Plant regeneration from in vitro leaf tissues of <Emphasis Type="Italic">Viburnum dentatum</Emphasis> L

Shoots were regenerated from in vitro leaf tissues of two genotypes of Viburnum dentatum, a popular shrub species for landscape use. Adventitious shoots were induced when leaf tissues were cultured on woody plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Effects of cytokinin concentration, indole-3-butyric acid (IBA), and dark treatment on shoot regeneration were investigated. Dark treatment for the first 4 weeks of leaf explants cultured in the regeneration medium significantly increased the frequency of regeneration. The highest frequency of shoot regeneration (70%) for ‘Synnesvedt’ was obtained when leaf tissues were cultured in the medium with 40 μM BA or 8 μM TDZ with 4 weeks dark treatment. The highest frequency of shoot regeneration (90%) for ‘MN34’ was found in the 4 μM TDZ medium with 4 weeks dark treatment. Addition of IBA significantly enhanced shoot regeneration. Ethyl methanesulfonate (EMS) treatment inhibited callus proliferation, particularly in the early stage of callus recovery; however, no significant difference in shoot regeneration among different treatments was observed, indicating that the inhibitory effect of EMS was minimal after calluses re-acquired their capacity to grow and regenerate in the regular medium. Regenerated shoots (>1.5 cm) were rooted in the half-strength MS medium containing 5-10 μM IBA or naphthalene acetic acid (NAA). Rooted plants were transferred to the potting medium and grown in the greenhouse.     

In vitro propagation for the conservation of a rare medicinal plant <Emphasis Type="Italic">Justicia gendarussa</Emphasis> Burm. f. by nodal explants and shoot regeneration from callus

Justicia gendarussa is a valuable medicinal plant and various parts of this plant are pharmaceutically used for the treatment of different diseases. In vitro regeneration of shoot buds was obtained from culture of nodal cuttings as well as shoot regeneration from callus. The nodal cuttings differed in shoot proliferation in terms of percentage of explants that responded and average shoot length with various concentrations (4.4, 8.9, 13.3, 17.7, 22.2 μM) of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron. In all treatments, one shoot was invariably present. Optimum 87% of cultures responded with an average shoot length of 4.4 cm on Murashige and Skoog (MS) medium supplemented with 17.7 μM BA. Callus was induced from the mature leaf segments on MS medium supplemented with Kn (4.7, 13.9, 23.2 μM) alone or in combination with 2, 4-dichlorophenoxyacetic acid (2, 4-D; 2.3 μM, 4.5 μM). Optimum callus induction (78%) was obtained on MS medium supplemented with 14 μM Kn and 4.5 μM 2, 4-D. When the callus was subcultured on MS medium fortified with BA (8.9, 17.7, 26.6 μM) or Kn (9.3, 18.6, 27.9 μM) alone or in combination with α naphthalene acetic acid (NAA; 2.7, 5.4 μM), shoot regeneration was obtained. The highest response (92%) was observed on MS medium containing 17.7 μM BA and 5.4 μM NAA. On this medium, an average number of 12.2 shoots were obtained per responding callus. The shoots obtained from callus and nodal cuttings were rooted with a frequency of 73% on MS medium augmented with 9.8 μM indole-3-butyric acid. The rooted shoots were successfully transplanted to soil and sand mixture (1:1) with 90% survival rate. The protocol standardized for shoot proliferation and regeneration in J. gendarussa from nodal cuttings and leaf-derived callus is suitable for micropropagation and conservation of this essential medicinal plant.     

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.     

High frequency shoot organogenesis and somatic embryogenesis in juvenile and adult tissues of seabuckthorn (<Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.)

