Callus Explants Research Articles

Induction of kopyor coconut embryogenic callus using 2.4-D and TDZ

This research was conducted to obtain an effective method to produce true to type coconut kopyor seeds and determine the effect of 2,4-D with or without TDZ in inducing callus to produce somatic embryos. This research was conducted in the Lampung State Polytechnic laboratory with concentrations of 2,4-D 0, 2.5, 5, 7.5, and 10 mg.L-1 with or without the addition of 0.5 mg.L-1. Further analysis of the data used the 5% LSD test. The parameters observed in this study were: percentage of callus explants, callus diameter, callus weight. The results showed that increasing the concentration of 2,4-D and 7.5 mg. L-1 could increase the percentage of embryogenic callus, callus diameter, and callus weight and the addition of 0.5 TDZ concentration to the 2,4-D medium resulted in a decrease in parameters. The best 2,4-D concentration to induce callus embryogenic in kopyor embryos is 7.5 mg. L-1. The use of 2,4-D for embryogenic callus formation can accelerate callus growth and obtain kopyor coconut seeds true to type.

In Vitro Regeneration of Stevia Rebaudiana Bertoni from internode and leaf explants using different concentrations of BAP (6-Benzyl Amino Purine)

The aim of this study was to determine the response of the growth of internode explants and leaves of Stevia (Stevia Rebaudiana Bertoni), which were cultured at several BAP concentrations. This study was designed using a non Factorial Complete Randomized Design that is by testing the internode and leaf explants at 3 different BAP concentration levels of 2, 3 and 4 ppm and will be repeated 10 times. The parameters observed related to callus growth include: emergence, type, color and percentage of callus formation. In addition, observations were also made on the parameters of bud growth including the appearance of shoots, number and length of shoots. The results of this study indicate that 100% of leaf and internode explants are able to form callus. The color of the callus explants on average leaves are yellowish green while the internode explants are yellowish white and all have compact textures. In shoot growth parameters, only internode explants were able to grow shoots while leaf explants were not. The emergence of shoots (3 DAP), number of shoots (8.70), and shoot length (2.81cm) were produced by MS media with the addition of 3ppm BAP.

Enhancing the production of scopoletin and quercetin 3-O-β-d-glucoside from cell suspension cultures of Tilia americana var. mexicana by modulating the copper and nitrate concentrations

The aerial tissues of Tilia americana var. mexicana produce compounds with anxiolytic activity, such as quercetin 3-O-β-d-glucoside and tiliroside, in addition to ones with anti-inflammatory properties, such as scopoletin. These three compounds were initially identified in callus cultures of apical buds. In the present study, suspension cultures from leaf explant callus were established; the accumulation of scopoletin and quercetin 3-O-β-d-glucoside in these cultures were found to be cell-growth-associated using cell growth and active compound-production kinetics assays. The effects of varying the nitrate and copper concentrations in Murashige and Skoog (MS, 27.4 mM total nitrates and 0.01 µM copper) medium on the growth of a suspension culture of T. americana cells and on the production of active compounds were tested by means of central composite design (CCD) generally used in the response surface methodology (RSM). Cell growth, measured as maximal biomass, improved when the total nitrate concentration decreased in the MS medium to 13.7 mM (p < 0.01) regardless of the copper concentration. As a phytoalexin, scopoletin accumulated rapidly in plants after pathogen infection, in the suspension cultures scopoletin yield was stimulated by increased copper concentration to 1.2 μM (p < 0.01). According to the C:N hypothesis, the carbon excess generated by nitrates reduced to 8.03 mM (p < 0.01) stimulated the production of quercetin 3-O-β-d-glucoside. Cell suspension of T. americana represents a potential biotechnological alternative for industrial exploitation in a stirred-tank bioreactor using a two-phase process: (1) the first step will be to grow the cell suspension, (2) the second stage will consist in handle the suspension culture towards the production of anxiolytic compounds or towards the production of anti-inflammatory compounds. As well as to evaluate another elicitors to stimulate tiliroside production in the T. americana suspension cultures.

