Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

Summary Chile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N⁶-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N⁶-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.     

Micropropagation of mature <Emphasis Type="Italic">Crataegus aronia</Emphasis> L., a medicinal and ornamental plant with rootstock potential for pome fruit

The effects of culture media and cytokinin types on micropropagation of mature Crataegus aronia L. were investigated. Using single-axillary bud explants, the growth of cultures on MS, WPM, DKW and NRM containing 4.44 μM benzyladenine (BA) plus 0.05 μM indole-3-butyric acid (IBA), and on NRM containing thidiazuron, meta-Topolin (mT) or BA at 1.25, 2.5, 5.0 or 7.5 μM plus 0.05 μM IBA were compared. The culture medium had significant effects on shoot number and length. In comparison with MS, DKW and WPM, shoot production was greater on NRM (5.7 shoots per explant). Shoot production on MS, DKW and WPM (4.2, 4.2 and 4.1, respectively) were statistically similar to each other. Thidiazuron was detrimental to shoot formation and caused formation of rosette shoots and/or large callus to form on explants. In the presence of mT, only some of the explants developed into shoots. Benzyladenine was the only cytokinin that promoted both shoot proliferation and shoot elongation. Higher shoot numbers were obtained at 5.0 and 7.5 μM BA compared to lower concentrations of BA. Over 80% of microshoots rooted and rooted shoots were successfully acclimatized to ex vitro conditions.     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

Thidiazuron promotes adventitious shoot regeneration from pothos (Epipremnum aureum) leaf and petiole explants

Summary Regeneration of adventitious shoots of pothos (Epipremnum aureum Linden and Andre) ‘Jade’ was obtained using leaf and petiole explants preprated from shoot tips of 3-yr-old greenhouse-grown plants. Explants were cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), 6-(4-hydroxy-3-methy-trans-2-butenyl-amino)purine (zeatin) or N-isopentenylaminopurine (2iP) individually with α-naphthaleneacetic acid (NAA) in 18 combinations. Callus was initiated from cut surface and along the midrib or major vein of leaf sections. Shoot regeneration from leaf and petoole explants occurred in 30d on medium containing 1, 5 or 10μM TDZ with 0.5 or 1.0μM NAA except petioles on medium with 10 μM TDZ and 1.0 μM NAA where regeneration failed. More time (50d) was needed for shoot regeneration when explants were cultured on medium containing either 2iP or zeatin with NAA. Regeneration frequencies were up to 20% and 50% for leaf and petiole explants, respectively. Shoot numbers per responding explant attained 30 for leaf and petiole explants on medium containing TDZ but only one to four on medium containing either 2iP or zeatin. These results indicate that TDZ is a more effective cytokinin for in vitro regeneration of pothos than either zeatin or 2iP.. Shoots elongated readily and rooted well on MS basal medium, without plant growth regulators. Plantlets acclimatized rapidly and grew vigorously in the greenhouse after transfer to pots containing a commerecial potting medium.     

Micropropagation of chinese redbud (<Emphasis Type="Italic">Cercis yunnanensis</Emphasis>) through axillary bud breaking and induction of adventitious shoots from leaf pieces

Summary Factors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.     

High frequency shoot regeneration from immature embryo explants of Hungarian vetch

An in vitro propagation protocol has been developed from mature trees of Pittosporum napaulensis. The best bud proliferation (83.1%), shoot number (21 axillary shoots/ explant) and shoot length (5.5 cm) was achieved in Murashige and Skoog (MS) medium supplemented with 5.0 μM N⁻⁶ benzyladenine and 0.1 μM α- naphthalene acetic acid. Of the three cytokinins tested (N⁻⁶ benzyladenine, kinetin and thidiazuron), N⁻⁶ benzyladenine proved to be the best for shoot induction. Shoot regeneration potential varied among genotypes. Regenerated shoots rooted after 48 hours treatment on half-strength MS liquid medium supplemented with 20 μM indole-3-butyric acid. Rooted shoots transferred to 120 g (w/v) soilrite + sand + soil (1:1:1) mixture showed 70% survival. Twenty-one plantlets are growing well in green house conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Direct shoot regeneration from foliar explants of an epiphytic orchid,Acampe praemorsa (Roxb.) Blatter and McCann

