<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.     

An efficient micropropagation system for <Emphasis Type="Italic">Eclipta,alba</Emphasis>—A valuable medicinal herb

Summary An efficient rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Eclipta alba (Asteraceae) by enhanced axillary shoot proliferation in cotyledonary node segments was designed. The medium type, various carbon sources, plant growth regulators, and coconut water markedly influenced in vitro propagation of Eclipta alba. An in vitro plantlet production system has been investigated on Murashige and Skoog (MS) medium with the synergistic combination of benzyladenine (4.4μM), kinetin (4.6μM), 2-isopentenyladenine (4.9μM), gibberellic acid (1.4μM), 5% coconut water, and 3% sucrose which promoted the maximum number of shoots as well as beneficial shoot length: Subculturing of cotyledonary node segments on a similar medium enabled continuous production of healthy shoots with similar frequency. Rooting was highest (94.3%) on full strength. MS medium containing 9.8 μM indolebutyric acid. Micropropagated plants established in garden soil, farmyard soil, and sand (2∶1∶1) were uniform and identical to the donor plant with respect to growth characteristics as well as floral features. These plants grew normally without showing any morphological variation.     

<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.     

Plant regeneration through shoot formation from callus of Areca catechu L.

In order to establish and optimize an in vitro micropropagation protocol of Venus fly trap (Dionaea muscipula Ellis), a carnivorous plant, the effects of medium type, MS medium concentration, pH, and cytokinin and auxin types on shoot proliferation and root formation were investigated using 3-month-old shoots. The shoot proliferation was most effective in 2.3 M kinetin-supplemented 1/3MS medium at pH 5.5. The best conditions for rooting were 1/3MS medium supplemented with 0.5 M IBA. All subcultured shoots produced extensive root systems after 5–6 weeks culture. When plantlets after rooting were planted in plastic pots filled with 1:1 peat moss and sand, the survival rate of plantlets was almost 100%, exhibiting normal development. With subculture every 8 weeks, hundreds of the plants were propagated from a single plant within a year.     

Somaclonal variation as a tool to develop pest resistant plants of Torenia fournieri ‘Compacta Blue’

Callus cultures of Torenia fournieri Compacta Blue were initiated on a modified Murashige and Skoog salt medium (MS) with 2.26 M 2,4-dichlorophenoxyacetic acid. Shoots were regenerated from these cultures using MS medium amended with 2.46 M indolebutyric acid and 8.88 M benzyladenine. These shoot cultures were subjected to two-spotted spidermite (Tetranychus urticae Koch.) and the greenhouse whitefly [Trialeurodes vaporariorum (Westwood)]. Pests were allowed to feed until such time that their populations started to decrease due to lack of food. The remaining live tissue of the Torenia was placed on MS medium amended with 2.28 M zeatin to induce new adventitious shoots and plantlets. Newly regenerated plantlets were acclimated to greenhouse conditions and evaluated for resistance to the pest to which they were subjected in vitro. Highly significant differences in pest numbers were found in somaclones for both the two-spotted spidermite and greenhouse whitefly when compared to control plants. A wide range of variability was observed among the somaclonal population. There were significantly fewer mite eggs laid on plants regenerated from in vitro cultures screened with two-spotted spidermites than on seed sown controls. Regenerants from cultures screened with whiteflies in vitro had fewer eggs, immatures and live adults than controls.Abbreviations BA benzyladenine - IBA indolebutyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog salt medium Storrs Agricultural Research Station Scientific Publication 1641.     

