Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

Rapid clonal propagation of Vitex trifolia

This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2-isopentenyladenine (2-iP) (0.25 – 10.0 μM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 μM BAP. Shoot cultures was established repeatedly subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 μM naphthaleneacetic acid (NAA). Rooted plantlets transferred to pots containing autoclaved soil and vermiculite mixture (1:1) showed 90 % survival when transferred to outdoor.     

A two-step procedure for in vitro multiplication of cloudberry (<Emphasis Type="Italic">Rubus chamaemorus</Emphasis> L.) shoots using bioreactor

Cultures of three cloudberry (Rubus chamaemorus L.) clones collected from natural stands in Newfoundland and Labrador, Canada were established in vitro on a modified cranberry (Vaccinium macrocarpon Ait.) tissue culture medium containing 8.9 μM 6-benzylaminopurine (BAP). Clones were compared for in vitro shoot proliferation on gelled medium supplemented with varying levels of BAP and thidiazuron (TDZ). Addition of 5.8 μM gibberellic acid (GA₃) in 8.9 μM BAP-contained medium improved shoot proliferation. TDZ supported rapid shoot proliferation at low concentration (1.1 μM) but induced 20–30% hyperhydricity in a plastic airlift bioreactor system containing liquid medium. Bioreactor-multiplied hyperhydric shoots were transferred to gelled medium containing 8.9 μM BAP and 5.8 μM GA₃ and produced normal shoots within 4 weeks of culture. Genotypes differed significantly with respect to multiplication rate with ‘C1’ producing the most shoots per explant. Proliferated shoots were rooted on a potting medium with 65–75% of survivability of rooted plants. Present results suggested the possibility of large-scale multiplication of cloudberry shoots in bioreactors.     

Multiple Shoot Regeneration from Immature Embryo Explants of Papaya

A simple and rapid method for multiple shoot formation in vitro from immature embryo axis explants of Carica papaya L. cvs. Honey Dew, Washington and Co2 is described. Multiple shoot regeneration was achieved by culture of the explants on modified Murashige and Skoog (MS) medium supplemented either with thidiazuron (TDZ; 0.45–22.7 μM) or a combination of benzylaminopurine (BAP; 0.2 – 8.84 μM) and naphthalene acetic acid (NAA; 0.5 – 2.64 μM). Highest frequency of shoot regeneration occurred on medium supplemented either with 2.25 μM TDZ or a combination of BAP (4.4 μM) and NAA (0.5 μM). Composition of the basal media influenced the frequency of multiple shoot initiation. Stunted shoots regenerated at 4.5 μM and higher concentrations of TDZ. Such shoots could, however, be elongated by transfer to medium containing 5.7 μM GA₃. Rooting of the regenerated shoots was achieved in presence of indolebutyric acid (IBA; 4.92 – 19.68 μM), however, least response was in presence of 14.7 μM IBA. Rooted plants were hardened and transferred to pots. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis and shoot organogenesis from leaf explants of <Emphasis Type="Italic">Primulina tabacum</Emphasis>

Primulina tabacum is a rare and endangered species that is endemic to China. Establishing an efficient regeneration system is necessary for its conservation and reintroduction. In this study, when leaf explants collected from plants grown in four ecotypes in China are incubated on Murashige and Skoog (MS) medium containing 5.0 μM thidiazuron (TDZ) for 30 days, then transferred to medium containing 5.0 μM 6-benzyladenine (BA), adventitious shoots are then observed. Conversely, when leaf explants are incubated on medium containing 5.0 μM BA for 30 days, then transferred to medium containing 5.0 μM TDZ, somatic embryogenesis is induced. This indicates that somatic embryogenesis and shoot organogenesis could be switched simply by changing the order of two cytokinins supplemented in the culture medium. Histological investigation has revealed that embryogenic cells are induced within 30 days following incubation of explants in medium containing TDZ. Only if embryogenic cells were induced, TDZ could enhance somatic embryogenesis and BA could stimulate shoot organogenesis. When comparing explants from different ecotypes, leaf explants from Zixiadong in Hunan Province could induce low numbers (1–2) of either somatic embryos or adventitious shoots on medium containing either 5.0 μM TDZ or 5.0 μM BA, respectively. Whereas, leaf explants from plants collected from the other three ecological habitats could induce 50–70 somatic embryos/adventitious shoots per explant. Moreover, somatic embryos could induce secondary somatic embryogenesis and adventitious shoots on different media. All regenerated shoots developed adventitious roots when these are transferred to rooting medium, and over 95% of plantlets have survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite).     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of Indian Kino (<Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.) using Thidiazuron

