In vitro propagation ofCereus peruvianus mill. (cactaceae)

Summary Cereus peruvianus seedlings were used as a source of stem explants to determine the effective conditions for inducing and maintaining callus tissues in a state of rapid growth, as well as to obtain plants regenerated from callus cultures. Factorial combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in MS medium were tested, and we concluded that the 18.1µM 2,4-D and 18.6 or 27.9µM kinetin combinations were suitable for callus induction. The cactus shoots were produced from the friable callus; root elongation occurred within 2 wk in medium without 2,4-D and with 18.6µM kinetin. This method can be used to rapidly produce manyC. peruvianus plants.     

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.     

In vitro propagation of endangered Lippia filifolia mart. and schauer ex schauer

Summary This work describes an efficient micropropagation protocol of Lippia filifolia. Nodal segments cultivation in MS medium supplemented with 6-benzylaminopurine (4.5 μM)/α-naphthaleneacetic acid (NAA; 54nM) induced multiple shoots (in average 27 shoots per explant). Elongated shoots were rooted with NAA (0.11 μM) and they maintained ploidy level of the in vitro produced explants. The basic chromosome number were 2n=2x=24. Regenerated rooted shoots were successfully acclimatized under shading house conditions. This is the first report involving the establishment of a protocol for shoot multiplication and rooting for endangered L. filifolia, contributing for germplasm preservation of this species.     

Rapid in vitro propagation of Eclipta alba (L.) Hassk. through high frequency axillary shoot proliferation

An efficient protocol has been developed for rapid in vitro propagation of Eclipta alba L. (Asteraceae) through axillary bud multiplication. Murashige and Skoog (MS) medium supplemented with BA (10 M) was found to be most effective in breaking bud dormancy. An average number of 23 ± 0.57 shoots per explant was recorded after 30 days. Culture of node segments on fresh medium with lower concentration of BA (2 M) enhanced the multiplication rate. A maximum of 79 ± 1.90 mean number of shoots were obtained after three subcultures without any decline in multiplication rate. The regenerated microshoots showed the most efficient rooting on half strength MS medium augmented with 0.5 M IBA. Plantlets went through a hardening phase prior to ex vitro transfer and established in earthen pots containing garden soil; survival of about 90%. The established plants were uniform and exhibited morphological characters identical to mother plants.     

In vitro propagation of Fibigia triquetra (DC.) Boiss., a rare stenoendemic species

A rapid clonal propagation method for Fibigia triquetra (DC.) Boiss. (Brassicaceae), a rare Croatian stenoendemic species, has been developed. Shoots originated from aseptically germinated seeds were used for culture initiation. The highest multiplication rate of 9.2 shoots per explant was achieved in a 4-weeks-culture period in the third subculture on half strength Murashige and Skoog medium supplemented with 0.5 μM 6-benzylaminopurine and 2.9 μM gibberellic acid. Excised shoots were rooted best with addition of 8.6 μM indole-3-butyric acid on the same basal medium. Rooted plantlets were successfully transferred to potting soil and acclimatized to outdoor conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro clonal propagation of tea (Camellia sinensis (L.) O. Kuntze)

A system for in vitro clonal propagation has been developed in tea plants. Shoots obtained from primary explants were induced from terminal buds and axillary buds of mature field-grown plants. Cultures were initiated from both types of explants on Murashige and Skoog (MS) medium supplemented with 10% coconut milk (CM), 200 mg l^-1 of yeast extract (YE), 1.4 M indoleacetic acid (IAA) and 17.8 M benzyladenine (BA). The shoot tips were multiplied on 1/2 strength MS medium containing 10% CM, 2.9 M IAA and 17.8 M BA. The larger shoots were separated after multiplication and rooted on 1/2 MS medium supplemented with 11.4 M ascorbic acid and 34.5 M indolebutyric acid (IBA). A pretreatment of the plants with an aqueous solution of 493 M IBA greatly increased the frequency of rooting. More than 60% of the rooted plants have been transferred to soil successfully.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - YE yeast extract - CM coconut milk - MS Murashige and Skoog medium (1962)     

