Half-strength Murashige & Skoog Research Articles

Assessing the genetic fidelity of somatic embryo-derived plantlets of finger millet by random amplified polymorphic DNA analysis.

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal because of its mineral-nutrition value. With the increasing demand, there is a pressing need to conserve it through biotechnological approaches. High-frequency somatic embryogenesis from seed-derived callus of E. coracana was developed on Murashige-Skoog (MS) medium supplemented with a combination of auxins [Indole-3-acetic acid (IAA), 2,4-Dichlorophenoxy acetic acid (2,4-D)] and cytokinins [6-Benzylaminopurine (BAP), kinetin (KN)] in different concentrations, ranging from 0.1 to 5.0mg L-1. Seeds cultured on this medium produced three different types of primary callus. Type I callus was very compact and dark brown, type II callus was light brownish and type III callus appeared whitish and light brown. All three types of calli had differential proliferation responses. Type II compact brown calli were obtained on the MS medium supplemented with 1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg kinetin L-1. Friable yellowish embryogenic calli with a large number of somatic embryos were developed within 60days after being transferred to auxins and cytokinin (1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg Kinetin L-1) along with 200mg casein hydrolysate L-1. Germination of somatic embryos on a half-strength MS medium supplemented with 0.1% Kinetin led to the development of healthy plantlets within 30days. Genetic fingerprinting using random amplified polymorphic DNA (RAPD) revealed high levels of genetic fidelity. The study provides methods and hormonal concentrations required to develop somatic embryos in E. coracana for its genetic improvement and conservation.

Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Ranunculus wallichianus is a medicinally important plant and an endemic species to the Western Ghats of South India. An efficient and reliable indirect regeneration protocol system for R. wallichianus was developed from leaf explants in the present investigation. Leaf explants were cultured on both full-strength and half-strength MS (Murashige & Skoog) medium supplemented with different concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of 2,4-D and NAA. Among the different concentrations tested, the highest percentage of yellowish-green compact nodular callus formation was observed on a half-strength MS medium with 2.0 mg L− 1 of 2, 4-D. Then, the in vitro raised organogenic callus was cultured on a half-strength MS medium containing various concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of BA, KIN, and TDZ with 0.5 mg L− 1 NAA and 10 % CW for in vitro shoot regeneration. The highest percentage of regeneration response (97 %) and a maximum number of shoots formation (11.1 ± 0.13 shoots/culture with 9.2 ± 0.21 cm mean shoot length) were obtained from MS medium containing 2.5 mg L− 1 BA with 0.5 mg L− 1 NAA and 10 % CW. The well elongated in vitro raised shoots were effectively rooted in a half-strength MS medium with 2.5 mg L− 1 IBA + 250 mg L− 1 activated charcoal. The well-rooted plantlets were successfully hardened and acclimatized with a survival rate of 94 %. Clonal fidelity of in vitro raised plantlets was assessed by using DNA-based RAPD and ISSR molecular markers. A total of 56 and 47 monomorphic bands were obtained from RAPD and ISSR markers respectively. This present in vitro propagation protocol system could be effective for the conservation of R. wallichianus with their genetic purity and its further investigations.

Correction to: Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl2 for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L−1 resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L−1), along with IAA (0.5 to 2 mg L−1), or Kn (0.5 to 5 mg L−1), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR.

High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 μM BA. The presence of 4.0 μM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 μM and 2.0, 4.0 μM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 μM IBA or 4.0 μM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 μM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative 1H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ≤ 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.

Ex situ Conservation Using In vitro Methods of an Endangered Plant Sterculia urens Roxb.: A High Volume Trade Plant for Gum Karaya

