In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

Mass propagation of <Emphasis Type="Italic">Plectranthus bourneae</Emphasis> Gamble through indirect organogenesis from leaf and internode explants

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.     

Subcellular localization of endogenous IAA during poplar leaf rhizogenesis revealed by in situ immunocytochemistry

Poplar 741 [Populus alba × (P. davidiana + P. simonii) × P. tomentosa] leaves were rooted within 8 days when cultured on 1/2 MS medium. The subcellular localization of endogenous indole-3-acetic acid (IAA) in the rhizogenesis was investigated, using an immunocytochemical approach. The results of IAA subcellular localization revealed organelle-specific distribution. Three days after root induction, IAA in vascular cambium cells of the basal region of the petiole was distributed mainly in the plasma membrane, endoplasmic reticulum (ER), and nucleus, with a lesser amount in the cytoplasm. In phloem of the basal region of the petiole, IAA was detected in the plasma membrane and ER of the companion cell and in the plasma membrane of the sieve element. In xylem of the basal region of the petiole, no IAA gold particles were labeled. In mesophyll cells IAA was distributed in the chloroplast starch grains before root induction, and the amount in the chloroplast starch grains increased after 3 days after root induction. This suggests that the plasma membrane and nucleus of cambium cells may be the target sites where IAA performs its physiological activities during poplar leaf rhizogenesis. IAA polar transport from lamina mesophyll to the basal region of the petiole during rhizogenesis is mediated by phloem. The starch grains of mesophyll chloroplasts appeared to accumulate IAA and may be a source of IAA during poplar leaf rhizogenesis. Novel and direct evidence regarding the function of IAA during rhizogenesis is provided in this study.     

An efficient callus proliferation protocol and rhaponticin accumulation of Rheum franzenbachii Munt., a medicinal plant

An efficient callus proliferation system for Rheum franzenbachii Munt., a rare medicinal plant, has been developed. Callus induced from leaf explants incubated on Murashige and Skoog (MS) medium with appropriate supplements of plant growth regulators. In the 6-benzylaminopurine (6-BAP) in combination with α-naphthalene acetic acid (NAA) treatments, different concentrations of NAA showed different induction effects on explants. When concentration of 6-BAP was as high as 2.0 mgl^?1 in combination with 0.5 mgl^?1 NAA, the callus induction rate reached 58.3%. N-phenyl-N’-1,2,3-thiadiazol-5-ylure (TDZ) in combination with NAA was very suitable for callus proliferation compared to TDZ in combination with 2,4-dicholorophenoxy acetic acid (2,4-D) or TDZ in combination with indole-3-acetic acid (IAA). Fresh and dry weight of callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ in combination with 0.2 mgl^?1 NAA increased 26.3 and 15.0 times within 35 days culture, respectively. Quantitative analysis of rhaponticin by HPLC showed that the phytochemical profile of callus was similar to that of wild plants, and the content of rhaponticin in callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ and 0.2 mgl^?1 NAA was 16.6 mgg?¹DW compared to that of 4.0 mgg^?1 DW in wild plants.     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

杨树试管苗嫩茎生根过程中内源IAA的免疫金银定位

生长素类物质在木本植物生根过程中发挥重要作用。杨树生根与生长素的关系及生根过程中内源激素的变化已有大量报道, 而生根过程中生长素的组织定位分析则尚未见报道。该文应用免疫化学分析方法对741杨(Populus alba × (P. davidiana × P. simonii) × P. tomentosa)嫩茎生根过程中内源IAA在组织中的分布进行了研究。结果显示, 741杨的嫩茎在无外源激素的1/2MS培养基上诱导10天后可生根, 14天后生根率达100%。诱导前, 嫩茎基部组织中几乎没有IAA信号; 诱导8天后, 嫩茎基部维管组织中有大量的IAA积累, 而且中部的维管组织中也有明显的IAA信号(主要分布在韧皮部和维管形成层); 10天后, 形成不定根原基, 此时IAA主要分布在根原基; 12天后, 根原基分化成不定根并突破表皮, IAA在不定根中的分布主要集中在根尖和中柱。该文对741杨的嫩茎生根过程中IAA的组织分布特点及运输途径进行了讨论。     

