<Emphasis Type="Italic">In vitro</Emphasis> propagation and cryostorage of <Emphasis Type="Italic">Syzygium francissi (Myrtaceae)</Emphasis> by the encapsulation-dehydration method

Summary An efficient procedure for the in vitro propagation and cryogenic conservation of Syzygium francissi was developed. The maximum number of shoots per explant was obtained on a Murashige and Skoog (MS) medium supplemented with 4.5 μM benzyladenine and 0.5 μM indole-3-butyric acid (IBA). The in vitro-propagated shoots produced roots when transferred to MS medium containing IBA, indold-3-acetic acid, or naphthaleneacetic acid at various concentrations. Rooted microshoots were transferred to a coco-peat, perlite, and vermiculite (1∶1∶1) mixture, and hardened off under greenhouse conditions. Ninety-five percent of rooted shoots successfully acclimatized in the greenhouse. Shoot tips excised from in vitro-grown plants were successfully cryostoraged at −196°C by the encapsulation-dehydration method. A preculture of formed beads on MS medium containing 0.75 M sucrose for 1 d, followed by 6 h dehydration (20% moisture content) led to the highest survival rate after cryostorage for 1h. This method is a promising technique for in vitro propagation and cryopreservation of shoot tips from in vitro-grown plantlets of S. francissi germplasm.     

<Emphasis Type="Italic">Acmella radicans</Emphasis> var. <Emphasis Type="Italic">radicans: In vitro</Emphasis> culture establishment and alkamide content

Summary Acmella radicans var. radicans propagation was established in vitro. This plant belongs to the Asteraceae from which some species are known for their insecticide, fungicide and antibacterial activity. The complete Murashige and Skoog (MS) medium was the best in assisting seed germination. In order to obtain shoots in vitro, a complete MS medium and half-strength MS medium were assayed with explants from leaves, nodes, and internodes. The best medium for shoot production was the half-strength MS medium with no addition of plant growth regulators, and the highest shoot propagation was from single-node explants. Regeneration of roots on shoot explants in the medium was obtained without the addition of growth regulators. Of the plantlets that were acclimated, 90% of them were obtained from rooted shoots with completely expanded leaves. The alkamide content was evaluated for each tissue and the higher concentration was observed in flower heads. The main alkamides present in the leaves and the flower heads were N-(2-phenylethyl)-2Z,4E-octadienamide and 3-phenyl-N-(2-phenylethyl)-2-propenamide. This study describes the methodology for the establishment and propagation of Acmella radicans in vitro and the evaluation of different tissue alkamide contents in vitro and in the field.     

In vitro propagation of Notocactus magnificus

Most commercially grown cacti can be easily propagated by seed and/or cuttings. A group of rare and endangered species does not fit into this category and is therefore a good candidate for in vitro propagation productions as a tool to overcome habitat and plant-destruction. The number of rare and endangered species of Cacti goes into about 100. Many show a low production and germination of seeds and plantlets are prone to damping-off, making the in vitro propagation a feasible alternative for the multiplication and conservation of their germplasm. The aim of the present investigation is to establish a protocol for the in vitro culture and plant regeneration of Notocactus magnificus, the blue cactus, a highly ornamental species, native to Brazil. The surface sterilization of the explants was achieved with immersion for 10 min in sodium hypochlorite solution for either seeds (0.25% v/v) or ribs segments (1% v/v). Callus formation was observed when explants were cultured on MS medium supplemented with sucrose at 2% (w/v), 2,4-dichlorophenoxyacetic acid 0.5 μM, benzylaminopurine 4.4 μM, thiamine HCl 0.4 mg l⁻¹ and i-inositol 100 mg l⁻¹. The regeneration of shoots was carried out on MS medium supplemented with either different concentrations of benzylaminopurine and 1-naphthaleneacetic acid, or kinetin and indole-3-acetic acid. The highest number of shoots occurred when MS medium was supplemented with benzylaminopurine 22.2 μM, sucrose 3% (w/v) and agar 0,6% (w/v). In vitro spontaneous rooting of shoots was observed after eight months under culture on MS medium. Only in vitro rooted shoots developed into normal plants under glasshouse culture conditions. This in vitro protocol should be useful for the conservation as well as mass propagation of Notocactus magnificus.     

