In vitro propagation of limonium wrightii (Hance) Ktze. (Plumbaginaceae), an ethnomedicinal plant, from shoot-tip, leaf- and inflorescence-node explants

Summary Rapid in vitro propagation of Limonium wrightii (Hance) Ktze. (Plumbaginaceae), an endangered medicinal plant, was achieved by culturing the shoot-tip (primary and lateral), leaf- and influorescence-node explants. MS (Murashige and Skoog, 1962) medium supplemented with 8.87 μMN⁶-benyladenine (BA) and 1.07 μM α-naphthaleneacetic acid (NAA) supported induction of adventitious shoots from the shoot-tip, inflorescence-node and middle and basal parts of leaf explants after 60 d of culture. Adventitious shoots were multiplied by subculturing on MS medium supplemented with BA (2,21–17.75 μM) in combination with NAA (1.07 μM). The percentage of explants forming shoots and the average number of adventitious shoot buds produced per explant were stimulated by increasing the strength (1/4x, 1/2x, 1x, 2x) of the MS medium. Shoots were rooted on MS basal medium with 4.92 μM indole-3-butyric acid. Plantlets with a morphologically normal appearance produced from adventitious shoots were transferred to soil and acclimated in the growth chamber for 1 mo.     

Micropropagation of <Emphasis Type="Italic">Clitoria ternatea</Emphasis> Linn. (<Emphasis Type="Italic">Fabaceae</Emphasis>)— An important medicinal plant

Summary An efficient protocol was established for in vitro shoot multiplication from nodal explants of Clitoria ternatea on semisolid Murashige and Skoog (MS) basal medium supplemented with 8.9μM 6-benzylaminopurine (BA). Inclusion of 1-naphthaleneacetic acid (NAA) in the culture medium along with BA promoted higher rates of shoot multiplication than BA alone. The rate of shoot multiplication was maximum (5.21) after 4 wk of culture on MS basal medium supplemented with 8.9μM BA and 1.34μM NAA. The elongated shoots rooted within 7–8d in half-strength MS basal salts supplemented with 1.34μM NAA and 2% (w/v) sucrose. About 85% of the rooted plantlets were acclimatized and transferred to the greenhouse.     

Adventitious shoot regeneration from ovaries of Hosta ‘Golden Scepter’

Summary The objective of this study was to evaluate the ability ofHosta Golden Scepter (GS) ovary explants to generate adventitious shootsin vitro. Ovaries were transversely cut into halves and transferred to petri dishes containingHosta initiation medium supplemented with naphthaleneacetic acid (NAA) at 2.5 μM and N⁶-benzyladenine (BA) at 10 μM. GS produced adventitious shoots from the ovary base via organogenesis. The number of adventitious shoots regenerated from callus increased linearly with repeated subculturing on Murashige and Skoog (MS) medium supplemented with 2.5 μM NAA and 10 μM BA. The number of multiple shoots developing from callus (15.8), shoot tip (8.4), leaf (6.7), and root (4.3) occurred on MS medium supplemented with 2.5 μM NAA and 20–30 μM BA. There were significant differences in the number of shoots regenerated from shoot tips and callus on MS medium with 50 and 100 mgmyo-inositol per l. Similarly, there were significant differences in the number of axillary shoots and adventitious shoots produced with 20 g/l sucrose treatment.     

<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> 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.     

Organogenesis and somatic embryogenesis in <Emphasis Type="Italic">Ariocarpus kotschoubeyanus</Emphasis> (Lem.) K. Schum. (Cactaceae), an endemic and endangered Mexican species

Summary Shoots by direct and indirect organogenesis and somatic embryos were induced from tubercles excised from Ariocarpus kotschoubeyanus seeds germinated in vitro. Shoot formation was greatest (6.3 per explant) when explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA; 13.3 μM) and α-naphthaleneacetic acid (NAA; 5.4 μM). Individualized shoots were rooted in half-strength MS; the addition of activated charcoal (1 g l⁻¹) and the use of sun cap closures to seal containers improved the rooting of shoots. Nearly 20% of the explants produced somatic embryos on media containing combinations of BA (8.9–22.2 μM) and NAA (0.5–5.4 μM). The establishment of plantlets in soil presented no significant problems. In vitro culture is a useful option for mass propagation of A. kotschoubeyanus and contributes to its conservation.     

