Half-strength Murashige-Skoog Medium Research Articles

Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.

This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2mg/L and kinetin 2mg/L; for shoot initiation 6-benzylaminopurine 2mg/L, kinetin 0.2mg/L, and indole-3-acetic acid 0.2mg/L; for shoot multiplication 6-benzylaminopurine 3mg/L and indole-3-acetic acid 0.2mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.

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.

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.

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.

Polysaccharides extracted from Moroccan seaweed: a promising source of tomato plant growth promoters

The present study investigates the use of seaweed polysaccharides as biostimulants of seed germination and plant growth. Seventeen species of Moroccan seaweeds were collected and screened for their crude polysaccharide content which ranged from 0.3 to 34% (dry weight basis). Chemical analyses of the obtained polysaccharide-enriched extracts (PEEs) were performed. Two bioassays were carried out to study seed germination and growth-promoting activity of PEEs. In the first experiment, the effect of PEEs on tomato seed germination was carried out under in vitro conditions. Half-strength Murashige-Skoog medium was supplemented with different PEEs at the concentration of 0.2 mg mL−1 (w/v). The germination percentage was recorded daily over 4 days. In a second experiment, tomato plants were grown in greenhouse conditions and watered with aqueous solution of seaweed PEEs (0.1 mg mL−1). After 30 days, the evaluated plant growth parameters were plant length, fresh and dry weight of plants shoots and roots, as well as chlorophyll content. Results showed that six seaweed species were a promising source of PEEs with beneficial effect on germination, plant biomass, and chlorophyll content. Two green seaweeds: Ulva rigida and Codium decorticatum, two red seaweeds: Gigartina sp. and Chondracanthus acicularis, and two brown seaweeds: Fucus spiralis and Bifurcaria bifurcata enhanced seed germination and plant growth. Nevertheless, the PEEs obtained from the red seaweed Gigartina pistillata inhibited seed germination. Principal component analysis of the results showed positive correlation between the sulfate content of PEEs and shoot dry weight as well as chlorophyll content.

Effect of La(NO3)3 and Ce(NO3)3 on shoot induction and seedling growth of in vitro cultured anoectochilus roxburghii

Anoectochilus roxburghii, a highly valuable medicinal plant species, is threatened in its native habitat. To ensure the sustainability of this useful resource, we studied the effect of La(NO3)3 and Ce(NO3)3 on plant tissue culturepropagated A. roxburghii. Apical buds, mid-stem segments, and basal rhizome segments were taken from aseptic seedlings of A. roxburghii and cultured in vitro on half-strength Murashige-Skoog medium containing different concentrations (up to 5.0 mg/L) of La(NO3)3 or Ce(NO3)3. After 100 d of culturing, average heights of plantlets derived from apical buds were 6.0 cm with 5.0 mg/L La(NO3)3 and 6.2 cm with 2.0 mg/L Ce(NO3)3, which respectively increased by 28.0% and 32.0% as compared with that in the non-treated control group. The optimum concentration for shoot induction from mid-stem segments was 1.0 mg/L Ce(NO3)3 which had a better proliferation times of 1.5-fold and an average length of 3.0 cm compared with 1.0-fold and 2.2 cm in the control group. Optimum growth from basal rhizome segments was achieved on media supplemented with 3.0 mg/L Ce(NO3)3, which provided a better proliferation times of 5.1-fold and an average shoot length of 4.5 cm compared with corresponding control values of 2.0-fold and 3.5 cm. Our results showed that La(NO3)3 and Ce(NO3)3 can accelerate A. roxburghii regeneration, which was probably due to the effect on chlorophyll contents, enzymes activity (superoxide dismutase, catalase and peroxidase) and malonldialedhyde contents caused by the addition of La(NO3)3 or Ce(NO3)3.

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.

