Micropropagation and Cryopreservation of Yukon Draba (Draba yukonensis), a Special Concern Plant Species Endemic to Yukon Territory, Canada

Yukon Draba (Draba yukonensis) is a small, short-lived perennial mustard species that is endemic to southwestern Yukon in Canada. This plant has been categorized as a species of Special Concern. It faces the threat of habitat loss due to natural and man-made causes and a population that is unevenly distributed to a few large and several small subpopulations in the area. It will therefore be judicious to undertake investigations on the conservation of this species to save it from further deterioration which may lead to its extinction. In this study, a protocol was developed for in vitro propagation and cryopreservation of Yukon Draba. The micropropagation protocol was optimized using shoot tips which enabled clonal propagation and in vitro storage of the species. Shoots grew best in the medium containing MS basal salts and had the highest multiplication with the addition of 2 µM 6-benzylaminopurine or 5 µM Kinetin with 3% sucrose. The addition of 10 µM Indole Butyric Acid (IBA) produced the highest number of adventitious roots on the shoots and the longest root length was observed at 2 µM IBA. The rooted plantlets were transferred to greenhouse and the highest survival (87.5%) was observed for the plantlets treated with a lower concentration of IBA (2 µM). Cryopreservation protocol was developed using the droplet-vitrification method for in vitro shoot tips. Two-week-old shoots had the highest survival and regrowth following exposure to plant vitrification solution 3 (PVS3) for 30 min, prior to direct immersion of the droplets into the liquid nitrogen. The optimized protocols for the micropropagation and cryopreservation may be useful for the long-term germplasm conservation and reintroduction of this species in its natural habitat.

Factors Influencing Somatic Embryo Maturation in Sugi (Japanese Cedar, Cryptomeria japonica (Thunb. ex L.f.) D. Don)

This paper presents the results of several experiments identifying basal salts (BS) contained in maturation medium, polyethylene glycol (PEG) concentration, abscisic acid (ABA) concentration, additional supplementation with potassium chloride (KCl), amino acid (AA) concentration, and proliferation culture medium (PCM) as the main culture factors affecting somatic embryo maturation in sugi (Japanese cedar, Cryptomeria japonica, Cupressaceae). Highly efficient embryo maturation was achieved when embryogenic cell lines (ECLs) were cultured on media supplemented with a combination of PEG, ABA, and AAs. More than 1000 embryos per gram of fresh weight (FW) can be produced on EM maturation medium supplemented with 175 g L−1 PEG, 100 µM ABA, 2 g L−1 glutamine, 1 g L−1 asparagine, and 0.5 g L−1 arginine.

In vitro development of the Mexican endemic twig epiphyte Erycina hyalinobulbon (Orchidaceae) to promote its conservation

Background and Aims: Orchids in Mexico are mainly threatened by deforestation, changes in land use, illegal trade, deficiencies in environmental policy and legislation, and a lack of community participation in the conservation of their forests. Erycina hyalinobulbon is an endemic twig epiphyte orchid with a short life cycle and with large flowers in relation to its size, for which it has been harvested from its wild populations. The objectives of this work were to evaluate the in vitro development of E. hyalinobulbon in culture media with organic supplements, to compare sucrose vs. N’Joy Stevia® as a carbon source for its initial stages of development, and to evaluate the development of its seedlings in media enriched with plant growth regulators (PGR).Methods: For the sowing of seeds, PhytamaxTM and MS medium at 30% of its basal salts were used in combination with organic supplement (coconut milk, pineapple puree and banana puree), along with the Phy medium used as control. In order to measure the effect of sucrose vs. N´Joy Stevia® as a carbon source, these two treatments were used, with the PhytamaxTM medium. To evaluate the development of seedlings with PGR, three treatments were tested: the 100% PhytamaxTM control, 30% PhytamaxTM with 1.166 ml/l of Maxi-grow and the medium Chiu.Key results: PhytamaxTM medium added with banana promoted germination by 9.3%, being a low cost and easy production option. Ninety days after sowing, N´Joy Stevia® as a carbon source promoted germination by 8%. The best development of the seedlings was registered in the medium PhytamaxTM without PGR.Conclusions: With this study, it was possible to develop an accessible in vitro propagation system for E. hyalinobulbon, in order to sustainably manage it and favor its conservation.

