Cryopreservation of asian Dioscorea bulbifera l. and D. alata l. by vitrification: importance of plant growth regulators.

The aim of this study was to develop cryopreservation protocols for Asian races of Dioscorea bulbifera and D. alata with high survival and plant regeneration after cryopreservation. Using a vitrification procedure, survival of shoot tips postcryopreservation of up to 89% in D. bulbifera and up to 82% in D. alata were recorded when excised shoot tips were pretreated overnight with 0.3 M sucrose in MS medium, followed by loading with 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degrees C, exposure to PVS2 solution for 90 min at 0 degrees C, immersion in liquid nitrogen for 1 h, rewarming at 40 degrees C for 2 min, unloading in medium with 1.2 M sucrose for 20 min and culturing on growth recovery medium. During growth recovery, 58% shoot regeneration was obtained in D. bulbifera when cryopreserved shoot tips were initially cultured for 40 days on MS medium with 1.5 mg/L BAP, 0.15 mg/L NAA and 0.2 mg/L GA3 followed by culturing on a medium with 0.05 mg/L BAP and 0.15 mg/ L NAA. However, a maximum of 39% shoot regeneration was recorded in D. alata when cryopreserved shoot tips were initially cultured for 40 days on medium M2 (MS containing 1/5 NH4NO3 and 40 g/L sucrose) supplemented with 1.0 mg/L BAP, 1.0 mg/L zeatin, 0.15 mg/L IAA and 0.2 mg/L GA3. Subsequently, the regenerating shoots were cultured for 30 days on medium M2 with 1.0 mg/L BAP, 0.3 mg/L zeatin, 0.02 mg/L NAA and 0.2 mg/L GA3 followed by culturing for another 30 days on medium with 0.5 mg/L BAP, 0.02 mg/L NAA and 0.2 mg/L GA3. Finally, transfer onto medium with 0.05 mg/L BAP and 0.15 mg/L NAA stimulated production of fully grown plantlets. Alteration of post-thaw culture media with plant growth regulators and their application at various stages of growth recovery was crucial for regeneration of shoot tips and formation of plantlets in D. alata.

Cryopreservation of embryogenic cultures of Dioscorea bulbifera l. by encapsulation- dehydration.

Embryogenic cultures of Dioscorea bulbifera were cryopreserved using an encapsulation- dehydration procedure with subsequent plant regeneration. Embryogenesis was induced by culturing in vitro grown axillary bud meristems on MS medium supplemented with 2.0 mg per liter 2,4-D. After cryopreservation, recovery growth of embryogenic culture up to 53.3 percent was recorded when excised proliferating embryogenic cultures of 1.5-2.0 mm in diameter were: encapsulated in 3 percent calcium alginate containing 0.15 M sucrose followed by preculturing with 0.5 M sucrose for 3 d; dehydrated in the laminar air flow for 4 h, thereby reducing the bead moisture content to 19.4 percent ( fresh weight basis); plunged into liquid nitrogen; thawed at 40 degree C; and cultured on recovery growth medium, i.e. MS supplemented with 2.0 mg per liter 2,4-D and 0.3 mg per liter BAP. However, preculturing for an extended period of 7 d increased the recovery growth further to 67.8 percent. During recovery growth the embryogenic tissue protruded out of the beads without loss of structural integrity of the cryopreserved embryos. Subculturing of these cultures on to embryo conversion medium, i.e. MS medium with 0.5 mg per liter zeatin and 400 mg per liter glutamine, resulted in production of plantlets through embryo conversion. The regenerated plantlets exhibited the same morphology as that of originally maintained in vitro plantlets and were established in vivo, in a net house with 80 percent success.

Cryopreservation of Dioscorea rotundata poir.: a comparative study with two cryogenic procedures and assessment of true-to-type of regenerants by RAPD analysis.

The aim of this study was to develop a cryopreservation protocol for Dioscorea rotundata with maintenance of genetic stability of regenerated plants after cryopreservation. In vitro shoot tips were cryopreserved using vitrification and encapsulation-dehydration to compare the efficacy of the two methods. Both methods produced high levels of plant regeneration from cryopreserved shoot tips. The regeneration level obtained using vitrification (71%) was not significantly different from that obtained using encapsulation-dehydration (67%). Genetic stability of plants derived from cryopreserved shoot tips was evaluated using RAPD markers. Analysis of 50 cryopreserved-derived and 20 in vitro- maintained (control) plantlets showed that 10 primers produced 77 clear, reproducible bands, with the amplification products being monomorphic for all the plantlets tested. A total of 5,390 bands obtained from this study exhibited no aberration in RAPD banding. Thus, the present study showed that both vitrification and encapsulation-dehydration methods are equally applicable to D. rotundata for cryopreservation. The in vitro plantlets derived from cryopreservation were genetically stable at the molecular level tested.