An efficient and reproducible procedure is described for the large-scale propagation of an epiphytic orchid,Acampe praemorsa (Roxb.) B latter and McCann using foliar explants. Shoot buds were induced in basal parts of foliar explants on Murashige and Skoog medium supplemented with N⁶-benzyladenine (BA), kinetin (Kn) or thidiazuron (TDZ), the latter being most effective at 1.0 mg/1. Shoots formed to a TDZ-containing medium elongated following transfer to a substrate supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA) and 0.5 mg/1 BA. NAA at lower concentrations had no beneficial effects on shoot regeneration, whether added to the medium along with BA, Kn or TDZ. However, it promoted shoot elongation and leaf expansion. Higher concentrations of NAA suppressed shoot regeneration. The frequency of shoot regeneration was greatly influenced by the developmental stage and orientation of the leaf. Shoots regenerated from the foliar explants were rooted successfully on MS medium containing 1.0 mg/l indole-3-butyric acid. The plantlets were acclimated and eventually transferred to a garden.Abbreviations BA N⁶-Benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)     

Rapid plant regeneration through <Emphasis Type="Italic">in vitro</Emphasis> axillary shoot proliferation of butterfly pea (<Emphasis Type="Italic">Clitoria ternatea</Emphasis> L.)—a twinning legume

This study describes a reproducible protocol for rapid mass propagation of a multipurpose legume, Clitoria ternatea L., using cotyledonary node explants derived from axenic seedlings. Multiple shoots were induced in Murashige and Skoog (MS) medium supplemented with N ⁶-benzyladenine (BA), zeatin riboside, or thidiazuron. N ⁶-Benzyladenine at 1.0 mg l⁻¹ (4.44 μM) was most effective for shoot proliferation. Multiple shoots were also induced in nodal segments of in vitro-raised shoots grown on MS medium containing 1.0 mg l⁻¹ (4.44 μM) of BA. Rooting was best induced in shoots grown on half-strength MS medium with 0.25 mg l⁻¹ (1.42 μM) of indole-3-butyric acid. Plants were acclimatized in vermicompost and established in soil where they flowered and formed mature seeds.     

Contents of Macroelements and Growth of Sweet Cherry Rootstock in vitro

Rhododendron shoot regeneration was accomplished using either flower explants (each consisting of ovary with pedicel) of Rhododendron cvs. Nova Zembla and Irina or leaves isolated from in vitro grown Rhododendron catawbiense Michx. Multiple shoot tip clumps were obtained on Anderson's medium containing 0.5 to 1.5 mg dm⁻³ thidiazuron (TDZ) in combination with 12 to 15 mg dm⁻³ N⁶-[2-isopentenyl]adenine (2iP) and 1 to 3 mg dm⁻³ indole-3-butyric acid (IBA). After 16 weeks on the regeneration media, explants with shoot tip clumps were transferred for shoot elongation to Anderson's medium with 3 mg dm⁻³ 2iP. Two months later, the shoots have reached 5 to 40 mm in length and were fit for subcultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of adult Swainsona formosa (Leguminosae: Papilionoideae: Galegeae)

Summary Explants of axillary buds excised from mature adult stems of Swainsona formosa (G. Don) J. Thompson (syn. Clianthus formosus) were cultured on Murashige and Skoog medium supplemented with a range of auxins, cytokinins, and sucrose concentrations. Auxins did not increase shoot or bud numbers above controls, and 2,4-dichlorophenoxyacetic acid was the only auxin to significantly increase callus production. Benzyladenine or thidiazuron incorporated into the medium at 0.1 μM stimulated shoot and bud production, and shoot growth occurred following removal of cytokinins from the medium after 4 wk. Shoot number increased linearly with sucrose concentration up to 40 g l⁻¹, but shoot height and the number of cytokinin-induced buds were optimal at sucrose levels of 20–30 g l⁻¹. Roots were initiated in vitro following treatment of cuttings with 0.1% indole-3-butyric acid and 0.1% α-naphthaleneactic acid. Plantlets were successfully established in soil but were plagiotropic and exhibited distichous phyllotaxy.     

