Half-strength MS Medium Research Articles

Chemical Constituents and Antimicrobial Activity of Essential Oils from Micropropagation and Field-Grown Plants of Wedelia biflora [L.

An efficient regeneration protocol through axillary and inter nodal explants was developed from Wedelia biflora. Explants were placed on MS medium supplemented with different treatments of cytokinins, 6-benzyladenine (BA) and kinetin (KIN) for adventitious shoot production. The highest multiple shoots (95 %; 26.4 shoots/ explant) and elongation (2.75 cm shoot length), was obtained in 1.0 BA mg L-1 in axillary node explants. The shoot was transferred to half-strength MS medium supplemented with 2.0 mg L-1 IBA showed the best root formation (85 %; 22.2 roots/shoot; 2.65 cm root length; propagate leaf d.w 1.42 g L-1 and root d.w 1.10 g L-1) and plantlets were successfully acclimatized in the green house. The compositions of essential oils were studied in in vitro and in vivo leaf explants and to determine their absolute and relative concentrations using GC-MS and GC-FID, respectively. In vitro and in vivo variations in composition among the essential oils were detected. Sesquiterpene and monoterpene are major components in the oil of in vitro and in vivo leaves of W. biflora. Essential oil quantification showed α-pinene (44.03 %); verbenone (15.37 %); δ-cadinene (5.48 %) of in vivo leaves; whereas α-pinene (5.02 %); verbenone (1.40 %) and δ-cadinene (3.13 %) of in vitro leave respectively. The in vitro and in vivo essential oils were analyzed by bacterial and fungal organism through disc diffusion and minimum inhibitor concentration (MIC) methods. The results were compared with reference drugs ciprofloxacin and nystatin respectively. In vitro developed plants exhibited higher sesquiterpene contents and antimicrobial activity than field grown plant.

The effects of different concentration of sucrose and various auxin on in vitro shoot and microtuber formation of red potato (Solanum tuberosum, L. var Desiree)

The experiment was aims to study the effect of different sucrose concentrations (30, 90, 120 and 150 g.L−1) and various types of auxin (IAA, IBA, NAA) on in vitro shoot and microtuber formation of Red Potato (Solanum tuberosum, L. var Desiree). Young stem cutting from planlet cultured on half strength MS medium. All types of auxin use the concentration of 0.25 mg.L−1. The research conducted a completely randomized design (CRD) factorial. The data obtained were analyzed by analysis of variance (ANOVA) and DMRT test at 0.05. Parameters observed included growth of microtuber and shoot growth, such as number of shoots and shoot length. The observations result for 3 months, show that the higher the concentration of sucrose, can stimulate tuber formation but suppress shoot growth. Early tuber initiation (36,7 DAP) and optimum number of tuber was observed in 150 g.L−1 sucrose, whereas at 30g.L−1 had no effect to tuber induction. In addition, high number of microtuber was observed in NAA compare to other auxin. Observational data on shoot growth showed that the higher concentration of sucrose will inhibit bud formation. The 90 g.L−1 sucrose concentration showed the best number of shoots, which averaged 1.5 shoots/plantlet. But at a concentration of 30g/L sucrose, explants are only able to form callus. In the bud length parameter, IAA gave the best response that is an average of 3cm/shoot.

Effect of additives on enhanced in vitro shoot multiplication and GC-MS analysis of Lawsonia inermis L.

Investigations were undertaken to explore a successful and reproducible protocol for plant regeneration of Lawsonia inermis L. Shoot tip and nodal segment were excised and cultured upon MS medium supplemented with various cytokinines i. e. BAP, kinetin, zeatin, GA3 and spermidine. The explant responded successfully at a concentration of BAP (2 mg/l). Additives like silver nitrate (AgNO3), adenine sulphate (AdSO4) and casein hydrolyzate (CH) were tried for improving shoot regeneration abilities. A complete success was achieved using BAP (2 mg/l) along with kinetin (0.25 mg/l) and adenine sulphate (20 mg/l) after 45 days. For root induction, surgically separated microshoots were cultured upon half strength MS medium containing 0.5 mg/l indole-3-butyric acid (IBA) with an average of 76.66%. After one month, the in vitro regenerated plantlets were transferred to greenhouse condition in clay pots containing a mixture of sand: soil: vermicompost (1: 1: 1). The regenerated plantlets were subjected to GC-MS analysis and compared with the mother explants. The higher beta-glucopyraoside, methyl (59.75%) and alpha linoleic acid (11.25%) were observed indicating the application of plantlet in skin aliments and heart problems. The increase in active phytochemical components of in vitro regenerated plants over its mother plants showed the applicability of this technique over conventional method.