Optimized shoot regeneration for Indian soybean: the influence of exogenous polyamines

A simple and efficient regeneration protocol was established for soybean [Glycine max (L.) Merrill]. Cotyledonary node explants from 7-day-old in vitro seedlings were used as explants. The effect of different plant growth regulators [N ⁶ –benzyladenine (BA), kinetin (KT), thidiazuron (TDZ), gibberellic acid (GA₃), zeatin riboside (ZTR), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA)] along with polyamines (Spermidine, spermine, and putrescine) were investigated at different stages of regeneration using direct organogenesis system. Exogenous spermidine (137.69 μM) in shoot induction medium containing optimal BA concentration (2.22 μM) induced maximum number of shoots (39.02 shoots/explant) compared to BA (2.22 μM) alone. Regenerated shoots elongated well in shoot elongation medium containing GA₃ (1.45 μM) and spermine (74.13 μM), and developed profuse roots in root induction medium containing putrescine (62.08 μM). Rooted plantlets were successfully hardened and acclimatized with a survival rate of 92 %. The amenability of the standardized protocol using cultivar PK 416 was tested on four more Indian soybean cultivars JS 90–41, Hara soy, Co1, and Co2 of which PK 416 was found to be the best responding cultivar, with a maximum of 96.94 % shoot induction.     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l^?1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l^?1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l^?1 glycine, 690 mg l^?1 proline, and 1.0 g l^?1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l^?1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l^?1 6-benzylaminopurine; addition of 2.0 mg l^?1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l^?1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.     

Adventitious shoot regeneration from Sinningia speciosa leaf discs in vitro and stability of ploidy level in subcultures

Summary Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N⁶ benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26.     

Direct embryo formation in leaves of Camillia japonica L.

Summary The culture conditions for direct embryo formation in leaves of Camellia japonica L. were established. An auxin treatment followed by incubation during 11 days in darkness on diluted Murashige and Skoog modified basal medium induced direct morphogenesis. The number of subcultures, subculture interval and leaf age affected in vitro leaf response. The results showed that the cells from a cultured leaf respond differently to the same culture conditions by forming embryos, roots, and non-morphogenic as well as organogenic callus. Direct embryo formation occurred only in the marginal leaf regions. Direct root formation only occurred in a well-defined region of the midrib whereas callus was preferentially formed on the leaf basis. The results suggest the existence of differences in morphogenic competence according to leaf regions. Plantlet regeneration was successfully achieved from somatic embryos and from leaf basisderived callus, via shoot bud induction.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Indirect organogenesis from leaf explants of Hemidesmus indicus (L.) R. Br.: an important medicinal plant

Hemidesmus indicus (Asclepiadaceae) leaf explants were utilized for establishing culture in MS medium fortified with individual cytokinins, auxins, and their combinations. Optimum response (80%) was observed in N⁶-benzyladenine (BA, 20 μM) + indole-3-acetic acid (IAA, 1 μM) with 19.67 ± 0.81 shoots per explant. Roots were induced in ¼MS + indole-3-butyric acid (IBA, 20 μM).     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Effect of plant growth regulators on morphogenesis and forskolin production in Plectranthus barbatus Andrews

