Solid Murashige And Skoog Medium Research Articles

First Report on Mesophyll Protoplast Isolation and Regeneration System for the Duboisia Species.

The Duboisia species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, employing both conventional breeding methods and advanced biotechnology tools. The primary objective of this research was to establish a highly efficient method for isolating mesophyll protoplasts and facilitating their regeneration, thereby laying a robust foundation for the application of various advanced plant biotechnology tools in the pursuit of genetic enhancement. The mesophyll protoplast isolation process was developed for hybrid D. myoporoides × D. hopwoodii with careful optimisation of the following parameters: leaf strip size; incubation conditions; physical treatment; and enzyme concentration. The optimal parameters were combined in each individual step; the best enzyme concentration was determined to be 2% (w/v) cellulysin and 0.5% (w/v) macerase. Protoplast yield was found to be greatly affected by the enzyme concentrations. The isolated protoplasts were cultured at a density of 0.5 × 105 to best sustain the highest cell division (33.2%) and a microcalli induction frequency of 17.9%. After 40 days of culture in a modified KM8P medium at 25 °C in darkness, visible microcalli were transferred to a solidified Murashige and Skoog (MS) medium with 1 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction under a 16 h photoperiod. After 30 days of culture, compact organogenic calli were transferred into a solid MS medium with 6-benzylaminopurine (BA) alone or thidiazuron (TDZ) alone or in combination with BA or naphthalene acetic acid (NAA) for shoot regeneration. The maximum shoot regeneration frequency (63.3%) was observed in the medium with 1.5 mg L-1 TDZ alone. For the first time, a reliable protoplast isolation and regeneration system from mesophyll cells was established for Duboisia with high protoplast viability, successful microcalli formation, and intact plant regeneration. This innovation will significantly contribute towards the genetic enhancement of the Duboisia species.

An Investigation into Using Temporary Immersion Bioreactors to Micropropagate Moringa oleifera Lam. Callus, Roots, and Shoots

Moringa oleifera Lam., a tree naturally grown in the tropics, is becoming increasingly popular as an industrial crop due to its multitude of useful attributes. Therefore, this study tested the effect of temporary immersion system (TIS) bioreactors for mass micropropagation of Moringa oleifera Lam. callus, roots, and shoots. TIS are tissue culture systems that make use of timers to periodically immerse and drain plant cultures in a liquid nutrient medium instead of using solidified media. In initial studies, Moringa oleifera seeds were germinated in vitro, and in vitro seedling leaflets were then used as explant material for callus production on the pre-culturing media. Two experiments were conducted to improve the protocol for TIS bioreactor production. The first experiment investigated the effect of 6-benzylaminopurine (BA) and kinetin, whereas experiment 2 was conducted to improve shooting production. For the first experiment, leaf material was cultured onto a solidified medium consisting of half-strength Murashige and Skoog (MS) basal salts and 0.5 ppm 1-naphthaleneacetic acid (NAA) to initiate callus production before splitting it between solidified media and bioreactors for shooting. The shooting media consisted of full-strength MS basal salts and different treatments of kinetin and BA. A significant increase in callus production was observed with the use of TIS bioreactors, compared to solidified media, whereas root production had a highly significant interaction effect between the media and the cytokinin treatments. With shoot proliferation in mind, experiment 2 was performed, where microcuttings from in vitro-grown seedlings were excised and cultured onto a solidified MS medium, consisting of a control (0 ppm BA) and two different concentrations of 6-benzylaminopurine (BA) (0.1 ppm BA and 0.2 ppm BA) in the pre-culturing phase. Microcuttings were again excised after two weeks and transferred to the shooting media containing 0.1 ppm BA in TIS bioreactors and semi-solidified medium. Results showed TIS bioreactors to be effective in increasing both the amount and length of shoots produced. Shoot and callus fresh weights were also higher in explants cultured in TIS bioreactors. The results of this study also suggest M. oleifera sensitivities to plant growth regulators (PGRs). In conclusion, this study successfully produced callus, roots, and shoots in both the solidified media and TIS bioreactors, emphasizing the prospect of using TIS bioreactors for mass micropropagation of M. oleifera callus, roots, and shoots.