Sterculia urens is a vanishing non timber tree species of India, known for its gum exudates having high worldwide trade value and hence needs to be protected for its sustainable supply in nature. In present research, an efficient regeneration protocol is standardized using cotyledonary node of Sterculia urens (Gum Karaya) seedlings. In addition, appearance of different anomalies such as high callogenesis, yellowing of leaves, translucent shoots, shoot etiolation, reduced leaf number, and leaf abscission was observed during the study. Thus based on the assumption that these abnormalities can be a result of ethylene accretion, effect of silver nitrate (AgNO3; ethylene inhibitor) to overcome such abnormalities and enhancement of shoot quality was studied. Among different media compositions tried, full strength Murashige & Skoog (MS) medium supplemented with 0.33 mg/L thiadiazuron (TDZ), was found to be optimum resulting highest shoot multiplication (19.62 ± 0.27) after five weeks incubation with 81.9% regeneration response recorded. After mass regeneration shoots were transferred to full strength MS medium fortified with 0.2 mg/L N6-Benzyladenine (BAP), 1.5 mg/L 6-furfurylaminopurine (Kn), and 2.0 mg/L gibberellic acid (GA3) in combination, which resulted optimum shoot elongation (5.06 ± 0.41 cm) within 2 weeks. Treatments, AgNO3 (1.0 mg/L) with Kn (1.0 mg/L) in MS medium gave adequate shoot quality having dark green & shiny leaves, strengthened stem (dark green), reduced inter-nodal length (de-etiolation) and reduced callus formation. The optimum rhizogenesis (5.67 ± 0.58) was obtained on half strength MS fortified with IBA (2.0 mg/L) when shoots were given pulse treatment using IBA (200 mg/L) for 3-4 min. Further, process of biotization at hardening stage using Glomus sp. enhanced the plant survival rate (80%) at acclimatization stage. Mycorrhizae treated plants were healthy dark green in color having improved root system and less susceptibility to infections in comparison to untreated plants. Tissue culture raised plants revealed 95% genetic homogeneity through Jaccard’s silmilarity coefficient using RAPD markers. The established protocol can significantly be used to generate quality plantlets in elevated amounts to overcome declining abundance of S. urens in natural environment.

Standardization of protocol for in vitro seed germination of Citrus indica Tanaka

An experiment was conducted with different concentrations of cytokinins and auxins, either singly or in combinations with full and half strength Murashige & Skoog (MS) medium for in-vitro seed germination of Citrus indica Tanaka-an endangered Citrus species of Meghalaya. Among the various treatments tried in full strength MS medium earliest germination (4.33 days) was recorded in BAP 0.5 mg/l + NAA 0.5 mg/l and Kn 1.0 mg/l+ NAA 0.5 mg/l. Cent per cent germination was noted in treatments BAP 2.0 mg/l + IBA 0.5mg/l and Kn 0.5 mg/l+ NAA 0.5 mg/l. Longest shoot of 2.93 cm was observed in Kn1.5mg/l + IBA 0.5 mg/l while longest root of 2.9cm was was observed in Kn 0.5 mg/l+ NAA 0.5 mg/l. Similar treatments were tried in half strength MS medium and observed that BAP 1mg/l showed earlier (4.33 days) and complete germination (100%) with higher shoot length (3.13 cm) and root length (3.15 cm). Comparing the results obtained from full strength and half strength MS medium it can be concluded that half strength MS medium supplemented with 6-benzylaminopurine @ 1.0 mg/l exhibited the best response for in vitro seed germination of Citrus indica.

Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl2 for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L−1 resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L−1), along with IAA (0.5 to 2 mg L−1), or Kn (0.5 to 5 mg L−1), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

An Efficient Somatic Embryo Liquid Culture System for Potential Use in Large-Scale and Synchronic Production of Anthurium andraeanum Seedlings.

Anthurium andraeanum Lind. is the second most important tropical flower in the world flower market. Somatic embryogenesis and plant regeneration in Anthurium has been reported previously; however, a stable and effective method for its commercial use has not been available. In this study, an efficient somatic embryogenesis and liquid culture system for large-scale production of A. andraeanum seedlings was achieved. Building on previous research, this study investigated the main factors for proembryogenic mass (PEM) proliferation, somatic embryo (SE) development, and SE germination in Anthurium. The results showed that relatively low concentrations of plant growth regulators, mineral nutrition, and sucrose promoted PEM proliferation, SE formation, and germination in a liquid culture system. This system can be described as follows: PEMs were induced from leaf blade explants on Murashige & Skoog (MS) medium with half-strength MS macronutrients (1/2 MS) containing 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L-1 kinetin (KT), and 3% sucrose and were proliferated in ½ MS liquid medium containing 1.0 mg L-1 2,4-D, 0.5 mg L-1 KT, and 3% sucrose. The highest proliferation coefficients were 5.11–5.16. PEMs were then transferred to MS medium with 1/8 MS macronutrients (1/8 MS) liquid medium containing 1% sucrose to develop into globular embryos and mature embryos. Finally, the mature embryos were placed on four layers of absorbent filter paper saturated with 1/8 MS liquid medium containing 1% sucrose for germination, and an average of 60 seedlings per gram SEs was obtained. This liquid culture system can be used in large-scale and synchronic production of Anthurium seedlings.

Storage and in vitro germination of orchids (Orchidaceae) seeds from Atlantic Forest - Brazil.