<Emphasis Type="Italic">In vitro</Emphasis> clonal propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> (L.) wall. through nodal explants from mature trees

Summary In vitro clonal propagation of 18–20-yr-old Holarrhena antidysenterica tress has been achieved by employing nodal explants. The tree explants showed marked seasonal variation in their morphogenic response under in vitro conditions. Maximum response was obtained from the beginning of May to the end of July, followed by a gradual decline, finally dropping to zero from October to February. The explants induced multiple shoots only on cytokinin-containing medium. Several cytokinins [N⁶-benzyladenine (BA), N⁶-(2-isopentenyl) adenine (2ip), 6-furfuryl aminopurine (Kn), and adenine sulfate (Ads)] were assayed. The best response was achieved with 15 μM BA in which 62.5% of cultures produced 2.75±0.2 shoots per explant with 3.56±0.2 cm average length. Amongsth the three heavy metals assayed, silver nitrate (AgNO₃) significantly improved the response. This compound enhanced both the percentage of responding cultures (86.6%) and the average shoot number (4.73±0.2) at a concentration of 20mgl⁻¹. Further improvement in the morphogenic response occurred when explants from in vitro shoots were employed instead of mature trees. In this case, the percentage of morphogenic cultures was increased to 100% at the third subculture with an average of 11.45±0.3 shoots per explant. Regenerated shoots were rooted in half-strength Murashige and Skoog medium with 10 μM indole-3-acetic acid. The plantlets were successfully acclimatized in soil.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Pelecyphora aselliformis</Emphasis> erhenberg (cactaceae)

Summary In vitro propagation of Pelecyphora aselliformis, a Mexican cactus which is considered rare and is highly valued in the commercial market, was initiated using seeds as explants. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium containing 8.8 μM benzyladenine (BA) or 4.6 μM kinetin at pH 7.0. After 120 d, each explant gave rise to five shoots and this number of shoots increased 20–25% after subculture. The hyperhydricity was similar in both media, but callus formation was lower on the medium with BA. The shoot development, in terms of epicotyl length, and fresh and dry weight after 6 wk, was also recorded. The epicotyl length was similar on shoot-forming media but the quality of shoots was better on media containing BA. In about 1 yr, 500–600 well-defined shoots were obtained. The rooting of shoots was very slow and a vigorous radical system was observed after 1 yr of culture.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of four lupin species

The complete protocols for long-term micropropagation of some cultivars of four lupin species: Lupinus luteus, L. albus, L. angustifolius and L. mutabilis were elaborated. The shoots were regenerated in vitro via induction of axillary buds development. Plantlets were multiplicated on lowered salts MS-derived media containing BAP in diverse and generally low concentrations. Significant differences in regeneration capacity between species and cultivars were observed. The highest multiplication ratio revealed L. mutabilis and L. luteus. Regenerated shoots were rooted in vitro on low-salts MS-derived media with B5 vitamins. Media were supplemented with different auxins that affected roots formation of particular species and cultivars. Rooting ability of regenerated shoots decreased rapidly through in vitro culture. For that reason, grafting was applied as an alternative method of transfer of shoots to in vivo conditions. This method turned out to be successful for the majority of studied species and cultivars. Complete rooted or grafted plantlets were cultivated in pots with perlit in greenhouse. An erratum to this article is available at .     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Regeneration of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) from root explants

Summary Eggplant (Solanum melongena L.) was efficiently regenerated from cultured roots of 15-d-old seedlings on Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron and 13.3 μM 6-benzyladenine. Within 28d of culture initiation, induction of organogenic calluses and subsequent differentiation into shoot buds were observed. Shoot buds upon subculture to MS basal medium elongated into healthy shoots. Excised shoots (2–4 cm) were rooted on Soilrite^? irrigated with water either in vitro or in vivo. Plants with well-developed root systems were established under field conditions after hardening in the glasshouse, where they developed into flowering plants and produced mature fruits with viable seeds.     