An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2 ± 0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4 ± 0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4 ± 0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.     

Plant regeneration from in vitro leaves of mature black cherry (Prunus serotina)

A regeneration system was developed for Prunus serotina from a juvenile (F) and two mature genotypes (#3 and #4). Adventitious shoots regenerated from leaves of in vitro cultures on woody plant medium with thidiazuron (TDZ) and naphthaleneacetic acid (NAA). The best regeneration for genotype F (91.4%) was observed on medium with 9.08 μM TDZ and 1.07 μM NAA. The highest mean number of shoots (8.2) was obtained on medium containing 9.08 μM TDZ and 0.54 μM NAA. Genotype #3 had the highest regeneration (41.7%) with a mean number of shoots (4.8) on 9.08 μM TDZ and 1.07 μM NAA, whereas genotype #4 had a 38.8% regeneration with a mean of 3.3 shoots. Genotype #4 had the highest mean number of shoots (4.8) on 4.54 μM TDZ and 1.07 μM NAA. Silver thiosulphate at 60 or 80 μM increased the percent regeneration of the mature genotypes #3 (75%) and #4 (58%). Adventious shoots were rooted (70–76%) and rooted plantlets survived after acclimatization to the greenhouse. The effect of kanamycin concentration on adventitious shoot regeneration was also evaluated.     

In vitro regeneration of sesame (Sesamum indicum L.) from seedling cotyledon and hypocotyl explants

The goal of this study was to develop an efficient regeneration protocol to be used for genetic transformation of sesame. Published regeneration methods using benzyladenine (BA) and 1-naphthalene acetic acid (NAA) were unsuccessful for the cultivars used herein. Experiments were carried out using cotyledon and hypocotyl explants from the cultivar Mtwara-2. Later the optimised culture conditions were used to investigate the regeneration response of different genotypes. There was significant interaction between hormone treatments and macronutrients for shoot and root regeneration. Results also showed that shoot regeneration was significantly influenced by explant type. Shoots were only obtained from cotyledons whereas both cotyledons and hypocotyls could produce roots. Modified Murashige and Skoog (MS) medium with N6 macronutrients resulted in twice the shoot regeneration frequency obtained with ½MS macronutrients in the presence of thidiazuron (TDZ). The shoot regeneration frequency was significantly reduced when BA was used in place of TDZ. On shoot regeneration medium containing BA and NAA, only roots were formed. Replacing NAA with indole-3-acetic acid (IAA) greatly improved the regeneration of shoots. The optimum growth regulator combination for shoot regeneration was 20 μM TDZ together with 2.5 μM IAA, which gave a frequency of 63% and 4.4 shoots per regenerating explant for the best cultivar Ex-El. Genotypic differences were significant both for the number of explants regenerating shoots and the number of shoots produced per regenerating explant.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

Plant regeneration from protoplasts of <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA) via somatic embryogenesis

