The influence of micrografting in vitro on tissue culture behavior and vegetative propagation of old European larch trees

The influence of a new micrografting method in vitro was tested by using long shoot meristems of adult 140 year old European larch trees grafted onto larch seedling rootstocks in sterile peat pellets. Five months after grafting and transfer of grafts to ex vitro conditions, new shoots from sprouting scions were re-established into tissue culture. The propagation behavior of shoots derived from these explants was compared with shoots established via bud culture from the same donor trees. Shoot explants from micrografts multiplied more rapidly than shoots explants from the original adult donor trees. Rooting experiments with cuttings from six micrografted clones resulted in 49.9 ± 11.9% rooting. Cuttings from donor trees invariably showed no root formation. The results confirm a rejuvenating influence of micrografting in larch.     

Establishment of juvenile-like shoot cultures and plantlets from 4–16 year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees

A method was developed for the establishment of shoot cultures from Douglas-fir trees selected for outstanding growth and form in a 12-year-old genetic test. Vegetative buds from the lower crown were sterilized and grafted in vitro onto juvenile clonal rootstock. The rootstocks were produced from adventitious buds induced on cotyledons, and were maintained through micropropagation. Buds that established grafts slowly elongated into shoots, which were harvested and multiplied through micropropagation. Grafts often grew several new shoots which in turn could be harvested. In 1987, 2830 buds were grafted from 18 superior trees. Twenty nine grafts (1%) produced shoots which established 11 of the 18 trees in culture. Their appearance and behavior in vitro became more juvenile over 1–3 years, as indicated by shoot and needle morphology, disappearance of episodic growth pattern, increase in multiplication rates, and ability of needles to produce adventitious buds.The five most prolific of the 11 clones were given a pre-rooting treatment and planted in soil under fog. The success of rooting and subsequent establishment in soil varied from 5 to 17% depending on clone. In contrast, trees multiplied in vitro for 1–2 years longer showed soil establishment rates from 8–60%. This technique allows establishment, multiplication, and maintenance in vitro of cultures from high value Douglas-fir genotypes. Such cultures may serve as a starting point for further research on rejuvenation and cloning.     

Micropropagation of Taxus mairei from mature trees

Micropropagation of mature Taxus mariei was achieved using bud explants derived from approximately 1,000-year-old field-grown trees. Bud explants derived from 1-year-old stecklings raised from rooted cuttings of these trees were included in this study for comparison. Steckling-derived explants collected in different seasons showed high survival rates and good growth performance in vitro, whereas the survival of mature tree-derived explants was highly season dependent, with satisfactory survival in early spring only. During successive subculturing, tissue browning was observed in cultures of both origins, but the problem was circumvented by supplementing the basal medium with 1 g l^-1 activated charcoal and 100 mg l^-1 silver nitrate. The steckling-derived cultures performed better than mature tree-derived cultures in terms of shoot multiplication and rooting ability. New shoots of both origins exhibited plagiotropic growth, but steckling-derived shoots had a smaller plagiotropic angle (near vertical) than mature tree-derived shoots. In both cases, the plagiotropic angles decreased after these subcultures were maintained in shoot multiplication conditions for half a year. Orthotropic growth was restored in 25% and 10% of the steckling- and mature tree-derived cultures, respectively.     

Regeneration and genetic transformation of <Emphasis Type="Italic">Hagenia abyssinica</Emphasis> (Bruce) J.F. Gmel. (Rosaceae) with <Emphasis Type="Italic">rolB</Emphasis> gene

The effects of growth regulators, wounding and antibiotics on regeneration of Hagenia abyssinica were investigated and the rolB gene was introduced into this species by Agrobacterium-mediated transformation. Regeneration was affected by type of growth regulators, wounding and antibiotics. Up to 100% regeneration could be obtained. Three transformed clones (T1, T2.1, T2.2), confirmed by PCR and Southern blot, were obtained only by excluding kanamycin from the selection medium 6 weeks after culture, followed by selection during shoot multiplication. RT-PCR revealed strong expression of rolB gene in shoots and roots of all the transgenic clones, but from leaf samples, it was detected only in T1. Rooting frequency was 77% (T1), 50% (T2.1), 57% (T2.2) and 0% for control shoots on growth regulator-free rooting medium.     

