High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis

An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented with 4.65 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23 μM gibberellic acid (GA₃) and 0.46 μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic stability of these regenerant plantlets.     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

Regeneration of <Emphasis Type="Italic">Clivia miniata</Emphasis> and assessment of clonal fidelity of plantlets

An efficient in vitro micropropagation system for Clivia miniata Regel was developed using basal tissues of young petals and young ovaries as explants. For callus induction, explants were incubated on Murashige and Skoog (MS) medium containing either 2.22 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 4.44 μM BA, 5.37 μM α-naphthaleneacetic acid (NAA), and 9.05 μM 2,4-D. Moreover, callus was induced from young ovaries when these were incubated on MS medium containing 8.88 μM BA, 10.74 μM NAA, and 9.05 or 18.10 μM 2,4-D. Subsequently, callus was transferred to MS medium supplemented with kinetin (KT) and NAA for shoot organogenesis. Frequency of shoot regeneration from petal-derived callus was highest when callus was transferred to medium containing 2.69 μM NAA with either 9.29 or 13.94 μM KT. Shoot regeneration frequency from ovary-derived callus was highest when this callus was transferred to medium containing 9.29 μM KT and 10.74 μM NAA. Overall, different explant types exhibited different organogenic capacities wherein, young petals had higher shoot regeneration frequencies than young ovaries. The highest rooting frequency (98.25 ± 3.04%) was obtained when shoots were transferred to half-strength MS medium without plant growth regulators. Regenerated plantlets were transplanted to soil mix and acclimatized, yielding a 96.80% survival frequency. Only 0.6% of regenerated plantlets exhibited morphological changes. The diploid status (2n = 22) of regenerated plantlets was determined using chromosome counts of root-tips. Moreover, inter-simple sequence repeats were used to assess the genetic fidelity of regenerated plantlets. Overall, regenerated plants shared 90.5–100.0% genetic similarities with mother plants and 89.0–100.0% similarities with each other.     

Regeneration of daylily (<Emphasis Type="Italic">Hemerocallis</Emphasis>) from young leaf segments

Calli were initiated from leaf segments (~0.5 × 0.5 cm) of daylily incubated on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-dichlorophenoxyacetic acid (2.4-D), and either benzyladenine (BA) or thidiazuron (TDZ). The highest frequency of callus induction was observed on medium with 6.79 μM 2,4-D plus either 4.55 or 6.81 μm TDZ. A period of callus maintenance on medium containing 5.37 μM naphthaleneacetic acid (NAA) plus 2.22 or 4.44 μM BA was necessary following induction to improve the quality of the callus, and significantly increase the frequency of embryogenic-like callus formation and shoot regeneration once calli were transferred to light. Over 70% of the regenerated shoots produced roots on ½ strength MS medium lacking plant growth regulators. The regenerated plantlets were successfully transferred into soil and acclimatized in growth rooms. This is the first report showing that leaf segments can be used for daylily regeneration.     

In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Callus induction and plant regeneration in <Emphasis Type="Italic">Dorem ammoniacum</Emphasis> D., an endangered medicinal plant

Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l⁻¹, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l⁻¹ for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l⁻¹ NAA and 2 mg l⁻¹ BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l⁻¹) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l⁻¹ for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l⁻¹ BA and 0.2 mg l⁻¹ IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l⁻¹ IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.     

Direct shoot organogenesis from hypocotyl explants of Jatropha curcas L. an important bioenergy feedstock

Multiple shoots were induced on hypocotyl segments reared from in vitro germinated seedlings of Jatropha curcas L., using Murashige and Skoog (MS) medium supplemented with N⁶‐benzyl adenine (BA) and kinetin (kin) either alone or in combination with indole‐3‐acetic acid (IAA). The combined treatment of 7.05 μm kin and 1.425 μm IAA resulted in maximum shoot production with an average shoot bud initiation of 12.1 per explant. The regenerated microshoots were transferred to root induction medium containing half‐strength MS salts supplemented with either indole‐3‐butyric acid (IBA) or IAA. Rooting of microshoots was best achieved on half‐strength MS medium supplemented with IBA (9.8 μm ). Rooted plantlets were subsequently acclimatized under green house condition and the plantlets showed 70% survival.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

High frequency in vitro propagation of <Emphasis Type="Italic">Trichopus zeylanicus</Emphasis> subsp. <Emphasis Type="Italic">travancoricus</Emphasis> using branch–petiole explants

Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 μM 2,4-dichlorophenoxyacetic acid upon subculture to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot morphogenesis. Medium with 13.3 μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower concentrations of BA (4.4, 6.6 and 8.8 μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 μM BA containing medium, but with reduced number of shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6 μM IBA, the whole branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium containing 2.7 μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix: a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar to that of the source plants flowered normally and set fruits.     