Regeneration of plants from isolated cotyledons of salgareño pine (Pinus nigra Arn. ssp.Salzmannii (Dunal) Franco)

Summary Plantlet regeneration of salgare?o pine (Pinus nigra Arn. ssp.salzmannii (Dunal) Franco) was achieved from cotyledons. The data showed that the best differentiation response occurred when cotyledons, from 1-d-old embryos germinated in darkness, were cultured on Murashige and Skoog medium (half-strength macroelements) with 22 μM N⁶-benzyladenine (BA) and 0.05 μM α-naphthaleneacetic acid (NAA) for 2–3 wk. Shoot development was obtained by subculturing treated explants on the same medium without growth regulators. Shoots were successfully micropropagated by sequential subculturing them on medium containing growth regulators (5 μM BA and 0.005 μM NAA) and on hormone-free medium for 2 and 12 wk, respectively. To induce adventitious roots, shoots were treated with 3 μM NAA, and 8 μM indole-3-butyric acid for 2 wk, followed by transfer to Murashige and Skoog medium (1/4-strength macroelements, 20 g·L⁻¹ sucrose) without growth regulators. After 3–4 wk, almost all the rooted shoots (65%) could be successfully transplanted and acclimatizated in the greenhouse, where the plants exhibited normal growth habit.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Salvia nemorosa</Emphasis> L. from shoot tips and leaf explants

Summary Shoot tips and leaves excised from in vitro shoot cultures of Salvia nemorosa were evaluated for their organogenic capacity under in vitro conditions. The best shoot proliferation from shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with 8.9 μM 6-benzylaminopurine (BA) and 2.9 μM indole-3-acetic acid (IAA). Leaf lamina and petiole explants formed shoots through organogenesis via callus stage and/or directly from explant tissue. The highest values for shoot regeneration were obtained with 0.9 μM BA and 2.9 μM IAA for lamina explants. No shoot organogenesis was obtained on leaf explants cultured on MS medium supplemented with α-naphthaleneacetic acid (NAA). The regenerated shoots rooted the best on MS medium containing 0.6 μM IAA or 0.5 μM NAA. In vitro-propagated plants were transferred to soil with a survival rate of 85% after 3 mo.     

<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 for shoot regeneration (85%) and maximum number of shoots per explant (9.5) were obtained on the 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 original 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 (IBA) after 25 d of culture. Fifty percent of shoots were also directly rooted as microtuttings on a peat moss, soil, and compost mixture (1∶1∶1). About 52% of plantlets were successfully acclimatized and established in pots.     

Micropropagation of <Emphasis Type="Italic">Pseudoxytenanthera stocksii</Emphasis> Munro

Summary An efficient and reproducible procedure for the large-scale propagation of Pseudoxytenanthera stocksii is described. High-frequency multiple shoot induction was achieved from nodal shoot segments collected from superior/elite genotypes on Murashige and Skoog (MS) liquid medium supplemented with 1-naphthaleneacetic acid (NAA; 2.68 μM) and 6-benzylaminopurine (BA; 4.40 μM) at 28±1°C and 60 μmol m⁻² s⁻¹ light intensity under 12h photoperiod. In vitro-differentiated shoots were multiplied on MS liquid medium fortified with NAA (2.68 μM), BA (2.21 μM) and additives: ascorbic acid (283.93 μM), citric acid (118.10 μM), cysteine (104.04 μM), and glutamine (342.24 μM). Subculturing was carried out every 2wk on fresh shoot multiplication medium. About 125–150 shoots per culture flask were harvested within 45–50d. In vitro-differentiated shoot clumps (three or four shoots) were successfully rooted on half-strength MS basal liquid medium with indole-3-butyric acid (4.90 μM), BA (0.44 μM), and additives. This is the first report where in vitro- and in vivo-(through tillers) raised clonal plants were acclimatized and established in the field, where they exhibited normal growth.     