Tissue culture studies and estimation of camptothecin from Ophiorrhiza prostrata D. Don

Camptothecin is an important molecule in the synthesis of some of major anticancer agents such as irinotecan and topotecan. Traditional sources of camptothecin are mainly woody plants, some of which are being overexploited to a level that threatens the existence of the respective species. Alternate sources are being identified. In the current study, a tissue culture protocol was developed for the mass multiplication of Ophiorrhiza prostrata, an herbaceous camptothecin-containing plant, using seed explants, in order to evaluate its potential as an alternate source of commercial camptothecin. The germination of seeds was standardized on half-strength solid basal MS medium (94.8 ± 2.65% success rate). Seedlings subcultured on half-strength MS agar medium supplemented with 1.5 mg l−1 benzylaminopurine (BAP) produced the most shoots (28.0 ± 2.58) from the nodal region of a single seedling. Mass multiplication of in vitro shoots was achieved by subculturing a single shoot as well as shoot clumps. Subculturing shoot clumps containing a minimum of 4–6 shoots yielded fewer shoots per node (32.2 ± 2.03) than when single shoots were subcultured (25.0 ± 2.0). Shoot elongation was achieved on half-strength MS basal medium in a period of 2–3 weeks. Various concentrations of auxins were tested for rooting, and 1 mg l−1 NAA gave the highest number of roots (44.0 ± 4.13). Rooted shoots were successfully established under field conditions. High-performance thin-layer chromatography (HPTLC) was employed to measure the camptothecin levels in wild-grown plants (1.46 ± 0.02 µg g−1 dry wt), in-vitro-regenerated plants (1.10 ± 0.02 µg g−1 dry wt), and in-vitro-derived hardened plants (1.83 ± 0.02 µg g−1 dry wt). The results of this study indicate that in vitro methods could be a potential path to the rapid expansion of plant biomass, which could serve as a potential source of commercial camptothecin.

Highly Efficient Proliferation and Regeneration of Protocorm-like Bodies (PLBS) of the Threatened Orchid, Phalaenopsis bellina

Phalaenopsis bellina is an important indigenous fragrant orchid threatened with extinction. In this study, we evaluated the effect of medium strength, sucrose, nitrogen (NH4NO3) and potato extract on proliferation of P. bellina protocorm-like bodies (PLBs) to improve micropropagation in this species. Optimal treatment for PLBs proliferation rate with an average fresh weight (FW) of 0.97±0.16 g was obtained through culturing on half strength (½) MS medium containing 20 g/L sucrose, 15 mM NH4NO3 and 20% w/v potato extract supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2,4-D). The optimal treatment produced large, healthy and greenish PLBs with reduction in the occurrence of culture browning. In contrast, treatments with high potato extract (>20% w/v) or NH4NO3 (>30 mM) concentrations tend to have inhibitory effect and resulted in low PLBs proliferation rate, with an average FW of 0.77±0.15 g and 0.69±0.15 g, respectively. Plant regeneration of PLBs was achieved on plant growth regulator (PGR)-free ½ MS medium. In total, 60 healthy PLBs from the optimal treatment were successfully regenerated, acclimatized with 100% survival percentage and grew well in a mixture of soil, sand and vermicompost (8:4:2 (w/w/w)). With the optimal treatment, PLBs proliferation rate was enhanced by 27.63%. Our findings offer an improved micropropagation protocol of the endangered P. bellina for conservation and commercial production.

INDIRECT ORGANOGENESIS OF BOUCERSIA PROCUMBENS (GAMBLE) PLOWES - A RARE SUCCULENT PLANT

Efficient protocols of callus culture indirect organogenesis were established for mature internodal segments of Boucerosia procumbens (Asclepiadoideae). When MS medium was supplemented with different concentration of auxins, the texture of the callus varied according to the nature of auxin. Optimum callus was developed on MS medium supplemented with 3mg/l IAA. Best response (65%) of callus proliferation was obtained when MS medium fortified with 2iP 2mg/l + Zeatin 0.5 mg/l. IAA was most effective in producing the highest frequency of organogenic culture. Regeneration of callus into plantlets could not be achieved inthe present study. The regenerated shoots were rooted on half strength MS medium fortified with 0.1 mg/l NAA. 57% of the rooted shootlets survived in the field.