Impact of Stevia rebaudiana Culturing in Liquid Medium: Elevation of Yield and Biomass, Mitigation of Steviol Glycosides

An efficient micropropagation system is developed by culturing nodal segments containing the axillary shoot buds on liquid Murashige and Skoog (MS) medium. Direct shoot and root formation are highly effective in both solid and liquid MS media without any plant growth regulators (PGRs). Interestingly, a significant difference in yield is obtained between solid and liquid cultures. It is revealed that a relatively higher amount of plant biomass is obtained after culturing for 4 weeks in a liquid MS medium. However, the shoots produced on solid MS medium produce a remarkable decline in all physiological parameters. On contrary, the bioactive steviol glycosides (rebaudioside-A and stevioside) content is higher in shoots grown in solid MS on a comparative basis, which could be compensated by higher yield. After hardening off, all the regenerants are effectively grown in the field with a negligible loss (<1 %), and steviol glycosides spectra is again obtained by conducting high-performance liquid chromatography (HPLC) analysis after 10 weeks of the plantation. This method has great potential to be applied on large scale in bioreactors.

Development of Somaclones through Callus Culture in Fenugreek (Trigonella foenum-graecum) Variety Gujarat Methi-2

Background: Fenugreek (Trigonella foenum-graecum) a widespread aromatic herb, holds an important place in Indian traditional medicine system. A fenugreek or methi seed commonly used to flavour food contains various alkaloids, different saponins and fixed oil content. The steroidal nature of natural saponins has high market potential as alternative to Dioscore species, the current declining resource of commercial saponins. The current experiment designed to induce and identify somaclone variants of fenugreek resistant to powdery mildew, rich in saponins and fixed oil content with acceptable yield.Methods: Seeds of fenugreek var. Gujarat Methi-2 (GM-2) were used for in vitro regeneration during 2019 laboratory experiment. Explants cultured on Murashige and Skoog (MS) media supplemented with different concentrations of 2,4-D and NAA. The induced calluses were further transferred in to the regenerated semi solid MS medium with various levels of BAP for the development of calliclones. For root induction, shoots were transferred in White’s rooting media with different concentrations of Indole Acetic Acid.Result: Our experimental findings shows induction and detection of somaclonal variants, as a possible source of genetic improvement in fenugreek with limited information on genetic diversity. This approach could generate new genotypes useful to pharmaceutical industries with competitive yield and resistance to powdery mildew.

Effects of Benzyladenine on in vitro ‘Hom Rangsi’ Rice and Induction of Aluminum Acid Tolerance Lines by Gamma Irradiation

‘Hom Rangsi’ was the non-photoperiod aromatic mutant rice which derived from fast neutron radiation KDML 105. ‘Hom Rangsi’ seeds were cultured on MS solid medium without any supplemented for a week. And then, all explants were placed on MS (Murashige and Skoog, 1962) medium supplemented with 0, 5, 10, 15, 20, 25 mg/L BA (benzyladenine) for multiple shoot induction. The optimal concentration of BA for induced multiple shoot induction of ‘Hom Rangsi’ line was MS + BA 25 mg/L, the highest number of shoots were 5.38 shoot/seed. The following experiment was done, irradiated ‘Hom Rangsi’ seeds with 0, 100, 200, 300, 400 Gy gamma ray which cultured on MS solid medium supplemented with 400 mg/L Al3+ pH 2.9 were selected for acid tolerance lines. After six weeks cultured, the survivals of irradiated plantlets were 86.32, 77.78, 58.95, 58.95, 21.87% and the height of irradiated plantlets were 8.4, 8.3, 6.7, 6.6, 6.1 cm respectively without any shoot budding. All survival plantlets were transferred to suitable MS + BA 25 mg/L medium which discovered from the first experiment for multiple shoot budding. After six weeks cultured, the maximum of 5.24 shoots/plantlet were found from 300 Gy irradiation significantly and followed by 400, 200, 0 and 100 Gy irradiation treatments which gave 4.55 and 4.41, 4.37 and 4.31 shoots/planlet respectively.

