In vitro adventitious shoot regeneration from leaves of Prunus spp.

A high level of adventitious shoot regeneration was obtained from proliferating shoots in vitro for a range of Prunus spp. There was a significant variability in clone response to a range of adventitious shoot regeneration treatments. Treatment of apricot clone H.152 with Quoirin macroelements (C.R. Rech., Stu. Cult. Fruit. Maraîchères Gemblaux (1977) 93–117), and both apricot clone H.146 and hybrid plum clone P.1869 with half-strength Murashige and Skoog medium, consistently induced regeneration. Thidiazuron (TDZ) alone, or in combination with naptthaleneacetic acid (NAA), was most effective in stimulating adventitious shoot production, the optimum concentration being clone-dependent. Addition of silver nitrate (AgNO₃) to regeneration media enhanced regeneration by 10–40% and reduced the variability between experiments. Regeneration with AgNO₃ was obtained also for three other plum clones belonging to the P. marianna, P. domestica and P. insititia species.     

In vitro adventitious shoot formation from embryonic and cotyledonary tissues of Pinus brutia Ten.

Adventitious buds were induced when isolated whole embryos, and excised cotyledons from treated seeds of Calabrian pine (Pinus brutia Ten.) were cultured on a cytokinin-supplemented medium. The adventitious buds formed directly from the cotyledons. The highest number of bud primordia were formed, with both embryos and excised cotyledons, after 6 weeks on a BAP — supplemented Schenk and Hildebrandt medium under a 16 h photoperiod. The buds, when separated and maintained individually on a full-strength medium without growth regulators, developed into well-formed shoots within 4 weeks. The average number of harvested shoots obtained (>1 cm in height) per seed over 24 weeks was 55; however, a maximum number of 152 shoots was obtained from one individual over the same period. The shoot forming capacity of the meristematic tissue was not lost after seven harvests.     

In vitro adventitious shoot formation on mature-phase leaves and petioles of Liquidambar styraciflua L.

Adventitious shoots were induced to form on leaves and petiole segments of mature-phase Liquidambar styraciflua L. Shoot organogenesis occurred directly, without the formation of a distinct callus stage, and well-defined shoots were visible in 6–9 weeks. Prolific shoot production was supported by Woody Plant Medium supplemented with relatively high levels of benzyladenine (2.5 mg/l). Changes in benzyladenine concentration and the addition of 0.1 mg/l naphthaleneacetic acid to the medium altered the relative abundance of shoots on various parts of a leaf. Shoot formation occurred most frequently at or near breaks in major vasculature. Wounding of the leaves by slashing across the lamina and vasculature significantly increased the total number of shoots formed per explant and also altered the pattern of organogenesis. Differences in organogenic response were seen between the cultivars ‘Moraine’ and ‘Variegata’. Shoots derived from leaves were easily rooted and acclimated to greenhouse conditions. Three new variegation types arose in vitro as a result of adventitious shoot formation on ‘Variegata’ leaves.     

In vitro adventitious shoot formation from leaf cultures of Clerodendrum inerme (L) Gaertn.

In vitro adventitious shoots (about 28) of Clerodendrum inerme were regenerated from leaf segments on MS medium containing BA (4 mg/L). These shoots developed directly from the leaf explants without callusing after 5 weeks. Leaf explant when cultured in MS medium containing BA (2 mg/L) and NAA (0.5 mg/L) developed compact callus that became nodular and regenerated shoots (about 50) after 5 weeks. The in vitro developed shoots were rooted in MS medium supplemented with IAA (2 mg/L). The hardened plantlets were successfully established in the field with 90% survival.     