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

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

Micropropagation ofCereus peruvianus mill. (cactaceae) by areole activation

Summary Currently,Cereus peruvianus plants can be rapidly clonedin vitro via adventitious organogenesis using callus cultures; however, somaclonal variation is a problem. A method is described herein using lateral bud explants to produce multiple shoots for clonal propagation. Apical and lateral explants were cultured on MS (Murashige and Skoog, 1962) media with factorial combinations of the auxins indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and cytokinins 6-ben-zyladenine (BA) and N-(2-furanyl-methyl)-1-purine-6 amine (kinetin) at the concentrations 0.0, 0.01, 0.1, 1.0 mg“l⁻¹. Positive results were obtained from the lateral explants in all conditions tested, but apical explants did not respond toin vitro multiplication ofC. peruvianus cactus at all growth regulator combinations tested. Formation of axillary shoots inC. peruvianus seems most frequent in medium containing BA at 1.0 mg·l⁻¹ (4.44 μM) and IAA or NAA at 1.0 mg·l⁻¹ (5.71 μM or 5.37 μM respectively), but the frequency of shoot formation in the BA or kinetin and NAA or IAA combinations indicated that any of the combinations tested can be used for multiplication ofC. peruvianus plants regenerated from callus tissue culture. Root formation occurred in all (100%) of the cactus shoots after 9 wk in the same culture medium. All the cacti that developed at the different auxin and cytokin combinations continued growth after transfer to a potting mix of red earth (Paleudult) and ground river sand (1∶1).     

Influence of phenylacetic acid on clonal propagation of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> wight &; ARN: An endangered shrub

Summary We have developed a highly efficient two-stage protocol for induction of multiple shoots from single node in vitro shoot tip explants of Decalepis hamiltonii. It was found that phenylacetic acid (PAA) had a synergistic effect on shoot multiplication when treated with N⁶-benzyladenine (BA). This protocol used PAA for both multiple shoot induction from nodal explants, elongation of primary shoots, and initiation of adventitious shoot formation from primary shoots. Murashige and Skoog medium containing BA (2.22–31.08 μM) and α-naphthaleneacetic acid (0.27–10.74 μM) or PAA (7.34–36.71 μM) was used to initiate shoot formation from nodal explants. The maximum number of shoots per culture was produced on a medium containing 31.08 μM BA and 14.68 μM PAA, while the longest shoot length and nodes were obtained on medium containing 22.2 μM BA and 14.68 μM PAA. Shoots subcultured on MS medium containing 22.2 μM BA and 14.68 μM PAA elongated along with secondary shoot formation. The shoots were rooted on medium containing 9.7 μM indole-3-butyric acid. The plantlets were acclimatized in soil with an 80–90% survival rate under field conditions.     

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.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

Plant regeneration through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

in vitro propagation of Ochreinauclea missionis (Wall. EX G. Don), an ethnomedicinal endemic and threatened tree

Summary Biotechnology has offered a nonconventional method of plant propagation and has been intensively applied as a conservation strategy for sustaining biodiversity for rare plants. In vitro conservation through micropropagation of Ochreinauclea missionis, a rare, endemic and medicinal tree species of Western Ghats in Karnataka region of India is reported. Multiple shoots were initiated from nodal explants on Murashige and Skoog (MS) medium supplemented with 8.8 μM 6-benzylaminopurine (BA) and 0.3% (w/v) activated charcoal. Shoots were elongated in MS medium with a combination of 2.2 μM BA and 5.3 μM α-naphthaleneacetic acid (NAA) or growth regulator-free medium. Individual shoots with a minimum of one node were excised and rooted in vitro on MS medium with 0.3% activated charcoal or ex vitro rooted by treatment with 49 μM indole-3-butyric acid (IBA) for 30 min. Regenerants acclimated in Soil-rite exhibited 65% survival in the greenhouse.     