In vitro and in vivo propagation of Monotheca buxifolia (Falc.) A. DC. An economical medicinal plant

Monotheca buxifolia an economic and medicinal plant is restricted to limited areas due to deforestation, overgrazing, low regeneration, slow rate of germination, unsuccessful germination from cuttings and dormancy. Hence to cope with the issue of seed dormancy various invitro and invivo experiments were designed. The accidental breaking of seed through fungi (Rhizophus stolonifer) is also reported for the first time in current study. Different techniques used for breaking of dormancy are mechanical, chemical treatments i.e. (priming, scarification, stratification, ethanol, sulphuric acid, organic matter treatment, hot water) and tissue culture. Seeds were treated through various experiments in field, green house and laboratory. The scarified seeds placed on Murashige and Skoog medium for proliferation and callus production was 60% proliferation. Explants (Meristematic tissues) from field and tubes were inoculated on M. S (Murashige and Skoog medium) + 2, 4-D+ Kinetin. Meristematic tissues collected from field shows 10% callus formation and meristematic tissues from tubes show significant result (70% callus formation). Our study concludes that the best suitable media for callus preparation of Monotheca is Murashige and Skoog medium. Moreover, propagation of Monotheca via cutting is not possible. Development of callus explant from test tube was found to be more promising than field. Interestingly, Rhizophus stolonifer fungi can break dormancy of Monotheca seeds and found to promote growth significantly.

Thidiazuron and Explant Type Effects on High-frequency In Vitro Mass Propagation of Cherry Tomato

Cherry Tomato is one of the most important vegetable crops cultivated for export in Egypt. In vitro culture response was assessed in tomato (Solanum lycopersicum L. var. cerasiforme) cv. (Summer Cherry) for optimum callus induction and plantlet regeneration. Callus induction was achieved within eight to 12 days directly on the cut surfaces of hypocotyl, cotyledon and leaf disc explants cultured on Murashige and Skoog (MS) basal medium supplemented with various concentrations of Thidiazuron (TDZ)] and benzyl adenine (BA) alone, but not in hormone free-medium. The highest callusing index (3.9 and 3.7) was obtained on hypocotyl explants cultured on MS medium supplemented with TDZ(1.0 and 2.0 mgl-1) followed by an index of 3.5 obtained from the same explant by using 0.5mgl-1 BA. However, for the leaf disc explants, the highest callusing index (3.1) was obtained on MS medium supplemented with BAat 2.0 mgl-1. After 8 weeks of culture, organogenesis was observed only on the explants cultured on medium containing different concentrations of TDZ and BA. The best shoot formation (93%) was obtained from leaf disc explant callus induced on MS medium containing TDZ. The highest number (13.4) of shootsexplant-1 was found when cotyledon explant callus was sub cultured on MS medium supplemented with 2.0 mgl-1 TDZ. Half strength of MS was found to be the best rooting medium, however, addition of IAA at 1.0 mgl-1 and IBA at 2.0 mgl-1 were found necessity to induce highest number of roots (22.5) and longer roots (11.0 cm), respectively. Acclimation of in vitro rooted plant is important for testing the post culture behavior of tissue culture regenerated plants. Cherry tomato derived from different explant sources under different concentration of TDZ and BA were not significantly different in their vegetative characters to those obtained from seed. However number of (raceme plant-1, flower raceme-1, fruit raceme-1, fruit plant-1) which produced by seed-derived plants was significantly less than those derived from in vitro propagated plants. This protocol would be valuable to create somaclonal variation and develop transgenic approaches for varietal improvement of cherry tomato.

Callus formation and production of secondary metabolites by seedling explants of Chenopodium quinoa

QUINOA (Chenopodium quinoa Willd) which is considered a pseudocereal or pseudograin, has been originated from the Andean region in South America, and is belonging to family Amaranthaceae. The highest percentage germination of seeds was achieved in MS medium with full strength in the full strength MS medium (100%). Best callus production from seedling explants was obtained on MS medium supplemented with 2mg/L 2, 4-D+0.05mg/L Kin. Callus was also obtained when MS medium was fortified with 1mg/L NAA+0.5mg/L BA, 1mg/L 2, 4-D+0.5mg/L BA and 3mg/L PCIB. However, the percent culture response on these concentrations was lower. The lowest total amount of callus was found to be on MS medium containing 1mg/L PCIB. Callus of explants were grown on MS media with 2mg/L 2,4-D+0.05 mg/L Kin gave the significant highest value (22μg/g fresh wt) of tocopherols content, followed by seedling on half strength MS medium (15.6). While the lowest value (2.1) was observed with Callus obtained from seedling planted on MS medium contained 0.5mg L-1 NAA+0.05 mg L-1 BA. Protein extraction and enzymatic assay protein extraction and measurement of tyrosine aminotransferase (TAT) activity were performed. Enzyme activity in leaves was twofold higher than that in seeds. In callus cultures, activity was about onefold lower than in leaf extracts.

