Regeneration of `juvenile' plants of black plum,Syzygium cuminii Skeels,from nodal exp of mature trees

Nodal explants, excised from young shoots from mature trees of Syzygium cuminii, were cultured on MS medium supplemented with different concentrations of BA or kinetin. Among these, BA (0.5 or 1.0 mg l^–1) induced greening and opening of the incipient shoot buds, which however did not elongate. Elongation of the shoot buds was facilitated on MS medium with 1.0 mg l^–1 BA supplemented with casein hydrolysate (1.5 g l^–1) or glutamine (200 mg l^–1). Nodal explants (microcuttings), taken from shoots developed in vitro, also developed multiple shoots when cultured on MS with1 mg l^–1 BA. These explants did not require an additional supply of reduced nitrogen, for further normal development. Shoots developed from explants from mature trees and microcuttings were rooted by sub-culturing them on Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The plants that developed in vitro were planted in soil and were transferred to the field after an acclimatization period of 7–8 months. These plants have been thriving well for more than three years and have no apparent phenotypic aberrations.     

Micropropagation of Capparis decidua through In Vitro Shoot Proliferation on Nodal Explants of Mature Tree and Seedling Explants

In vitro clonal propagation of Capparis decidua was achieved using nodal explants from mature trees, and cotyledonary node, cotyledon and hypocotyl explants taken from the seedlings. Explants cultured on MS medium supplemented with BAP showed differentiation of multiple shoots and shoot buds in 4–5 weeks in the primary cultures. The medium with BAP (5 mg/l) was the best for shoot bud proliferation from the nodal as well as seedling explant. Shoot multiplication was best on cotyledonary node. In the nodal explants shoot multiplication was best on medium supplemented with 5 mg/l BAP and after second subculturing further multiplication of shoot buds was highest on the medium containing 3 mg/l BAP. Shoots were separated from mother cultures in each subculturing for rooting. Rooting was best achieved using 1 mg/l IBA in the medium. Rooted plantlets were transferred td earthen pots with garden soil and peat moss mixture.     

Plant regeneration from callus cultures of Psoralea corylifolia Linn

Plantlet regeneration via organogenesis was achieved in callus cultures derived form mature leaves, stems and leaves, petioles and roots of young seedling of Psoralea corylifolia on Murashige and Skoog medium supplemented with 2.5–3.0 mg L^-1 BA, 1.0 mg L^-1 NAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more readily from juvenile explants (seedling source) as compared to the mature explants. Addition of adenine sulphate (5 mg L^-1) to the culture medium increased the growth of shoot buds. Optimum responses were obtained in hypocotyl and leaf explants using NAA in combination with BA, the highest rate of shoot bud regeneration being in hypocotyl explants. Rooting was readily achieved on the differentiated shoots on MS basal media without growth regulators. Regenerated plantlets were successfully established in the greenhouse.     

Seasonal and micro-environmental factors controlling clonal propagation of mature trees of marwar teak [<Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem]

A simple method has been developed for clonal propagation of mature trees of Tecomella undulata (Sm.) Seem, a medicinally important deciduous timber tree of hot arid regions, via multiple shoot proliferation from axillary buds after examining the role of season influences and physico–chemical conditions on micropropagation. Spring season (March–April) was the best period for contamination free establishment of explants and maximum sprouting of healthy axillary buds. Shoots proliferated directly from the explant nodes cultured on Murashige and Skoog’s medium containing cytokinins, BAP supporting better growth compared to kinetin during shoot induction as well as multiplication phase. Cytokinin concentration influenced the bud induction frequency and optimal response of 2.6 buds per explant was achieved in 86.66% explants on media supplemented with 10 µM BAP. Stunted shoot buds with excessive callus were observed when cytokinin concentration was increased beyond optimal levels. Ascorbic acid (50 mg/l), arginine and citric acid (25 mg/l each) were added to proliferation and multiplication media for reducing callus proliferation and better shoot growth. Among the media (B5, MS, NN, WPM and SH) tested, SH was best for shoot multiplication. Shoot cultures were multiplied by regular subculture of axillary shoots on SH medium containing 5.0 µM each of BAP and kinetin. Shoots produced roots when cultured on ½× SH medium + 10 μM IBA. Regenerated plantlets were successfully transferred to field after hardening and acclimatization. Genetic homogeneity of tissue culture raised plants was confirmed by generation of monomorphic DNA fragments with Start codon targeted and intersimple sequence repeat (ISSR) markers.     