Effect of CPPU on in vitro axillary shoot proliferation and adventitious shoot regeneration in kiwifruit

Abstract

The effect of CPPU, N‐(2‐chloro‐4‐pyridyl)‐N‐phenylurea, on the development of axillary buds and on adventitious shoot regeneration was investigated in mature leaves of in vitro‐cultured shoots of Actinidia chinensis Planch (Sel. K190) and Actinidia deliciosa A. Chev. cultivars Hayward and Tomuri. In the multiplication phase, 2.2 mM CPPU induced proliferation rates comparable to 4.4 mM benzyadenine (BA) both in Hayward and in Tomuri, while a higher CPPU concentration reduced proliferation. In A. chinensis, significant differences in multiplication rates were not detected between BA and CPPU. However, shoots developed on CPPU appeared hyperhydric, and had very short internodes, reduced leaf laminas, higher water, carotenoid and phenol contents and considerably lower chlorophyll level in comparison with the BA‐grown shoots. On the other hand, in adventitious shoot regeneration CPPU was more effective than zeatin (ZEA) and BA in A. deliciousa cultivars and the best morphogenic response was obtained with the lowest concentration (10 mM) in cv Hayward, while 16 mM was the most efficient treatment in cv Tomuri. In A. chinensis, CPPU was as efficient as ZEA when the highest concentration was used.     

In vitro shoot proliferation and production of sets from garlic and shallot

A procedure is described to regenerate shoots and bulbs in vitro with high frequency from shoot tips of garlic and shallot plants using benzyladenine or thidiazuron. Regenerated shoots were induced to form bulbs in Murashige and Skoog medium (1962) containing 5 g l^-1 activated charcoal and 120 g l^-1 sucrose under a long-day photoperiod. Bulbs formed in vitro were transferred to soil without acclimatization and produced viable plants. This method could be useful to produce low-cost bulbs, which are easy to handle and store until needed.Abbreviations AC activated charcoal - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog's (1962) medium - NAA naphthaleneacetic acid - TDZ thidiazuron     

Influence of light on adventitious root formation in birch shoot culturesin vitro

The influence of light of different spectral composition and levels of irradiance (2-40 Wm-²) on adventitious root formation (ARF) in birch shoot segments was investigated. Spontaneous rooting of shoot segments occurred in segments with intact apical or axillary meristems. Concerning ARF shoot meristems could be substituted by application of auxin. The very low rooting percentage of shoot segments in darkness was improved considerably by auxin application. Irradiation of cuttings was a requirement for a high percentage of spontaneous rooting. The promoting effect of light was dependent on its spectral composition and was the highest under red followed by white and blue light. The low rooting response under blue light was enhanced almost to the red light level by shielding the root-forming cutting base from light.     

High frequency adventitious shoot regeneration in sainfoin

A procedure is described for the rapid and efficient adventitious shoot regeneration from leaflets, petioles and stems of field-grown sainfoin plants. All explants formed shoots on a range of media supplemented with 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). Stem explants appeared to have better regeneration capacity than leaflet and petiole explants in most media tested. The highest frequency of shoot regeneration was achieved from stem segments on a medium containing 20 μM BA and 0.5 μM NAA. Regenerated shoots rooted in half-strength Murashige and Skoog medium containing 5 μM indole-3-butyric acid and later established well under greenhouse conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine

Adventitious buds were induced on isolated immature cotyledons of Pinus pinea L. in the presence of benzyladenine (BA). The response to different BA concentrations also depended upon the culture medium used (modified MS, SH and GD). A wide range of BA concentrations (5, 25 or 50 M) can be applied to the GD and SH media, which are the media with the lower nitrogen content, without damaging effects. In the MS medium, which has the highest nitrogen concentration, the range of BA that can be applied was narrower and the highest BA concentration was lethal. The addition of indolebutyric acid (0.05, 0.25 or 0.5 M) to the induction medium, decreased the response of cotyledons. The increase in the concentration of sucrose from 3% to 5% did not increase the number of responding cotyledons. The addition of activated charcoal (0.5 and 3 g l^-1) or indolebutyric acid (1.5 or 3 M) did not speed up the elongation of explants. Elongation of the buds produced shoots with two different phenotypes, each phenotype having a different multiplication rate.Abbreviations BA benzyladenine - GD Gresshoff & Doy medium - IBA indolebutyric acid - MS Murashige & Skoog medium - SH Schenk & Hildebrandt medium     

In vitro production of adventitious roots containing asiaticoside from leaf tissues of Centella asiatica L.