Regeneration of Dioscorea floribunda plants from cryopreserved encapsulated shoot tips: effect of plant growth regulators.

The encapsulation-dehydration protocol for the cryopreservation of in vitro shoot tips of Dioscorea floribunda was optimized. Maximum survival of 87% was obtained when overnight pretreatment with 0.3 M sucrose was followed by encapsulation, preculture in 0.75 M sucrose for 4 d, dehydration in a laminar air flow for 5.5 h, quenching in liquid nitrogen and thawing at 40 degrees C. During recovery growth, 29% shoot formation was obtained when cryopreserved shoot tips were initially cultured for 25 d on a medium with 1.5 mg per liter (-1) BAP, 0.2 mg per liter(-1) NAA and 0.2 mg per liter(-1) GA3 followed by culturing for 15 d on a medium with reduced BAP (1 mg per liter(-1)) but increased NAA (0.5 mg per liter(-1)) and GA3 (0.3 mg per liter(-1)). Finally, transfer on to a medium with further reduced doses of BAP (0.05 mg per liter(-1)) and NAA (0.15 mg per liter(-1)) but without GA3 stimulated production of fully grown plantlets. All plants regenerated without callus formation. Modification of post-thaw culture media with plant growth regulators was essential for regrowth of shoot tips to plantlets.

Cryopreservation of in vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant: effect of cryogenic procedure and storage duration.

In vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant, were successfully cryopreserved using the vitrification and the encapsulation-dehydration techniques with subsequent high frequency plant regeneration. Using vitrification, post-liquid nitrogen (LN) shoot regeneration up to 83% was recorded when excised shoot tips were pretreated overnight on MS medium containing 0.3 M sucrose followed by loading with MS containing 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degree C, dehydration with PVS2 for 90 min at 0 degree C and quenching in LN. After 1 h of storage in LN, the shoot tips were rewarmed in a water-bath at 40 degrees C, unloaded with 1.2 M sucrose solution for 20 min and cultured on recovery growth medium. While using encapsulation-dehydration, the highest regeneration frequency recorded was 76% when sucrose-pretreated shoot tips were encapsulated with 3% calcium alginate, precultured in 0.75 M sucrose for 3 days, dehydrated to 25% moisture content (FW basis) under the laminar air flow, stored in LN for 1h and rewarmed at 40 degree C. The cryopreserved shoot tips maintained their viability and an unaltered level of regeneration capability after up to one year of storage in LN.

Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera l. Using RAPD, biochemical and morphological analysis.

Embryogenic tissues of Dioscorea bulbifera were cryopreserved using the encapsulation-dehydration technique. Genetic stability of plants regenerated from cryopreserved embryogenic tissues was assessed using molecular, biochemical and morphological analysis. The random amplified polymorphic DNA (RAPD) analysis of 60 cryopreserved-derived and 20 in vitro grown (control) plantlets showed that 10 primers produced 62 clear reproducible DNA fragment profiles. The amplification products were monomorphic for all the plantlets except one. A total of 4960 DNA fragments were obtained from this study showing no variation in RAPD profiles. The diosgenin content of cryopreserved-derived plants, analyzed using HPLC, was similar to that of control plants. Morphology and the ability to form microtuber were also found to be unaltered in cryopreserved embryo-derived plantlets. Thus, the D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genetically stable at the molecular, biochemical and morphological levels.

Role of hla class-I antigens in delusional disorder.

The investigation was conducted to find out whether there is any association between delusional disorder and HLA antigens. The sample comprised 50 patients with delusional disorder and 282 control samples collected from normal controls. Statistical analysis revealed that the frequency of A3 antigen of the locus A are significantly higher. In case of HLA - B locus significantly higher frequency of B5 and B21 antigens have also been observed. The present study shows that there may be some association of HLA class-1 antigens with delusional disorder.