Activity of thidiazuron inin vitro shoot cultures ofPrunus sp. andMorus alba

Thidiazuron incorporated into MS medium stimulated rosettes formation only in some treatments. This effect was more pronounced in cultures ofMorus alba thanPrunus sp. Mulberry cultures responded to the optimal concentration of thidiazuron (0.2 mg I^?1) not only with shoot formation but also, with growth of large leaves and poor development of callus tissue. In cultures of both investigated genera the shoot elongation was inhibited. Shoots of mulberry cultures growing on proliferation medium supplemented with thidiazuron formed roots, in many cases.     

High-frequency plant regeneration by <Emphasis Type="Italic">in vitro</Emphasis> shoot proliferation in cotyledonary node explants of grasspea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.     

Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes

A protocol is presented for micropropagation of an economically important timber-yielding forest tree, Dalbergia sissoo Roxb. (Sissoo). Multiple shoots were induced from cotyledonary nodes derived from 1-week-old axenic seedlings on Murashige and Skoog's medium containing either N ⁶-benzyladenine (BA), kinetin (Kn), isopentenyladenine (2iP) or thidiazuron (TDZ), with BA being the most effective growth regulator. High-frequency shoot proliferation (99%) and maximum number of shoots per explant (7.9 shoots) were recorded with BA at an optimum level of 8.9 μM. Concentrations of all cytokinins tested above the optimum level markedly reduced the frequency of shoot proliferation. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary node on shoot multiplication medium after each harvest of the newly formed shoots. Primary shoots were multiplied as nodal explants, and from each stem node 2 or 3 shoots developed. Thus, 60–70 shoots were obtained in 3 months from a single cotyledonary node. About 91% of the shoots developed roots following transfer to half-strength MS medium containing a combination of 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. Eighty percent of the plantlets were successfully acclimatized and established in soil. Received: 1 October 1997 / Revision received: 31 March 1998 / Accepted: 7 April 1998     

Shoot regeneration from adventitious buds induced on juvenile and adult almond (Prunus dulcis mill.) explants

Summary Adventitious shoot regeneration was achieved from almond leaves, cv. Boa Casta, excised fromin vitro cultures of juvenile and adult material. Murashige and Skoog (1962) medium (MS) was found to be more efficient for adventitious shoot induction than a modified medium of Quoirin et al. (1977) when using identical growth regulator supplements. Thidiazuron (TDZ) at 4.54, 5.90, 6.81, and 9.08 μM was used in all induction media, together with indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), or a combination of IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). When N⁶-benzyladenine (BA) was used instead of TDZ, no adventitious shoots were induced. Leaf explants of juvenile origin yielded the highest regeneration rates (40.0 and 38.2%) and required higher concentrations of TDZ for shoot induction than leaves of adult origin. An increase from 15.0 to 35.3% in the regeneration ability of adult leaf explants, tested on one of the induction media, modified medium of Quoirin et al. (1977) supplemented with 5.90 μM TDZ and 2.85 μM IAA], was achieved when donor shoots were subcultured twice on a medium with a low BA concentration of 1.33 μM.     