The orchid seed banks of Atlantic Forest may be considered a key strategy for the conservation of species threatened with extinction by indiscriminate collection or habitat destruction. The aim of this study was to evaluate the seed viability, to choose the best culture medium for the asymbiotic germination and evaluate germination, after storage for different periods and temperatures for the Brazilian native orchids: Gomesa praetexta (Rchb.f.) M.W.Chase & N.H.Williams, Gomesa forbesii (Hook.) M.W.Chase & N.H.Williams, Gomesa recurva R.Br. and Grandiphyllum divaricatum (Lindl.) Docha Neto. Knudson C (KC), Murashige & Skoog (MS), half-strength MS (1/2 MS macro- and micro-nutrients) and Woody Plant Medium (WPM) culture media were tested for germination. The WPM culture medium was the best for asymbiotic germination of all species evaluated, with high germination percentages and improved seedling development. Seeds of G. divaricatum, G. praetexta, G. recurva and G. forbesii indicated orthodox behavior, with high viability rates after 12 months of storage, being recommended the storage temperature of -80°C for the first three species and -20°C for G. forbesii. The protocol developed in the present study was efficient for seed bank storage, in vitro germination and seedling production of G. divaricatum and G. praetexta, contributing to conservation strategies of these species.

Development of an efficient regeneration system for the precious and fast-growing timber tree Toona ciliata.

Toona ciliata (Chinese mahogany) is an important timber species and secondary protected plant due to excessive exploitation in China. Here we developed a robust and efficient regeneration system for adventitious shoot induction using hypocotyl explants of T. ciliata. To facilitate plant growth, different regulators were added to Murashige-Skoog (MS) medium (0.5 mg/l 6-BA, 1.0 mg/l KT and 0.1 mg/l IBA). A regeneration frequency of 58.67% with four shoots per explant was achieved by horizontal setting of hypocotyls on MS medium and following a 20-day seeding period. MS medium supplemented with 0.3 mg/l 6-BA and 0.2 mg/l NAA was optimal for shoot multiplication and elongation, with a multiplication coefficient of 3.06. A rooting frequency of 93.33% was achieved using the half-strength MS containing 0.1 mg/l NAA. After acclimatization, plantlets were transplanted to sterilized nutrient soil containing a 2 : 1 ratio of vermiculate with 90% survival frequency. Thus, the regeneration system developed in this study would be useful for genetic transformation and other biotechnology endeavours in T. ciliata.

Activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and Padina gymnospora as growth promoters of tomato and mung bean plants

Although marine seaweeds have been used as biostimulants since the beginning of modern agriculture, studies have only recently focused on the ability of seaweed extracts and their polysaccharides to enhance growth of plants. In this work, two bioassays were carried out to study the growth-promoting activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and Padina gymnospora, obtained in neutral and alkaline conditions. Initially, the effect of seaweed extracts and polysaccharide-enriched extracts on seed germination and growth-promoting activity on tomato (Solanum lycopersicum cv. Rio Grande) plants under in vitro conditions was studied. Half-strength Murashige-Skoog (MS) medium with or without sucrose was supplemented with different concentrations of seaweed extracts (2, 4, and 10 mg mL−1) or polysaccharide-enriched extracts (0.2, 0.4, and 1.0 mg mL−1). The parameters evaluated were germination percentage, radicle and shoot length, and dry weight. In a second experiment, polysaccharide-enriched extracts at 1.0 mg mL−1 and indole-3-butyric acid as the control were studied for root inducer activity in mung bean (Vigna radiata). The majority of seaweed extracts had an inhibitory effect on seed germination. However, a significant effect (P ≤ 0.05) on tomato seedling growth (except for dry weight) was shown with seaweed extracts at 2 mg mL−1 included in half-strength MS medium with sucrose (30 g L−1). Moreover, 10 mg mL−1 neutral and alkaline seaweed extracts had an inhibitory effect on the parameters evaluated. In contrast, polysaccharide-enriched extracts obtained from U. lactuca and P. gymnospora promoted germination and stimulated growth of tomato plants compared to the controls. Additionally, treatment of mung bean hypocotyl cuttings with polysaccharide-enriched extracts of U. lactuca and P. gymnospora induced rooting more rapidly and in greater number compared to the controls. These results provide evidence that polysaccharide-enriched extracts act as an effective growth-promoting treatment.