Optimization of in vitro regeneration of Haloxylon salicornicum: a keystone species of extreme arid regions.

Haloxylon salicornicum is an economically important perennial woody shrub of family Amaranthaceae and is a good source of food additives, fodder, fuel and nutrients in Indian Thar Desert. In vitro regeneration of H. salicornicum through nodal shoot segments is achieved in present investigation. Young green shoots in horizontal orientation were found to bemore responsive than vertical orientation. Axillary shoot buds were activated on MS medium containing 10µM BAP. Adventitious roots with intense root hairs were induced on horizontally placed shoots on half strength MS medium containing 2µM NOA + 100mgL-1 activated charcoal. This is a first report on micropropagation of H. salicornicum. This protocol can be a useful means for in vitro characterization, mass propagation and conservation of the plant for futureprospects.

Callusing and regeneration response of in vitro derived leaf explants of gerbera (Gerbera jamesonii)

Gerbera (Gerbera jamesonii Bolus ex. Hookes F.) is one of the most popular commercial flowers worldwide used both as cut flower and potted plant. Conventionally, it is propagated through vegetative means such as divisions of clump which is very slow process and not commercially viable. Rapid multiplication of elite hybrids/genotypes with improved quality traits, has been achieved by using both direct and indirect tissue culture methods. Gerbera hybrid seeds obtained by half-sib mating were raised in half strength MS medium. The earliest days for seed germination(2.00), first leaf development (3.00 days) and rooting (3.00 days) was recorded line IIHR1-1, IIHR 5-4 and IIHR1-1, IIHR1-2, IIHR1-5, IIHR3-2 and IIHR5-4. The response of in vitro leaf explants to callusing and regeneration was recorded when cultured on full strength Murashige and Skoog's media supplemented with different concentrations of 2,4-D or BAP or IAA and Kinetin. Leaf explants cultured on MS medium fortified with equal concentration of BA (1 mg l-1) and 2, 4-D (1 mg l-1) produced green and greenish white granular callus within 25 days, however, leaf explants cultured on MS medium supplemented with 2,4-D produced yellowish callus. Plant regeneration was also found earliest in the same media within 14 days.

Cytokinin is involved in TPS22-mediated selenium tolerance in Arabidopsis thaliana.

Excess selenium (Se) is toxic to plants, but relatively little is known about the regulatory mechanism of plant Se tolerance. This study explored the role of the TPS22 gene in Se tolerance in Arabidopsis thaliana. Arabidopsis wild type and XVE mutant seeds were grown on half-strength MS media containing Na2SeO3 for screening of the Se-tolerant mutant tps22. The XVE T-DNA-tagged genomic sequence in tps22 was identified by TAIL-PCR. The TPS22 gene was transformed into the mutant tps22 and wild type plants using the flower infiltration method. Wild type, tps22 mutant and transgenic seedlings were cultivated on vertical plates for phenotype analysis, physiological index measurement and gene expression analysis. We identified an Arabidopsis Se-tolerant mutant tps22 from the XVE pool lines, and cloned the gene which encodes the terpenoid synthase (TPS22). TPS22 was downregulated by Se stress, and loss-of-function of TPS22 resulted in decreased Se accumulation and enhanced Se tolerance; by contrast, overexpression of TPS22 showed similar traits to the wild type under Se stress. Further analysis revealed that TPS22 mediated Se tolerance through reduction of Se uptake and activation of metabolism detoxification, which decreased transcription of high-affinity transporters PHT1;1, PHT1;8 and PHT1;9 and significantly increased transcription of selenocysteine methyltransferase (SMT), respectively. Moreover, loss-of-function of TPS22 resulted in reduced cytokinin level and repression of cytokinin signalling components AHK3 and AHK4, and upregulation of ARR3, ARR15 and ARR16. Exogenous cytokinin increased transcription of PHT1;1, PHT2;1 and SMT and decreased Se tolerance of the tps22 mutant. In addition, enhanced Se resistance of the tps22 mutant was associated with glutathione (GSH). Se stress downregulated TPS22, which reduced endogenous cytokinin level, and then affected the key factors of Se uptake and metabolism detoxification. This cascade of events resulted in reduced Se accumulation and enhanced Se tolerance.

