Agrobacterium-Mediated Transformation of the Recalcitrant Vanda Kasem’s Delight Orchid with Higher Efficiency

The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem’s Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusAgene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD’s PLBs. Based on the results, 3-4mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit a

Effects of ascorbic acid on PVS2 cryopreservation of dendrobium Bobby Messina’s PLBs supported with SEM analysis.

Regrowth of the cryopreserved protocorm-like bodies (PLBs) of Dendrobium Bobby Messina was assessed based on the plant vitrification solution 2 (PVS2) optimisation conditions. The optimized protocol obtained based on TTC spectrophotometrical analysis and growth recovery were 3–4 mm of PLBs size precultured in 0.2 M sucrose for 1 day, treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min and subsequently dehydrated with PVS2 at 0 °C for 20 min prior to storage in liquid nitrogen. Following rapid warming in a water bath at 40 °C for 90 s, PLBs were treated with unloading solution containing half-strength liquid MS media supplemented with 1.2 M sucrose. Subsequently, the PLBs were cultured on half-strength semi-solid MS media supplemented with 2 % (w/v) sucrose without any growth regulators and resulted in 40 % growth recovery. In addition, ascorbic acid treatment was used to evaluate the regeneration process of cryopreserved PLBs. However, growth recovery rates of non-cryopreserved and cryopreserved PLBs were 30 and 10 % when 0.6 mM ascorbic acid was added. Scanning electron microscopy analysis indicates that there are not much damages observed on both cryopreserved and non-cryopreserved PLBs in comparison to PLBs stock culture

Effect of PVS2 vitrification on Brassidium shooting star orchid using protocorm-like bodies (PLBs)

A cryopreservation procedure was developed to preserve protocorm-like bodies (PLBs) of Brassidium Shooting Star using the PVS2 vitrification technique. The optimised protocol involved the preculture of 3-4mm PLBs in half-strength Murashige and Skoog (MS) semi-solid medium supplemented with 0.8M sucrose, followed by dehydration in PVS2 solution for 20 minutes at 0°C, prior to storage in liquid nitrogen. The viability of non-cryopreserved and cryopreserved PLBs was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) assay, after two weeks of recovery. The chlorophyll contents, total soluble protein and peroxidase activities of both non-cryopreserved and cryopreserved PLBs were assayed after three weeks of recovery. The results from the biochemical analyses indicated that control PLBs produced the highest viability, followed by treatment on non-cryopreserved PLBs (-LN) and cryostored PLBs (+LN), except in the peroxidase activity assay. The peroxidase activity was detected as the highest in cryostored PLBs followed by treated but non-cryopreserved PLBs, and control PLBs

Preliminary study on cryopreservation of Dendrobium Bobby Messina protocorm- like bodies by vitrification

Protocorm-like bodies of Dendrobium Bobby Messina were cryopreserved by vitrification method. In this study, protocorm-like bodies (PLBs) with the size range of 1-2 and 3-4 mm were selected from 4 weeks old culture, pretreated with half strength semi-solid Murashige and Skoog (MS) media supplemented with 0.5 M sucrose at 25°C for 24 h. Pretreated PLBs were then treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half strength liquid MS media at 25°C for 20 min. Osmoprotected PLBs were then dehydrated with plant vitrification solution 2 at 0°C for 20 min before storage in liquid nitrogen. After rapid warming in water bath at 40°C for 90 s, the PLBs were washed with half strength liquid MS media supplemented with 1.2 M sucrose and then cultured on half strength semi-solid MS media supplemented with 2% sucrose without the presence of any growth regulators. Survival of the cryopreserved PLBs was assessed based on triphenyl tetrazoliumchloride (TTC) spectrophotometrical analysis. The PLBs with 3-4 mm size range showed better viability comparative to size range 1-2 mm for both cryopreserved and non-cryopreserved PLBs. The best pretreatment concentration used in pretreatment media was 0.6 M sucrose and 1.2 M sorbitol, respectively

Preliminary study on cryopreservation of Dendrobium Bobby Messina protocorm- like bodies by vitrification