HERITABILITY OF TRAITS RELATED TO GERMINATION AND MORPHOGENESIS IN VITRO IN ORNAMENTAL PEPPERS

The species Capsicum annuum L. has a low seed viability and is recalcitrant in vitro. Therefore, studies on the genetic control of traits favorable for in vitro development obtained from a diallel analysis might be interesting. Here we investigated the heritability of germination percentage and plant morphogenesis in in vitro seedlings of ornamental peppers. Four lines of ornamental pepper (UFPB 132, UFPB 134, UFPB 137, and UFPB 390) and their crosses were used. The seeds were germinated in half-strength MS (Murashige Skoog) medium. On the 7th day of cultivation, the percentage of germinated seeds (GS) was analyzed. After 30 days, the percentage of deformed plants (PD); presence of root (PR); length (RL) and number of roots (NR); hypocotyl length (HL) and diameter (HD); and number (NL), length (LL) and width of leaves (LW) were analyzed. Data were subjected to analysis of variance and Hayman’s diallel analysis. The effects of treatment (parents, F1 hybrids, and -reciprocals) were significant at the level of 1% for all traits, indicating existence of genetic variability. In the sufficiency test of the additive-dominant model, variables GS, NR and NL showed non-significant values for the regression coefficients. This demonstrated the validity of the imposed assumptions and indicated the feasibility of this model for the study of these genetic traits. Correlations Wi+Vi and Yii were negative for GS, PR and RL, ranging from -0.3137 to -0.9760, indicating these traits were controlled by dominant alleles. Positive correlations for other traits were seen, suggesting that the observed increase in the traits’ values are mostly caused by recessive alleles. Additive effects were more important than dominant effects for all traits, except NL, which indicates that these traits will be transmitted to the next generations.

Direct shoot regeneration from immature inflorescence cultures of Chlorophytum arundinaceum and Chlorophytum borivilianum

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L−1 BA, 150 mg L−1 Ads, 0.1 mg L−1 NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L−1 IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.

Effects of plant growth regulators on the rapid proliferation of shoots and root induction in the Chinese traditional medicinal plant Atractylodes macrocephala

We developed an efficient plant regeneration protocol for rapidly propagating Atractylodes macrocephala Koidz, an important traditional Chinese medicinal plant, via shoot organogenesis. Shoot multiplication was induced on Murashige-Skoog (MS) medium supplemented with various concentrations of N-phenyl-N-1, 2, 3-thidiazol-5-ylurea (TDZ), 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA). Rooting was induced on half-strength MS medium supplemented with NAA and indolebutyric acid (IBA). The maximum mean number of shoots (5.61) was obtained from a single explant by the combined effect of 1.08 μmol/L NAA and 2.25 μmol/L TDZ. The longest roots and a minimum number of roots were produced when they were cultured in a medium without plant growth regulators. The shortest roots and the largest number of roots were observed in the medium supplemented with 2.7 μmol/L NAA.

Direct shoot regeneration from epicotyl explants of grasspea (Lathyrus sativus)

A reproducible procedure is described for adventitious shoot organogenesis in epicotyl segments resulting in prolific plant regeneration of a grain legume grasspea (Lathyrus sativus L.). Among seedling explant types examined, epicotyl segments were most responsive. The highest percentage of direct shoot regeneration was elicited on Murashige–Skoog (MS) medium augmented with 4.0 mg L–1 6-benzyladenine (BA) + 2.0 mg L–1 α-naphthaleneacetic acid (NAA). Compared with four other genotypes examined, IC-120487 showed the highest shoot regeneration frequency (approximately 80%) with maximum shoot numbers (averaging eight shoots per explant) and longest average shoot length (approximately 4 cm). Rhizogenesis was induced in ~78% of the regenerated shoots in half-strength MS medium containing 0.5 mg L–1 indole-3-acetic acid (IAA). Plantlets were acclimated in vermi-compost and 75% of those transferred to soil survived and set viable seeds.

High Frequency Plant Regeneration from Callus Culture of Pleione formosana Hayata

High frequency plant regeneration was induced from protocorm-derived callus cultured on half-strength of Murashige—Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D, 0–5 mg l−1) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl, 0–1 mg l−1) urea (TDZ) in the dark. Twelve totipotent callus lines were selected within 76 callus lines regenerated on half-strength of Murashige—Skoog (MS) medium with 0.5 mg l−1 TDZ. The proliferation rate was 4–5-fold in fresh weight after 30 days of culture on half-strength MS medium containing 5 mg l−1 2,4-D and 0.5 mg l−1 TDZ in the dark. The maximum number of shoot buds generated by 0.01 g callus explant was 134 after 4 months of culture. These calli were regenerated to plantlets via protocorm-like bodies (PLBs) after 75–150 days of culture. The shoots, with two true leaves, were transferred to hormone-free medium, rooting and eventually formed plantlets. Totipotent callus lines of Pleione formosana Hayata have been successfully established in this study.