Bulb organogenesis of Tulipa tarda in vitro cultures in relation to light environment

ABSTRACTHere organogenesis of tarda tulip (Tulipa tarda Stapf.) from callus explants is presented. The callus tissue was cultivated on MS media containing 3% or 6% sucrose and either no addition of BAP (6-benzyl-aminopurine) or supplemented with 0.5 μM BAP. The cultures were maintained under a 16 h photoperiod under white, red or blue fluorescent light, at 20 ± 2°C for 12 weeks. This study aimed to determine the most suitable light conditions and medium composition for seed-derived callus explants in order to obtain an efficient formation of adventitious bulbs. There were no differences between the spectra of light in differentiating adventitious bulbs. Explants cultured in darkness (control), on 0.5 µM BAP and 3% sucrose, formed the highest number of adventitious bulbs. The efficiency of adventitious organogenesis amounted to 36.6 bulbs per 1 g of callus tissue. The fresh weight of biomass, cultured in these conditions, increased within 12 weeks from 1 to 6.99 g. Supplementation with BAP of the medium containing 3% sucrose promoted the formation of bulbs, but in the case of the medium with 6% sucrose, BAP had an adverse influence under every type of light. The obtained results provide a useful protocol for the micropropagation of T. tarda, which can be used commercially for rapid and cost-effective production of the tulip.

Development of an efficient in vitro mutagenesis protocol for genetic improvement of saffron (Crocus sativus L.).

Saffron (Crocus sativus L) is a triploid (2n = 3x = 24), sterile geophyte which can only be propagated by means of underground vegetative corms. Since corm multiplication does not induce genome variations, therefore, the entire saffron population is expected to have a similar genetic makeup. Keeping in view the economic importance of the plant and the factors responsible for its low yield, the present investigation has been undertaken to establish an in vitro ethyl methanesulfonate (EMS) mutagenesis protocol followed by characterization of the induced variability in the advanced generations. The present report is limited to standardization of in vitro mutagenesis protocol only. Among the mutagenic treatments tested, concentrations ranging from 0.1 to 0.5% EMS showed a varied survival of explants. Based on various growth parameters, the LD50 was calculated to be 0.3% EMS for 3h. Among the two types of explants analyzed, the corm explant gave better results for in vitro survival and the growth parameters than callus explant. An average of 57.33 and 92.00 daughter cormlets in all EMS treatments as compared to 47.67 and 57.67 daughter cormlets in control, obtained from callus and corm explants respectively, were transferred to the field. The maximum, average daughter cormlet weight was obtained in control (3.01g, corm explant) followed by 0.1% EMS (2.8g, corm explant). In general, the growth parameters showed decreasing trend with an increase in EMS concentration in both the explants. The present study has been a significant achievement in the sense that the first mutagenesis protocol for C. sativus has been standardized.

Response of Pineapple Callus (Ananas comosus Merr.) through In-Vitro Colchicines Treatment

Pineapple Plant ( Ananas comosus (L.) Merr) is a fruit plant that has a high economic value. Increased variety of pineapple preparations lead to increased demand for pineapple fruit. Polyploidation is one way to increase the varieties using colchicine. This study aims to know the pineapple explants of callus response after being treated by colchicine using in vitro method. This research used Group Randomized Design (GRD) which variations concentration of Colchicine 0%, 0.01%, 0.05%, and 0.1%. Parameters were observed to response and thickness of callus of pineapple explants. Data were analyzed using F test and DMRT test in which significance level of 5% with SPSS 17 program. The results showed that pineapple plant explant able to respond colchicine concentration marked with thicker callus. The best colchicine treatment to induce pineapple explants callus was concentration 0.1% of colchicine.