Effect of plant growth regulators on in vitro culture of pineapple (Ananas comosus L. Merr) MD2 variety

Pineapples (Ananas comosus L. Merr) are fruits that belong to the Bromeliaceae family. Pineapple variety MD2 is one of the varieties that has gained a place in the market among pineapple farmers due to its high value and quality. However, it is difficult to meet the demand for planting materials using conventional propagation techniques. Hence, plant tissue culture technology is one of the methods that has been widely used in the agriculture industry that boosts up the production of pineapple planting materials within a short period and is cost-efficient. The objective of this study was to determine the effect of plant growth regulator concentration to in vitro culture of MD2 variety pineapple. In this study, the various concentrations of 6-Benzylaminopurine (BAP) and α-Naphthalene Acetic Acid (NAA) for in vitro culture of MD2 pineapple were studied. The plantlets were effectively initiated from MD2 pineapple crown on Murashige and Skoog (MS) basal salt containing 30.0 g/L sucrose, and 2.0 mg/L BAP in two months of culture. Next, the pineapple plantlet was subculture on shooting medium containing full strength solid Murashige and Skoog (MS) medium supplemented with vitamins with various concentration BAP (0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mg/L) and NAA (0, 1.0, and 2.0 mg/L). The result obtained showed that the solid MS medium added with 30.0 g/L sucrose, without any BAP and NAA (T1) had the highest in vitro shoot regeneration. Meanwhile, the solid MS medium with 30.0 g/L sucrose with 1.0 mg/L NAA (T1) recorded the highest plantlet height (cm). The mean value for in vitro shoot regeneration in T1 and plantlet height (cm) in T1 were 2.80 (±0.5) and 4.40 (±0.3). To conclude, less amount plant hormone regulator required to obtain the mass quantity of in vitro clonal pineapple that can help solve the problem of lack of plant material in the pineapple crop industry.

The effects of thidiazuron and 2,4-D on the regeneration of Melastoma malabathricum L. cultured leaves

Melastoma malabathricum L. has potential for exploitation of its pharmacological and phytoremediation properties. We cultivated M. malabathricum leaves using in vitro cultures in Murashige and Skoog (MS) media supplemented with thidiazuron (TDZ) (0, 0.1 and 0.2 mg.L−1), 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 1, 2 and 3 mg.L−1) or both. We studied the effect of TDZ alone or combined with 2,4-D on the growth and development of cultured leaves. Explants were cultured on solid MS medium supplemented with TDZ (0, 1, 2 and 3 mg.L−1) or 2,4-D (0, 0.1 and 0.2 mg.L−1) or both. All treatments induced callus with different colours and textures. TDZ, 2,4-D or both induced callus formation in approximately 75–95 %, 95–100 % or 45–90 % of explants. Adventitious root was produced in the presence of 0.1 mg L−1 (70 %) and 0.2 mg.L −1 (60 %) 2,4-D. Adventitious shoot formation was initiated in the presence of 1 mg.L−1 (15 %), 2 mg.L−1 (5 %) and 3 mg.L−1 (5 %) TDZ. Callus formation was induced by 0.1 mg.L−1 2,4-D (63 %), 0.2 mg.L −1 2,4-D (50 %), 2 mg.L−1 TDZ (42 %) and 3 mg.L−1 TDZ (50 %), which were higher than other treatments. Callus, adventitious root or adventitious shoot was induced from leaves using 12 different media.

The interplay between light, plant growth regulators and elicitors on growth and secondary metabolism in cell cultures of Fagonia indica

Manipulation in the light regimes combined with the effects of plant growth regulators (PGRs) and elicitors through plant cell culture technology is a promising strategy for enhancing the yield of medicinally important secondary metabolites. In this study, the effects of interplay between PGRs, elicitors and light regimes on cell cultures of F. indica have been investigated. The results showed that callus cultures resulted in maximum biomass formation (13.2 g/L) when incubated on solid MS (Murashige and Skoog) medium containing 1.0 mg/L BA under continuous light for 4 weeks. Among the other PGRs, compared with the auxins such as 2,4-D, and IBA, TDZ resulted in higher biomass accumulation (12.1 g/L). Elicitors (Me-J and PAA) resulted in a lower growth response, when compared with cytokinins and a higher response than auxins under all the light regimes on solid MS media. However, in liquid media, no significant increase in biomass was observed in response to the combined effects of PGRs and photoperiod regimes. Further, the highest phenolic content (TPC = 6.8 mg) and flavonoid content (TFC = 5.2 mg) were detected in the dark-grown cell cultures raised in vitro at 0.5 mg/L Me-J. The highest antioxidant activity (88%) was recorded in the dark-grown cell cultures harvested from LOG phase of the growth cycle supplemented with 0.5 mg/L Me-J. Furthermore, BA resulted in considerable flavonoids production (TFC = 4.7 mg) in the cell cultures grown under continuous light. However, overall dark treatment and elicitation with Me-J resulted in the optimal metabolic response in terms of secondary metabolites accumulation in cell suspension cultures of F. indica.

