Inducinduction of Protocorm-like Bodies, Synthetic Seed Production and Cryopreservation in Phalaenopsis bellina (Rchb.f.) Christenson

Phaleanopsis bellina (Rchb.f.) Christenson is one of the important orchid species originating from Malaysia. It is an orchid which is known to be difficult to propagate even using in-vitro techniques. This study was carried out to establish an in-vitro system for induction and proliferation of Phaleanopsis bellina. Furthermore attempt was made to convert the PLBs into synthetic seed as well as to establish a method to store the synthetic seeds both for short and long-term. An in-vitro culture procedure was established to induce protocorm-like bodies (PLBs) from leaf segments of Phalaenopsis bellina directly from epidermal cells without intervening callus on half-strength modified Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA; 0, 0.1, 1.0 mg/L) and thidiazuron (TDZ; 0, 0.1, 1.0, 3.0 mg/L). The best response was established at 3 mg/l TDZ which induced 78% of leaf segments to form a mean number of 15.5 PLBs per explant after 16 weeks of culture. No PLBs were found when leaf segments were cultured on half-strength modified MS medium supplemented with 0.1 and 1 mg/l NAA. The best induction percentage for auxin: cytokinin combination was using 1.0 mg/l NAA and 3.0 mg/l TDZ which gave 72% induction with 11 PLBs per explant. Once successfully induced, it is important to maximize the proliferation and utilization of the PLBs. In this regard, semi-solid half-strength MS and liquid Vacin and Went (VW) media with and without sucrose were used in order to find the highest survival and number of PLBs proliferation after three months in culture. Half-strength MS medium showed an average of 9 PLBs with 60% survival and mean fresh weight of 0.5g in comparison with VW medium with and without sucrose which showed an average of 4.8 and 5.3 PLBs per explant followed by 55 and 57.5% of survival respectively. Histological observations revealed that the adaxial surfaces near wounded regions had the highest number of PLBs compared to other regions of explants. Also, SEM micrographs showed that leaf derived PLB (LDP) were formed from leaf segment after 16 weeks of culture. Twelve decamer RAPD primers were used to estimate the somaclonal variation among the mother plant, the initially induced PLBs and proliferated PLBs after 3 and 6 months in culture. Eight out of twelve primers produced 172 bands with 18 polymorphic bands in all the treatments. The amplified products varied between 125 to 8000 bp. Among the primers used, P 16 produced the highest number of bands (29) while primer OPU 10 produced the lowest number (15). The range of similarity coefficient was from 0.83 to 1.0 among the different sub-cultures and mother plant. It was found that minimal or no changes occurred between the mother plant and the PLBs produced after 3 months of induction. The induced PLBs were then subcultured for six months for proliferation and this resulted in about 17% dissimilarity with mother plant. Micropropagation of Phalaenopsis bellina can be carried out successfully using ½ strength MS media for 6 months but further proliferation may result in somaclonal variation which might change the prolific characteristic of this orchid. In-vitro PLBs of Phalaenopsis bellina obtained from PLBs explants on modified semi-solid half-strength MS medium, 4-5 mm in diameter, were selected for encapsulation with different concentration of sodium alginate (3, 4 and 5%) and calcium chloride (25, 50, 75 and 100 mM) and none encapsulated PLBs as a control. PLBs encapsulated with 4% sodium alginate in 75 mM calcium chloride showed the best encapsulation combination on survival of PLBs after two weeks of incubation at 5°C giving the survival of 70 and 65%. Subsequently, PLBs encapsulated with 4% sodium alginate + 75 mM calcium chloride were used for evaluating the storage durations (15, 30, 45 and 60 days) and temperatures (5, 15, and 25°C). The highest PLBs survival of 70% was observed after 15 days storage at 5°C followed by 30 days of storage at 5°C with 50% of survival frequency. The best survival percentage in case of storage temperature for the synthetic seeds were 5°C >15°C > 25°C. The highest PLB fresh weight was 0.36g which belong to the encapsulated PLBs stored for 15 days at 5°C. In addition the lowest fresh weight of 0.19g belonged to encapsulated PLBs stored for 60 days at 25°C. The moisture content (MC) of the PLBs decreased sharply with increasing storage temperature and duration to 10.2% after 60 days at 25°C. As synthetic seeds should technically function as normal seeds, it would be useful to establish a method to store them both for short and long time. This study indicated that Phalaenopsis bellina PLBs were successfully cryopreserved by encapsulation-dehydration method. The highest re-growth of cryopreserved explants was observed when PLBs were pretreated in half-MS medium supplemented with 30 g/l sucrose for 3 days followed by preculturing on 0.75 M sucrose for 3 days. In addition, the highest sucrose concentration at 0.78 g/l was measured using HPLC in 100g of PLBs precultured on 0.75 M for 3 days. Encapsulated PLBs were dehydrated with silica gel for 6 h prior to immersion in liquid nitrogen for 1 h. Protocorm viability was tested by the 2, 3, 5-triphenyltetrazoliumchloride (TTC) assay and re-growth ability was assessed by determining the survival percentage after 2 weeks recovery. The survival rate of cryopreserved PLBs was 30% while highest viability percentage by TTC assay was 46.6%. Finally, dehydration time after freezing and thawing affected positively electrolyte leakage (EL) of the PLBs. Non-dehydrated PLBs showed the highest EL (85.3%) while the lowest amount (53%) was achieved after 6 h dehydration with silica gel

Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb.f.) christenson by encapsulation-dehydration

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulation-dehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth

Precision Agriculture Application for Sustainable Nitrogen Management of <i>Justicia brandegeana</i> Using Optical Sensor Technology

Over-fertilization is a common practice in ornamental nursery production. Oftentimes, visual analysis is used to determine plant nutrient levels, leading to less accurate estimates of fertilizer application. This study focused on exploring the suitability of two non-destructive sensors, Soil Plant Analysis Development (SPAD-502) and GreenSeekerTM, for measuring plant tissue nutrient uptake. Florikan Top-Dress fertilizer 12N-6P-8K was applied to Justicia brandegeana in various increments (0, 10, 20, 30, 40, and 50 g) to simulate plants with deficient to excessive nitrogen rates. Various parameters were recorded including Normalized Difference Vegetation Index (NDVI) and SPAD readings, soil leachate analysis (nitrates and phosphate), and total leaf carbon:nitrogen (C:N). The NDVI and SPAD readings were recorded biweekly for three months after the initial controlled release fertilizer (CRF) treatments. Leaf C:N was analyzed through dry combustion while nitrates and phosphate were determined from soil leachate. Results suggest that the smaller amount (20 g) of CRF is as effective in providing N to J. brandegeana as larger amounts (30, 40, 50 g). Implementation of this fertilizer regimen will result in reduced agricultural nutrient runoff and overall negative environmental impacts. Application of optical sensor technology using SPAD and GreenSeekerTM showed promising results in determining the fertilizer requirements of J. brandegeana. This method could serve as a guideline for nursery producers and landscape personnel as a fast and non-destructive tool for sustainable fertilizer management practices within the ornamental plant industry

Precision Horticulture: Application of Optical Sensor Technology for Nitrogen Monitoring Status in Cocoplum, a Native Landscaping Plant