In vitro propagation ofHolostemma annulare (Roxb.) K. Schum., a rare medicinal plant

Summary A rapid micropropagation system was established forHolostemma annulare (Roxb.) K. Schum., (H. ada-kodien R. Br. ex Schult; Asclepiadaceae), a rare medicinal plant. Shoot tips (0.5–0.8 cm) and terminal and basal nodes (1.0–1.5 cm) harvested from actively growing shoots of conventionally raised plants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). Multiple shoot formation (3.8) was observed in 68% of basal nodes cultured on medium with optimum concentration of 4.43 μM BA and 0.54 μM NAA after 8 wk. Terminal nodes were not suitable for inducing multiple shoots. Irrespective of the orientation (vertical/horizontal), all shoot tip explants responded with a single shoot in all the combinations of plant growth regulators tried. Effects of other cytokinins (kinetin and 2-isopentenyladenine) and auxins [indole-3-acetic acid and indole-3-butyric acid (IBA)] to enhance the regeneration potential of basal nodes were analyzed. Shoots were multiplied by subculture of basal nodes and stumps (the original explant tissue free of shoots, but with remnant axillary, meristem and two or three protruding buds) in a reduced concentration of BA (2.21 μM) and NAA (0.27 μM). Liquid medium for multiplication was found to be ineffective due to a high degree of hyperhydricity. To make the multiplication process cost effective, culture bottles with polypropylene, caps were used for multiplication. The best root induction (75%) and survival (80%) was achieved on 0.5 strength MS medium supplemented with 1.48 μM IBA. Field-established plants had uniform growth habit traits in terms of height of plants and number, length, and weight of the tuberous roots.     

Developing an improved In vitro propagation system for slow-growing species using garcinia mangostana L. (mangosteen)

Summary This investigation disclosed that evaluation of tissue culture parameters of slowly developing species (e.g. Garcinia mangostana) requires monitoring of treatments through two or more successive, relatively long passages. Two 8-wk passages were necessary to observe differences in phytohormone effects. Photoperiod and temperature effects were not clearly evident until tissues had been cultured through three passages; the optimal photoperiod and temperature for shoot proliferation could not be established until after the fifth passage. Our investigation revealed that no auxin supplementation was necessary for bud primordium differentiation in cotyledon explants or proliferation of regenerated shoots. The optimum N⁶-benzyladenine concentration for primordium differentiation was 13.3 μM, and for shoot proliferation ranged from 4.4 to 13.3 μM. Continuous culturing in an 8-h photoperiod at 30°C resulted in progressively intensified degeneration of shoots after three passages. In contrast, successive passages in a 16-h photoperiod/26°C regimen enabled sustained regeneration of shoots. The shoots rooted at a rate of 85% when precultured for 3 d in a medium containing 4921.3 μM indole-3-butyric acid, or 10 d at 492.1 μM, then cultured for two 8-wk passages in phytohormone-free medium. Following acclimatization by gradually lowering the relative humidity in the growth chamber, rooted shoots survived transfer to the greenhouse at a rate of 95%.     

An optimized method for in vitro propagation of African baobab (Adansonia digitata L.) using two-node segments

Adansonia digitata L. (African baobab), is an important multi-purpose tree, whose distribution is at present limited to wild or semi-domesticated individuals widespread in Africa. Its distribution is threatened by seedling clearance for other land use and potentially by overharvesting induced by growing commercial use of baobab fruit. Recently, efforts have been made to establish baobab domestication and conservation strategies, with mixed results due to the low germinability of baobab seeds, a factor that hinders the possibility of developing commercial A. digitata plantations. Here, micropropagation was tested as a method for clonal propagation of explants from in vivo-grown seedlings. In vitro shoot multiplication was achieved by enhanced axillary bud proliferation of sterilized two-node segments. Bud break was dependent on cytokinin supply, but the combination of 1.0 or 10.0 μM zeatin riboside and 10.0 μM indole-3-butyric acid (IBA) increased the formation of microshoots after 8 weeks of culture. Regenerated microshoots rooted successfully in in vitro nutrient medium containing 10.0 μM IBA and normally grew in a greenhouse after acclimatization.     