Azospirillum brasilense increased salt tolerance of jojoba during in vitro rooting

Jojoba and Azospirillum brasilense can survive at high NaCl concentrations but with some undesirable effects. The aim of this work was to evaluate whether A. brasilense inoculation reduced the negative effects of salinity on several clonal lines of the jojoba plant. Shoots were induced in half-strength Murashige–Skoog medium with 24.6μM or 49.2μM indole-3-butyric acid for 6 days and grown in an auxin-free medium with 0, 40, 80, 120 or 160mM NaCl and inoculated or not with 107cfu of A. brasilense Cd or Az39 for 45 days. Rooting percentage, shoot and root biometric parameters, and root index were determined. Regressions of rooting percentage and root index with respect to NaCl concentration were performed for each combination of bacterization and indole-3-butyric acid induction, and the 50% inhibitory concentration (IC50) was determined. A. brasilense Cd and Az39 promoted rhizogenesis and increased root index and IC50, reducing the undesirable effects of NaCl on jojoba rooting. Both strains were successfully reisolated at the end of the experiment. Here, we establish a useful model for evaluating plant-bacteria interactions and selecting strains that promote plant growth under saline conditions.

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.

In vitro cultures of Drosera aliciae as a source of a cytotoxic naphthoquinone: ramentaceone

A protocol for the in vitro propagation of Drosera aliciae to increase the yield of the naphthoquinone, ramentaceone, was developed. The highest micropropagation coefficient was obtained using half-strength Murashige-Skoog medium supplemented with 0.4 μM 6-benzyladenine (BA). The genetic fidelity and stability of the regenerated plants was confirmed with RAPD markers. The activity of the isolated ramentaceone was determined against four human tumor cell lines: U937, HeLa, MCF-7, HCT-116 with the highest cytotoxic activity towards the leukemic U937 cell line with an IC(50) value of 3.2 μM.

PLANT REGENERATION FROM CALLUS CULTURE OF AN ENDANGERED ORCHID, GEODORUM DENSIFLORUM

An in vitro method was developed to regenerate plantlets from protocorm-derived callus of Geodorum densiflorum (Lam.) Schltr. cultured on half-strength Murashige-Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D) at 3 to 5 mg/L and 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) at 0.1 to 0.5 mg/L in the dark. The proliferation rate of the pale-yellow embryogenic callus was 3.9 to 4.2 fold in mediums containing 2,4-D 3 mg/L and TDZ 0.1 to 0.5 mg/L after one month of culture in the dark. The callus was regenerated to plantlets via protocorm-like bodies (PLBs) on medium containing 0.5 mg/L TDZ after 6 months of culture, and 644 PLBs and 56 shoot buds per 0.1 g callus were obtained after 12 months of culture. The well-rooted plantlets with pseudobulbs were transferred to sphagnum-containing pots and acclimatized in a greenhouse. The embryogenic callus of the endangered orchid, Geodorum densiflorum has been established.

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.

培養増殖中の温度と継代がスターチス・シヌアータの開花に及ぼす影響

1. スターチス・シヌアータ(Limonium sinuatum Mill.) 'Early Blue'の花穂切片を外植体とし, 4週間の初代培養を20℃ならびに27℃下で行った後, 20℃4週間の発根培養に移し, 器外に植え出して20℃で栽培した.20℃区では67%が抽だいしたのに対し, 27℃区では13%にとどまった.2. 初代培養後, 20℃での4週間の継代培養を繰り返し, 最後に発根培養を行って植え出したところ, 初代培養後ただちに発根培養に移した場合, 抽だい率は67%であったのに対し, 1回継代培養を行うと抽だい率は43%と低くなり, 2回以上継代培養を行うと全く抽だいが観察されなくなった.3. 継代培養中に2℃4週間の低温処理を行い発根培養に移した場合, また発根培養中に同様の低温処理を行った場合, いずれも植え出し後の抽だい率が著しく高くなった.4. 継代培養中に2℃4週間の低温処理を行っても, その後20℃で増殖のための継代培養を繰り返すと, 植え出し後の抽だい率が低下した.5. 以上より, スターチス・シヌアータの培養苗生産においては, 高い培養温度や継代培養の繰り返しが培養苗の開花能力の消失を引き起こす原因となること, 培養中に低温処理を行うことにより培養苗に開花能力を再付与しうることが示された.