In vitro regeneration protocol of Rauvolfia serpentina L.

An efficient in vitro regeneration system was developed for Rauvolfia serpentina L. through direct and indirect organogenesis from nodal and leaf explants. Among the different growth regulators, MS medium supplemented with 2.0 mg/l BAP, 0.5mg/l IAA and 0.02mg/l NAA found best for the multiple shoot formation from nodal segments. In this combination 98% explants produced multiple shoots and the average number of shoots per explants is 13∙4. The frequency of callus induction and multiple shoot induction from leaves was highest 88% in MS medium supplemented with 2.0 mg/l BAP, where mean number of shoots/explants was 12.5. The highest frequency of root induction (80%) and mean number of roots/plantlets (10) were obtained on half strength of MS medium containing 0.2 mg/l IBA. The rooted plantlets were transferred for hardening following acclimatization and finally were successfully established in the field.Bangladesh J. Sci. Ind. Res.53(2), 133-138, 2018

Successful plant regeneration system via de novo organogenesis in Syzygium cumini (L.) Skeels: an important medicinal tree

Syzygium cumini L. (Myrtaceae) is an evergreen medicinal tree confined to tropical and subtropical regions of the world and is mainly recognized for healing of type, two diabetes. An efficient indirect regeneration system has been standardized for this tree using cotyledonary node, inter-node, nodal and leaf segment explants excised from 10 days old aseptic seedling. For callus induction explants were incubated on Murashige and Skoog (1962) medium supplemented with different concentrations of 2, 4-Dichlorophenoxy acetic acid (2, 4-D) or 2, 4, 5-Trichloro phenoxy acetic acid (2, 4, 5-T). All the tested concentrations facilitated callus initiation with different types and varying regeneration potentialities. The regenerated callus on different treatments showed variation in color and texture and best callogenesis (75%) was observed on (7.5 µM) 2, 4-D after 4 weeks. Different morphogenic response was observed when callus was transferred to a secondary organogenic medium fortified with (5.0 µM) benzyl adenine or Kinetin. The yellow white friable (YWF) callus obtained from CN explants exhibited appearance of small leafy structures on secondary medium. Not all types of calli originating from various types of explant were organogenic. Best shoot induction (93%) were observed in (YWF) type of callus obtained from CN explants on MS medium enriched with 5.0 µM BA + 0.25 µM NAA + 10 mg/l AgNO3 with maximum mean shoot number (10.19 ± 0.05) and mean length (4.12 ± 0.32 cm) per clump after 4 weeks of transfer. However the shoot number got increased up to 48 shoots/clump on subsequent subculturing after 12 weeks. For ex vitro rooting shoot lets (4 cm) were excised and pulse treated in full or half strength MS medium augmented with varied concentrations of IBA. Best rhizogenesis (80%) was achieved on 200 µM IBA treated shootlets after 4 weeks of transfer to sterilized soilrite. The regenerants were successfully acclimatized and established in earthen pots with 90% survival.

In vitro sterilization technique on embryo of black Toraja rice

Toraja black rice has a high anthocyanin content, a water-soluble pigments, with antioxidant activity. Toraja black rice has a variety of seeds colour in one panicles such as full black (the outside and inside the rice), medium black (the outside and slightly inside rice) and a little black (only the outside of rice). Embryo culture in vitro is one way to grow plants in sterile conditions. The presence of contamination and the death of the embryo require in vitro embryo culture. The sterilization technique is a very important first step to eliminate contamination and the death of embryos. This research aims to determine the right material composition for sterilization of black rice’s embryo. The experiment was done by growing black rice on half strength MS media with the treatment of three method of sterilization, i.e.: S1 (70% alcohol for 5 minutes, 3% and 2% Chlorox each for 10 minutes,), S2 (70% alcohol for 3 minutes, 2% Clorox for 10 minutes) and S3 (70% alcohol for 3 minutes and 1% Clorox for 15 minutes). The materials used are rice seedlings that have been cut in two and opened the pericarp of paddy grain, leaving a piece of rice that has a complete embryo. The best sterilization for Toraja black rice embryo culture was using the S3 composition. Best germination was seen on the seeds with full and medium black color.