Instructions for Authors

Efficient shoot regeneration of Vanda coerulea was achieved using thin shoot tip sections and thidiazuron. Protocorm-like bodies or proliferating shoot buds was observed when thin shoot tip sections were cultured on Vacin and Went's (VW) (1949) basal medium supplemented with 11.35 µM thidiazuron. The highest percentage of protocorm-like bodies (95%) survived and ultimately produced healthy shoots with 2 – 3 leaves when subjected to a 4 week thidiazuron treatment. A culture period longer than 8 weeks with thidiazuron resulted in the formation of fasciated or distorted shoots. Shoots produced roots when cultured on half strength VW basal medium supplemented with 11.42 µM IAA. The well rooted shoots were transferred to pots containing charcoal chips, coconut husk and broken tiles (2:2:1) and a 98% survival rate was achieved.     

Micropropagation of Eucalyptus tereticornis Smith.

Axillary shoot bud multiplication has been achieved in Eucalyptus tereticornis Smith. using explants from different regions of 8–10 years old elite trees, growing in the field. Results showed that addition of NAA at 0.1 mgl^-1 and BAP at 1.0 mgl^-1 to modified MS medium induced maximum number of shoot buds. For inducing axial growth in regenerated bud promordia, the hormone concentration of the medium was lowered. The addition of charcoal and gibberellic acid to the medium were beneficial. Rooting was best in Knop's medium containing 1.0 mgl^-1 IBA. The key factor in root induction was primarily a dark incubation for a short period. The percentage of both rooting of shoots and survival of the rooted shoots was 60–80.Continuous trials using explants from the elite trees throughout the year showed that the period between July–September was the best season for the explant source for rapid and increased multiplication of axillary buds. Phenolic exudation was also minimum at this period. The experiments were repeated using 50 populations from different plantations. It was observed that during culture, genotypically different populations responded differently in spite of optimal growth conditions.     

Promotion ofin vitro shoot formation from excised roots of silktree (Albizzia julibrissin) by an oxime ether derivative and other ethylene inhibitors

Summary This report describes the regeneration response of excised seedling roots of silktree (Albizzia julibrissin) to added ethylene precursors/generators (1-amino-cyclopropane-1-carboxylic acid [ACC], 2-chloroethylphosphonic acid [CEPA]), biosynthesis inhibitors (aminoethoxyvinylglycine [AVG], an oxime ether derivative [OED={[(ispropylidene)-amino]oxy}-acetic acid-2-(methoxy)-2-oxoethyl ester], CoCl² [Co⁺⁺]), and an ethylene action inhibitor (AgNO₃ [Ag⁺]). When placed on B5 medium, about 50% of the control explants formed shoot buds within 15 days. Addition of ACC or CEPA (1–10 µM) to the culture medium decreased both the percentage of cultures forming shoots and the number of shoots formed per culture. In contrast, AVG and OED (1–10 µM) increased shoot formation to almost 100% and increased the number of shoots formed per culture. Likewise, both Co⁺⁺ and Ag⁺ (1–10 µM) increased shoot regeneration, but the number of shoots produced after 30 days was less than with AVG or OED. The inhibitors of ethylene biosynthesis were partially effective in counteracting the inhibitory effect of ACC on shoot formation. These results suggest that modulation of ethylene biosynthesis and/or action can strongly influence the formation of adventitious shoots from excised roots of silktree.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CEPA 2-chloroethylphosphonic acid - OED oxime ether derivative     