Plant regeneration from organogenic callus and assessment of clonal fidelity in Elephantopus scaber Linn., an ethnomedicinal herb

An efficient callus induction and plant regeneration system has been standardized for an ethnomedicinal plant, Elephantopus scaber Linn. Two explants i. e. seeds and leaf segments were used for callus induction. Murashige and Skoog (MS) medium supplemented with 5.0 μM 2, 4-dichlorophenoxy acetic acid (2, 4-D) and 0.5 μM kinetin (Kn) gave the optimum frequency (89 %) of callus induction from seed explant. The results showed that the highest response in terms of percent callus regenerating (91 %) and number of shoots (56) per culture was recorded on MS medium supplemented with 6.0 μM N₆-benzylaminopurine (BA) and 1.5 μM α naphthalene acetic acid (NAA). The best rooting of regenerated shoots was obtained on half strength MS medium supplemented with 6.0 μM indole-3- butyric acid (IBA). On this medium, 100 % of the shoots produced roots with a mean number of 3.2 roots per shoot. The positive role of vesicular arbuscular mycorrhizae (VAM) along with potting mix has been well established in the present study. Of the various potting mix employed for plant acclimatization, the highest response of 100 % plant survival was noticed when autoclaved garden soil, sand (2:1) and VAM was utilized as potting mix. Inter-simple sequence repeats (ISSR) were used to establish the clonal fidelity of regenerated plantlets and the banding profiles from callus derived plants were monomorphic and similar to those of mother plant, thus ascertaining the true-to-type nature of these plants.     

In vitro propagation,genetic and phytochemical assessment of Thymus persicus — a medicinally important source of pentacyclic triterpenoids

Thymus persicus (Ronniger ex Rech. f.) Jalas is a valuable and endangered natural source of antitumor pentacyclic triterpenoids, i.e., betulinic acid, oleanolic acid and ursolic acid, which grows in northwest Iran. As the plant has a low propagation rate in nature, a suitable method for in vitro-propagation is needed. With the aim of identifying a suitable system for regenerating T. persicus via direct organogenesis, Murashige & Skoog (MS) medium supplemented with different plant growth regulators (PGRs) was tested. In vitro-grown shoot tips were exposed to the cytokinins 6-benzylaminopurine (BAP), kinetin (KN), and thidiazuron (TDZ), alone or in combination with the auxins 1-naphthalene-acetic acid (NAA), 2,4-dichlorophenoxyacetic (2,4-D), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). The highest shoot formation (7.1 ± 0.9) was obtained with a medium fortified with 8.9 μM BAP plus 2.7 μM NAA. Regenerated shoots were easily rooted on the different tested media, with the most abundant (16.6 ± 1.4) and strongest roots obtained on half-strength MS medium containing 2.5 μM IBA. The rooted plantlets were successfully acclimatized (76.6%) in a greenhouse before transference to natural conditions. Homogeneity and phytochemical productivity of the in vitro regenerated plantlets were confirmed by random amplified polymorphic DNA (RAPD) profiles and high performance liquid chromatography (HPLC), respectively.     

High Frequency Plant Regeneration from Astragalus melilotoides hypocotyl and stem explants via somatic embryogenesis and organogenesis

An efficient and reproducible procedure is established for the plant regeneration from hypocotyl explants and hypocotyl-or stem-derived calli in Astragalus melilotoides. High frequency somatic embryo formation (98.3%) occurred direct on hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.69 µM NAA and 4.44 µM BA within 5 weeks. Three types of calli were induced from the hypocotyl and stem segments on MS medium containing 9.05 µM 2,4-D and 2.22–4.44 µM BA. Both somatic embryos and adventitious buds were initiated from hypocotyl-derived calli while only adventitious buds were formed from stem-derived calli in MS medium supplemented with 2.69 µM NAA and 4.44–8.89 µM BA. Somatic embryos or adventitious buds developed into plantlets following being cultured for 3 weeks on MS medium without any growth regulators or with 14.78 µM IBA, respectively. All the regenerated plants were normal with respect to morphology and growth characters, and produced fertile seeds after planting in soil.     