In vitro propagation ofPhebalium equestre andPhebalium hillebrandii (rutaceae)

Summary The endangeredPhebalium equestre D. A. Cooke and the rarePhebalium hillebrandii J. H. Willis were propagated in vitro using shoot tips and nodal segments as explants. For each species, shoot proliferation was initiated on de Fossard MZZM (Medium levels of minerals, Zero auxins, Zero cytokinins and Medium levels of sucrose, growth factors, and amino acids) medium supplemented with 1 μM benzyladenine. ExcisedP. equestre shoots initiated roots when cultured on MZZM medium containing 60 μM 2,4-dichlorophenoxyacetic acid, whileP. hillebrandii shoots required LZZL (Low levels of minerals, Zero auxins, Zero cytokinins and Low levels of sucrose, growth factors, and amino acids) medium containing 10 μM 2,4-D for maximal root initiation. Both species required transfer to MZZM medium without growth regulators after 2 wk to allow root initials to develop and grow. Plantlets were successfully transferred to soil with 80% survival after 2 mo.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Feronia limonia</Emphasis> (L.) Swingle from hypocotyl and internodal explants

The hypocotyl and internodal segments from in vitro grown seedlings of Feronia limonia (L.) Swingle (wood apple) were cultivated on Murashige and Skoogs (1962, MS) medium supplemented with N⁶-benzyladenine (BA) or adenine (ADE) or kinetin (KN) at 0.5 to 5 µM. The optimum response was recorded on the medium containing 2 µM BA. An average of 12 and 8 shoots were developed from hypocotyl and internodal explants, respectively, after eight weeks of culture. The shoots were excised, and the residual explants were transferred to fresh medium where again they developed shoots. Up to three such passages resulted in the production of shoots from repeatedly subcultured explants and an average of 24 – 36 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to half strength MS medium supplemented with 1 µM 1-naphthaleneacetic acid (NAA). The developed plantlets were successfully transferred to mixture of soil, sand and coco-peat (1:1:1) and hardened in controlled environment. Hardened plants were transplanted to soil in greenhouse.     

In vitro response of apical bud explants from mature trees of jackfruit (Artocarpus heterophyllus)

A tissue culture technique for rapid vegetative propagation of mature jackfruit trees using apical bud cultures has been developed. Shoot-tip cultures were established on MS medium with 5–10 mm explants dissected from terminal buds of new growth from trunk. After initial culture of bud explants, one- to two-node pieces were taken from the microshoots formed and used to proliferate further axillary shoots for multiplying and maintaining shoot cultures. Benzyladenine and kinetin (4.5–9.0 µM), either separately or together, supported shoot proliferation; higher concentrations of the cytokinins inhibited bud breaking and favoured callus formation at the explant bases. Bud explants taken from emerging trunk sprouts invariably produced clumps of multiple shoots, whereas buds obtained from actively growing top branches generally elongated to form a solitary shoot. November to January was the best season for initiation of cultures from field-grown trees. Shoots proliferated at the initial subcultures had mature morphology and were difficult-to-root. Shoots assumed to be juvenile-like developed at the later passages and could be rooted with 60–80% success using 1/2-MS salts and 10 µM of indolebutyric acid or naphthaleneacetic acid. Regenerated plantlets were transferred to the soil and about 50% survived.     