Comparative studies of cytokinins on in vitro propagation of Bacopa monniera

A mass in vitro propagation system for Bacopa monniera (L.) Wettst. (Scrophulariaceae), a medicinally important plant, has been developed. A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants. Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (6.8 μM) and 6-benzyladenine (8.9 μM) proved superior to other treatments. Optimum adventitious shoot buds induction occurred at 6.8 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation. However, subculture of leaf explants on medium containing 2.2 μM benzyladenine yielded a higher number (129.1) of adventitious shoot buds by the end of third subculture. The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting. In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 4.9 μM IBA. After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro adventitious bud regeneration from internode segments of beech

Internode explants collected from in vitro grown shoots of two clones of Fagus sylvatica L. (European beech) and five clones of F. orientalis Lipski (Oriental beech) were used to evaluate their bud regeneration capacity. Adventitious shoot-buds formed on callus, which developed from internode segments cultured in a Woody Plant Medium supplemented with different concentrations of either thidiazuron (TDZ) or benzyladenine (BA). After 4 weeks of culture on induction media, the explants were transferred to a proliferation medium supplemented with 2.2 μM BA, 9.1 μM zeatin and 2.9 μM indole-3-acetic acid (IAA) for another 8 weeks. Medium containing TDZ was much more efficient than medium containing BA in inducing adventitious buds, the optimal TDZ concentration being 4.5 μM and the optimal BA concentration 17.8 μM. Genotypic variation in shoot regeneration capacity was observed among the two Fagus species and between clones within each species, with a significant interaction between TDZ concentration and genotype regarding mean bud number. Thidiazuron induction medium supplemented with a range of individual auxins was investigated, and it was found that IAA or indole-3-butyric acid at 2.9 μM enhanced the bud forming capacity of explants. Morphogenic response varied significantly with the position of the internode along the stem. The highest regeneration potential was obtained from apical internodes, while those distal to the apex were the least productive. Elongated shoots of adventitious origin can be readily proliferated by axillary branching. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of benzylaminopurine and thidiazuron onin vitro shoot proliferation ofTilia cordata MILL.,Sorbus aucuparia L. andRobinia pseudoacacia L.

Benzylaminopurine and thidiazuron stimulated shoot proliferation ofTilia, Sorbus andRobinia. Low concentration of BAP (0.2—1.0 mg I^?1) promoted axillary bud formation and shoot elongation. Thidiazuron displayed high cytokinin activity at very low concentrations (0.002—0.05 mg I^?1). Shoot number induced on media containing thidiazuron was large. Numerous shoots were produced on the media containing BAP together with thidiazuron. Shoots produced on media containing thidiazuron or BAP together with thidiazuron rooted after transfer to medium supplemented with low concentration of auxin (IBA or NAA).     

Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system

 In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 2.3 μmol·l^–1) and thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20 μmol·l^–1) was evaluated. Multiple shoot induction medium with 1 μmol·TDZ l^–1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainung 1, Tainung 2, and Everglades 71). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced multiple shoots on the induction medium with 1 μmol·TDZ l^–1. Multiple shoot clumps formed after 2 weeks in culture without callus formation. Shoots elongated and rooted within 2 weeks after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots from the shoot apex. Received: 2 February 2000 / Revision received: 30 March 2000 / Accepted: 22 June 2000     

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus

Protoplasts isolated from lateral shoot buds of cotyledon-free pea embryo axes were regenerated to callus. Protoplast derived calluses with a diameter of about 1cm were transferred to shoot induction media, containing different concentrations (1–50µM) of thidiazuron. Shoot formation was observed after 16 weeks up to 12% efficiency. Thidiazuron (10µM) was the most effective concentration in all experiments. Shoot buds elongated in medium supplemented with N-isopentenyl adenine and indole-3-butyric acid. Since rooting was almost impossible in these thidiazuron-induced shoots, shoots were grafted onto young pea seedlings and regenerated to fertile plants.Abbreviations 2ip N-isopentenyl adenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MES 2-(Nmorpholino)ethane acid - NAA naphthaleneacetic acid - TDZ thidiazuron - BAP 6-benzylaminopurine - PEG polyethylene glycol