Adventitious Root Cultures of Boesenbergia rotunda as a Source of Pinostrobin

Pinostrobin is the major bioactive compound from the roots of Boesenbergia rotunda, and it possesses activities against HIV,  Helicobacter pylori,  ulcer, leukemia and inflammatory. The present study aimed to optimize the conditions for the production of adventitious roots from B. rotunda on Murashige and Skoog (MS) solid medium. The effects of the MS strength and sucrose concentration on adventitious root growth were studied as well as the production of pinostrobin. Adventitious roots were initiated from bud explant of  B. rotunda , on MS solid medium supplemented with 2.0 mg/L 1-Naphthaleneacetic acid (NAA) and were used to establish root suspension cultures. The optimum biomasses of 1.83 ± 0.21 g fresh weight were obtained with a 1/2 strength of MS medium. High pinostrobin production (3.54 mg/g) was obtained with 50 g/L of sucrose concentration. These results suggest that adventitious root cultures are suitable for the raw material for the large-scale production, which contain a high yield of the pinostrobin compound. The results also revealed that the culture condition is a good strategy for enhancing the pinostrobin content in the adventitious root cultures.

Ultrastructure of somatic embryo development and plant propagation for Lachenalia montana

Embryogenesis for plant propagation by cell suspension culture was achieved for the first time using friable embryogenic callus (FEC) from leaf explants of Lachenalia montana Schltr. ex W.F. Barker. FEC was established with solid (8gL−1 agar) MS (Murashige and Skoog, 1962) medium containing various concentrations and combinations of sucrose and plant growth regulators (PGRs). The somatic embryos (SEs) were developed from FEC in liquid MS (MSL) medium with or without PGRs. A higher number of SEs of different developmental stages (26.0–19.4, globular to cotyledonary-stages respectively) were obtained on MSL medium with 0.5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1μM thidiazuron (TDZ). The germination frequency (33.7%) was highest in MSL medium containing 1μM 2,4-D and 2μM TDZ. The different stages of SEs germinated (92%) on solid full-strength MS medium containing 15gL−1 sucrose and 10μM phloroglucinol (PG). The plantlets were successfully acclimatized in the greenhouse. Developmental stages and features of embryoids were confirmed by histological and ultrastructural studies using light and transmission electron microscopy (TEM). Many mitochondria, lipid bodies together with starch grains, chloroplasts, Golgi apparatuses, vacuoles and nuclei were detected. The system developed offers controlled large-scale clonal propagation which ensures germplasm conservation, confirms viability of embryogenesis by ultrastructural studies and provides a system for genetic transformation studies for horticultural and ornamental improvement.

The influence of cytokinins on proliferation and polyphenol accumulation in shoot cultures of Scutellaria altissima L.

S. altissima shoots were cultivated on MS (Murashige and Skoog) solid medium supplemented with IAA (indole-3-acetic acid, 0.57μM) and various concentrations of cytokinin: zeatin, kinetin, BAP (6-benzylaminopurine) (1, 2, 4, 8μM) or TDZ (thidiazuron) (0.2, 0.5, 1μM). The effects on shoot proliferation and their accumulation of pharmacologically valuable phenolic compounds (baicalin, wogonoside, luteolin, luteolin-7-O-glucoside, verbascoside) were evaluated. Ultra-high performance liquid chromatography (UHPLC) was used for quantitative analysis of the compounds accumulated in the plant biomass. The metabolites were identified by comparing their retention time, UV spectra and mass spectra with those of the standard compounds and published data. The highest metabolite contents were recorded in shoots treated with TDZ at a concentration of 1μM; under these conditions, the total level of all evaluated flavones expressed as the sum of baicalin, wogonoside, luteolin and luteolin-7-O-glucoside (17.35mg/g dry wt) was more than twice that of those grown on MS cytokinin-free medium (7.55mg/g dry wt). Verbascoside accumulation was also stimulated by 1μM TDZ; its level was about six times higher than that found on the control medium (6.2mg/g dry wt vs. 1.03mg/g dry wt). The highest number of shoots (5.5–6.4 per explant within five weeks) was achieved with 2–8μM BAP.