A protocol for plant regeneration from protoplasts of Musa acuminata cv. Mas (AA) via somatic embryogenesis was developed. Viable protoplasts were isolated from embryogenic cell suspensions at a yield of 1.2 × 10⁷ protoplasts/ml packed cell volume (PCV). Liquid and feeder layer culture systems with medium-A and medium-B were used for protoplast culture. In liquid culture system, medium-B was more efficient for inducing cell division (17.5% at 14 days) and colony formation (6.7% at 28 days) than medium-A. However, all protoplast-derived cell colonies (PDCC) obtained from liquid culture system could not develop further. In feeder layer culture system, there was no significant difference between medium-A and medium-B on cell division and colony formation of the cultured protoplasts, and the cell division frequency at 14 days and colony formation frequency at 28 days were 24.5% and 11.2%, respectively, in medium-B. Comparative study on the effects of BAP (2.2 μM, 4.4 μM, 8.8 μM), zeatin (0.4 μM, 0.8 μM, 1.2 μM) and TDZ (0.2 μM, 0.4 μM, 0.6 μM) on embryo formation of PDCC from feeder-layer culture indicated that TDZ was best. TDZ at 0.4 μM induced 7906 mature embryos per ml PCV PDCC, which was 4-fold the frequency as with BAP at 4.4 μM, 7.5-fold as with zeatin at 0.8 μM and 150-fold as control medium (no mentioned cytokinins) after 45 days on M3 medium. About 44% of the mature embryos were converted into plantlets with poor root system after subculture on M4 medium. Root further development of regenerated plantlets was promoted by addition of activated charcoal (AC) to MS basal medium.     

In vitro plant regeneration from organogenic callus of <Emphasis Type="Italic">Curcuma kwangsiensis</Emphasis> Lindl. (Zingiberaceae)

A novel protocol for callus-mediated shoot regeneration was established for an important medicinal and ornamental plant native to South China, Curcuma kwangsiensis, using shoot base sections excised from seedlings in vitro as explant sources. The frequency of callus formation reached 91% for explants cultured on MS medium containing 1.4 μM TDZ, 4.4 μM BA and 2.3 μM 2,4-D. 8.2 shoots per callus was achieved on MS medium supplemented with 1.4 μM TDZ, 17.8 μM BA and 2.7 μM NAA. Single shoots transferred into MS medium free of plant growth regulator rooted well. Regenerated plants acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Direct somatic embryogenesis and shoot organogenesis from leaf explants of Primulina tabacum

An efficient propagation system via somatic embryogenesis and shoot organogenesis and plant regeneration system for endangered species Primulina tabacum Hance was established. Thidiazuron (TDZ) was the key plant growth regulator for inducing somatic embryogenesis and kinetin (KIN) and 6-benzylaminopurine (BAP) were the key cytokinins for inducing shoot organogenesis from leaf explants. TDZ combined with BAP or KIN in the induction Murashige and Skoog medium induced both somatic embryos and adventitious shoots. Leaf explants with abaxial site in contact with the medium induced less somatic embryos or adventitious shoots compared to inversely placed leaf explants and the optimum pH was 6.5–7.0. Secondary somatic embryos or adventitious shoot could be induced from primary somatic embryos using TDZ and BAP. Shoots developed adventitious roots on rooting medium containing 0.5 μM indole-3-butyric acid and 0.2 % activated carbon. Over 90 % of plantlets survived following acclimatization and transfer to potting mixture (sand:Vermiculite:limestone; 1:2:1).     

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented <Emphasis Type="Italic">Pelargonium capitatum</Emphasis> cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

In vitro regeneration of Acacia mangium via organogenesis

Plant regeneration of Acacia mangium was achieved through organogenesis in callus cultures. Calli were induced from five types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaflets, petioles and stems of seedlings) of A. mangium on MS (Murashige and Skoog, 1962) basal medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 13.95 μM kinetin (KT). Green or green purple compact nodules containing clusters of meristematic centers were induced in these calli after transfer to MS basal medium containing 1.14–22.75 μM thidiazuron (TDZ) and 1.43–2.86 μM indole-3-acetic acid (IAA). A combination of 4.55 μM TDZ and 1.43 μM IAA promoted the highest percentage of calli to form nodules, in 8–11% of calli derived from cotyledons, embryo axes, leaflets or petiole and in 4% of calli derived from stems. Twenty-two percent of the nodules formed adventitious shoots on MS basal medium containing 0.045 μM TDZ. Shoots were elongated on MS medium containing 0.045 μM TDZ supplemented with 7.22 μM gibberellic acid. The medium containing 10.75 μM NAA and 2.33 μM KT promoted rooting of 10% of the elongated shoots. Plantlets grew up well in the green house. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adventitious shoot regeneration of oriental lily (Lilium orientalis) and genetic stability evaluation based on ISSR marker variation