Organogenesis of seedling explants of Carum copticum

ABSTRACTOrganogenesis of Carum copticum from hypocotyl explants of in vitro seedlings was examined. The highest callus induction was observed from hypocotyl explants on both Murashige and Skoog (MS) and Nitsch and Nitsch (NN) media. A maximum of 92% of hypocotyl explants produced callus on MS medium supplemented with 1.0 mgL–1 N6-benzyladenine (BA)and 0.5 mgL–1 naphthaleneacetic acid (NAA), whereas, 84% of hypocotyl explants produced callus on NN medium with 1.0 mgL–1 BA and 0.5 mgL–1 NAA. Hypocotyl explants-derived light green and friable callus responded to shoot organogenesis on both MS and NN media with plant growth regulators. A maximum of shoot regeneration frequency (76%) was observed on MS with 1.5 mgL–1 BA, whereas, 55% shoot regeneration frequency was observed on NN medium with 1.5 mgL–1 BA. A maximum number of 8.4 shoots per callus explant having a shoot length of 4.05 cm was observed on MS medium with 1.5 mgL–1 BA, whereas, 5.5 shoots per callus explant with a shoot length of 3.5 cm was observed on NN medium with 1.5 mgL–1 BA. Regenerated shoots were cultured on full strength MS and NN medium for root induction. The highest root regeneration frequency of 96% was obtained on MS medium with 0.25 mgL–1 NAA inducing 7.6 roots per shoot with root length of 4.25 cm, whereas, 83% root regeneration frequency was observed on NN medium with 0.25 mgL–1 NAA inducing 6.4 roots per shoot with a root length of 3.81 cm. In vitro derived plantlets were hardened and acclimatized successfully.

RETRACTED: Development of enhanced hypericin yielding transgenic plants and somaclones: High throughput direct organogenesis from leaf and callus explants of Hypericum perforatum

Abstract Hypericum perforatum is well known for its anti-depressent properties due to the presence of hypericins and hyperforins. The direct regeneration protocol from leaf explants and Agrobacterium tumefaciens mediated genetic transformation is a prerequisite for gene transfer studies in this valuable plant system as H. perforatum has shown its recalcitrance to these studies previously. The aim of generating transgenic shoots or somaclones in the present study is to select high hypericine yielding H. perforatum plants. High throughput direct bud organogenesis from leaf explants has been achieved for the first time in this plant system on media combination of MS+ 5.0 mg/l BAP + 1.0 mg/l. This medium interestingly also worked as the indirect regeneration media for generation of somaclones from callus cultures. Thus far optimized single media can be used for direct and indirect regeneration in H. perforatum . The direct regeneration frequency of plantlets from leaf explants was found to be 80% while 100% was the regeneration frequency noted for indirect plantlet regeneration from callus cultures. The optimized direct regeneration protocol from leaf explants was utilized for A. tumefaciens (harbouring p CAMBIA 1301) mediated genetic transformation studies with the aim of establishing transgenic lines of H. perforatum . The resultant study successfully gave rise to kanamycin resistant GUS positive shoots with transformation efficiency of 33.36 ± 3.6. A total of four transgenic H. perforatum clones namely T2, T3, T5 and T8 with different growth parameters were established, where highest of fivefold increase in hypericin content was recorded by T5 (276.8 ±9.2 μg/g dry wt.) in comparison to control non transformed plants (35.6 ± 2.7 μg/g dry wt.). Interestingly the presence of serotonin and melatonin was also detected in transgenic plants on TLC basis. The transgenic nature of the plants was confirmed by PCR with amplification of uidA gene. Apart from it, from indirect regeneration from callus, total of ten somaclones were generated. The ISSR profiling of somaclones scored a total of 156 bands, among which 118 were polymorphic in nature. The glasshouse established somaclones showed up to twofold increase in hypericin content on HPLC analysis.