Seabuckthorn (Hippophae rhamnoides) is a multipurpose small tree with unique berries of high nutritional and pharmaceutical values. A clonally propagated plant originating from a 20-year-old tree of H. r. rhamnoides × mongolica hybrid cultivar Julia and seedling offspring of this cultivar were investigated regarding induction of shoot organogenesis in leaf explants and in roots of intact seedlings, and induction of direct somatic embryogenesis in explants from shoot tissue. The highest percentage of leaf explants showing shoot organogenesis was achieved (juvenile explants, 65%; adult explants, 75%) when incubated in Murashige and Skoog (MS) medium supplemented with either 4.5 μM of the phenylurea cytokinin thidiazuron (TDZ) or 2.25 μM TDZ plus 2.2 μM 6-benzyladenine (BA), for juvenile and adult explants, respectively, both supplemented with 0.53 μM α-naphthaleneacetic acid (NAA). Juvenile explants developed on average 18 shoots per explant in the MS medium supplemented with 4.5 μM TDZ, a four fold increase over those incubated on the medium supplemented with 2.25 μM TDZ and 2.2 μM BA. Adult leaf explants grown on medium containing 2.25 μM TDZ and 2.2 μM BA medium produced 12 shoots per explant, while those grown on medium containing 4.5 μM TDZ produced 5 shoots per explant. Shoot organogenesis was observed in roots of intact seedlings pre-cultured on plain medium lacking nutrients (PM) or woody plant medium (WPM) salts and then grown on WPM salts supplemented with 4.4 μM BA, 0.29 μM gibberrelic acid (GA3), and 57.0 μM indoleacetic acid (IAA). The number of shoots formed on each seedling root system was ten fold higher when the pre-culture was in WPM medium indicating a promoting effect of mineral nutrients in the pre-culture medium. Somatic embryogenesis was induced in both juvenile and adult leaf explants in 65 and 78% of the explants, respectively, in MS-based medium supplemented with 2.0 μM N-(2-Chloro-4-pyridyl)-N ¹-phenylurea (CPPU), 0.53 μM NAA and varying concentrations of BA. There was an interaction effect between MS salt strength and BA concentration. The most effective medium for inducing somatic embryogenesis in juvenile explants contained half strength MS salts and 2.2 μM BA and full strength MS salts and 13.2 μM BA for adult explants.     

Regeneration of salvia (<Emphasis Type="Italic">Salvia officinalis</Emphasis> L.) via induction of meristematic callus

Nodular meristematic callus was induced on the basal cut surface of apical shoot explants of salvia cultured on Murashige and Skoog (MS) medium supplemented with 4.5, 13.5, or 22.5 μM thidiazuron (TDZ). Cultures were incubated in the dark for 1 wk and then transferred to light conditions for 4 wk. A higher percentage of explants developing callus was observed on medium containing either 4.5 or 13.5 μM TDZ, although explants on 4.5 μM developed larger calluses. The callus was maintained on medium containing 4.5 μM TDZ and 0.45 mM ascorbic acid. Shoot differentiation, after each of three successive maintenance passages, was induced from callus grown on medium containing either 4.4 or 8.8 μM benzyladenine (BA). A greater number of shoots were harvested from callus differentiated on BA (4.4 or 8.8 μM) medium with 0.45 mM ascorbic acid added. Shoots developed roots on MS medium supplemented with 4.9 μM of indole-3-butyric acid. The addition of ascorbic acid to the shoot differentiation medium enhanced rooting, number of roots per shoot, and survival rate. Approximately 75% in vitro plantlets were acclimatized to ex vitro conditions. Histological investigations confirmed both adventitious meristem initiation during the callus induction phase, and subsequent organogenic shoot development on the differentiation medium. The novel protocol for the meristematic callus induction and plant regeneration in this study may be useful for biotechnological applications for salvia improvement via genetic transformation or mutagenesis and in vitro propagation approaches.     

In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.     

<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.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

<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 rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

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.     

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.     

Direct adventitious shoot induction and plant regeneration of <Emphasis Type="Italic">Embelia ribes</Emphasis> Burm F.