DKW basal salts improve micropropagation and callogenesis compared to MS basal salts in multiple commercial cultivars of Cannabis sativa

Existing Cannabis sativa micropropagation protocols use a limited number of cultivars and are often not reproducible. Currently, MS + 0.5 μM TDZ has been reported as the optimal medium for nodal micropropagation, yet our preliminary studies with this medium have resulted in abnormal morphology and high mortality rates in multiple cultivars. Following an initial screen of basal salt mixtures (MS, B5, BABI, and DKW), we determined that DKW produced the healthiest plants. In a second experiment, the multiplication rate and canopy area of explants grown on MS + 0.5 μM TDZ and DKW + 0.5 μM TDZ were compared using five drug-type cultivars. The combined multiplication average of explants grown on DKW + 0.5 μM TDZ was 1.5x higher than explants grown on MS + 0.5 μM TDZ. Similarly, the combined average of the canopy area was twice as large on DKW + 0.5 μM TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μM) on both MS and DKW and similarly, callus growth was superior on DKW. This study presents the largest comparison of basal salt compositions on the micropropagation of five commercially grown Cannabis cultivars to date.

Reductive Dechlorination of 1,2-dichloroethane to Ethylene by Anaerobic Enrichment Culture Containing Vulcanibacillus spp.

Bioremediation has been widely used for clean-up of 1,2-dichloroethane (DCA) at contaminated sites. Only a small number of specific anaerobes, halorespiring bacteria (HRB), have been reported to degrade DCA. The goals of this research were the screening and isolation of HRB with capable dechlorination of DCA. HRB were screened and isolated from 7 enrichment cultures (ES1-ES7), from DCA-contaminated soils, on bicarbonate-buffered basal salts medium containing 10 mM acetate and 250 µM DCA under anaerobic conditions and analyzed by gas chromatography. The results showed that the mixed cultures of ES3 and ES5 could reductively dechlorinate DCA to ethylene via a direct reductive dihaloelimination pathway. In particular, ES5 showed rapid transformation of 250 µM DCA to ethylene within 6 days at 30 °C. Specific microbial populations of Vulcanibacillus spp., elucidated by polymerase chain reaction-denaturing gradient gel electrophoresis, were found only in ES3 and ES5, which was related to reductive dihaloelimination activities on DCA. A 16S rRNA gene analysis of isolate es5d8 from mixed culture ES5 revealed 2 strains of Vulcanibacillus spp. (KKU-DCA1 and KKU-DCA2) concerned with dechlorinating DCA. These findings suggested that Vulcanibacillus spp. were HRB that have the potential for detoxifying DCA.

DKW basal salts improve micropropagation and callogenesis compared to MS basal salts in multiple commercial cultivars of Cannabis sativa

Micropropagation of Cannabis sativa is an emerging area for germplasm storage and large-scale production of clean plants. Existing protocols use a limited number of genotypes and are often not reproducible. Previous studies reported MS + 0.5 μM TDZ to be optimal for Cannabis nodal micropropagation, yet our preliminary studies using nodal explants suggested this media may not be optimal. It resulted in excessive callus formation, hyperhydricity, low multiplication rates, and high mortality rates. Following an initial screen of four commonly used basal salt mixtures (MS, B5, BABI, and DKW), we determined that DKW produced the healthiest plants. In a second experiment, the multiplication rate and canopy area of explants grown on MS + 0.5 μM TDZ and DKW + 0.5 μM TDZ were compared using five drug-type cultivars to determine if the preference for DKW was genotype-dependent. Four cultivars had significantly higher multiplication rates on DKW + 0.5 μM TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 μM TDZ. The canopy area was also significantly larger on DKW + 0.5 μM TDZ for four cultivars with the combined average being twice as large as the explants grown on MS + 0.5 μM TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μM) on both MS and DKW and similarly, callus growth was superior on DKW. This study presents the largest comparison of basal salt compositions on the micropropagation of five commercially grown Cannabis cultivars to date.

Factors Influencing in vitro Germination and Seed Storage Behavior of Couroupita guianensis Aubl. – A Useful Tropical Tree Species

Couroupita guianensis Aubl. popularly known as cannonball tree is widely distributed in the tropical regions. The tree parts are commonly used to treat wounds and tumors. Leaves, flowers, and fruits contain active phytochemicals with significant biological activity. In the recent years, destruction of natural habitats by mankind has reduced its distribution. Natural propagation of C. guianensis by seeds is greatly hindered by poor seed germination and viability. Therefore, the present study was undertaken to optimize the conditions for in vitro embryo germination and to investigate the seed storage behaviour. Mature seeds inoculated on MS basal medium germinated within 10 d with a frequency of 61.6%. Supplementation of plant growth regulators (PGRs) to MS medium improved the embryo germination frequency (100%). Seedlings with highest shoot length (8.10±0.11 cm) and root length (6.27±0.14 cm) were produced in MS medium supplemented with 1.0 mg/l kinetin and 0.1 mg/l indole-3-butyric acid. Among different strength liquid MS basal salts, quarter-strength produced a greater number of secondary roots (8.00±0.28) with average root length of 17.83±0.58 cm. Seed storage behaviour studies clearly proved the recalcitrant nature as only freshly harvested mature seeds retained the germination potential upon storage at 15 ºC for up to 45 d. Desiccation of seeds on exposure to air-dry storage resulted in rapid deterioration of germination. Pre-conditioning of germinated seedlings in liquid MS basal salts was required for their survival under field conditions. Plantlets with well-developed roots were successfully acclimatized to the field with 100% survivability. This protocol facilitates conservation, sustainable utilization and re-introduction of C. guianensis into its natural habitats.