Performance of NAA, 2iP, BAP and TDZ on Callus Multiplication, Shoots Initiation and Growth for Efficient Plant Regeneration System in Chrysanthemum (Chrysanthemum morifolium Ramat.)

Three series of plant growth regulators experiments in Chrysanthemum tissue culture, each arranged in completely randomized design (CRD), were studied to formulate efficient plant regeneration system for developing Chrysanthemum plant cell engineering. In the first experiment, 100 mg callus were cultured in liquid Murashige and Skoog (MS) medium supplemented with different combinations of NAA and BAP concentrations treatments. After 30 days of culture, heaviest callus (1.55 g) was obtained in NAA (1.0 mg L-1) + BAP (0.5 mg L-1). Consequently, shoot regeneration experiment with different kinds and concentrations of auxin, i.e. NAA and 2iP were each combined with different concentrations of cytokine BAP. Callus grown on solid MS medium supplemented with both NAA and BAP at 0.5 mg L-1 showed fastest shoot initiation (30 days), largest number of shoots (5 shoots), longest shoot length (2.88 cm). While combination of 2iP (0.5 mg L-1) and BAP (2.0 mg L-1) also produced same response on shoot initiation but shorter (1.88 cm) and only one shoot. Largest number of shoots (3 shoots) was obtained in treatment BAP (0.5 mg L-1) without 2iP, although shoot initiation was slower (39 days) with shorter length (1 cm). In a separate experiment, effect of single treatment of potential synthetic cytokine for shoot regeneration, Thidiazuron (TDZ), at different concentrations was examined. However, callus grown on TDZ incorporated medium did not produce any shoots and changed from green to brown at end of study (90 days). It was assumed that TDZ inhibited formation of shoots in Chrysanthemum callus culture.

Somatic embryogenesis of Sandalwood (Santalum album L.)

Sandalwood (Santalum album L.) is native species of Indonesia, especially in East Nusa Tenggara, is oneof the twenty two species of the genus Santalum in the world. Sandalwood is an important tree because it hashigh economic value can produce sandal oil these can be used for perfumes, cosmetics, pharmaceuticals, andare often used in religious ceremonies. In vitro particularly somatic embryogenesis has been widely appliedin the propagation of sandalwood. The Objective of this research is to obtain regeneration of sandalwoodthrough somatic embryogenesis using leaves explant from various clones. Medium for embryo induction is MS(Murashige and Skoog, 1962) solid medium containing treatment of 2,4-D (2,4-Dichlorophenoxyacetic acid)at various concentrations. To the media 0,15 mg /l kinetin, 40 g/l sucrose, and 2,5 g/l gelrite were added.Culture were incubated in the dark. Medium for Embryo development (maturation) is MS solid mediumcontaining treatment of BAP (Benzyl-amino-purine) at various concentrations. To the media 0,01 mg /l NAA(Napthalene-acetic-acid), 40 g/l sucrose, and 2,5 g/l gelrite were added. Culture were incubated in the light. Tostudy the specifi c structure of sandalwood somatic embryo early detection was conducted using histologicalanalysis. Results of anova showed that the clones, media, and interaction between clones with media did notsignifi cantly affect the development of sandalwood callus percentage. Results of anova showed that the clonesand BAP concentration signifi cantly effect to the embryo development of sandalwood.

Germination and storability of calcium-alginate coated somatic embryos of mango ginger (Curcuma amada Roxb.)

