The effect of the gaseous state on bud induction and shoot multiplication in vitro in eastern white cedar

The effect of vessel type and the gaseous phase on the morphogenic response of Thuja occidentalis L. explants in vitro was studied. Explants were cultured in container types that varied in their degree of gas exchange. Traps for ethylene and CO₂ were employed. During shoot bud induction from embryonic explants, the number and elongation of shoot buds improved significantly when gastight, serum-capped flasks were used compared to the foam bung-capped flasks or the regularly used Petri dishes. Elimination of the two gases from the headspace of the flasks either singly or together reduced shoot bud induction and especially elongation of shoots. A similar response was seen during axillary bud development from cultured shoots. Ethylene and CO₂ accumulation promoted development and elongation of axillary shoots. An increase in the zeatin concentration in the medium produced a greater number of axillary shoots and higher levels of ethylene in the culture vessels. Removal of CO₂ caused gradual death of the shoots, while removal of ethylene alone reduced axillary shoot lengths significantly. Inclusion of aminoethoxyvinylglycine in the medium combined with ethylene traps produced an effect similar to the use of ethylene traps alone.     

In vitro clonal propagation of Lagerstroemia flos-reginae Retz

Multiple shoots were obtained from nodal segments of young and mature trees of Lagerstroemia flos-reginae Retz on MS medium with 7.50–20 mg/l of benzyl amino purine. Rooting was achieved on transfer of the excised shoots to MS medium with 1 mg/l of indole butyric acid. The plantlets have been successfully transferred to soil.     

In vitro multiplication ofVanilla planifolia using axillary bud explants

A clonal propagation method has been developed for efficient multiplication ofVanilla planifolia. Multiple shoots were developed from axillary bud explants using semi-solid Murashige and Skoog (MS) medium supplemented with N⁶-benzyladenine (BA, 2 mg l^–1) and -naphthaleneacetic acid (NAA, 1 mg l^–1). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg l^–1 and NAA at 0.5 mg l^–1 for 2–3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots were obtained from a single axillary bud explant over a period of 134 days. Use of an intervening liquid medium has been found to enhance multiplication of shoots inV. planifolia.Abbreviations BA N⁶-benzyladenine - DMRT Duncan's multiple-range test - KC Knudson (1946) medium - KCB KC basal medium - Kn kinetin - MS Murashige and Skoog (1962) medium - MSB MS basal medium - 1/2 MSB half-strength MSB - MS-D double-phase MS medium - MS-L liquid MS medium - MS-S semi-solid MS medium - NAA -Naphthaleneacetic acid     

Survival and growth of eastern white pine shoot apical meristemsin vitro

Shoot apical meristems of seedling and mature eastern white pine trees were excised and grownin vitro. Placing the meristems on filters instead of directly on agarose-solidified nutrient medium enhanced survival of both juvenile and mature meristems. Applying forcing treatments to mature branches improved survival and growth of dissected meristems compared with meristems from non-forced branches in experiments conducted over two years. No consistent differences were observed among 2-, 4-, and 6-week forcing treatments. Including 5.37 nM (0.001 mg l^-1) l-naphthaleneacetic acid in the culture medium did not affect meristem survival or growth. Some meristems from seedlings grew rapidly, produced primary leaves, underwent internode elongation, and in three cases, produced adventitious roots. Meristems from mature trees did not grow as rapidly as seedling meristems. The leaves produced by mature meristems appeared to be scale leaves and a few of these had brachyblast primordia in the axils. The shoots derived from mature meristems did not produce adventitious roots.     

In vitro clonal propagation of Pisum sativum L.

A system of in vitro clonal propagation has been developed in Pisum sativum L. (cv. Bohatýr). A modified MS-medium supplemented with 20 M 6-benzylaminopurine (BAP) and 0.1 M -naphthaleneacetic acid (NAA) was used to induce multiple shoot formation from shoot apices, axillary buds of the first normal leaf, axillary buds of the first and second primary scales and axillary buds of cotyledons of 4 to 6 day old pea seedlings. Meristem explants maintained a high proliferation ability in each subculture in the course of 20 months of the culture. Regenerated shoots were rooted in the same basal medium containing 5 M NAA. Rooted plants were cultured in hydroponic pots filled with half-strength MS-medium to attain anthesis and seed maturity. The phenotypic uniformity of the regenerants was evaluated. Cytological investigation confirmed the diploid stage (2n=14) of regenerants and their progeny. Histological studies revealed that proliferating shoots originated from axillary and adventitious buds. In vitro propagation is discussed as related to pea breeding.     