The Role of Abscisic Acid in Acclimation of Plants Cultivated in vitro to ex vitro Conditions

The content of endogenous free abscisic acid (ABA) in the shoots of in vitro cultivated tobacco (Nicotiana tabacum L. cv. White Burley) and its changes during ex vitro acclimation of these plants to the greenhouse or growth chamber were estimated. The content of free ABA significantly increased at the 1^st and/or 2^nd day after plant transfer from in vitro to ex vitro. The ABA content of plants covered with transparent foil to maintain higher relative humidity (RH), did not significantly differ from ABA content of plants cultivated under ambient RH. Transfer to fresh medium also transiently increased the content of endogenous ABA. The ABA content in plants, which had been acclimated for 1 week to ex vitro conditions, decreased to the content found in the in vitro plants. Acclimation to ex vitro conditions affected the stomata on adaxial and abaxial sides differently: stomata on the adaxial side were less open than those on the abaxial one. The exogenous application of 5 μM ABA increased transiently its endogenous concentration in shoots of in vitro plants more than ten fold, but after 1 week the concentration in the shoots decreased. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of <Emphasis Type="Italic">in vitro</Emphasis> methods for <Emphasis Type="Italic">ex situ</Emphasis> conservation of <Emphasis Type="Italic">Eucalyptus impensa</Emphasis>, an endangered mallee from southwest Western Australia

A method is described for in vitro propagation of the critically endangered ‘Eneabba mallee’ (Eucalyptus impensa) from southwest Western Australia. Half-strength MS medium supplemented with 0.25 μM 6-benzylaminopurine and 2.5 μM kinetin resulted in the best combination of shoot multiplication and shoot quality compared to other treatments. Shoots of this species tended to be very compact under in vitro conditions. Shoot length was significantly enhanced with the addition of 0.5 or 1.0 μM gibberellic acid (A₄ isomer) when compared to basal medium (no hormone supplements) or basal medium containing only cytokinin (0.5 μM zeatin). Up to 97.0 ± 3.0% of shoots produced roots on 1/2 MS medium supplemented with a combination of 5 μM indolebutyric acid and 0.5 μM α-naphthaleneacetic acid. Over 70% of shoots transferred to potting mixture remained viable after 3 months. This study has significantly progressed ex situ conservation initiatives for Eucalyptus impensa.     

Micropropagation of <Emphasis Type="Italic">Teucrium fruticans</Emphasis> L., an ornamental and medicinal plant

An efficient protocol for in vitro propagation of the valuable ornamental and medicinal plant Bush germander (Teucrium fruticans L.) was developed through axillary shoot proliferation. A Murashige and Skoog agar medium supplemented with benzylaminopurine (6.6 μM), α-naphthaleneacetic acid (0.053 μM), and sucrose (3%) significantly improved the production of multiple shoots directly from nodal segment explants, resulting in an average of 2.8 shoots per segment with an average of 6.8 nodes per shoot that would be potential newly formed explants. The new shoots were developed without a marked decrease in the average height of the shoots. Shoots treated with 2.5 μM indole-3-butyric acid showed the highest average root number (7.9) and the highest percentage of rooting (94%). Plantlets were hardened off and transferred to jiffy pots for acclimatization under greenhouse conditions, resulting in a 100% survival rate.     

The influence of micrografting in vitro on tissue culture behavior and vegetative propagation of old European larch trees

The influence of a new micrografting method in vitro was tested by using long shoot meristems of adult 140 year old European larch trees grafted onto larch seedling rootstocks in sterile peat pellets. Five months after grafting and transfer of grafts to ex vitro conditions, new shoots from sprouting scions were re-established into tissue culture. The propagation behavior of shoots derived from these explants was compared with shoots established via bud culture from the same donor trees. Shoot explants from micrografts multiplied more rapidly than shoots explants from the original adult donor trees. Rooting experiments with cuttings from six micrografted clones resulted in 49.9 ± 11.9% rooting. Cuttings from donor trees invariably showed no root formation. The results confirm a rejuvenating influence of micrografting in larch.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency of responding explants (85%) and maximum number of shoots per explant (9.5) were obtained on MS medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the orginal cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on peat moss, soil, and compost mixture (1∶1∶1). About 52% plantlets rooted under ex vitro conditions were successfully acclimatized and established in pots.     