Plectranthus barbatus (syn. Coleus forskohlii) is the only known source of forskolin, a compound with a wide range of pharmacological activities. Here, an efficient protocol for adventitious root regeneration from leaf explants of P. barbatus was developed. Different concentrations of plant growth regulators individually and in combination were used to induce roots in vitro. Morphogenic responses and forskolin production varied depending on the concentrations of plant growth regulators added to the medium. Lower concentrations of auxins trigger callus proliferation while higher concentrations induced adventitious root regeneration. Of all the auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2 (2,4,5-trichlorophenoxy) propionic acid (2,4,5-TP), and 4-amino-3,5,6-trichloropicolinic acid (picloram) induced callus, whereas α-naphthaleneacetic acid (NAA), indole-3-acetic acid, and indole-3-butyric acid induced rhizogenesis. Use of picloram at 1.0 and 0.5 mg l⁻¹ resulted in the formation of friable callus, and when combined with 0.5 mg l⁻¹ 6-benzylamino purine (BA), rhizogenic callus was produced. The cytokinins BA and kinetin produced a mixed response of multiple shoot regeneration, callus proliferation, and rhizogenesis. The maximum forskolin content of 1,178 mg kg⁻¹ dry weight was found in root cultures initiated on Gamborg’s B₅ medium supplemented with 0.5 mg l⁻¹ NAA. The biosynthesis of forskolin was differentiation dependent, and rhizogenic cultures exhibited the maximum biosynthetic potential for forskolin.     

Plant regeneration from apple seedling explants and callus cultures

Leaf, cotyledon, and hypocotyl explants were obtained from 3-week-old seedlings of open-pollinated ‘Golden Delicious’ (Malus domestica bork H.) grown in vitro. They were placed on modified Murashige and Skoog (MS) medium containing B5 vitamins, sucrose and agar, supplemented with 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA), and maintained at 25°C±2 in the light or in the dark to assess morphogenetic responses. Leaf and cotyledon explants cultured in the dark for an initial 3 weeks, then transferred to light for 4 weeks, produced 5- to 20-fold more adventitious shoots than those cultured for 7 weeks in the light. Conversely, light did not significantly influence the number of adventitious shoots formed on hypocotyl explants. Five-minute daily exposures of leaf explants to red light (651 nm) suppressed adventitious shoot formation by 80%; five-minute exposure to far-red light (729 nm) immediately following the red light counteracted the red suppression. Seedling explants, immature fruit halves and immature embryos were also cultured on Schenk and Hildebrandt (SH) medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D), p-chlorophenoxyacetic acid (CPA) and kinetin. Light inhibited callus formation on leaf and cotyledon explants, but not on hypocotyl explants. The derived callus was placed on MS + BAP or MS + BAP + NAA for shoot regeneration. Both shoots and roots regenerated from callus placed in the dark but not in the light; the frequency of shoot regeneration was 5% or less. Regenerated shoots were rooted on MS macronutrient salts (1/3 concentration), micronutrients, i-inositol, thiamine HCl, sucrose and agar with or without indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or NAA under a light intensity of 5.0 W.m^-2 (16 h per day). Auxin concentration strongly influenced root morphology.     

Micropropagation of Rehmannia glutinosa Libosch.: production of phenolics and flavonoids and evaluation of antioxidant activity

Rehmannia glutinosa Libosch., a valuable medicinal plant, was successfully propagated in vitro using shoot tip explants. Shoot multiplication was performed in glass tubes and in a nutrient sprinkle bioreactor. A mixture of 0.1 mg L^?1 indole-3-acetic acid (IAA) and 1.0 mg L^?1 of 6-benzylaminopurine in Murashige and Skoog (MS) agar-solidified medium proved the best combination for multiple shoot induction, yielding 8.2 shoots per explant after 4 weeks of culture in glass tubes. The number of shoots increased to 21 per explant when the same combination of growth regulators was used in a nutrient sprinkle bioreactor. The shoots rooted with a frequency of 93 % after 6 weeks of culture on MS agar medium supplemented with IAA (0.1 mg L^?1) before being acclimatized in the greenhouse. The antioxidant activities of methanolic extracts from the leaves and roots of the in vitro-regenerated plants of R. glutinosa cultivated in the greenhouse were evaluated using four in vitro assays: scavenging of free radicals (DPPH and ABTS), transition metal reduction and total antioxidant activity phosphomolybdenum test. In all cases, the methanolic extract from leaves demonstrated better antioxidant activity than those taken from roots. A strong correlation was found between total phenolic and flavonoid content, and the antioxidant capacity of the studied extracts.     