Production of biomass and useful compounds through elicitation in adventitious root cultures of Fagonia indica

Fagonia indica is an important medicinal plant highly traded for its multitude of potentials against breast cancer. The natural availability of this high-valued herb is not fulfilling the ever-increasing demand. Alternative options should be adopted to balance the supply with demand. Adventitious root culture provides a preferred platform to produce commercially important secondary metabolites. In the present report, adventitious roots (AR) were induced from callus explants in F. indica for the first time. Higher adventitious root induction frequency (88%) and biomass accumulation (6.9 DW g/L) were observed when callus explants were treated with 0.5mg/L Methyl Jasmonate (Me-J) for 2h and subsequently incubated on solid Murashige and Skoog (MS) media containing 1.0mg/L Naphthalene acetic acid (NAA). Variations in growth of roots in suspension cultures were analyzed to estimate the impact of elicitors on adventitious rooting in F. indica. In response to 1.0mg/L NAA, a lag phase of 6days, logarithmic (log) phase of 15days and stationary phase of 6days were observed in the growth curve of AR in suspension culture. Maximum biomass (9.5g DW g/L) was recorded on the 27th day in log phase of culture. Analysis of AR samples derived from different growth stages showed that at 0.5mg/L Me-J, maximum Total Phenolic Content (TPC; 6.0mg GAE/g of DW) and Total Flavonoid Content (TFC; 5.0mg QE/g of DW) were produced, respectively in the lag phase compared to other growth lines. Quantification of vital plant secondary metabolites through High-Performance Liquid Chromatography (HPLC) revealed a higher amount of Gallic acid (148.0±4.8μg/mg of DW) detected in Control (CTR) samples raised in vitro at 1.0mg/L NAA. This was followed by Rutin (122.3± 3.8μg/mg of DW) in LOG phase and Myricetin (32.3±0.7μg/mg of DW) in LAG phase during growth of AR suspension cultures. In response to 0.5mg/L Me-J, more Apigenin (25.3±0.6μg/mg of DW), Caffeic acid (25.3±0.6μg/mg of DW) and Catechin (9.4±0.07μg/mg DW) were accumulated in the LOG phase in comparison to other growth stages. Thus, a feasible protocol was developed for induction and proliferation of adventitious roots with sustainable metabolite profiles through elicitation.

Embryogenic Callus Induction from Leaf Tip Explants and Protocorm-Like Body Formation and Shoot Proliferation of Dimorphorchis lowii: Borneon Endemic Orchid

Dimorphorchis lowii is a rare epiphytic orchid endemic to Borneo and very popular due to its ornamental value. For the purpose of mass propagation, procedures for in vitro propagation through callus were developed. The objectives of this study were to determine the effects of plant growth regulators (PGRs) on callus induction from leaf tip explant and the effects of complex additives on PLBs formation and shoot development . The best combination of PGR was 3.0 mg L -1 Thidiazuron (TDZ) with 0.046 mg L -1 NAA supplemented in half-strength medium (½ MS) (Murashige & Skoog, 1962). The percentage of survival and callus formation obtained were 96.0 % ± 19.8 and 52.0 % ± 16.5 respectively. Maximum shoot proliferation from PLBs was observed in Knudson C (KC) medium enriched with 15 % (v/v) coconut water. In this treatment, 10.2 ± 6.2 shoots were produced from one callus explant. Shoots were rooted on KC medium containing 15 % (v/v) coconut water and transferred in a medium mixture containing sphagnum moss, charcoal, brick and coco peat with the ratio of 1:1:1:1. After 2 months being acclimatized in the glasshouse, 78 % of plantlets survived. Histology observations showed that embryogenic callus derived from leaf tip explants might originate from epidermis and mesophyll cells and was capable of developing into complete plantlets.