Protocorm-like bodies of Dendrobium Bobby Messina were cryopreserved by vitrification method. In this study, protocorm-like bodies (PLBs) with the size range of 1-2 and 3-4 mm were selected from 4 weeks old culture, pretreated with half strength semi-solid Murashige and Skoog (MS) media supplemented with 0.5 M sucrose at 25°C for 24 h. Pretreated PLBs were then treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half strength liquid MS media at 25°C for 20 min. Osmoprotected PLBs were then dehydrated with plant vitrification solution 2 at 0°C for 20 min before storage in liquid nitrogen. After rapid warming in water bath at 40°C for 90 s, the PLBs were washed with half strength liquid MS media  supplemented with 1.2 M sucrose and then cultured on half strength semi-solid MS media supplemented with 2% sucrose without the presence of any growth regulators. Survival of the cryopreserved PLBs was assessed based on triphenyl tetrazoliumchloride (TTC) spectrophotometrical analysis. The PLBs with 3-4 mm size range showed better viability comparative to size range 1-2 mm for both cryopreserved and non-cryopreserved PLBs. The best pretreatment concentration used in pretreatment media was 0.6 M sucrose and 1.2 M sorbitol, respectively.Key words: Cryopreservation, vitrification, Dendrobium Bobby Messina, Pretreatment

Optimization of different auxin and cytokinin combination in nutrient medium for establishment of optimal in vitro multiple plantlet in Ficus carica L. cv Siyah Orak

Ficus carica Linnaeus is a flowering plant under the Moraceae family, usually propagated conventionally from cuttings due to the seeds being non-viable. However, this method is prone to diseases, and pests, time-consuming and space-intensive. Therefore, other methods are needed to overcome these issues. This study was conducted to induce callus and multiple shoots via plant tissue culture techniques enabling mass production of fig plants. Initially, leaf segments of Ficus carica L. cv Siyah Orak were cultured on different MS media strengths (¼, ½, ¾,1 MS) to induce callus. The highest callus means weight was observed on explant cultured in ¾ MS media (875±0.036). Callus was proliferated by subculturing explant into ¾ MS media supplemented with different concentrations of TDZ (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L). MS media (3/4) supplemented with 2.0 mg/L TDZ (920±0.03) shows the best result for callus proliferation. Callus induction using transverse and longitudinal thin cell layers from nodal segments cultured on different MS media strengths (¼, ½, ¾,1 MS) shows ¼ MS as the optimum media for both tTCL (100±0) and lTCL (96.7±0.15). Friable callus (%) was observed the highest on ½ MS (63.33±0.55) and ¼ MS (76.67±0.50) media for both tTCL and lTCL, respectively. As for the number of leaves produced, both tTCL (0.83±0.0.28) and lTCL (1.00±0.33) explant showed the best results in ¼ MS media. Apical buds produced the highest mean for both the number of leaves and length of the shoot on 1MS media supplemented with 2.0 mg/L BAP (3.5±0.20, 13.73±0.66), respectively. For root formation (%) and number of roots, both show the best results in media supplemented with 2.5 mg/L IAA (10±0.31, 0.83±0.50). It can be concluded that the best shoot growth performance was observed from apical bud cultured on 1MS media supplemented with 2.0 mg/L BAP+ 2.5 mg/L IAA

Characterization of the Second Generation Cryopreserved Dendrobium Bobby Messina Using Histological and RAPD Analyses

This study was conducted to detect the morphological, histological and molecular diff erences in the second generation of the PVS2 cryopreserved Dendrobium Bobby Messina [DBM] (18 months old culture) plantlets. Morphological analyses indicated that similarities and diff erences in cryopreserved DBM plantlets comparing to control stock culture based on selected morphological criteria. Morphological criteria, such as root length, number of shoot per explant and shoot length displayed diff erences, while the other three criteria, leaf diameter, leaf length and PLBs size were similar in cryopreserved compared to the control stock culture plant. Higher amount of homogenous cell population and denser cytoplasm were observed in cryopreserved PLBs compared to control stock culture PLBs based on histological analysis. This suggests the existance of somatic embryogenesis development mechanism taking place during the recovery and regeneration of the cryopreserved PLBs. However, RAPD analyses based on 10 primers indicated that cryopreserved DBM regenerated from vitrifi cation method generated a total of 20 to 39.9% polymorphic bands as compared to stock culture indicating potential somaclonal variation. Hence, an increase percentage of polymorphics bands in cryopreserved plantlets 18 months post cryopreservation as compared to previous report of 10% polymorphic bands in cryopreserved DBM 3 months post cryopreservation