Micropropagation of the Mongolian medicinal plant Zygophyllum potaninii via somatic embryogenesis

The Mongolian medicinal plant Zygophyllum potaninii has been assessed as an endangered species with regional status. We applied the somatic embryogenesis technique using aseptic in vitro germinants of the plant as an effective propagation technology. The seed germination rate in vitro was 16.5% after 2 weeks of culture. Embryonic calli (EC) and somatic embryos (SEs) were induced using the cotyledon or hypocotyl segments of the germinants. Calli were effectively induced on MS medium supplemented with 0.1 mg/L 2, 4-dichlorophenoxy acetic acid (2, 4-d) and 0.5 mg/L 6-benzylamino purine (BA). The callus was composed of pale yellow or pale green friable cells. SE formed from EC only on Murashige and Skoog medium (MS) with 0.5 mg/L abscisic acid (ABA). Other concentrations of ABA failed to induce SE formation. All SEs germinated in MS medium with different salt levels. However, normal plant conversion was achieved only on half-strength MS medium. The converted plantlets were effectively acclimatized in vitro in sand and transferred to a mixture of sand and perlite (1:1 v/v) in the greenhouse. After 8 weeks of culture, 55.4% of the plants survived. This is a first report of propagating the medicinal desert plant Z. potaninii via somatic embryogenesis and plant regeneration.

Enhanced growth and development of Chrysanthemum morifolium in microponic system under light-emitting diodes

This paper investigated the growth and development of Chrysanthemum shoots in microponic systems in comparison with shoots in micropropagation system. A microponic culture system, combining micropropagation and hydroponics, could reduce the drawbacks of micropropagation system such as reduction of infection, saving of labor, material, space, etc. In this study, Chrysanthemum morifolium shoots with 3 cm in length were cultured in MC (microponic system with circular container - 12 cm diameter at top, 9 cm diameter at bottom and 8.5 cm in height), MR (microponic system with rectangular box - 8.5 cm in height, 35 x 28 cm at top and 30 x 25 cm at bottom) and micropropagation system (MO - rectangular plastic box with 800 ml of half-strength MS medium containing 30 g/l sucrose and 8 mg/l agar). The results indicated that shoots pretreated with 500 ppm IBA, cultured in MC (15 shoots per container) and ventilated with millipore membrane (MillisealTM, pore size 0.5 µm of diameter 2 cm) under 70% red LED combined with 30% blue LED gave thebest plant height, number of roots, fresh weight, and chlorophyll content (a, b and a+b) (5.18 cm, 12.50, 0.52 g, 28.19 µg/g, 13.56 µg/g and 41.75 µg/g, respectively). The survival rates of plants derived from MC and MR in the greenhouse were higher than those in MO (100%, 100% and 85%, respectively). This study indicated that MR was an effective and simple system for large-scale production of Chrysanthemum morifolium.

In vitro Regeneration of Sugarcane (Saccharum officinarum) Varieties GUI 11 and PMA 96/48

The present investigation is deals with in vitro regeneration of two sugarcane varieties from Yezin, Pyinmana, Myanmar. The young suckers of two sugarcane varieties GUI11 and PMA 96/48 were used as explants. In this research, both sugarcane varieties gave high shoot proliferation rate on MS medium plus hormone BAP and NAA combinations. The shoot regeneration results of both varieties were not significant on MS basal medium supplemented with BAP + IBA (1+0.2, 1+0.5, 1+0.7, 0.5+0.1, 0.5+0.2, 0.5+ 0.3 mg/l). The maximum shoot formation was observed in MS medium supplemented with BAP 1mg/l + IBA 0.2 mg/l for both GUI 11 and PMA 96/48. The rooting experiment was conducted in half strength MS medium with various concentrations of NAA. The root formation from regenerated shoot was observed in half strength of MS medium supplemented with NAA 4, 5, 6 mg/l for both sugarcane varieties. Among three levels of NAA tested, the highest root formation was found at 5mg/l in GUI 11 and 6mg/l in PMA 96/48.