English

The first objective of this study was to assess and optimize somatic embryo production in a genetically diverse range of cacao genotypes. The primary and secondary somatic embryogenesis response of eight promising cacao clones and a positive control was evaluated using modified versions of standard protocols. The second objective was to optimize the efficiency of primary somatic embryogenesis for a commercially important cacao clone, CCN 51, which has proven to be quite recalcitrant to standard protocols, relative to CCN 10, a clone also included in our analysis. The efficiency of the overall process was assessed by determining the number of somatic embryos produced per starting somatic tissue explant, as well as the quality of embryos (normal vs. abnormal) produced. Donor floral explants were subjected to five tissue culture steps, each 15-25 days in duration. Although all studied genotypes produced primary somatic embryos, most of them originated only from brown or brown-white callus. Overall, flower petals performed better than staminodes, and our best performing genotype yielded an average of 7-10 embryos produced in brown callus explants with embryogenic response during primary somatic embryogenesis procedures. In conclusions our analysis from a pilot in a small-scale are: 1) it is possible to achieve a high production of plants by somatic embryogenesis, although the efficiency is highly genotype-dependent; it is therefore necessary to optimize hormone balance and hormone type, as well as the explant type for each genotype, 2) through the use of secondary somatic embryogenesis, it is possible to increase somatic embryogenesis production at least ten-fold, and 3) the observed response variation between genotypes may reflect differences in endogenous and exogenously-supplied hormones. The importance of adapting the tissue culture protocol to the genotype is discussed. Key words: Somatic embryogenesis, 2,4-D, cellular competence, propagation, abnormalities, ethylene, cacao.

Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 105 protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5 × 105 Pp ml−1. Protoplast-derived microcalli became visible after 3–4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2–3 weeks, with an overall shoot organogenesis efficiency of 78–93 %. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.

Induksi Kalus Jeruk Kasturi (Citrus microcarpa) Menggunakan Hormon 2,4-D dan BAP dengan Metode in vitro

Calamansi fruits ( Citrus microcarpa ) or musk lime is one horticulture crops has been known as beverage and food flavorings. This research aimed to determine the effect of 2,4-D and BAP on callus induction of explants calamansi fruits ( Citrus microcarpa ). Parameters observed is percentage of explants, time appeared callus, callus width, and texture of the callus. Research results and a growing percentage of the width callus explants were analyzed by ANOVA and a further test DMRT at 5% level. The next to appear callus timing parameters and texture of callus analyzed in description. The result shows that a combination of hormones D 4 B 1 and D 4 B 2 produces a callus emerged fastest of 3.3 days after culture. The highest percentage of callus formation that is 100% contained in the treatment of D 2 B 0 , D 2 B 0.5 -D 4 B 0 . The mean width of callus was highest in treatment D 4 B 2 with a width of 0.97 cm. Embryogenic callus resulting by treatment of D 2 B 1 , D 2 B 2 , and D 3 B 0 . However, the best a combination of hormones is treatment D 2 B 2 (2 mg/l, 2,4-D, dan 2 mg/l BAP) producing embryogenic callus edvanced to suspension cultures.

Callus induction treatments influence antimicrobial effect of tissue culture-derived Thuja orientalis L.

Thuja orientalis belonging to the family Cupresseaceae is an important forest tree species for timber and wood production. Its wood is light in weight and highly resistant to decay. The leaf of explants of this plant was cultured for callus induction and plant regeneration. The explants of this plant were cultured onto Murashig and Skoog (MS) medium supplemented with different concentrations of (α- naphtalene acetic acid (NAA), pchlorophenoxyisobutyric acid (PCIB) alone and in combination with 6- benzyladenine (BA) for callus induction. The highest callus was induced in medium containing 3 mg/l PCIB. The lowest callus was found to be on MS medium containing (3 mg/l NAA +0.1 mg/l BA). Callus of explants were grown on MS media contained 3 mg/l PCIB +0.1 mg/l BA extract is the most active fraction against E. coli also it is active against Streptococcus pneumonia and Bcilis subtilis. Callus of explants were grown on MS media (3 mg/l NAA +0.1 mg/l BA) has higher activity against Streptococcus pneumonia, Callus of explants were grown on MS media supplemented with (3 mg/l PCIB +0.1 mg/l BA) has higher activity against Bacilis subtilis. These results provide a basis for future studies on genetic improvement and could be applied to production of secondary metabolites through cell culture in Thuja orientalis.