We developed a protocol to produce synthetic seeds of mango ginger (Curcuma amada Roxb.) for large-scale propagation, storage, and germplasm exchange. The seeds were produced by encapsulating somatic embryos in a calcium–alginate matrix. Embryogenic callus was induced from leaf sheath explants on 0.8% agar solidified with full-strength Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose, 2.0 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg·L-1 6-benzyladenine (BA). Somatic embryos were induced by transferring the calli to 1/2 MS liquid medium supplemented with 3% (w/v) sucrose and 0.3 mg·L-1 BA. A synthetic seed coat for the somatic embryos that is uniform in size and shape was produced by incubating the embryos in 3% (w/v) sodium alginate in 1/2 MS liquid medium and exposure to 100 mM calcium chloride (CaCl2.2H2O) for 15 min. The highest germination percentage of synthetic seeds (91.66%) was achieved on 1/2 MS solid medium supplemented with 3% (w/v) sucrose and 0.25 mg·L-1 gibberellic acid (GA3) in darkness (24 h) for 35 days. Sucrose-dehydrated synthetic seeds showed better storability than fresh seeds. Synthetic seeds that were dehydrated in 8.55% (w/v) sucrose solution and stored at 4?C showed germination percentages of 88.10% after 30 days of storage and 54.16% after 120 days of storage. Plantlets were successfully acclimatized to ex vitro conditions (survival rate, 82.66%) and showed normal phenotypes. In a random amplified polymorphic DNA (RAPD) analysis, a monomorphic banding profile was obtained from the plants derived from synthetic seeds that had been stored at 4°C for 120 days. This confirmed the genetic fidelity of the plants derived from the stored artificial seeds.

In Vitroseed Germination And Micropropagation Of Dendrobiumlindleyisteud. An Indigenous Orchid Of Bangladesh

The seeds of Dendrobium lindleyi were germinated on 0.8% (w/v) agar solidified Murashige and Skoog (MS) as well as Phytamax (PM) basal media. The germination rate was found to be better on PM medium. The germinated seedlings underwent rapid elongation on Plant Growth Regulators (PGRs) supplemented media and the highest growth was recorded on MS with 3% (w/v) sucrose + 2.0 mg/1 6-Benzyl Amino Purine (BAP) + 0.1mg/1 Indole-3-Butyric Acid (IBA). The seed derived seedlings developed strong and stout roots on half strength MS medium with 1.5% sucrose. For micropropagation the pseudobulb segments were used as explant and the lower part of pseudobulb segment produced highest number of multiple shoot buds (9-10/segment) on agar solidified MS medium supplemented with 3% (w/v) sucrose + 2.5 mg/1BAP + 0.1% (w/v) Activated Charcoal. The highest rate of elongation and rooting was found from multiple shoot bud derived seedlings as well as same seed derived seedlings. Seed and multiple shoot bud derived rooted seedlings were finally transferred to outside natural environment by successive phases of acclimatization.
 Asiat. Soc. Bangladesh, Sci. 41(2): 163-171, December 2015

Effects of liquid, temporary immersion bioreactor and solid culture systems on micropropagation of Lilium ledebourii via bulblet microscales— An endangered valuable plant with ornamental potential

Lilium ledebourii (Baker) Boiss. (Liliaceae) is a critically endangered lily species native tonorthern Iran, where it is protected by law. In order to develop a cost effective method for largescalepropagation, the effects of three culture systems (solid, liquid and temporary immersion)and two types of cytokinins [6-Benzyladenine (BA) and Thidiazuron (TDZ)] were studied onthe in vitro plant regeneration of L. ledebourii. To establish the protocol, we used in vitroregenerated bulblets obtained from bulb scale segments that were cultured on solid Murashigeand Skoog (MS) media as starting material. The bulblet microscale transverse thin cell layerswere cultured on MS solid medium containing 3% sucrose and different combinations of plantgrowth regulators. Choice of both, the culture system and the type of cytokinin, affected thedifferentiation of explants. Two types of calli formed on explants: type I callus wasembryogenic, while type II callus was shoot organogenesis. The highest percentage (94%) ofembryogenic callus was obtained when calli were transferred on MS solid media supplementedwith 0.54 μM α-Naphthaleneacetic acid (NAA) and 0.44 μM BA. In addition, it was alsoobserved that the use of temporary immersion bioreactor resulted in a significantly loweramount of shoot organogenesis rather than solid culture systems. Seventy percent of theplantlets were successfully acclimatized to ex–vitro conditions and were phenotypically similarto the mother plants.

The preliminary attempts of in in vitro regeneration from petioles of recalcitrant species of Cephalotus follicularis Labill.