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

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

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Rosa damascena</Emphasis> Mill.

Summary A protocol for in vitro propagation using direct induction of shoot buds from leaf explants of in vitro-raised shoots of Rosa damascena var. Jwala is reported. The present study is the first report on direct shoot regeneration in scented roses. Elite plants raised from nodal explants and maintained for over 2yr in vitro on a static liquid shoot multiplication Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 3% sucrose were used. Petioles from fully developed young leaves, obtained after 4 wk of pruning of old shoots, were found to be ideal for regeneration of shoots. Initially the explants were cultured in an induction medium [half-strength MS+3% sucrose+6.8μM thidiazuron+0.27 μM α-naphthaleneacetic acid (NAA)+17.7 μM AgNO₃] and subsequently transferred to the regeneration medium (MS+2.25 μM BA+0.054 μM NAA) after 7, 14, 21, 28, and 35d. The highest shoot regeneration response (69%) was recorded when shoots were kept in the induction medium for 21 d and later transferred to regeneration medium. Histological studies revealed direct formation of shoot buds without the intervening callus phase. In vitro rooting of micro-shoots was accomplished within 2wk on half-strength MS liquid medium supplemented with 10.0 μM IBA and 3% sucrose for 1 wk in the dark and later transferred to hormone-free medium and kept in the light. Plantlets, remaining in the latter medium for 5–6 wk when transferred to soil, showed 90% survival.     

In vitro regeneration and micro-propagation of enset from Southwestern Ethiopia

Three clones of enset (Ensete ventricosum Welw. Cheesman) from southwestern Ethiopia (Keffa-Shaka zone) were investigated for their potential for micropropagation and regeneration in tissue culture. Corm and leaf tissue of greenhouse-grown plants as well as in vitro germinated zygotic embryos were used as starting material for micro-propagation and regeneration studies. Embryos were excised from disinfected seeds and cultured in vitro. Multiple shoots from both corm- and embryo-explants were obtained using regeneration medium supplemented with 10 μM or 20 μM BAP. Rooting of shoots was achieved using medium with 5 μM IBA, 1 μM BAP and 1 g l⁻¹ activated charcoal. Plantlets obtained by this process were transferred to soil under greenhouse conditions. Optimal conditions, which were determined for clonal propagation of three different genotypes of enset, allowing both in vitro micropropagation and regeneration, are described. This protocol makes for conservation of enset clones, rapid propagation of selected disease-free germplasm and more efficient breeding procedures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimization of <Emphasis Type="Italic">Renealmia mexicana</Emphasis> (Klotzsch ex. Petersen) cultivation <Emphasis Type="Italic">in vitro</Emphasis>

Renealmia mexicana (Klotzsch ex. Petersen) is a tropical plant found in southern México with an ornamental value and a potential source of curcuminoids. Its distribution in Chiapas has decreased because of deforestation and low propagation and germination rate, so a protocol for in vitro propagation was developed. An orthogonal experimental design of L₉ (3⁴) in triplicate was used to investigate the effect of 6-benzyl adenine (BA), indole butyric acid (IBA), silver nitrate (AgNO₃), and sucrose on shoot, root, and leaf development of plantlets grown in vitro. Plantlets with well-developed shoots and roots were transferred to pots containing a mixture of peat moss and agrolite for hardening before transfer to soil. The Murashige and Skoog (Physiol. Plant. 15:473–497, 1962) mineral medium (MS) supplemented with 4.4 μM BA, 2.5 μM IBA, 11.7 μM AgNO_3y and 5.5% (w/v) sucrose gave most shoots, 8.9 μM BA, 2.5 μM IBA, 17.7 μM AgNO₃ and 5.5% (w/v) sucrose most roots, and 8.9 μM BA, 4.9 μM IBA, 11.7 μM AgNO₃ and 3.0% (w/v) sucrose most leaves, although other combinations were statistically equivalent in each case. Sucrose was the factor that most explained the variation in the promotion of shoots, roots, and leaves. The protocol developed resulted in up to 100% survival when plantlets were transferred to soil using AgNO₃, confirming that hardening of plantlets in vitro using hormonal stimulation was a suitable strategy to improve acclimatization.