Cocoplum (Chrysobalanus icaco) is an ecologically significant native species to Southern Florida. Application of precision agriculture technologies such as optical sensors reduces the cost of over-fertilization and nutrient runoff. The aim of this work was to establish a base line sensor value for fertilizer treatment in cocoplum by monitoring chlorophyll content using the Soil Plant Analytical Development (SPAD), atLEAF, and Normalized Difference Vegetation Index (NDVI) sensors. Initial slow-released fertilizer treatment 8N-3P-9K was used at 15 g (control), 15 g (supplemented with +15 g × 2; T1), 15 g (+15 g; T2), 30 g (+15 g × 2; T3), 30 g (+15 g; T4), and 45 g (+15 g × 2; T5). Evaluations were conducted at 0 (base reading), 30, 60, 90, 120, 150, and 180 days after treatment. Growth parameters, optical non-destructive chlorophyll meters, leaf and soil total nitrogen and total carbon, and total nitrogen of leachate were analyzed. The results demonstrated that the treatment using 30 g slow-released fertilizer (8N-3P-9K) supplemented twice with 15 g in November and March after the first fertilization in October provided the least contamination through runoff while still providing adequate nutrients for plant growth compared to higher fertilizer concentrations. These results demonstrate that the highest treatment of nitrogen can cause considerable losses of N, causing extra costs to producers and environmental damage due to the flow of nutrients. Thus, techniques that help in N monitoring to avoid the excessive use of nitrogen fertilization are necessary. This study can serve as a basis for future research and for nurseries and farms, since it demonstrated from the monitoring of the chlorophyll content by optical sensors and by foliar and substrate analysis that lower treatments of nitrogen fertilization are sufficient to provide nutrients suitable for the growth of cocoplum plants

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

Detection of somaclonal variation by random amplified polymorphic DNA analysis during micropropagation of Phalaenopsis bellina (Rchb.f.) Christenson

Phalaenopsis bellina (Rchb.f.) Christenson orchid species are known for their beautiful flower shape, graceful inflorescence and fragrance. Protocorm-like bodies (PLBs) of P. bellina were induced from leaf segments. The PLBs were then subjected to proliferation using ½ strength Murashige and Skoog (MS) media with two subcultures at three months intervals. Twelve decamer random amplified polymorphic DNA (RAPD) primers were used to study somaclonal variation among the mother plant, the initially induced PLBs and proliferated PLBs after 3 and 6 months in culture. Eight out of twelve primers produced 172 bands with 18 polymorphic bands in all the treatments. The amplified products varied between 125 to 8000 bp. Among the primers used, P 16 produced the highest number of bands (29), while primer OPU 10 produced the lowest number (15). The range of similarity coefficient was from 0.83 to 1.0 among the different sub-cultures and mother plant (MP). It was found that minimal or no changes occurred between the MP and the PLBs produced after 3 months of induction. The induced PLBs were then subcultured for six months for proliferation and this resulted in about 17% dissimilarity with MP. It is reported that micropropagation of P. bellina can be carried out successfully using ½ strength MS media for 6 months but further proliferation may result in somaclonal variation which might change the prolific characteristic of this orchids

Preliminary analysis of cryopreservation of Dendrobium Bobby Messina orchid using an encapsulation-dehydration technique with Evans blue assay

In vitro grown protocorm-like bodies (PLBs) of Dendrobium Bobby Messina hybrid were cryopreserved in liquid nitrogen (LN) at -196°C by an encapsulation-dehydration technique. PLBs (1 to 2 and 3 to 4 mm) were precultured in half strength semi-solid MS media supplemented with six different concentrations of sucrose (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 M). The PLBs were then encapsulated to form the beads in halfstrength liquid MS media supplemented with different concentrations of sodium alginate (2.5, 3.0 and 3.5%). The beads were placed in 2 ml cryovials and plunged into LN for 24 h. The beads were then thawed in a 40°C water bath for 90 s and were placed in recovery media composed of half strength semisolid MS media supplemented with 2% sucrose for four days under dark condition. After 12 days, the Evans blue dye assay was carried out to determine the viability of the PLBs. The highest viability was found in 1 to 2mm PLBs precultured in half strength semi-solid MS media supplemented with 1.0 M sucrose and encapsulated in 2.5% sodium alginate. Biochemical content analyses (chlorophyll, total soluble protein and peroxidase activities) were done to investigate the physiological responses of the PLBs after cryopreservation