Rapid propagation through shoot tip culture of Trichopus zeylanicus Gaertn., a rare ethnomedicinal plant

Rapid micropropagation of Trichopus zeylanicus Gaertn. subsp. travancoricus Burkil ex Narayanan, a rare ethnomedicinal herb endemic to the Western Ghats of southern India, was achieved by culturing shoot tips (0.3–0.5 cm) of 2-month-old axenic seedlings on Woody Plant Medium. Among the cytokinins tested, only BAP induced callus-free multiple shoot bud formation, with a maximum of 8.5±0.4 buds per explant being obtained with 2.0 mg.l^–1 BAP after 8 weeks of culture. Shoot tips containing proliferated buds were divided and subcultured on medium containing 0.2 mg.l^–1 BAP to produce 12.0±1.0 shoots per explant in 6 weeks. Excision of buds after culture initiation, with subculture of the debudded basal tissue in 2 successive passages yielded 20.0±1.0 and 13.5±0.5 buds per explant respectively. Each bud cultured in turn for 4 weeks on WPM with 1.0 mg.l^–1 BAP formed 3.8±0.4 secondary buds which were repeatedly recultured to increase bud production. Altogether this method enabled an estimated harvest of 7848 buds from a single shoot tip in 28 months. Shoots (3–5 cm) developed from bud cultures were rooted in half-strength WPM medium with 0.5 mg.l^–1 each of NAA and IBA, and 90–100% of the rooted plants were established in the field after hardening. Micropropagated plants were grown to maturity free of defects in growth, morphological, flowering and seed set characteristics.Abbreviations WPM Woody Plant Medium (Lloyd and `McCown 1980) - BAP 6-benzylaminopurine - 2-ip 2-iso-pentenyladenine - Kinetin 6-furfurylaminopurine - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid     

In vitro regeneration of adult Pinus sylvestris L. trees

The propagation of adult conifer trees by tissue culture has been studied for the last twenty years, but problems related to the juvenile to adult phase change of trees have limited the practical applications of these tissue culture procedures. This paper describes a micropropagation protocol for the in vitro propagation of mature Scots pine trees. In this study, dormant shoot buds, which had not started to elongate, were collected from twenty-one adult Pinus sylvestris trees (> 15 years old) during the winter. The sampled buds were cut transversely into slices of 0.5 to 1 cm in thickness and were cultured on three types of culture media (DCR, WP and LPm) supplemented with four cytokinins (BA, mT, Tdz and Z), at two different concentrations (25 and 50 µM), except for Tdz, whose concentrations were diluted to 5 µM and 2.5 µM. The evaluated culture media did not show significant differences in the bud organogenesis capacity. In fact, the highest organogenic response was obtained with buds cultured on DCR and WP media and by explants cultured on medium supplemented with 25 µM meta-topolin. This protocol is a successful and efficient biotechnological approach to the micropropagation of adult P. sylvestris trees.     

Morphogenesis in Sitka spruce (<Emphasis Type="Italic">Picea sitchensis</Emphasis> (Bong.) Carr.) bud cultures – tree maturation and explants from epicormic shoots

The adventitious bud forming ability of Sitka spruce (Picea sitchensis (Bong.) Carr.) buds in vitro was found to be dependent upon the age of tree from which the explants were taken. Bud formation declined exponentially with increasing tree age when 1.0 and 10 μM 6-benzylaminopurine (BA) were used to induce adventitious buds. When less BA was used (0.1 μM) bud production was much lower with all ages of tree and no mathematical relationship between declining bud production and tree age was found. By a tree age of 6 years bud-forming ability had declined severely. Even the few buds that developed on older tree tissues failed to elongate into shoots, became necrotic and eventually died, indicating that adventitious bud induction in this species is not a rejuvenative process. Callusing of bud explants also declined with increasing tree age when 0.1 μM BA was used whilst very little callusing occurred at the higher cytokinin concentrations (1.0 and 10 μM BA). Tissue necrosis in vitro increased with tree age, whilst the ability of BA to retard necrosis declined with increasing tree age. Buds from epicormic shoots, formed on the lower trunks of 20-year-old trees when these were exposed to light, were not significantly better at forming adventitious buds in vitro than buds taken from the lower branches of the crown.     