Application of Tissue Culture Technique on Marrubium vulgare L. Plant

This study was carried out at the Plant Tissue Culture Laboratory, Faculty of Environmental Agricultural Sciences (FEAS), Arish University, North Sinai, Egypt during 2012 to 2017. The aim of the study was to use tissue culture technique for the micropropagation of Marrubium vulgare plant. One node cutting and shoot tip were cultured on MS, NN and B5 media supplemented with 100 mgl-1 myo-inositol and 3% sucrose. The results showed that shoot tip cultured on the MS medium had the best effect on the establishment stage of mother plants. Multiple shoots were obtained on MS medium supplemented with 1.00 mgl-1Kin in combination with 0.10 mgl-1 NAA. MS medium supplemented with 0.30 mgl-1 pyridoxine achieved the best shoot development compared with the other additive treatments and control. The highest rooting of shoots was with full strength MS medium supplemented with 1.00 mgl-1IBA and half strength MS medium with 1.50 mgl-1 NAA. Hardening the rooted cuttings was done in a greenhouse in pots containing a mixture of peatmoss, vermiculite and washed sand at the rate of 1:1:1 (v/v/v). Plantlets were successfully acclimated with 90% survival.

Optimization of factors affecting in vitro regeneration, flowering, ex vitro rooting and foliar micromorphological studies of Oldenlandia corymbosa L.: a multipotent herb

The conditions were optimized for efficient in vitro regeneration of shoots and roots of Oldenlandia corymbosa L. using nodal shoot explants. Murashige and Skoog’s (MS) medium augmented with additives and 2.0 mg L−1 BAP was recorded optimum for shoot bud induction from the nodal meristems. The shoots were proliferated by subsequent subcultures on half strength MS medium fortified with 1.0 mg L−1 BAP and 0.5 mg L−1 kinetin + additives. This media combination yielded maximum number of shoots (223 ± 4.12 shoots/culture bottle) with 13.4 cm average length. Flower buds were induced (4.2 ± 0.28 flowers) from the in vitro multiplied shoots on MS medium contained 1.0 mg L−1 BAP and 0.5 mg L−1 of kinetin and IAA under 50 µmol m−2 s−1 SFPD light intensity for 12 h/day photoperiod. In vitro regenerated shoots were rooted on half strength MS medium conjunct with IAA, IBA and NAA singly at different concentrations. The best rooting response was observed on half strength MS medium containing IBA at 2.0 mg L−1 with activated charcoal. Roots were also induced from the cut ends of the shoots using ex vitro rooting techniques in O. corymbosa by pulse treating the shoots with 300 mg L−1 IBA for 4 min. Better roots were achieved in this method than the in vitro roots in terms of numbers (14.7 ± 0.21) and firmness. The foliar micromorphological studies could help to understand the structural adaptations of micropropagated O. corymbosa plantlets towards field environments. The in vitro induced anomalies in stomatal apparatus were repaired during hardening of plantlets in the greenhouse and after field transfer. Decrease in stomatal density (from 70.78 ± 0.55 to 55.6 ± 0.10), and increase in veins, trichomes and crystals/raphides densities revealed the developments of structural changes in the leaves to withstand in the harsh field conditions. The acclimatized plantlets with well developed root systems were successfully shifted to the natural soils with 98% survival rate.

A system for large scale production of chrysanthemum using microponics with the supplement of silver nanoparticles under light-emitting diodes

Abstract A microponic culture system, combining micro-propagation and hydroponics, could reduce the drawbacks of micro-propagation system such as being prone to contamination, being resource-intensive and require large areas, etc. In this study, chrysanthemum shoots (3 cm in length) were cultured in a microponic system and micro-propagation system. The growth of shoots cultured on half-strength sugar-free liquid MS medium supplemented with 7.5 ppm silver nanoparticles under 70% red LED combined with 30% blue LED were the highest among tested concentrations. Results of qualitative and quantitative tests of microbial contents in the microponic culture by 4 testing methods indicated that at concentration of 7.5 ppm silver nanoparticles effectively reduces microbial counts of 8 tested bacteria (Corynebacterium sp., Enterobacter sp., Arthrobacter sp., Agrobacterium sp., Xanthomonas sp., Pseudomonas sp., Bacillus sp. and Micrococcus sp.) and 3 fungi (Aspergillus sp., Fusarium sp. and Alterneria sp.). After 12 weeks at the nursery stage, the chrysanthemum plants derived from microponics began to bear flower-buds. Chrysanthemums produced by the microponic system started flowering after 15 weeks, which was 1 week earlier than those produced by in vitro micropropagation. Taken together, a microponic system was developed and tested successfully. The shoots derived from microponic culture could flower normally at 1-week earlier than the micropropagation-derived ones. The approach was proved to be both cost and time saving.