High frequency plant regeneration and accumulation of tropane alkaloids in regenerated plants of Scopolia parviflora

An efficient in vitro plant regeneration system was established from callus culture of Scopolia parviflora. Callus was induced from adventitious roots on B5 medium with 0.45–9.04 μM 2,4-dichlorophenoxyacetic acid (2,4-D). In vitro plantlet regeneration was achieved on B5 medium supplemented with 44.38 μM benzyladenine (BA), 3% sucrose, and 0.38% gelrite. Plantlets were transplanted to artificial soil and grown to maturity successfully in a greenhouse. The tropane alkaloid contents in regenerated plants were analyzed using high-performance liquid chromatography (HPLC), and were found to be higher than those of adventitious roots, native growing plants, and acclimated plants. Regenerated plants from organogenic callus cultures produced a greater amount of tropane alkaloids.     

Plant regeneration via somatic embryogenesis in Drimia robusta

A simple efficient in vitro plant regeneration system was developed by direct and indirect somatic embryogenesis of Drimia robusta, a medicinal plant extensively used in South African traditional medicine. Different developmental stages of somatic embryos (SEs: globular embryos, partial pear-shaped embryos and club-shaped embryos), club-shaped cotyledon initiation, plumule initiation and plantlets were directly obtained from leaf explants on Murashige and Skoog (MS) medium containing 3.5 % (w/v) sucrose and different plant growth regulators (PGRs). In MS medium containing 3.5 % (w/v) sucrose and supplemented with 10 μM picloram, 1 μM thidiazuron (TDZ) and 20 μM glutamine, a higher number of SEs and plantlets were achieved. These were established onto half-strength MS medium followed by successful acclimatization (100 %) in the greenhouse. Liquid somatic embryo medium (SEM_L) containing 500 mg of friable embryogenic callus on MS medium supplemented with different concentrations and combinations of PGRs and organic elicitors produced different stages of SEs. Somatic embryo production was enhanced by 0.5 μM picloram, 1 μM TDZ and mebendazole treatment. The highest number of plantlets (9.0 ± 0.70) was obtained in SEM_L containing 0.5 μM picloram, 1 μM TDZ and 25 mg l^?1 haemoglobin. All the cotyledon and plumule embryos germinated on half-strength MS medium, however 90 % of SEs germinated on half-strength MS medium containing 0.5 μM naphthaleneacetic acid. All plantlets were successfully acclimatized in the greenhouse. This first report of D. robusta somatic embryogenesis provides an opportunity to control extinction threats, ensure germplasm conservation and provides a system for analysis of bioactive compounds and bioactivity.     

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

Plant regeneration from suspension cells induced from hypocotyls derived from interspecific cross Alstroemeria pelegrina × A. magenta and transformation with Agrobacterium tumefaciens

Embryogenic cell suspension cultures were established using the ovule culture of an interspecific cross, Alstroemeria pelegrina var. rosea × A. magenta. Ovules harvested 14 days after pollination were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGRs); calli were produced on the hypocotyl surface in germinating zygotic embryos. Suspension cells were induced from the calli by using liquid MS media containing 2,4-dichlorophenoxyacetic acid or 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram). Adventitious embryos developed from the suspension cells on half-strength MS medium supplemented with 0.5 mg l⁻¹ of both α-naphthaleneacetic acid and N⁶-benzylaminopurine; they grew into plantlets on the same medium. The plantlets formed rhizomes following transfer to half-strength MS medium without PGRs, and acclimatized plants were easily established. Subsequently, Agrobacterium-mediated transformation system was applied. The suspension cells were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which contain neomycin phosphotransferase II, hygromycin phosphotransferase and intron-containing ?-glucuronidase (intron-GUS) genes. Seven days after co-cultivation, the cells were subjected to GUS assay; staining was most pronounced in the cells subcultured in a picloram-containing liquid medium and co-cultivated with EHA101/pIG121Hm. The co-cultivated cells were transferred to the MS medium containing picloram and 20 mg l⁻¹ hygromycin; 1 month later, several hygromycin-resistant callus lines showing GUS activity were obtained. Transgenic plants were obtained through our plant regeneration system, and foreign gene insertion into the regenerated plants was confirmed by polymerase chain reaction.     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.