A protocol toward multiplication of the medicinal tree,Eucommia ulmoides oliver

Summary A protocol based on shoot cultures of 1-mo.-old seedlings was developed for rapid asexual multiplication of eucommia, the source of an antihypertensive medicinal. The explant is an excised shoot tip, 3–5 mm tall. MS basal medium supplemented with 1 mg/liter BA is employed to establish primary cultures and subsequently multiply shoots. Shoots are subculturable on the same medium and can be increased at a rate of 7.5 new shoots per 2-shoot sector every 3 wk. Rooting is achieved in a Gelrite medium with the MS salts reduced to 1/3 strength and the BA replaced by 0.1 mg/liter NAA. The method is not directly applicable to mature trees. Applicability will require explants from rejuvenated sources, possibly attainable by the method of repeated grafting of shoot apices onto juvenile rootstocks, repeated subculturing of shoots, or culturing shoot apical meristems.     

Effect of controlled carbon dioxide on in vitro shoot multiplication in Feronia limonia (L.) Swingle

The effect of controlled carbon dioxide environment on in vitro shoot growth and multiplication in Feronia limonia (a tropical fruit plant, Family- Rutaceae) was studied. Carbon dioxide available in the ambient air of the growth room was insufficient for in vitro growth of the shoots alone. Also, the presence of sucrose only as the C-source in the medium (without CO₂), was found to be inadequate for sustainable growth and multiplication of shoots. The carbon dioxide enrichment promoted shoot multiplication and overall growth. The promotory effect of CO₂ was independent of the presence of sucrose in the medium. In the presence of both CO₂ and sucrose, an additive effect was observed producing maximum shoot growth. In the absence of sucrose a higher concentration of CO₂ (10.0)g m⁻³ was required to achieve photoautotrophic shoot multiplication comparable to ambient air controls. Highest leaf area per shoot cluster promoting shoot growth and multiplication was recorded under this treatment. Shoots growing on sucrose containing medium under controlled CO₂ environment of 0.6 g m⁻³ concentration evoked better response than ambient air controls (shoots growing on sucrose containing medium) in growth room. This treatment produced the overall best response. The present study highlighted the possibility of photoautotrophic multiplication which might prove useful for successful hardening and acclimatization in tissue culture plants.     

Density-dependent growth responses in two clonal herbs: regulation of shoot density

Summary It has been shown in clonal perennial herbs that shoot natality decreases, and shoot mortality increases, in stands of increasing density. In a two-year garden experiment, we have tested Hutchings' (1979) hypothesis that these responses are the result of physiological integration, i.e. the exchange of resources and growth substances between shoots of a single clone. Dense monocultures of two rhizomatous graminoids, Brachypodium pinnatum and Carex flacca, were created that differed more than 10-fold in the density of clones (genets), but that had similar densities of shoots. A more effective shoot density control was expected in stands with the smaller clone densities (larger clones) due to more extensive clonal connections. Shoot turnover was evaluated by counting living and dead shoots at different times. In the summer of the second year, when shoot densities and stand structure were similar between treatments, shoot natality (the number of shoots born per plot) and shoot mortality (the number of shoots that died per plot) were usually unrelated to clone density in either species. If there was a significant treatment effect, it could be attributed to (small) differences in shoot density. Over the whole range of shoot densities, natality was negatively density-dependent. The number of shoots that died in a given growth period was proportional to the number of shoots present, suggesting that mortality rates were density independent. In Carex, however, there were some indications that mortality rate increased with increasing density. Our study confirms that clonal herbaceous species can effectively prevent an overproduction of shoots, but in contrast to Hutchings' (1979) propositions, we found no evidence that physiological integration may be the responsible mechanism. An alternative explanation for the observed patterns is proposed.     

Effect of various growth regulators on growth in vitro of cherry shoot tips

An investigation was undertaken to examine the effects of nine different growth regulators on growth in vitro of shoot cultures of the semi-dwarfing cherry rootstock Colt (Prunus avium × P. pseudocerasus). The effects of each supplement on shoot extension and proliferation and also leaf and callus production were noted, and it was found that BAP has the ability to proliferate shoots, IBA nullifies this effect and that kinetin, ABA, GA₃ and ethylene inhibit the growth of colt cultures. Conditions were established which resulted in a) optimum shoot growth prior to subsequent rooting or grafting; b) maximum shoot proliferation for rapid clonal multiplication and c) minimum shoot growth. This study will form the basis of an investigation into germplasm conservation of temperate fruit trees by both cryogenic storage and minimal growth techniques.     