Fundamental concept of cryopreservation using Dendrobium sonia-17 protocorm-like bodies by encapsulation-dehydration technique

This study was carried out to evaluate the potential of using the encapsulation-dehydration technique on cryopreservation protocorm-like bodies (PLBs) of Dendrobium sonia-17. The survival of the PLBs was assessed based on the effects of 4 dehydration periods (0, 1, 3 and 5 h) and 4 different concentrations of 24-h sucrose pretreatment (0, 0.3, 0.5 and 0.7 M). Upon dehydration, moisture content was determined and the PLBs were evaluated for survival using absorbance values from 2, 3,5- triphenyltetrazolium chloride (TTC) assay at 530 nm and regeneration observations. Moisture content declined with the dehydration time, but the decline was not significant for encapsulated PLBs. All cryopreserved PLBs gave very low survival irrespective of the dehydration period. The best survival percentage in the cryopreservation of the PLBs of Dendrobium sonia-17 was obtained when the combination of 0.5 M sucrose pretreatment and 3 h dehydration time was applied in the experiment

Evaluation of the virulence of entomopathogenic fungus, Isaria fumosorosea isolates against subterranean termites Coptotermes spp. (Isoptera: Rhinotermitidae)

The entomopathogenic fungus Isaria fumosorosea Wize, formerly known as Paecilomyces fumosoroseus is reported as a promising biocontrol agent for controlling subterranean termites, particularly those belonging to the family Rhinotermitidae. In Malaysia, the family Rhinotermitidae includes two species of subterranean termites with extremely high economic importance; namely Coptotermes curvignathus Holmgren, and the Asian Subterranean Termite (Coptotermes gestroi Wasmann). To comprehend the potential control of this soil-dwelling fungus against these subterranean termites in Malaysia, an investigation was carried out by testing the pathogenecity of 11 isolates against these termite species. All isolates showed pathogenic potential against the termite (Mortality rate of C. curvignathus: 84.4%; C. gestroi: 67.3%). Isolate PF49 was the most effective against both species of termites and was further tested for its virulence and mycosis. The LC50 values of PF49 against C. curvignathus and C. gestroi were 7.55 × 103 and 1.09 × 102 conidia/ml, respectively. The average number of days required to complete the mycosis process in C. curvignathus and C. gestroi were 4.7 and 8 days, respectively. These fungi are believed useful for protecting living trees, plants, wood, wood structures, and other cellulosic materials susceptible to termite infestation and damage

Effects of plant growth regulators and activated charcoal on somaclonal variations of protocorm-like bodies (PLBs) of Dendrobium Sabin Blue orchid

Protocorm-like bodies (PLBs) of Dendrobium orchids are emerging as a potential source of valuable secondary metabolites. This study examined the effect of four additives namely 1-naphthaleneacetic acid (NAA), kinetin, thidiazuron (TDZ), and activated charcoal (AC) used in culture medium on genetic variability in PLBs of Dendrobium Sabin Blue. Nine (9) ISSR primers and eleven (11) DAMD primers were used to assess the genetic variability of PLBs that were subcultured over a period of two years. We confirmed that the use of kinetin in culture medium for two years resulted in the highest rate of somaclonal variation in PLBs. On the other hand, TDZ and activated charcoal registered the lowest genetic variability in PLBs. The findings of this study suggest the importance of selecting additives used in the culture medium to maintain stable genetic lines of PLBs. We recommend that the assessment of somaclonal variations should be performed for long term maintenance of tissue cultures