An efficient micropropagation system via direct shoot organogenesis from hypocotyl segments of Embelia ribes Burm F. was developed. A high frequency (84%) of adventitious shoot induction was obtained on Murashige and Skoog (MS) medium supplemented with additives (283.85 μM ascorbic acid [AA], 118.96 μM citric acid [CA], 142.33 μM cysteine, and 684.22 μM glutamine) and 1.13 μM of thidiazuron (TDZ) after 4 weeks following culture. Further development of shoot primordia into well-grown shoots of 4–5 cm in length was achieved by sub-culturing explants along with shoot primordia on MS medium supplemented with 0.44 μM benzyl adenine (BA) and 0.49 μM indole butyric acid (IBA) for three sub-culture periods with an interval of 15 days between them. The highest shoot multiplication was obtained when explants were incubated on MS medium supplemented with 2.2 μM BA and 0.49 μM IBA in 4 weeks. All in vitro developed shoots, 3–4 cm in length, rooted when grown on half-strength MS basal medium along with 2.47 μM IBA within 4 weeks. Moreover, 100% of shoots developed roots when these were treated with 4.93 μM IBA for 20 min and then transferred to pots containing soilrite mix and grown in the greenhouse. In vitro and ex vitro rooted plants showed a survival of 85 and 95% respectively, during hardening in the greenhouse for a 6-week period.     

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.     

An improved plant regeneration system and ex vitro acclimatization of <Emphasis Type="Italic">Ocimum </Emphasis><Emphasis Type="Italic">basilicum</Emphasis> L.

An efficient, rapid and large scale propagation of a multipurpose herb, Ocimum basilicum through in vitro culture of nodal segments with axillary buds from mature plants has been accomplished. Among the cytokinins, 6-benzyladenine (BA), thidiazuron (TDZ), kinetin (Kin) and 2-isopentenyl adenine (2-iP) tested as supplements to Murashige and Skoog (MS) medium, 5.0 μM BA was optimum in inducing bud break. The highest rate of shoot multiplication was achieved on half-strength MS medium supplemented with 2.5 μM BA and 0.5 μM indole-3-acetic acid (IAA) combination. The shoots regenerated from TDZ supplemented medium when subcultured to hormone-free MS medium considerably increased the rate of shoot multiplication and shoot length by the end of third subculture. For rooting, MS medium supplemented with 1.0 μM indole-3-butyric acid (IBA) proved to be better than that supplemented with IAA or α-naphthalene acetic acid (NAA). The in vitro raised plantlets with well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 90% survival rate. Chlorophyll a and b, carotenoids and net photosynthetic rate were measured in leaves during ex vitro acclimatization at 0, 7, 14, 21 and 28 days. Firstly these parameters showed a decreasing trend but subsequently increased after 7 days of acclimatization. These findings indicate that the adaptation of micropropagated plants to ex vitro conditions is more extended in time than generally accepted.     

Comparison of in vitro regeneration pathways in <Emphasis Type="Italic">Punica granatum</Emphasis> L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA₃). However, adding 24 μM silver nitrate (AgNO₃) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l⁻¹ sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l⁻¹ sucrose.     

Shoot regeneration and determination of iridoid levels in the medicinal plant <Emphasis Type="Italic">Castilleja tenuiflora</Emphasis> Benth.