Enhanced multiplication and improved ex vitro acclimatization of Decalepis arayalpathra

The proposed work describes a protocol for high-frequency in vitro regeneration through nodal segments and shoot tips in Decalepsis arayalpathra, a critically endangered medicinal liana of the Western Ghats. Nodal segments were more responsive than shoot tips in terms of shoot proliferation. Murashige and Skoog’s (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA) was optimum for shoot initiation through both the explants. Among different combinations of plant growth regulators and growth additive screened, MS medium added with 5.0 μM BA + 0.5 μM indole-3-acetic acid + 20.0 μM adenine sulphate effectuated the highest response: 11.8 shoots per nodal segment and 5.5 shoots per shoot tip with mean shoot length of 9.2 and 4.8 cm, respectively. Half-strength MS medium with 2.5 μM α-naphthalene acetic acid was optimum for in vitro root induction. The plantlets with the well developed shoot and root were acclimatized in Soilrite™ with 92 % survival rate in the field conditions. During acclimatization, chlorophyll content, net photosynthetic rate, stomatal conductance, and transpiration rate were gradually changed in dependence of formation of new leaves. Further, the changes in activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) as well as activity of carbonic anhydrase were also observed: a continuous rise in SOD activity, but a rise and fall in the activities of CAT, APX, and GR were also noticed. Maximum fresh mass (3.1 g plant-1), dry mass (0.35 g plant-1) of roots and 2-hydroxy-4-methoxybenzaldehyde content of 9.22 μg cm-3(root extract) were recorded after 8 weeks of acclimatization.

Optimization of an in vitro Regeneration Protocol for Rough Lemon Rootstock (Citrus jambhiri L.) via Direct Organogenesis

Standardization of a reproducible protocol for in vitro rough lemon rootstock mass propagation was conducted at Tigray Biotechnology Center Plc., Plant Tissue Culture Laboratory, Mekelle, Ethiopia in 2015/2016 cropping season. Rough lemon is the frequently used rootstock both in the world and Ethiopia citrus fruit production, particularly in the Tigray region due to its superior performance over other rootstocks. However, seedlings produced through conventional ways are not recommended to be used in orchards due to variability problems caused by its polyembrony nature. To overcome such variations, in vitro regeneration of rough lemon rootstocks was performed using nodal segments and shoot tips as explant types. The explants were inoculated on MS medium supplemented with 5% scurose and 250 mg/L streptomycin followed to surface sterilization. The most effective and reproducible auxin (NAA), cytokinin (BA) and gebrillenllic acid (GA3) for in vitro shoot and root induction in rough lemon rootstocks were determined. Almost all IBA and BA treatments resulted in almost 100% shoot induction except for at 0.0 and 0.1 mg/L IBA and at 1.5 and 2.0 BA mg/L. Nodal segments induced a higher percentage of explant response with longer shoots in a shorter period of time than shoot tips, which produced more shoots and leaves than nodal segments. The effect different BA and IBA concentrations on various parameters of proliferation were studied. Full strength medium produced more regenerated shoots and leaves per shoot than half-strength MS medium. In addition, longer shoots formed with 0.1 mg/L GA3 than culture medium without this plant growth regulator. Root length decreased with higher concentration of NAA and the longest root (2.5 ± 0.22 cm) was found in the 1.0 mg/L NAA and followed by (1.95 ± 0.22 cm) at 0.5 mg/L of NAA. The rooted plants were successfully established in the greenhouse on the substrate called coco-peat and sand, and their survival rate was found to be 98%. These results suggest that standardization of these factors can help in development of a commercially viable tissue culture system for rough lemon. Moreover, it signifies the need of plant variety based in vitro protocol development and optimization across citrus species.