Cephalotus follicularis Labill. is representative of the extraordinary carnivorous group of plants. Carnivorous plants with pitcher traps grow in nutrient poor, sunny and wet habitats, they have adapted themselves to growth in bogs, sandy soils and obtain some nutrients (e.g., nitrogen and phosphate) from insects and other arthropods or protozoa, even from small mammals occasionally (Krol et al. 2012).C. follicularis belongs to the monotypic family Cephalotaceae, and it is the endemic plant of south-western Australia. This Australian pitcher plant is heterophyllous with different types of leaves, specialized either for prey capture or photosynthesis. In the spring, non-carnivorous leaves responsible for photosynthesis, while later, in the summer the second type – carnivorous pitcher-shaped leaves occur (Pavlovic 2011).In this paper we present the first observation of tissue culture of C. follicularis for regeneration protocol, taking into account the absence of organic and inorganic nitrogen as well. We kindly achieved the in vitro plants of C. follicularis clones: 00001/01 and 0004/03 from collection of Mr. Kamil Pasek (http://www.bestcarnivorousplants.net/) from Czech Republic.These plants have been maintained on MS (Murashige and Skoog) media, in sterile conditions for several years. Under these conditions the plants are smaller than in the nature, do not develop pitchers properly and do not produce flowers, and intensive multiplication of shoots is observed. Therefore we tried to ensure such conditions, which will enable the proper development of this species in tissue culture. Plants of 00001/01 and 0004/03 clones were transferred to MS solid medium modified by reducing the amount of MS major salts to ½ and ¼ (½ MS and ¼ MS respectively). Plants were placed on these media in two ways: in small groups and individually.These same rules were applied when using subsequent ½ MS based media supplemented with 8,2 µM 2,4-D with 9,3 µM KIN (first fodder) and 4,9 µM IBA with 18,2 µM TDZ (second fodder). In addition, on the same media we put photosynthetic and pitcher leaves separately. All cultures were kept in light conditions.It occurred, that in the long term culture (after 47 days) on the ¼ MS medium the plants growing in small groups, started to develop pitcher and non-carnivorous leaves similar to these in the nature. Plants growing individually on the same medium did not change their appearance. The similar situation was observed for 00001/01 genotype, with one difference, bigger quantity of plants created properly shaped pitcher leaves on ½ MS medium. Decreasing the quantity of MS major salts to ½ gave good results in the case of rooting of plants of the Drosera intermedia Hayne (Kromer et al. 2000) and in Cephalothus regeneration system from root mass (Ko et al. 2010).The first symptoms of morphogenesis were noted on 42 day of culture as the direct organogenesis and somatic embryogenesis. The adventitious shoots and young somatic embryos were observed in 0004/03 on photosynthetic leaves, on the medium supplemented with 2,4‑D and KIN. The pitcher leaves did not induce morphogenesis.Histological analysis confirmed direct morphogenesis. Somatic embryos and adventitious shoots appeared on the petioles without callus formation and originated from epidermal and subepidermal layers of the explants.Taking into account the specific way of nitrogen uptaking by Cephalothus plants, the photosynthetic and pitcher leaves were maintained on the conditions with the lack of organic source of nitrogen and lack of inorganic nitrogen in MS medium. The observations showed that in the absence of inorganic nitrogen in the medium the non-carnivorous leaves become white and slowly degenerate. At the same conditions the pitcher leaves growing in small groups develop well traps, but they are fragile and strong red. In this small group the non-carnivorous leaves are not present.When the medium is free of organic nitrogen the non-carnivorous leaves become white but still alive, while the pitcher leaves are weak and do not develop well shaped traps. Further investigations are necessary for improving the culture protocols for more efficient plant regeneration and improvement of shoots formation, as well as analysis of the nitrogen influence on leaves of this plant.

Efficient Cryopreservation of Lilium spp. Shoot Tips using Droplet-vitrification

Newly developed shoot tips from adventitious buds induced by tissue cultured bulb-scale segments of five accessions of Lilium spp. were successfully cryopreserved by a droplet-vitrification method. Bulb-scale segments cultured on Murashige and Skoog (MS) medium with 0.1 mg?L -1 IAA and 0.1 mg?L -1 zeatin were then cold-hardened at 4℃ for 7 days. The excised shoot tips were pre-cultured on solidified MS medium containing 0.3 M sucrose for 1 day at 23℃ and then soaked in a mixture of 0.7 M sucrose for a day at 23℃. Pre-cultured shoot tips were cryoprotected with two loading solutions, LD1 and LD2, which included 35% and 40% plant vitrification solution (PVS3), respectively, for 40~60 min at 23℃. The cryoprotected shoot tips were then soaked in PVS2, modified PVS2 and PVS3 for 90~120 min at 23℃. The shoot tips, frozen in microdroplets of vitrification solution, were wrapped with aluminum foil strips, which were immersed rapidly in liquid nitrogen. The shoot tips were then rapidly warmed using unloading solution, transferred to a regeneration medium, stored in the dark for two weeks at 23℃, and then cultured under white fluorescent light at an intensity of 2000 lux with a 16-h photoperiod at 23℃. The average post-cryo regeneration rates of five accessions ranged from 52.7% to 87.5%.