Embryogenesis and plant regeneration in anther culture of sunflower (Helianthus annuus L.)

A protocol for high frequency callus induction and plant regeneration from sunflower (Helianthus annuus L.) anthers is described. Different variables using Murashige & Skoog (MS) basal medium supplemented with 2.0 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/l N6-benzyladenine (BA) were tested for their ability to enhance the frequency of anther callusing and subsequent embryogenesis. Of these, agar concentration, sucrose concentration, carbohydrate source had significant effect on callusing, while differences due to incubation under dark vs light conditions, cold pretreatment of capitula for 1 to 6 days prior to anther inoculation and genotype on callusing were non-significant. However, all these factors exerted highly significant influence on embryogenesis when calli from the various media were transferred to medium supplemented with 0.1 mg/l NAA and 0.5 mg/l BA. With the procedure developed, callusing as high as 100% and embryo formation at a frequency of 44% was achieved. Although complete embryos were formed the frequency of their conversion to whole plantlets was low (14.3%). Hence, the embryogenic pathway was bypassed to obtain multiple shoots by transferring embryogenic calli with developing embryos to MS medium supplemented with 0.5 mg/l BA. Elongated shoots rooted on half-strength MS medium supplemented with 0.5 mg/l NAA. Cytological analysis of embryogenic callus and somatic embryos revealed haploids at a frequency of 30% while that of rooted plants showed haploid regenerants at a frequency of 8.3%. Nevertheless, the frequency of putative haploid plants could be enhanced through mass multiplication using nodal explants of the regenerants.

Triterpenoid Constituents of Tissue Cultures and Regenerated Organs of Taraxacum officinale

Complete plants were regenerated from callus cultures of Taraxacum officinale. Shoot cultures were first established on half strength Murashige-Skoog's medium containing 0.1ppm N6-benzyladenine with or without 0.1ppm α-naphthaleneacetic acid. These shoots regenerated roots on phytohormone-free medium, and then, upon transfer to soil in a pot, grew into complete plants with normal flowers and seeds. Triterpenoid constituents of the dedifferentiated callus cells, wild and regenerated shoot and root organs, and also of latex, were analyzed. Triterpene acids (oleanolic and ursolic acids) were found predominantly in callus cells. The composition of triterpen-3-ols was characteristically different between the organs: α-and β-amyrins were found in all the tissues, whereas taraxasterol and lupeol were detected in differentiated organs.

The effects of medium composition on biomass, sterols and triterpenols production by in-vitro cultures of Euphorbia characias

Calli of Euphorbia characias L. maintained on Murashige-Skoog (MS) medium (1962, Physiol. Plant., 15, 473-97) were grown on 20 other basal media. The highest callus biomass production was obtained on half-strength MS and the lowest on Cambell-Durzan medium (1975, Can. J. Bot., 53, 1652-6). Independently of the culture medium used, calli produced campesterol, β-sitosterol, Δ5-avenasterol and α-amyrin. The Gautheret medium (1942, Manuel Technique de Culture des Tissus Vegetaux, Masson Cie, Paris) was the most effective for the production of sterols and α-amyrin. The increase in sucrose supplementation increased callus biomass production but decreased the specific sterols and triterpenols content/dry weight. The increase in sucrose increased the specific phenolics content. The specific sterols and triterpenols content of in-vitro cultures increased during the exponential phase of biomass growth.

Photoautotrophic and photomixotrophic growth of strawberry plantlets in vitro and changes in nutrient composition of the medium

Explants excised from strawberry (Fragaria x ananassa Duch.) plantlets were cultured in vitro for 21 days on half-strength MS (Murashige & Skoog 1962) basal liquid medium with 20gl 1 sucrose and without sugar in the vessels capped with gas permeable microporous polypropylene film. The experiments were conducted under CO 2 nonenriched (350-450/zmol mol 1 in the culture room) and CO 2 enriched (2,000/xmolmol -I during the photoperiod in the culture room) conditions with a PPF (photosynthetic photon flux) of 200/xmol m-: s -1. The CO 2 concentration in the vessels decreased to approximately 200/xmol mol -~ during the photoperiod on day 21 under CO 2 nonenriched conditions. The fresh and dry weight, net photosynthetic rate (NPR) per plantlet, NPR per g leaf fresh weight, NPR per g leaf dry weight, the number of unfolded leaves, and ion uptake of PO]-, NO3, Ca2+, Mg2+ and K + on day 21 were the greatest under photoautotrophic (no sugar in the medium) and CO 2 enriched conditions. The residual percent of PO]- was 3% on day 21 under photoautotrophic and CO 2 enriched conditions.