Direct Differentiation of Shoot Buds from Leaf Explants of Cajanus Cajan L.

A protocol was developed for direct differentiation of multiple shoot buds from leaf explants of Cajanus cajan. In a modified Murashige and Skoog's medium supplemented with 2.22 µM benzyladenine (BA), 0.57 µM indole-3-acetic acid (IAA) and 41 µM adenine sulphate (AdS), the segments of basal halves of the first two leaves of a young seedling incubated on filter paper bridges in liquid medium took 20 – 25 d to differentiate shoot buds. The explants after transfer to solidified medium, with lower concentration of BA (0.22 M) resulted in fast growing healthy shoots. The developed shoots (measuring ca. 3 cm) were rooted in a medium supplemented with 1.42 µM IAA. They were subsequently grown in pots with soil with more than 80 % transplantation success.     

In Vitro Plantlet Formation in Mangosteen (Garcinia mangostana L.)

Optimum conditions were determined for in vivo growth and multiplicationof Garcinia mangostana L. using explants from aseptically germinatedseedlings and field-grown plants. Proliferating shoots wereobtained from cotyledon segments cultured on modified Murashigeand Skoog's (1962) medium with 6-benzylaminopurine. Juvenileleaf segments produced adventitious buds on Woody Plant Medium(Lloyd and McCown, 1981). Root segments gave few buds. Shoottip, nodal, and internodal explants gave multiple axillary andadventitious buds. Shoots were multiplied by enhanced axillaryand adventitious bud formation. The shoots were rooted withindolebutyric acid treatment. Rooted shoots were readily establishedin vermiculite: sand (1:1) mixture. Garcinia mangostana L., Mangosteen, tissue culture, shoot regeneration, bud development     

Rapid Micropropagation of Five Cultivars of Mulberry

Multiple shoots were initiated from nodal and shoot tip explants collected from mature trees of Morus alba L. cultivars Chinese White, Kokuso-27 and Ichinose, and M. multicaulis Perr. cultivars Goshoerami and Rokokuyaso after 2 weeks of culture. Nodal explants were more responsive than shoot tip explants. Murashige and Skoog basal medium was found to be most suitable medium and 6-benzylaminopurine was the most effective cytokinin for shoot induction. Explants collected between April and September evoked better response than the explants collected between October and March. Shoots were multiplied by transferring nodal explants excised from in vitro raised shoots onto a medium containing cytokinins. Sucrose was the most suitable carbon source examined for shoot multiplication. An increase in shoot multiplication rate was noticed upto 4 – 5 subcultures. Nodal explants rooted on an auxin-supplemented medium. The acclimatized plants were successfully transplanted in the field. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of mature <Emphasis Type="Italic">Pongamia pinnata</Emphasis> Pierre

Murashige and Skoog’s (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), and thidiazuron (TDZ) were tested for induction of multiple shoots from mature-tree-derived axillary meristems of Pongamia pinnata. Sprouting of buds was 64% on medium devoid of plant growth regulators (PGR). Incorporation of BA, KN, or Z was ineffective in enhancing sprouting frequency or induction of multiple shoots. Sprouting was completely suppressed in the presence of TDZ. Caulogenic buds appeared in nodal meristems of these explants after withdrawal of TDZ. The number of shoot buds was more on explants precultured in higher concentrations. At higher concentrations of this PGR, a swelling developed at the axil. Multiple shoot primordia appeared and differentiated from this swelling after culturing these explants on MS medium for six passages of 2 wk each. Shoots were harvested and cultured on 0.45 μM TDZ for further proliferation. Primary explants after harvesting of shoots were identified as ‘stump’. Reculturing of stumps on 0.45 μM TDZ produced more shoots. This step was followed for six cycles to obtain additional shoots in each cycle. Shoots maintained on 0.45 μM TDZ elongated and rooted (70%) on growth regulator-free medium. Rooted shoots (65%) survived transfer to a sand/soil mixture. This report describes the protocol for micropropagation of P. pinnata using mature-tree-derived nodal meristems. Recycling of mature stock to produce a stream of useable shoots for subculturing and eventual stabilization is of great value and can possibly be generalized as an isolation protocol especially for woody species. Repeated proliferation of caulogenic buds from the same origin may also find application in rescue of endangered germplasm.     