Regeneration of an anticarcinogenic herb, Curculigo orchioides (Gaertn.)

Summary Curculigo orchioides is an endangered anticarcinogenic herb. It is available only during the monsoon season, which lasts approximately 4 mo. each year. In vitro culture of the plant can ensure its availability throughout the year. Leaf explants of Curculigo orchioides cultured on a Murashige and Skoog (MS) medium without cytokinins produced a limited number of plantlets that originated directly from the cut end of the midrib. 6-Benzyladenine (BA) (0.44–6.66 μM) was needed to produce plantlets from rhizome explants. A higher concentration of BA (2.22–4.44 μM) resulted in nodular callus that when transferred to cytokinin-free medium formed shoots. The shoots were rooted on media supplemented with either (0.54–5.37 μM) of 1-naphthaleneacetic acid (NAA) (0.57–5.71 μM) of indole-3-acetic acid (IAA), or (0.49–4.90 μM) indole-3-butyric acid (IBA). Plantlets were kept in sterile sand for 3–4 d and then transferred to soil.     

Effect of auxins on indirect <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis and expression of <Emphasis Type="Italic">gusA</Emphasis> transgene in a lectinaceous medicinal plant, <Emphasis Type="Italic">Euphorbia nivulia</Emphasis> Buch.-Ham.

Summary The types of auxin in Murashige and Skoog (MS) medium containing N ⁶-benzyladenine (BA) determined indirect morphogenesis, i.e. development to bipolar somatic embryos or monopolar shoots in Euphorbia nivulia Buch.-Ham. Indirect in vitro morphogenesis depended on growth regulators, explant excision period, and light. Calli induced from explants collected in March–April were superior in the induction of indirect morphogenesis to those collected in July–August. Light enforced in vitro morphogenesis, while darkness was inhibitory. The presence of kinetin in the medium also inhibited morphogenesis. Calli developed on explants collected in March–April grown on MS medium fortified with α-naphthaleneacetic acid (NAA) and BA facilitated indirect organogenesis, while those developed on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and BA underwent somatic embryogenesis. MS medium with 13.3 μM BA and 2.69 μM NAA was the best for induction of shoots from callus, which developed a mean of 15.7 shoots. Shoots were best rooted on half-strength MS medium enriched with 2.46 μM indole-3-butyric acid with a mean of 5.1 roots per shoot. MS medium supplemented with 2.26 μM 2,4-D and 4.44 μM BA induced the highest number (mean of 13.4) of somatic embryos. Of the embryos transferred on half-strength MS medium containing 2.89 μM gibberellic acid, 78% of embryos developed to the cotyledonary stage. Most cotyledonary embryos (80%) underwent conversion to plantlets upon being transferred to half-strength MS basal medium in light. The survival rate of organogenesis and embryo-derived plants was 80 and 90%, respectively. Calli transformed with Agrobacterium tumefaciens showed expression of the gusA transgene and resistance to kanamycin, but did not undergo morphogenesis.     

In vitro propagation of pummelo (Citrus grandis L. osbeck)

Summary Several experiments were carried out to develop protocols for the in vitro propagation of pummelo (Citrus grandis L. Osbeck) using shoot-tip explants from seedlings. Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BA) and thidiazuron (TDZ), singly or in combination with α-naphthaleneacetic acid (NAA), was used to determine the rate of shoot proliferation. The response of explants to all concentrations of TDZ was very poor. After 6 wk culture, the most adventitious shoots per explant (average 5.2) were obtained on medium supplemented with 1.8 μM BA. NAA with cytokinin in the medium did not improve the rate of shoot multiplication significantly. Addition of 5.8 μM gibberellic acid in shoot-proliferation medium during the second subculture improved shoot elongation significantly. Shoot multiplication increased 3.5-fold in each successive subculture. NAA was superior to indolebutyric acid for in vitro root induction. Over 75% of the shoots developed roots when transferred to half-strength MS medium with 1.3, 2.7, or 5.4 μM NAA.