Centella asiatica (Apiaceae) is an important medicinal herb used in a variety of herbal medicines worldwide. Although the whole plant contains important triterpenoids, a significant quantity of pharmacologically important phytochemicals collectively known as centellosides can be extracted from leaf tissues and not from other parts of this plant. Asiaticoside is one of the major centellosides and is used in holistic medicine for treating a variety of human ailments. Genotypes of C. asiatica of Indian origin accumulate significant quantities of asiaticoside in their roots, while genotypes from other continents contain insignificant quantities of this chemical. The main purpose of this study was to manipulate the leaf-derived callus of C. asiatica using a combination of plant growth regulators to generate a large quantity of adventitious roots. The presence of asiaticoside in callus and regenerated roots of C. asiatica was detected by thin layer chromatography as well as by high-performance liquid chromatography, and the accumulation of a significant quantity of asiaticoside was demonstrated by spectrophotometric analysis. The protocol developed for the regeneration of roots was simple, reproducible, and reliable for the possible commercial production of root biomass enriched for asiaticoside.     

High frequency adventitious shoot regeneration from excised leaves ofPaulownia spp. cultured in vitro

High frequency, direct regeneration of shoots was induced in leaf cultures ofPaulownia tomentosa, P. fortunei x P. tomentosa andP. kawakamii. The optimum culture medium for the leaf explants derived from shoot cultures was Murashige-Skoog (MS) medium supplemented with 10 M indole-3-acetic acid and 50 M benzyladenine. Up to 40 shoots were obtained over a 4 month culture period from each leaf explant. Rooting occurred spontaneously in the shoots that were about 1 cm tall when subcultured on phytohormone-free MS medium. The plantlets could be transplanted successfully. Some of the transplantedP. tomentosa plantlets flowered in the greenhouse one year after transplanting. The protocol is suitable not only for rapid multiplication of the various species ofPaulownia, but also for analytical studies associated with adventitious shoot regeneration.     

Effect of genotype on in vitro adventitious shoot formation in Pinus radiata and correlations between pairs of phenotypic traits during in vitro shoot development

The influence of genotype at the family and clone levels on adventitious shoot formation in Pinus radiata was examined using the cotyledon tissue culture system. Twenty-nine full-sib and two selfed families were used. Family and clone influenced all in vitro traits assessed and final adventitious shoot formation significantly. Five families selected for the long-internode trait and two selfed families used in the study were of average performance when compared to all families. Correlations using family means or clone-within-family means showed that shoot production could be predicted based on measures of early culture health. Shoot production after 16 weeks was positively related to early embryo (6 days) and early culture (4 weeks) health. Shoot production could be improved by approximately 100% through family selection, but only limited additional improvements were obtained through early embryo selection.     

In vitro adventitious budding on Pinus pinaster cotyledons and needles

Adventitious budding can be induced on two types of Pinus pinaster Ait. organs. Cotyledons (10-mm-long), derived from 8 to 10-day-old seedlings, show morpho-genetic response when an appropriate mineral solution is used (NH₄⁺/K⁺= 1). Of the various cytokinin concentrations added to this optimal mineral medium, 0.8 μM BAP (6-benzylaminopurine), with 5 n M NAA (1-naphtaleneacetic acid), promoted organogenesis best. Buds were induced from outer mesophyll layers.
Short shoots and the elongating needles (70-mm-long) were collected from cuttings of a mature tree (10-years-old). These cuttings benefited from physiological advantages of a well-developed root system (by heating the substrate). In order to stimulate in vitro organogenesis, they had been sprayed every week from March to May with 10 μ M BAP.
When cultivated in the presence of 10 μ M BAP and 25 n M NAA, 79% of the explants produced buds from dome-shaped meristematic cell clusters that pre-existed at the top of the short shoots. Moreover, among these, 42% gave rise to adventitious buds induced from proliferating mesophyll cells at the needle base. The morphologies of the two kinds of shoots were similar. Adventitious budding on these two different explants should allow vegetative multiplication of selected seedlings and elite trees.     