&lt;i&gt;In vitro&lt;/i&gt; regeneration of &lt;i&gt;Hypochaeris radicata&lt;/i&gt; L. from sodium alginate-encapsulated synthetic seeds

Synthetic seeds were produced from in vitro derived leaf, root and callus explants of Hypochaeris radicata (hairy cat’s-ear) by encapsulating different concentrations of sodium alginate hydrogel (1-6%) containing MS medium. The texture, conversion frequency and the effect of temperature on shoot emergence were evaluated. Among three explants attempted, in vitro derived leaf segments encapsulated beads stored at 25°C observed successful shoot regeneration above 80% in the standardized MS medium supplemented with 2mg/L BAP. The regenerated shoots were rooted well (73.54%) on MS medium with 1mg/L NAA. The high frequency of plant re-growth was found in four month old leaf segment encapsulated beads. Generally, four months stored beads of all explants at 25°C found to have higher regeneration rate in comparison with 2 and 6 months old encapsulated beads.

In Vitro Callus Induction and Plantlet Regeneration of Saussurea lappa (Clarke.) from Ladakh Region of India

In vitro propagation protocol was developed for Saussurea lappa (Clarke.) species threatened by over exploitation due to medicinal importance and habitat destruction in Ladakh region of India. The aim of the present study was to examine the main aspects of in vitro callus induction (CI) and plantlet regeneration of S. lappa. Explants were cultured on Murashige and Skoog (MS) basal medium supplemented with various concentrations and combinations of auxins and cytokinins for in vitro CI and plantlets regeneration. Callus initiation and induction was observed within eight and fifteen days after inoculation. 3 mg/L 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 5 mg/L Kinetin (Kin) was found to be more effective for CI frequencies in all the explants as compared to other combinations. Root (79.6 ± 0.8) and stem (76.4 ± 0.6) explants were found with better callus frequencies and early response as compared to other explants. Maximum number of shoots (11.8 ± 0.7), highest shoot length (9.2 ± 0.6 cm), maximum root number (6.2 ± 0.7) and highest root length (6.6 ± 0.8 cm) were found on MS medium with 2 mg/L Kinetin (Kin) and 2 mg/L Indole-3-butyric-acid in root derived callus explant. Plantlets with 3–5 shoots were transferred to potting mixture containing sand: soil: perlite (1:1:1) for acclimatization to field conditions and further multiplication. For the first time, protocol has been developed in S. lappa for in vitro CI and plantlet regeneration that holds robust potential for metabolite production and large-scale propagation.

UNRAVELLING THE REGULATION OF SOMATIC EMBRYOGENESIS BY EXTRACELLULAR CALCIUM AND AUXIN EFFLUX BLOCKERS IN HYPOCOTYL EXPLANTS OF ALBIZZIA LEBBECK L.: SIMILARITY IN ACTION

Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cells towards embryo development that is crucial for cellular totipotency in higher plants. It is a prototype for comprehension of the physiological, biochemical and molecular biological events transpiring during plant embryo development. In this study, we analyzed the role of exogenous calcium and auxin during somatic embryogenesis in hypocotyl explants of Albizzia lebbeck L. Light microscopic studies revealed that anterior and posterior ends of hypocotyl explants had entirely different patterns of differentiation . Scanning electron microscopic analysis of cultured hypocotyl explants exhibited globular-, heart- and torpedo-shaped somatic embryos developing from the anterior cut surface and gradually proceeding towards the other end. Surface texture of these somatic embryos showed uni-or bi-celled trichomes. Presence of exogenous calcium (1-4mM) in hormone-free B5 medium is prerequisite for induction of somatic embryos in hypocotyl explants derived from 7-d-old, light grown seedlings of Albizzia lebbeck L. A two-fold increase in calcium concentration from 2mM (B 5 medium) to 4mM led to doubling of somatic embryogenic response whereas, embryogenic callus was induced in explants raised on medium containing 10 or 20mM calcium. Depletion or high dosage in the medium (20 mM) induce non-embryogenic callus in explants. A similar response induced on hypocotyl explants upon treatments with NPA and PCIB, (auxin transport blockers). Results indicate that polar auxin transport or basipetal movement of IAA triggers acropetal transport of calcium. This combination of inverse fluxes acts as the primary signal for embryogenic response at the cut ends proximal to shoot apex and callusing at the cut ends distal to shoot apex of explants. Moreover, a correlation exists between NPA and PCIB-induced disruption of basipetal IAA transport and the inhibitory effect of NPA and PCIB on calcium translocation in the induction of similar morphogenetic responses.