Evaluation of Endogenous Sugars, Chlorogenic Acid and Caffeine Associated with Direct Somatic Embryogenesis of Coffee (Coffea arabica L.)

Coffee is one of the most important cash crops cultivated in the world with great economic importance. During the induction of somatic embryogenesis, there are different endogenous compounds involved in the success or failure of the somatic embryogenesis response and these compounds determine the specificity of cellular responses. This present experiment identified and quantified endogenous sugars, chlorogenic acid and caffeine present during somatic embryogenesis of ‘Ruiru 11’. Laboratory experiments were set up at Coffee Research Institute, Ruiru-Kenya between 2014 and 2016. Third leaf pair explants were excised from 8-monthold greenhouse-grown mother plants and cultured in half strength Murashige and Skoog basal salts augmented with Thidiazuron. Once embryos had developed, the cultures were analysed for endogenous sugars, caffeine and chlorogenic acid using HPLC. Generally, green embryogenic cultures contained more and higher quantities of the compounds. Glucose and fructose were highest (38.95 mg/g and 45.43 mg/g respectively) in leaf discs of brown non-embryogenic cultures. Sucrose was highest (62.15 mg/g) in embryos of green embryogenic cultures. Embryos of green embryogenic cultures had the highest chlorogenic acid (5.3 mg/g), whereas caffeine was highest (0.55 mg/g) in embryos of brown embryogenic cultures. Endogenous fructose and glucose inhibited embryogenesis, while sucrose, chlorogenic acids and caffeine promoted embryogenesis and are potential biomarkers for embryogenesis. Other biochemical compounds such as organic acids should be identified and their role in coffee somatic embryogenesis determined.

Influence of gelling agent on micropropagation cost and in vitro conservation of Turmeric (Curcuma longa) germplasm

To examine the effect of different gelling agents on micropropagation and cost effective in vitro conservation of C. longa cv. Sona cultures, six gelling agents viz. 7 g l−1 Agar (Himedia PT Pure), 2.5g l−1 Clarigel (Himedia), 4.5g l−1 Clarigar (Himedia), 6g l−1 Gelzen (Sigma), Isabgol 3.5g l−1 (Baidyanath) and 2.5g l−1 Phytagel (Sigma) were tested. Shoot bud explants excised from in vitro established cultures were inoculated on basal salts of Murashige and Skoog (MS) medium supplemented with 2.5 mg l−1 BAP + 3% sucrose. Highest rate of shoot multiplication without hyperhydric transformation was recorded with 2.88± 0.03 shoots/ explant in the cultures grown on media solidified with Clarigar, compared to regularly used gelling agent agar (2.31± 0.38). After 12 months of conservation, highest 85% survival of cultures was also recorded in the medium solidified with Clarigar, whereas only 50% of cultures survived on agar supplemented media. Regenerated plantlets were successfully acclimatized and produced healthy rhizomes using soil: sand: Farm Yard manure (2:1:1) with 85% survival.

Development of a Cost Effective Medium for Enhanced Production of Bacillus thuringiensis ?-endotoxin

The present study was carried out to develop a sustainable production medium using locally available cheap raw materials for biopesticide production by Bacillus thuringiensis subsp. kurstaki (Btk) HD-73. In submerged fermentation (SmF) condition, the conventional Luria-Bertani (LB) medium which was enriched with nitrogen source (10% defatted soybean meal) supported 28.57% sporulation and 125% endotoxin increase over LB (alone). The effect of cystine on sporulation and endotoxin synthesis was highly pronounced in LB-soybean medium (LBS) with a range of 19.54% and 131.35% higher endotoxin yield respectively in SmF condition. While basal salts supplemented in soybean-cystine (SMc) medium, it resulted in 7.65% endotoxin yield compared to LB-soybean-cystine (LBSc) medium. Addition of molasses balanced the C: N ratio in the SMc medium thus helping 84.85% higher endotoxin synthesis after 24 hours fermentation. Substitution of basal salts with cost effective sea water yielded about 19.27% less endotoxin. The optimum medium thus obtained consisting of soybean extract-molasses-cystine with sea water was used in 3.0 L bioreactor cultivation for endotoxin synthesis by Btk HD-73 under 30% saturation of dO2 through cascade of agitation and aeration. The production rate obtained was 1.67 fold higher in bioreactor than in shake flask culture.Bangladesh J Microbiol, Volume 32, Number 1-2,June-Dec 2015, pp 1-6