Stimulation of in vitro shoot proliferation from nodal explants of cassava by thidiazuron, benzyladenine and gibberellic acid

Multiple shoots were produced from nodal explants of cassava (Manihot esculenta Crantz) by a two-step procedure: a 6- to 8-day exposure to 0.11–0.22 µM thidiazuron (TDZ) in liquid Murashige and Skoog (MS) medium followed by culture on agar-solidified MS medium supplemented with 2.2 µM 6-benzyladenine (BA) and 1.6 M gibberellic acid (GA₃). TDZ caused the nodal explants to expand and this expansion (growth) continued during culture with BA and GA₃. From this expanded explant, clusters of buds and fasciated stems developed continuously and these gave rise to shoots. The shoot proliferation process was open-ended, yielding an average of 31.5 shoots per nodal explant after 10 weeks of culture with genotype CG 1–56. A positive response was also obtained from seven other genotypes evaluated with this protocol.Abbreviations BA 6-benzyladenine - BM basal medium - DPU 1,3-diphenylurea - GA₃ gibberellie acid - 2iP isopentenyladenine - MSM multiple shoot medium - NAA 1-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - Z zeatin     

Adventitious shoot regeneration fromFagus sylvatica leaf explantsin vitro

Summary Adventitious shoots were induced on transversally divided expanding leaves fromFagus sylvatica shoot cultures of juvenile origin. Adventitious shoot buds formed mainly on callus that developed on the petiole stump or on the cut across the midrib of distal leaf halves. However, sometimes they arose directly from leaf tissue. An anatomical study confirmed both the direct and indirect origin of the adventitious buds. The best results were obtained by culturing proximal leaf sections on woody plant medium supplemented with 2.9 μM indole-3 acetic acid in combination with 8.9 μM benzyladenine or 2.3 μM thidiazuron (TDZ). Proximal explants were more responsive than distal explants in terms of both callus formation and bud regeneration, regardless of the induction medium or clone tested. Bud formation capacity was influenced by the genotype of the stock shoot culture and was enhanced by an initial 10 d darkness, but was inhibited by longer periods of darkness. Caulogenic competence was significantly affected by the duration of exposure to TDZ; in particular, adventitious shoot length was depressed by increasing the exposure period. Three weeks culture with TDZ was the most efficient treatment for shoot production and elongation. Further shoot development was promoted by subculturing the explants to the same medium used for the maintenance of the stock shoot cultures. Shoots so obtained were multiplied and rooted producing plantlets of adventitious origin.     

Direct adventitious shoot bud formation on hypocotyls explants in Millettia pinnata (L.) Panigrahi- a biodiesel producing medicinal tree species

A reproducible protocol developed for in vitro regeneration of Milletia pinnata using hypocotyl segments. Multiple shoots were induced from hypocotyl explants through direct adventitious shoot bud regeneration. The proximal end of hypocotyls was responsive for shoot bud induction. Silver nitrate and adenine sulphate had a positive effect on shoot bud induction and elongation. The maximum response and number of shoot bud produced in media supplemented with 8.88 μM BAP with 108.6 μM adenine sulphate and 11.84 μM silver nitrate. Elongated shoots were harvested and successful rooting of microshoots achieved on MS media supplemented with 9.84 μM IBA, with 81.1 % rooting. Remaining shoot buds sub-cultured for further multiplication and elongation. Each subculture produced eight to nine elongated microshoots up to four subcultures. The rooted microshoots were successfully hardened and transferred to field.     