Effects of N6-benzyladenine on shoots of five willow clones (Salix spp.) cultured in vitro

Growth and differentiation in shoot cultures of five willow clones on media of different BA concentrations were compared. The tendency of axillary shoots to develop on shoot cultures depended on the genotype, the type of shoot and the number of previous subcultures. The optimum concentration for shoot multiplication was either 5×10^-7 M or 10^-6 M. On BA concentrations of 10^-5 M or higher, browning and death of shoots occurred. Depending on the genotype, shoot elongation was best on media containing 0–5×10^-7 M BA. Rooting ability was also genotype dependent. Prolonged culture in vitro improved the rooting ability of the two most reluctant clones. BA concentrations of 5×10^-7 M or higher inhibited rooting almost completely, but this was not a permanent effect. All clones could be rooted on medium containing 10^-6 M NAA. Shoots were transferred to greenhouse conditions and rooted with varying degrees of success depending on shoot size and genotype.     

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.     

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.     

High-frequency induction of multiple shoots and clonal propagation from rhizomatous nodal segments of Houttuynia cordata Thunb.—An ethnomedicinal herb of India

Summary This study reports an efficient and direct shoot bud differentiation and multiple shoot induction from nodal segments of underground stoloniferous rhizomes of Houttuynia cordata Thumb. The frequency of shoot bud regeneration was influenced by the type of cytokinin and concentrations. Among the various concentrations used, benzylaminopurine (BAP, 17.74 &#x03BC;M) or kinetin (Kn, 18.58 &#x03BC;M) was found to be most effective for rapid and maximum shoot but differentiation. The number of shoots per explant was higher (20.00&#x00B1;2.61) on Murashige and Skoog (MS) medium supplemented with Kn (18.58 &#x03BC;M) compared to BAP and 6-&#x03B3;-&#x03B3;-(dimethyl-allylamino)-purine (2iP) during initial 40-d-old culture. Subsequent shoot differentiation and multiplication were achieved in MS medium containing 9.29 &#x03BC;M Kn and 15% (v/v) coconut milk. Elongation and growth of multiple shoots were also obtained on MS medium containing either 2.32 &#x03BC;M Kn or 2.46 &#x03BC;M 2iP alone. The rate of shoot multiplication during subcultures declined with an increase in the size of proliferating shoot cluster. Reducing shoot cluster size to three to four shoots and subculturing together in shoot multiplication medium resulted in a better shoot multiplication and growth, which could be maintained for 2 yr. The elongated shoots (&gt;20 mm) were successfully rooted on MS medium supplemented with 19.60 &#x03BC;M indole-3-butyric acid. Regenerated plants were successfully established in soil and were found to be healthy and uniform. The protocol reported in this study can be used for conservation and utilization of elite clone of H. cordata.     

Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of <Emphasis Type="Italic">Alyssum murale</Emphasis> Waldst et Kit

Shoot cultures of nickel hyperaccumulating Alyssum murale were established from epicotyl explants of seedlings aseptically germinated on hormone-free MS medium. They were further maintained on media with 0–0.92 μM kinetin. Optimal shoot multiplication was at 0.46 μM kinetin. Inoculation by shoot wounding was performed with overnight suspension of A. rhizogenes A4M70GUS which contains GUS gene cointegrated in pRiA4. After 30 days hairy roots were produced at the wounding site in 31 explant (25% out of 124). Hairy roots were excised and further propagated on hormone-free medium as separate clones. In the first passage clones 3 and 6 could be distinguished by fast growth and spontaneous shoot regeneration. In other clones (12, 23 and 25) shoot regeneration required presence of cytokinins. The five shoot culture clones regenerated from hairy roots were further cultured on media with 0.46 μM kinetin. These shoots were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus all plants regenerated from hairy root cultures manifested the characteristic Ri syndrome phenotype. They all had a strong positive GUS reaction. PCR analysis confirmed presence of uidA sequence from the gus construct. They were also tolerant to nickel accumulating up to 24,700 μg g⁻¹ dry weight.     

Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds

Cypripedium flavum, known as the rare lady’s slipper orchid, is one of the endemics with a yellow flower in China. Due to its conservation and commercial requirement, establishment of an efficient method for micropropogation is urgently needed. Multiple shoots were obtained by placing seedlings from seeds of C. flavum on Harvais media supplemented with two cytokinins (BAP or KIN) used alone or in addition to different concentration of potato homogenate. The effect of BAP was better than that of KIN on shoot multiplication. The Havais media supplemented with BAP (2.22 μM) and potato homogenate (20 g l⁻¹) was the most effective, providing high shoot multiplication frequencies (95%) associated with a high number of shoots per explant (2.55 shoots/plant). For root formation, high rooting and survival were achieved using 1/2 Harvais media supplemented with 0.6 g l⁻¹activated charcoals. High-level activated charcoal increased the number and the length of roots because the activated charcoal could absorb BAP in the media. This study demonstrated that C. flavum could be micropropagated by using multiple shoots of seedlings derived from mature seeds.     

Maturation and rejuvenation of Coriandrum sativum shoot clones during micropropagation

Three clones of Coriandrum sativum L. shoots were obtained from three seedlings and micropropagated alternately on modified MS media containing kinetin only and kinetin plus indolyl-3-acetic acid (IAA). During the first 9 months of culture the shoots possessed the juvenile phenotype after which a sharp transition to mature phenotype occurred. In 15–17 months this was followed by shoot necrosis and decrease in number of shoots in the clones, leading to death of the clones.Conditions of in vitro culture tripled the length of the juvenile period. Mature phase of the shoots was stable in that no reversion to the juvenile phase was observed. Partial rejuvenation of mature shoots took place owing to formation of adventitious shoots in the callus formed at the shoot base. However maturation of such rejuvenated adventitious shoots took place much more rapidly in comparison with micropropagated juvenile shoots derived from seedlings. Reduction of the morphogenic potential of the mature shoots after 15–17 months of subculturing, an increase in number of abnormal shoots and shoot necrosis indicated physiological ageing of the clones.Data presented in the paper provide evidence of the clone ageing phenomenon during prolonged subculture in vitro.     

Micropropagation of mature wych elm (<Emphasis Type="Italic">Ulmus glabra</Emphasis> Huds.)

Explants of mature vigorous donor trees of wych elm (Ulmus glabra Huds.) that had not been previously exposed to Dutch elm disease were investigated for the influence of phytohormones and media on shoot multiplication rates and organogenic capacity. The regenerates were micropropagated from cultures that originated from 15-year-old progeny of plus trees. Two plus trees aged over 70 years showed recalcitrant responses. Thidiazuron in combination with 6-benzylaminopurine (BAP) induced a significantly higher number of shoots per explant than the most optimal BAP treatment (5.88 vs. 3.05 shoots). Woody plant medium and Dubovský minimal medium had no significant effects on shoot formation and multiplication rates. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. Two experimental field plots with 3-year-old in vitro-propagated trees were established.Abbreviations DED: Dutch elm disease - BAP: 6-Benzylaminopurine - IBA: Indole-3-butyric acid - TDZ: Thidiazuron - WPM: Woody Plant Medium - DM: Dubovský Minimal Medium Communicated by D. Bartels     

Micropropagation of strawberry tree (<Emphasis Type="Italic">Arbutus unedo</Emphasis> L.) from adult plants

Arbutus unedo L. is a species of strawberry tree, widely represented in the Mediterranean climates of southern Europe. Fruits are used to make jellies and a spirit called “medronheira.” Shoot apices and nodal segments from epicormic and coppiced shoots of adult plants were used for plant propagation. Shoot apices from epicormic shoots, which were developed in a growth chamber, showed higher rates of in vitro establishment. The results also indicated that shoot apices are more effective for plant establishment than nodal segments, with rates of establishment significantly higher after 12 wk of culture. Of the three basal media used in combination with 9.0 μM benzyladenine and 0.087 M sucrose, the FS medium with the micronutrients of the Murashige and Skoog medium gave the highest rates of multiplication, especially when the parameter analyzed was the number of clusters formed. When shoot apices from selected adult plants (AL01–AL06) were tested, the multiplication rate was not significantly different among the plants. However, in the conditions tested, shoots from the clones AL1, AL2, and AL3 showed better development, whereas shoots from AL4, AL5, and AL6 showed an impaired development and could not be rooted. Rooting was achieved in all the conditions tested, even in the absence of auxin. The inclusion of an auxin significantly increased root formation, whereas the addition of charcoal did not improve root formation. Rooted plantlets were acclimatized, and some of them are now in the field for further study.     