Stimulation of Stem Callus and Regeneration of Ficus elastica "Decora" and from Apical Tips in vitro

Calli cultures of stems and leaves explants excised from field -grown rubber, Ficus elastica Decora, plants were formed on agar-solidified Murashige and Skoog (MS) medium.The results proved that MS medium supplemented with 1.0 mg L-1 benzyl adenin (BA) and 0.8mg L-1 2,4-dichloro-phenoxy acetic acid (2,4-D) was suitable to stimulate stem’s callus at ratio 87.5% . Whereas supplementation of MS medium with 0.5 mg L-1 of both BA and Indole -3-butyric acid (IBA)encouraged callus formation to reach76.6%. Leaves showed responses for callus initiation up to 75% at the same media. Stem calli showed limited ability to regenerate shoots on both agar solidified MS medium containing 1.0 mg L-1 2,4-D and kinetin (kin) 0.5 mg L-1 and MS medium supplemented with 0.5 mg L-1 of both BA and IBA. Transferring of shoots to differentiation medium (MS+0.5 mg L-1 BA+ 0.1 mg L-1 IBA) stimulated the growth and elongation of these shoots. Shoots were transferred to agar-solidified MS medium free from growth regulators failed to form roots. Because of the less number of shoots, other rooting media were not tested. The data showed, that shoot tips succeeded to regenerate shoots when they were cultured in different MS. The results proved clear increase in chlorophyll and protein content of the rubber shoots as compared with content of field grown rubber plants. It was noticed that agar-solidified MS medium supplemented with 4.0 mg L-1 BA was considered the optimum medium for shoots regeneration. All plants regenerated from shoot tips were readily rooted in agar-solidified MS medium with increasing Potassium Nitrate KNO3 from 1900 mg L-1 to 2000 mg L-1, and at the same medium supplemented with 3.0 mg L-1 IBA and 1.0 mg L-1 BA. All these plants were successfully acclimated and transferred to peat moss.

Tissue culture of Sophora tonkinensis Gapnep. and its quality evaluation

Background:Sophora tonkinensis Gapnep. is an important rare medicinal plant in China. There were only a few papers on the rapid propagation of S. tonkinensis through in vitro tissue culture, and still no report focuses on the quality analysis of in vitro tissue culture plantlets.Materials and Methods:The different concentrations of 6-benzylaminopurine (BAP), kinetin (KT), and indole-3-acetic acid (IAA) were used to establish and screen the optimal rapid propagation technology of S. tonkinensis by orthogonal test; the different concentrations of a-naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and ABT rooting power (ABT) were used to screen the optimal rooting technology. For quality evaluation of tissue culture plants, three different sites were chose to finish planting experiment. The leaf characteristics, radix ex rhizoma yield, and contents of matrine and oxymatrine were evaluated, respectively, to provide evidence of high yield and good qualities of tissue culture plants.Results:A large number of buds could be induced directly from epicotyl and hypocotyl explants on the Murashige and Skoog (MS) medium supplemented with 1.5 mg/l BAP, 0.5 mg/l IAA, and 0.5 mg/l KT; the best root induction medium was solid MS medium at half the macronutrient concentration supplemented with 1.0 mg/l NAA, 0.4 mg/l IBA, and 0.1 mg/l ABT. The rooting rate was 98%. All tissue culture plants showed normal leaf characteristics. Tissue culture plants from two sites possessed higher radix ex rhizoma yield and overall productivity of matrine and oxymatrine than those of seed plants.Conclusion:Tissue culture is a rapid, effective, and convenient propagation method for S. tonkinensis, and the quality of S. tonkinensis tissue culture plants meets the requirement of quality standard of China Pharmacopoeia (edition 2010), the crude drug from S. tonkinensis tissue culture plants will be suitable for substituting the crude drug from seed plants.