Micropropagation of licorice (Glycyrrhiza glabra L.) through shoot tip and nodal cultures

Summary Apical and axillary buds ofGlycyrrhiza glabra commonly known as licorice, a plant of repute in the Indian system of medicine, were used for induction of adventitious shoots. For induction of multiple shoots, Murashige and Skoog’s (MS) medium with N⁶-benzyladenine (BA, 0.88–8.87 μM) was used. Reduction in major salts of MS medium enhanced the multiplication ratio up to 1∶10. Plants transferred to the greenhouse showed 90% survival. The present work describes a stepwise protocol for production ofGlycyrrhiza glabra plants on simple minimal media, where very high multiplication rates with healthy root systems were obtained. Roots being the organ of commercial importance, the protocol has tremendous potential.     

In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.     

Genetic diversity and differentiation of core vs. peripheral populations of eastern white cedar, Thuja occidentalis (Cupressaceae)

? Premise of the study: Geographically peripheral (marginal) populations are expected to have lower genetic diversity and higher genetic differentiation than geographically core (central) populations as a result of supposedly lower effective population size (N(e)) and higher genetic drift, founder effect, fragmentation, and isolation in peripheral than in core populations. Here we address this issue for a long-lived plant species, eastern white cedar (Thuja occidentalis). ? Methods: Genetic diversity and population structure of 13 natural populations of eastern white cedar from its Canadian eastern peripheral and core natural ranges in New Brunswick, Nova Scotia, and Prince Edward Island were studied using six nuclear microsatellite DNA markers. ? Key results: The core populations of eastern white cedar had significantly higher allelic diversity (mean A = 8.83, A(r) = 8.13, A(e) = 4.03) and N(e) (428) than the peripheral populations (A = 6.64, A(r) = 6.15, A(e) = 3.12, N(e) = 198). However, expected heterozygosity was similar in the core (H(e) = 0.64) and peripheral (H(e) = 0.60) populations. Genetic differentiation was significantly higher among the peripheral (F(ST) = 0.089) than among the core (F(ST) = 0.032) populations. No genetic differentiation (F(ST)/Φ(RT) = 0.000) was detected between core and peripheral regions. ? Conclusions: Peripheral populations have significantly lower N(e) and genetic diversity in terms of allelic diversity (richness) and significantly higher genetic differentiation than the core populations of eastern white cedar in its Canadian eastern range. However, core and peripheral populations have similar levels of expected heterozygosity. Implications for conservation of eastern white cedar genetic resources are discussed.     

The effect of growth regulators on shoot propagation and rooting of common lavender (Lavandula vera DC)

Nodal segments from micropropagated plants were used to evaluate the effect of growth regulators on the in vitro shoot proliferation and rooting of Lavandula vera DC. The highest multiplication rate was obtained using MS medium supplemented with 1.0 mg l^-1 of TDZ (2.25 μM) or BA (2 μM). Hyperhydricity occurred at high concentrations of these growth regulators. Rooting of the plantlets was obtained in all the media evaluated. However, rooting rates and root growth increased with increased concentrations of NAA and the reduction of the salt strength of the media. The plantlets were successfully transferred to soil and grown to maturity, exhibiting a normal development, with high uniformity and no evidences of somaclonal variation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adventitious shoot production from immature embryos of white clover

Cotyledonary-stage embryos of Haifa white clover, collected 13 days after cross-pollination, were induced to form adventitious shoots primarily from the hypocotyl region. The culture medium used for the production of adventitious shoots contained 5 M thidiazuron and 0.5 M -naphthaleneacetic acid. Numerous shoot meristems were produced within the first week, discrete shoots developed by week three, small plantlets by week eight, and whole plants in soil by week ten. 95–100% of all embryos, regardless of genotype, produced adventitious shoots within four weeks with an average production of 17.5 shoots per embryo. The majority of shoots (on average 77%) were easily converted to whole plants in soil. The white clover regeneration system described is prolific, rapid and effective on a large number of genotypes.Abbreviations BA N⁶-benzylaminopurine - MS medium Murashige & Skoog medium (1962) - NAA -naphthaleneacetic acid - thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

In vitro propagation of the alkaloid producing plant Datura insignis Barb. Rodr.

A method is presented for the in vitro propagation of Datura insignis Barb. Rodr. Nodal explants were cultured on Murashige and Skoog medium supplemented either with BA alone or in combination with 2,4-D or IAA. Shoot multiplication and elongation were obtained in various growth regulator concentrations. Best results were obtained in a medium with 1.0 mgl^-1 of BA. Individual shoots were excised and transfered to growth regulator-free medium for rooting. Additional multiplication was obtained by single-node culture using explants from these in vitro rooted shoots. Rooted plantlets were successfully grown in soil.     