Castilleja tenuiflora is a medicinal plant that grows in pine–oak woods primarily in southern and central Mexico. It is highly valued for its medicinal properties, which have been attributed to aucubin-like iridoids. In the present study, we developed an efficient protocol for in vitro shoot proliferation and ex vitro rooting of C. tenuiflora. Using a colorimetric method, we determined total iridoid contents of various different tissues of propagated plants. The shoots were induced from nodal explants cultured on Murashige and Skoog (MS) (1962) medium supplemented with indole-3-butyric acid (IBA) (0 and 0.5 μM) and different concentrations of thidiazuron (TDZ), 6-benzyladenine (BA), or kinetin (KIN) (0–20 μM). Of the cytokinins tested, KIN was more effective for shoot induction than TDZ or BA, and the highest shoot proliferation rate was achieved with 5 μM KIN (4 shoots per explant). Plantlets were rooted on MS medium, nutrient solution, or potting mix, alone or in combination with auxins. The best responses (100% rooting efficiency) were obtained by dipping shoots in half-strength MS medium containing 7.5 μM IBA before transfer to potting mix. On average, each shoot formed 9 roots of 39.3 ± 3.8 mm in length after 21 days. These roots appeared to be more functional than those that developed in nutrient solution, and were associated with a high survival rate (95%) during acclimatization and cultivation in a greenhouse, where flowering occurred after 4 months. Propagated plants accumulated iridoids, thus representing a potential source of pharmacologically useful compounds.     

Globular embryo-like structures and highly efficient thidiazuron-induced multiple shoot formation in saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L.)

Saffron (Crocus sativus L.) is a monocotyledonous plant propagated via corms, but recently several alternative methods have been reported. To find the conditions suitable for saffron shoot formation from corms, the effect of different concentrations of the plant growth regulatory cytokinins N⁶-benzyladenine (BA) and N-phenyl-1, 2,3-thidiazol-5-ylurea, commonly known as thidiazuron (TDZ), were compared. In all corm explants, an average of 39.5 ± 5.1 shoots per corm were induced by 4.54 μM TDZ, whereas only 3.6-11.4% by BA. The outstanding result in the shoot formation stage is the generation of globular, translucent structures that are morphologically similar to globular embryos. To optimize the plant regeneration from the induced adventitious shoots obtained from the TDZ treatment, the shoots were transferred to MS and B5 media supplemented with different concentrations and combinations of NAA and BA. The highest rate of plant regeneration from developing shoots was observed in the B5 medium containing 2.22 μM NAA and 2.68 μM BA. With optimized hormonal conditions, an average of 19.55 ± 5.75 shoots and 3.18 ± 1.5 roots per explants were obtained. Based on this experiment, a simple, new and efficient protocol is presented to produce numerous plants from induced corm explants of saffron.     

Hypocotyl derived in vitro regeneration of pumpkin ash (<Emphasis Type="Italic">Fraxinus profunda</Emphasis>)

Pumpkin ash (Fraxinus profunda (Bush) Bush) is at risk for extirpation by an exotic insect, the emerald ash borer (EAB). Pumpkin ash is limited to wetland areas of the Eastern United States, and has been listed as an endangered species because of EAB activity. Pumpkin ash provides many benefits to the ecosystem, and its wood is used in the manufacturing industry. In vitro regeneration provides an integral tool for the mass propagation and genetic transformation of pumpkin ash to combat EAB. Therefore, a plant regeneration protocol was developed for pumpkin ash. Aseptically extracted hypocotyls formed adventitious shoots following 4 weeks on Murashige and Skoog (MS) medium supplemented with 0–22.2 μM 6-benzyladenine (BA) and 0–6.8 μM thidiazuron (TDZ) then transferred for an additional 4 weeks on MS medium with Gamborg B5 vitamins plus 0.2 g L⁻¹ glycine (B5G) containing 6.7 μM BA, 1 μM indole-3-butryic acid (IBA), and 0.29 μM gibberellic acid (GA₃). As adventitious shoots developed, these were transferred to a MSB5G medium with 13.3 μM BA, 1 μM IBA, and 0.29 μM GA₃ for shoot elongation. Elongated shoots were successfully micropropagated using MSB5 medium with 10 μM BA and 10 μM TDZ. Adventitious root formation was as high as 94% using woody plant medium supplemented with 4.9 μM IBA with shoots cultured for 10 days in the dark followed by culture under a 16-h photoperiod. Acclimatization to the greenhouse was successful and normal plant growth was observed. This protocol will provide a means for genetic transformation for EAB resistance and mass propagation for conservation.