In vitro propagation of Hosta sieboldiana using excised ovaries from immature florets

Ovaries from immature florets were selected as an improved explant source for in vitro cultures of Hosta sieboldiana. Culturing for 30 days on a modified Murashige & Skoog (MS) medium supplemented with thiamine (0.4 mgl^-1) and glycine (2.0 mgl^-1) induced the highest frequency of callus growth at 5.4 M NAA and 4.4 M BA, and the highest frequency of shoot initiation at 0.4 M BA. The severed basal portion of the ovary was the site of shoot growth. Eliminating phytohormones and adding 80 mgl^-1 of AdSO₄ significantly enhanced root initiation. Rooted plantlets were established in sterilized vermiculite containing MS mineral salts. A cold treatment (1°C) of 30 days in the dark was necessary to break dormancy of the rooted plantlets.Abbreviations AdSO₄ adenine sulfate - BA benzylaminopurine - NAA naphthaleneacetic acid     

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

Summary A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

In vitro propagation of Leucopogon obtectus Benth. (Epacridaceae)

Micropropagation was achieved with the rare and endangered Western Australian woody shrub Leucopogon obtectus Benth. (Epacridaceae). BA 10M, IBA 0.5M, 1/2 MS salts and various growth factors produced an approximate twelve-fold multiplication rate compared to controls. Micropropagation was not accomplished using auxin-supplemented 1/2 or 1/4-strength MS-based media. However, an average 70% rooting was achieved with only agar/water medium and a high percentage of these rooted plantlets were established in pots containing habitat soil.     

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.     

In vitro propagation of Ancistrocladus abbreviatus Airy Shaw (Ancistrocladaceae)

Ancistrocladus abbreviatus Airy Shaw (Ancistrocladaceae), a West African liana producing naphthylisoquinoline alkaloids, was successfully raised from seeds in vitro. Clonal propagation was best achieved growing nodal stem segments on 1/5 Linsmaier and Skoog medium with full strength organics and supplemented with 0.02 μM thidiazuron, 4.44 μM 6-benzylaminopurine and 0.05 μM 1-naphthaleneacetic acid. Detached axillary shoots were grown on Anderson's Rhododendron medium devoid of phytohormones and rooted within one month when dipped in 4.92 μM indole-3-butyric acid. Rooted plants became acclimatized to nonsterile greenhouse conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of Centaurea spachii from inflorescence stems

Procedures for micropropagation of Centaureaspachii (Compositae), an endangered rosulate plantendemic from the mediterranean Spain area, have beendeveloped using inflorescence nodal segments asexplants for in vitro establishment. Only 15%of explants remained contaminated using this materialto start the in vitro axenic cultures. Higherproliferation of shoots and multiplication coefficientwas obtained on Murashige and Skoog (MS) mineralmedium supplemented with 1.0 mg l^{minus 1}6-benzyladenine. However, shoot elongation decreasedwith the addition of this cytokinin.Rooting of shoots with only one auxin was very lowafter 6 weeks on the majority of rooting media tested.The best rooting result (60%) was obtained on MSmedium with a combination of 2 mg l^{minus 1}indole-3-acetic acid plus 2 mgl^{minus 1} indole-3-butyric acid. Moreover, in thisculture medium 50% of shoots rooted during the thirdweek of culture. High survival, over 80%, wasobtained when the plantlets were transferred togreenhouse conditions. The endangered Centaureaspachii can be successfully micropropagatedbeginning with a single inflorescence stem and withoutsignificant damage to the mother plant.     

Callus production, somatic embryogenesis and plant regeneration of Lycium barbarum root explants

A new micropropagation system for Lycium barbarum (L.) was developed using root explants as starting material. Callus can be produced from root explants on Murashige and Skoog (MS) medium containing 0.2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid. After three subcultures on the same medium, callus was then transferred onto the MS medium supplemented with 500 mg dm⁻³ lactalbumin hydrolysate to induce somatic embryogenesis (SE). After 20 d, about 60 somatic embryos per 0.25 g(f.m.) of embryogenic callus were obtained but only about 10 % of embryos converted into plantlets. After acclimated in the greenhouse, all of the randomly selected plantlets had survived and were similar phenotypically to zygotic seedlings. In addition, the effects of irradiance, photoperiod, growth regulators, explant age and cold treatment on SE of root-derived callus were evaluated.