Direct shoot organogenesis from <Emphasis Type="Italic">in vitro</Emphasis>-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl⁻¹ IAA and 2 mgl⁻¹ BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl⁻¹ BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl⁻¹ of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.     

Micropropagation of Ajuga bracteosa, a medicinal herb

For conservation and genetic transformation, a successful in vitro micropropagation protocol for Ajuga bracteosa, a medicinal herb has been established for the first time. MS medium supplemented with IAA (2 mg/L) and BA (5 mg/L) induced 100 % shoot regeneration with an average of 41.4 shoots of 8.4 cm per culture. Excised in vitro shoots when transferred to MS + IBA (0.5 mg/L) produced 20 roots/shoot of 20.2 cm average length in 100 % cultures. Of the three explants, leaf, petiole and root, leaf displayed quickest response followed by petiole while root was the slowest. Hardening of plantlets was achieved with 82 % survival. The hardened plants were maintained in pots with garden soil under controlled (Temp. 25?±?2 °C) conditions. RAPD exhibited genetic fidelity with 100 % monomorphism in regenerants.     

Callus mediated shoot organogenesis and regeneration of cytologically stable plants of Ledebouria revoluta: An ethnomedicinal plant with promising antimicrobial potency

Ledebouria revoluta are important ethnomedicinal plant found in India and South Africa. Micropropagation via indirect shoot organogenesis had been established from three types of explant (i.e. scale leaf, leaf lamina and root) of L. revoluta. Scale leaf was found superior as compared to leaf lamina and root explant with respect to their organogenic callus induction potentiality. Murashige and Skoog (1962) [MS] media supplemented with 3.0?mg?L^?1 2,4-dichlorophenoxyacetic acid, 0.75?mg?L^?1 β-naphthoxyacetic acid were best effective for inducing organogenic callus. Maximum 17.0?±?0.52 bulblets were induced from about 500?mg of callus within 42–46?days sub-culturing on a medium containing 0.75?mg?L^?1 kinetin. The bulblets were matured (86.7% success) after one month culture on the same medium composition. The best result of in vitro root induction with 100% response and 8.4?±?0.31 roots per bulb was achieved after 18?days of implantation on MS medium containing 2.0?mg?L^?1 indole-3-butyric acid. Plantlets were acclimatized with a 96.0% survival rate. Chromosomal studies revealed cytological stability of callus cells and all regenerants containing 2n?=?30 chromosomes, same as parental plants. Antimicrobial activity of L. revoluta was tested against two Gram-positive bacteria, three Gram-negative bacteria and two fungi. The methanol and ethanol extract proved more effective against bacteria, whereas acetone and chloroform extract shows potential anti-fungal activities. Present protocol can be applied reliably to produce uniform planting materials in large scale. In addition, this efficient indirect regeneration pathway via callus culture opens a way for improvement through genetic transformation.     

Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages

The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment “PLA”) cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0 μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0 μM) were tested for calli induction, and with 5 μM BA + 0.5 μM NAA or IAA for shoot development. The average number of shoots (≥5 mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5 μM IAA + 10 μM BA for calli induction and on 0.5 μM IAA + 5 μM BA for shoot development, and by culturing petiole explants on 0.5 μM NAA + 10 μM BA followed by a second culture on 0.5 μM NAA + 5 μM BA. The highest frequency of shoot rooting was achieved with 10.0 μM NAA and 1.0 μM gibberellic acid (GA₃). Rooted plants were acclimatised in a culture chamber, reaching 96 % survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100 % survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4 months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824 μg g^?1 DW total FC) and lowest in organogenic calli (up to 50 μg g^?1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage.