regenerated from control and cryopreserved axillary meristems of I. tinctoria
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The number of bands of each primer varied from three to six. RAPD fragment patterns for plantlets from cryopreserved materials were identical to those of control plants for all six primers tested. Figure 2 illustrates amplified band patterns produced by six primers in plantlets regenerated after cryopreservation and control plants. No differences were observed in the banding pattern of control and cryopreserved samples.

Figure 2.
figure 2

Pair wise comparison of RAPD banding pattern (six primers) of plants regenerated in vitro from cryopreserved and noncryopreserved shoot meristems of I. tinctoria.

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Discussion

Nodal segments cultured on inoculation medium gave maximum proliferation on media supplemented with BA and IAA. BA when used alone induced multiple shoots that were weak and fragile. The synergistic effect of BA and IAA in multiplication and growth of shoots has been established in this study. A similar trend of cytokinin–auxin synergism was noticed for in vitro cultures of the legumes Clitoria ternatea (Kalamani and Gomez 2002; Rout 2005) and Pterocarpus marsupium (Anis et al. 2005). However, a lower ratio of BA to IAA resulted in a decline in the number of shoots per culture, due to basal callusing. Though synergistic effects of BA in combination with Kn has been reported in many plant species (Jagatram et al. 2003; Karuppusamy et al. 2006), in this context BA–Kn combinations did not bring about any significant enhancement in the number of shoots produced per culture. However, the combination produced longer shoots compared to those with Kn alone. The culture medium supplemented with 1.0 mg l−1 BA and 0.1 mg l−1 IAA resulted in sturdy shoots, while that supplemented with BA alone produced succulent, brittle-appearing shoots.

Axillary shoot meristems from the in vitro cultures were subjected to a series of conditioning stages and could survive encapsulation, dehydration, and cryopreservation. Encapsulating the explants allows exposure to extreme treatments, including preculture with high sucrose concentrations and desiccation to low moisture content that would be highly damaging or lethal to non-encapsulated samples (Engelmann et al. 2008). In this study, encapsulated axillary buds to be cryopreserved were precultured in a medium supplemented with 0.75 M sucrose to provide a stress conditioning effect. Encapsulated noncryopreserved meristems recorded a survival rate of 93.3%. Only 4–5 h desiccation treatments resulted in survival and regeneration, while desiccation for lesser periods did not support any survival or regeneration after the cryopreservation process. The survival rate was zero percent for tissues that had up to 25.0% moisture content (resulting from 3 h desiccation). Maximum freeze tolerance, indicated by higher survival and regeneration rates, was observed after 4 h desiccation and a moisture content of 16.0%. The reduction of the water content of synthetic seeds to a minimal level is a necessary step for successful cryopreservation of the encapsulated meristems (Bouafia et al. 1996). Cryopreservation by the encapsulation–dehydration technique requires dehydration of the encapsulated meristems such that meristematic cells have greatly reduced cell water but do not reach the permanent wilting point, and the cells vitrify on exposure to liquid nitrogen (Benson 1999). Dehydration improved the freezing tolerance of the encapsulated meristems in this study. Similar observations were reported for encapsulated tissues precultured in high sugar, especially sucrose-enriched media, in certain plant species (Reed et al. 2005; Adela and Deliu 2006; Nair and Reghunath 2007).

It is important that cryopreserved meristems directly produce plants identical to the nontreated controls (Haskins and Kartha 1980; Towill 1984). In the present study, encapsulated cryopreserved meristems developed shoots in 4–6 wk of transfer to regeneration medium. Callus intervention did not occur when the encapsulated axillary shoot meristems resumed growth on transfer to recovery medium. Callus formation is very important concern in plant regeneration after cryopreservation, as it may provide for somaclonal variation (Fourré et al. 1997). No morphological abnormalities were observed during recovery and regeneration. Harding (1996) reported that PCR and RAPD technology are useful for the detection of genetic changes and the assessment of the genetic stability of plants recovered from in vitro cultures. Sadia et al. (2003) used two primers each of ten bases (OPA 2 and OPC 16) from Kits A and C (Operon Technology Inc., Alameda, CA) to screen for DNA modification by RAPD analysis in a cryopreservation study on Solanum tuberosum. Any genetic stability assessment should not be based only on molecular studies but also on physiological, morphological, and agronomic examination of the plants regenerated (Zhai et al. 2003). The RAPD technique, though considered to be a fast, simple, and efficient method for evaluating genetic stability of cryopreserved material, will only complement other genetic stability evaluation methods (Hirai and Sakai 2000). Within the primers used in this study, no differences were observed in RAPD analysis between cryopreserved and noncryopreserved (control) plantlets. However, further study is necessary to confirm their genetic stability by other analyses.

In conclusion, dehydration before immersing in liquid nitrogen is the major deciding factor for obtaining a higher rate of survival and plant regeneration in cryopreservation. Fully hydrated meristems did not survive freezing in liquid nitrogen (−196°C), and the best recovery rate was attained with encapsulated meristems with 16.0% moisture content. Properly dehydrated meristems, when subjected to freezing in liquid nitrogen and on transfer to recovery medium, developed shoots without intermediary callus formation.

Our results demonstrated that encapsulation–dehydration is a compatible technique for cryopreservation of axillary meristems of I. tinctoria. The procedure is simple, rapid, and reproducible. It could allow the establishment of Indigofera germplasm banks in the future. Further studies are still necessary to optimize the protocol established and to test the optimal protocol with other accessions of I. tinctoria.