Adventitious shoot formation is not inherent to micropropagation of banana as it is in maize

Despite their similar morphology, banana and maize shoot tips responded strikingly different with respect to the in vitro formation of homogeneous multiple shoot clusters. While up to 50 small shoots per maize explant could be induced within 1 month, zero to one additional shoot formed starting from a banana shoot tip. Subsequently, banana shoot tips were subjected to different combinations of five cytokinins (0–100 μM) and five auxins (0–5 μM). The cytokinins thidiazuron and benzylaminopurine stimulated multiplication to a higher extent compared to zeatin, kinetin and isopentenyl adenine. The addition of indoleacetic acid, naphthalene acetic acid or indolebutyric acid to cytokinin containing medium did not affect the in vitro response. In contrast, 2,4-dichlorophenoxyacetic acid (1 and 5 μM) and a higher concentration of picloram (5 μM) had a detrimental effect on shoot formation and resulted in explant death and globule development. When small (0.1 cm) shoot tips were grown on cytokinin medium without an auxin source, the average number of shoots was generally two to three times lower compared to bigger (0.5 cm) shoot tips. Based on our experience in maize and this large-scale study with banana shoot tips, we conclude that banana is extremely recalcitrant towards adventitious shoot formation. This recalcitrance could not be overcome by any of the 173 different plant growth regulator combinations tested. In vitro multiplication of banana thus appears solely restricted to axillary shoot formation.     

Micropropagation of Paulownia fortuneii through in vitro axillary shoot proliferation

Primary cultures were established with nodal segments from juvenile shoots of two- year-old Paulownia fortuneii trees from a clonal plantation in Andhra Pradesh. A medium containing half-strength MS salts + RAP (1 mg/L) + sucrose (2%) produced optimum bud break in nodal explants. The same basal medium with reduced hormone level (0.5 mg/L) supported maximum multiplication of secondary cultures of P. fortuneii (1:6 in 6 weeks). Specific treatments were tested to enhance this rate of multiplication. In one approach, five to six week old in vitro grown shoots were ratooned (cutting the main shoot at the bottom leaving one node). The stumps (ratooned basal node) produced 2 to 3 axillary shoots, which grew into 4 to 5 nodes by 3 weeks; thus, providing additional shoots from the same explant. This provided 30% additional shoots in 4 cycles. Secondly, reducing the light intensity to 1200 lux resulted in higher shoot elongation, i.e, formation of 8 nodes in 5 weeks with healthier shoots than the normal intensity of 3000 lux under which only 6 nodes were produced in 6 weeks. In vitro-grown shoots could be successfully rooted ex vitro in vermiculite + cocopeat mixture (1:1 v/v) under 90% humidity, transferred to soil in polybags for hardening in the green house for 2 weeks and shifted to shade net for further hardening. After one month, the plants could be successfully transplanted to field with 95% survival. Micropropagated plants showed an excellent growth in the field attaining a height of 1.5 m and a collar diameter of 2.8 cm in 3 months.     

In vitro clonal propagation of annatto (Bixa oreliana L.)

Summary A protocol for in vitro propagation of Bixa orellana is described. Plants were regenerated from shoot apex and nodal explants on B5 medium supplemented with 4.9 μM 2-isopentenyl adenine. The multiplication factor of shoot apex explants was higher (nine shoots per explant) than that of the nodal explants (five shoots per explant). Regardless of the position of the nodes, all the nodal explants gave similar responses. However, the size of the nodal explant was an important factor in producing multiple shoots: 0.5 cm nodal explants produced the maximum multiple shoots. Regenerated shoots from shoot apex explants rooted best on MS medium supplemented with 0.05 μM α-naphthalene acetic acid (NAA). whereas shoots regenerated from nodal explants needed 2.7 μM NAA for rooting. Eighty per cent survival of in vivo transferred plants occurred on the best potting substrate, coco peat. Since the multiplication factor was nine per explant, this protocol can be use for commercial microprogation. However, the regeneration capacity declined after 10 subcultures. Approximately, 3350 rooted plants could be generated in 10 mo. after eight subcultures, from one shoot with a shoot apex and four nodes.