Micropropagation of Alnus cordata (Loisel.) Loisel.

Procedures were developed for micropropagation of Alnus cordata through in vitro axillary shoot multiplication of axillary bud explants cultured in Murashige & Skoog (MS) medium. Establishment of cultures from plants grown in the field was very difficult due to bacterial contamination and phenolic oxidation in explants causing severe browning. Explants were first cultured on an MS medium containing 4.4 M 6-benzyladenine and 87.6 mM sucrose (initiation medium) for 7 days and then transferred to an MS medium containing 1.1 M 6-benzyladenine and 333 mM glucose (multiplication medium) for a further 20–25 days. It was necessary to transfer cultures from initiation medium to multiplication medium after 7 days to minimize excessive callus growth, abnormally thick and brittle leaves, inhibition of shoot elongation, and senescence. Shoot multiplication comparable to the above method was achieved by culture of axillary bud explants in MS medium supplemented with 1.1–4.4 M 6-benzyladenine and 333 mM glucose 4–5 weeks after culture. Shoots rooted in MS medium (1/2 x macro-nutrients) supplemented with 1.2–4.9 M indolebutyric acid. Also, 98% rooting was achieved when cultures were treated with 625 mgl^-1 indolebutyric acid for 24 h at the end of the shoot production stage and rooted in vivo as mini-cuttings. Plantlets established well in soil.     

Somatic embryogenesis inAesculus

Summary Somatic embryogenesis was observed with explants taken from four types ofAesculus tissue: (a) shoots of 4-wk-oldin vitro germinated excised embryos (seed fromA.×arnoldiana), (b) roots of 4-wk-oldin vitro germinated excised embryos (seed fromA.×arnoldiana), (c) shoots from newly forced 3-yr-old seedlings (A. glabra), and (d) newly forced shoots from a 30-yr-old tree (A.×arnoldiana “Autumn Splendor”). Shoots provided three types of explants, single node, shoot apex, and internodal section, and all exhibited embryogenesis. Proembryogenic masses developed in a few cases after 6 wk in culture but were more commonly seen after 3 mo. The yellow, friable proembryogenic masses emerged from proximal cut ends of explants. Almost all cultures that formed embryos possessed leaves, either from developing apical or axillary buds or from adventitious buds, prior to the emergence of proembryogenic masses. Only tissues that had begun to senesce and had been exposed to cytokinin (benzyladenine at 5 or 25 μM) formed somatic embryos. Embryos with distinct cotyledonlike structures and root/shoot axes developed during the 10 to 16 wk following the inital emergence of proembryogenic masses. Enhanced frequency of embryogenesis was obtained by dark culture of root and shoot explants from 4-wk-old germinated embryos (A.×arnoldiana) and by dark and cold (5°C) treatment of shoot tissue cultures derived from 3-yr-old seedlings (A. glabra). Embryogenic potential was greatest in the most juvenile tissue and least in the mature tissue. Five percent of shoot explants taken from the 30-yr-old select treeA.×arnoldiana “Autumn Splendor” produced somatic embryos.     

Shoot multiplication in cultures of mature <Emphasis Type="Italic">Alnus glutinosa</Emphasis>

Shoot cultures were initiated from mature trees of Alnus glutinosa. On medium containing 1–5 μM 6-benzylamino purine (BAP), the shoots elongated without branching, formed heavy callus at the base of the stems and readily formed roots. The possibility that these characteristics could be attributed to the strong influence of endogenous auxin was tested on media that contained two auxin transport inhibitors, 1-N- naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), at concentrations of 0.1–3 μM, in combination with 2 μM BAP. On these media, shoots produced numerous branches, less callus and no roots. After 30 weeks (five subcultures) on this medium, leaves were smaller and showed signs of vitrification. These problems were resolved without detriment to shoot proliferation, by reverting to medium without NPA or TIBA. Shoots rooted readily after transfer to medium without growth regulators and were successfully acclimatised after transfer to soil.