Micropropagation of Cunila galioides,a popular medicinal plant of south Brazil

Axillary buds of field plants of Cunila galioides Benth. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with 8.8 M of benzyladenine. Repeated subcultures of shoot tips and single nodes at 4-week intervals for eight months on the above medium enabled mass multiplication of shoots without any evidence of decline. The best conditions for rooting were MS medium plus 0.5 to 2.5 M of indolebutyric acid. The rooted plants were successfully transferred to soil, exhibiting a normal development.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

In vitro propagation of Iphigenia indica,an alternative source of colchicine

The present study involves in vitro propagation of Iphigenia indica (Kunth.) through multiplication of whole corms and corm buds. The whole corms produced very small micro-corms, which developed plants individually whereas corm buds multiplied to produce numerous shoots at variable rates in presence of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The best response in corm and bud multiplication was obtained in Murashige and Skoog's basal medium (MS) supplemented with 2.69 M NAA and 8.88 M BAP. The shoots regenerated were further cultured on MS medium containing NAA and indole-3-butyric acid (IBA) for initiation of roots. MS medium with 5.38 M NAA and 4.92 M IBA induced highest percentage of roots (81%) within 2 weeks in culture.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated DNA transfer to<Emphasis Type="Italic"> Aesculu</Emphasis>s<Emphasis Type="Italic"> hippocastanum</Emphasis> L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle     

In vitro plant regeneration from seed explants of wild groundnut species (Genus Arachis, Section Extranervosae)

In vitro regeneration of wild groundnut species from Section Extranervosae (Arachis villosulicarpa, A. macedoi, A. retusa, A. burchellii, A. pietrarellii, A. prostrata, A. aff. prostrata and a new species) was examined for the purpose of germplasm renewal and conservation. Seeds of different ages, stored at the seed bank of CENARGEN/EMBRAPA were either inoculated on culture medium or used as a source of embryo axis and cotyledon explants. Whole seeds failed to germinate on MS either without growth regulators (MS0) or supplemented with 10 M TDZ. Embryo axes cultured on MS0 produced only single plants. In the presence of 8.8 M BAP these explants showed multi-shoot formation. Cotyledons cultured on MS supplemented with 110 M BAP developed adventitious shoots through direct organogenesis. Plant regeneration was obtained from A. villosulicarpa, A. macedoi, A. retusa, A. burchellii and A. pietrarellii both from embryo axes and cotyledons. Explants from A. prostrata and A. aff. prostrata did not produce regenerants. Rooting of shoots was induced in the presence of 5.4 M NAA. Primary plants derived from these explants were further multiplied by culturing nodal segments on MS medium plus 2.7 M NAA.     

Rapid in vitro adventitious shoot propagation of <Emphasis Type="Italic">Scopolia parviflora</Emphasis> through rhizome cultures for enhanced production of tropane alkaloids

A rapid micropropagation system for Scopolia parviflora Nakai (Solanaceae), a rare medicinal plant native to Korea, was established using rhizome cultures. Shoots that originated from adventitious shoots of the rhizome were multiplied when the rhizomes were cultured on half-strength B5 liquid medium supplemented with various growth regulators. Optimum shoot multiplication was observed in half-strength B5 medium containing 3% (w/v) sucrose and 5.77 M gibberellic acid (GA₃). Each rhizome gave rise to an average of 12 shoots. Shoot elongation and root induction from multiple shoots occurred on growth regulator-free half-strength B5 solid medium. Healthy plantlets were transferred to a peat moss:vermiculite mixture for acclimatization, which was successful. The concentrations of tropane alkaloids, hyoscyamine and scopolamine were determined in different tissues of native growing plants, in vitro-propagated plants and acclimatized plants by high-performance liquid chromatography. The analysis revealed that the levels of hyoscyamine and scopolamine were higher in in vitro-propagated plants than in the native growing plants. When the rhizome was cut into segments and transferred to optimal culture conditions for multiple shoot propagation, only 12 weeks were required to produce a mature plant. We conclude that in vitro propagation techniques through rhizome cultures provide an efficient and rapid method for shoot propagation of S. parviflora.Abbreviations BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - HPLC High-performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Regeneration and large-scale propagation of Phragmites communis through somatic embryogenesis