In vitro shoot propagation of Dendrocalamus strictus nees

Multiple shoots were induced from seedling and axillary buds of mature plants of Dendrocalamus strictus on Murashige and Shoog's medium supplemented with BA and kinetin. About 35–45 shoots were obtained within 20–25 days from a nodal explant of seedling and 3–8 shoots were obtained from a nodal explant of mature plants in the primary culture. The seedling derived cultures were separated into groups of 5–7 and transferred to fresh subculture medium. Rooting of the shoots was achieved under in vitro and ex vitro conditions. 85–90% of rooting was achieved by the ex vitro method using IBA. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro shoot regeneration from olive cultivar tissues

Petioles, leaf discs and midribs of several olive (Olea europaea L.) cultivars, collected from potted greenhouse plants, field-grown and in vitro shoots, were used to test their morphogenic capacity. Adventitious shoots were induced only in petioles from in vitro-grown shoots of cultivars Moraiolo, Dolce Agogia and Halkidikis, grown on Olive Medium (OM) plus 18 M zeatin within 4 to 5 weeks. Regeneration was achieved, both on Murashige and Skoog (MS) and on modified OM, only in the dark. The highest regeneration was achieved directly from the proximal part of the petioles after 2 to 3 weeks in media containing 5 to 40 M thidiazuron, or with both 10 M 2-isopentenyladenine +2.2 M 6-benzyladenine with or without low auxin concentration (not more than 2.5 M). A few adventitious shoots were also regenerated from callus when it was shifted from auxin and cytokinin media to cytokinin only medium. The regeneration potential was higher in petioles collected from apical nodes than from basal ones. The adventitious shoots were transferred to solid half-strength MS medium supplemented with 4.5 M zeatin for further development. Several regenerated shoots were rooted and the plantlets hardened in the greenhouse. No apparent differences regarding morphological aspects were observed among the regenerated plantlets or with those obtained by stimulation of axillary buds.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea) - 2iP 2-isopentenyladenine - MS Murashige and Skoog medium - 1/2 MS half strength MS - OM Olive Medium - BN Bourgin & Nitsch     

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.     

In vitro adventitious root induction in Antirrhinum majus L.

A broadly applicable method for the successful induction of root systems in a number of cultivars of A. majus has been determined. This involves a double filter-paper bridge with a liquid medium for root induction and allows the transfer of culture-grown plantlets to a glasshouse environment with minimal disturbance to the plant as a whole. 100% survival of transferred plantlets has been achieved with the inclusion of a few simple precautions upon shoot transfer and during the initial stages of plant establishment in vivo.     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

In vitro cold storage of cork oak shoot cultures

Friable callus cultures were initiated from cotyledons and hypocotyls of Opuntia ficus-indica. Explants from cotyledons produced significantly more callus than those from hypocotyls. Optimum callus growth was observed on Murashige & Skoog medium supplemented with 0.9 μM 6-furfurylaminopurine, 2.3 μM 2,4-dichlorophenoxyacetic acid, 1.0 μM 4-amino 3,5,6-trichloropicolinic acid, 400 mg l^-1 casein hydrolysate and 3% sucrose. The same medium without agar was used for establishing cell suspensions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of carbon source on shoot multiplication and adventitious bud regeneration in in vitro beech cultures

Experiments were performed to determine the effects ofcarbon source and concentration on shootmultiplication in shoot cultures of Fagussylvatica (one clone) and F. orientalis (twoclones) and on the induction of adventitious shootbuds from leaf and internode explants of F.orientalis. In general, glucose was the best carbonsource for both axillary branching and adventitiousshoot regeneration. Shoot-tip explants grown on 3–4%glucose medium produced more shoots than those onsucrose or fructose. For maximum shoot length, glucosemedium was best for two of the three clones, and 4%sucrose for the other. The number of shoots was theparameter most influenced by glucose concentration inthe adventitious shoot regeneration experiments, thenumber increasing with sugar concentration. The lowesthyperhydricity rate occurred in the presence ofsucrose in both species. Shoot growth and quality wasnegatively affected by fructose supplied media. Theuse of filter-sterilized rather than autoclavedfructose neither stimulated shoot growth nor reducedthe incidence of hyperhydricity in all three clones.The response of shoot cultures to differentcarbohydrate treatments appears to some extent to begenotype dependent.