A regeneration system was developed for oriental lily (Lilium orientalis) based on both leaf and bulb scale. Adventitious shoots were regenerated from leaves of in vitro cultures on Murashige and Skoog medium containing thidiazuron (TDZ) or 6-benzylaminopurine (BA) and naphthaleneacetic acid (NAA). The highest percent regeneration from leaf explants was 74.2%, being observed on medium containing 10.8 μM TDZ and 0.54 μM NAA. The highest mean number of shoots generated was 4.4 and was obtained from bulb scale explants on medium containing 0.54 μM TDZ and 0.54 μM NAA. Adventitious shoots were successfully rooted at rates ranging from 79.2% to 100%. The rooted plantlets survived after acclimatization in the greenhouse. The effect of kanamycin concentration on adventitious shoot regeneration was also evaluated, a value of 100 mg l⁻¹ being suggested as a lethal dose for lily transformation. Eighteen ISSR markers were employed to determine the genetic stability of the regenerated shoots in comparison to their mother plant. Eleven primers in total produced 70 clear and reproducible bands. Genetic similarity indicators among the clonal derivatives and the mother plant ranged from 0.92 to 1.0. All 15 micropropagated progenies and the mother plant could be grouped together in one major cluster with a similarity level of 92%. The somaclonal variation rate across the plantlets was estimated as 4.2%, indicating that direct shoot formation from explant regeneration is a safe method for multiplication of “true-to-type” plants.     

High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro (15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l⁻¹ sucrose, 2.2 g l⁻¹ Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO₃). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA₃). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an average of 40 plants per leaf explant with a culture period of 98 days.     

Thidiazuron-induced high-frequency plant regeneration from leaf explants of Paulownia tomentosa mature trees

Attempts were made to study the effect of thidiazuron (TDZ) on adventitious shoot induction and plant development in Paulownia tomentosa explants derived from mature trees. Media with different concentrations of TDZ in combination with an auxin were used to induce adventitious shoot-buds in two explant types: basal leaf halves with the petiole attached (leaf explant) and intact petioles. Optimal shoot regeneration was obtained in leaf explants cultured on induction medium containing TDZ (22.7 or 27.3 μM) in combination with 2.9 μM indole-3-acetic acid (IAA) for 2 weeks, and subsequent culture in TDZ-free shoot development medium including 0.44 μM BA for a further 4-week period. The addition of IAA to the TDZ induction medium enhanced the shoot-forming capacity of explants. The caulogenic response varied significantly with the position of the explant along the shoot axis. The highest regeneration potential (85–87%) and shoot number (up to 17.6 shoots/explant) were obtained in leaf explants harvested from the most apical node exhibiting unfolded leaves (node 1). An analogous trend was also observed in intact petiole explants, although shoot regeneration ability was considerably lower, with values ranging from 15% for petioles isolated from node 1 to 5% for those of nodes 2 and 3. Shoot formation capacity was influenced by the genotype, with regeneration frequencies ranging from 50% to 70%. It was possible to root elongated shoots (20 mm) in basal medium without growth regulators; however, rooting frequency was significantly increased up to 90% by a 7-day treatment with 0.5 μM indole-3-butyric acid, regardless of the previous culture period in shoot development medium (4 or 8 weeks). Shoot quality of rooted plantlets was improved not only by IBA treatment but also by using material derived from the 4-week culture period. Regenerated plantlets were successfully acclimatized in the greenhouse 8 weeks after transplanting.