A protocol for large-scale propagation of Phragmites communis Trin. by somatic embryogenesis has been established. Plants were regenerated through somatic embryogenesis from stem segments of R5002-12, a salt-tolerant variant line of Phragmites communis Trin. Stem segment explants produced hard white callus on the semi-solid Murashige and Skoog (MS) medium supplemented with 9.05 M 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 weeks. The induction frequency was 36.7%. Then, the callus was transferred to MS medium supplemented with 4.52 M 2,4-D. After 4 weeks in culture, yellow embryogenic callus with some nodular structures was formed. When the embryogenic callus was transferred to differentiation medium (MS supplemented with 0.45 M 2,4-D), differentiation was initiated to form small green islands on the surface of the callus after 2 weeks in culture. Within 4 weeks, a large number of somatic embryos were formed with a frequency of 86.7%. Six weeks later, they developed into strong plantlets. When the plantlets (about 1 cm in length) were cultured on propagation medium (MS supplemented with 13.31 M BA+5.37 M NAA), a great number of regenerated plants were obtained. After the plants were cultured on liquid 1/2 MS medium with 2.69 M NAA added 2.46 M IBA roots developed. The rooted plants were transferred to soil with over 85% survival. Using this methodology, more than 20000 regenerated plants of salt tolerant variant line of Phragmites communis Trin. have been produced.     

An improved micropropagation protocol for teak

An improved micropropagation protocol has been developed for teak (Tectona grandis). Nodal explants placed on MS medium supplemented with 22.2 M benzylaminopurine and then serially transferred to fresh medium after 12, 24, 48 and 72 h gave maximum culture establishment (76.8%). Establishment was reduced when explants were retained in the initial culture medium longer than 12 h. Explants collected in May showed maximum (76.8%) response. Placement of the explants on MS medium supplemented with 22.2 M benzylaminopurine and 0.57 M indole-3-acetic acid resulted in the maximum average number of shoots. In vitro raised micro shoots were rooted ex vitro by dipping in indole-3-butyric acid (9.8 mM) for 2 min followed by planting in polyethylene pots containing a soil:vermiculite (1:1 v/v) mixture. This treatment resulted in 77.9% survival of the plantlets. They were weaned in a glasshouse and finally moved to an agro-net shade house.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

In vitro propagation ofMimosa tenuiflora (Willd.) Poiret,a Mexican medicinal tree

Summary Mimosa tenuiflora (Willd.) Poiret (Leguminosae) was micropropagated throughin vitro culture of axillary buds on Murashige and Skoog (MS) medium. Shoot formation was achieved when the media were supplemented with 0.1 mg.L^–1 IAA + 3 mg.L^–1 KN.In vitro rooting of regenerated shoots was achieved when 0.1 mg.L^–1 KN was combined with 1 mg.L^–1 IBA in the absence of IAA. Ninety-four percent of the rooted plants were succesfully adapted to field conditions and grown in the soil. A total of 180 trees grown under these conditions were obtained over a one-year period.Abbreviations KN (kinetin) - IAA (-indoleacetic acid) - MS (Murashige and Skoog (1962) medium) - IBA (indole-3-butyric acid) - NAA (anaphthaleneacetic acid)     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.     

Effect of ZnSO4 and CuSO4 on Regeneration and Lepidine Content in Lepidium Sativum L.

Significant amounts of lepidine was detected in mature and juvenile explants from both in vivo and in vitro grown plants. The yield, however, was variable depending upon the source and type of explant used. Mature in vivo plants at vegetative stage exhibited highest yield. Among all the explants, maximum lepidine was detected after 8 weeks in shoot apex callus on MS medium supplemented with 2 mg dm^-3 naphthaleneacetic acid and 5 mg dm^-3 benzylaminopurine. Addition of 900 M Zn^2- or 100 M Cu^2- further enhanced the yield of lepidine.     

Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators

A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and -naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 M BA and 1.0 M NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 M BA and 1.0 M NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 M indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.