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.     

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.     

In vitro tuberization in white yam (Dioscorea rotundata Poir)

Nodal cuttings of white yam were induced to produce microtubers on a MS-revised medium supplemented with various concentrations of sucrose, 20 mgl^–1 L-cysteine, 0.5 mgl^–1 kinetin and 0.7% agar. The frequency of tuberization was affected by the daylength, which is optimal at 12 and 16 h of light depending on the sucrose concentration. The microtubers were planted in a seed bed and grown to maturity. The importance of in vitro tuberization of yam as a means of international germplasm distribution or exchange as well as for the propagation of planting material is discussed.     

Micropropagation of yellow cedar (Chamaecyparis nootkatensis)

Mature yellow cedar (Chamaecyparis nootkatensis (D. Don) Spach) embroys were exposed to a range of N⁶-benzyladenine concentrations in a variety of culture media generally used for conifer caulogenesis. All seven media supported the induction of adventitious shoots but Schenk & Hildebrandt medium was the best. The best cytokinin level of N⁶-benzyladenine was 0.35 mg 1^-1. This resulted in an average of 4–5 large adventitious shoots per explant. Shoots arose primarily from the cotyledons regardless of whether they were in contact with the medium or not. Embryos from seeds stratified four weeks at 21°C and eight weeks at 5°C were more caulogenic than unstratified controls. An additional four weeks at 5°C caused a change in the pattern of shoot induction in that shoots arose from the hypocotyl as well as the cotyledons. Shoots elongated on basal Schenk & Hildebrandt medium. The best rooting response was obtained under non-sterile greenhouse conditions where approximately 60% of the shoots formed roots. Over a 12-month period the average shoot height ranged between 10–13.9 cm with a stem diameter of 2.29–2.68 mm. These propagules are still being grown under forest nursery conditions.     

In vitro axillary shoot proliferation of apple rootstocks under different ethylene conditions

Summary Ethylene effect on in vitro shoot proliferation of two apple rootstocks, MM111 and M9, was studied. Ethylene biosynthesis was proportionally stimulated by increasing concentrations of the precursor 1-aminocyclopropane-1-carboxylic acid (ACC). When 25 μM or more ACC was applied without any control of the headspace of culture vessels, shoot proliferation of both rootstocks was negatively affected. However, when shoot cultures were transferred to ACC-supplemented medium after the second week of culture, ACC had no effect. Supplementing the medium with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, together with the application of gas traps inside the flasks, significantly enhanced axillary shoot formation and elongation. Steady and high exogenous concentrations of ethylene in the culture flasks had negative effects on shoot proliferation. MM111 appeared to be more sensitive to ethylene than M9. For AVG a threshold dose was noticed, beyond which phytotoxic effects were induced.     

In vitro clonal multiplication of mature trees of Dalbergia sissoo Roxb.

Multiple shoots were obtained from nodal explants of 30-year-old trees of Dalbergia sissoo Roxb. on a defined medium, MS (Murashige & Skoog medium) supplemented with auxin-cytokinin combinations. IAA (Indole accetic acid) alone promoted 15% rooted shoot buds. A combination of IAA+Kn (Kinetin) gave 100% rooted shoot buds. A combination of NAA (napthaleneacetic acid) + Kn and NAA+BAP (6-benzylaminopurine) also gave high percentages of rooted shoot buds. Ascorbic acid in the medium prevented the death of callus and plantlets, which followed darkening of the medium.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass

Summary An efficient and reproduciblein vitro culture system has been developed for regeneration of multiple shoot clumps from intact seedlings of both lowland and upland cultivars of switchgrass (Panicum virgatum L.). The multiple shoots were induced on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-phenyl-3-(1,2,3-thiadiazol-5YL)-urea (thidiazuron or TDZ). Maximum response was obtained with 4.5 μM 2,4-D and 18.2 μM TDZ. These shoots proliferated and rooted efficiently on MS medium without growth regulators. The developmental pattern of the multiple shoots indicated their origin from the enlarged shoot apex via proliferation of axillary buds and subsequent reprogramming of shoot meristems followed by secondary differentiation of adventitious shoots The simplicity of the protocol and direct production of multiple shoots make this a potential system that is highly attractive and amenable for microprojectile-mediated gene transfer.     

In vitro plant regeneration in Holarrhena antidysenterica wall., through high-frequency axillary shoot prolieferation

Summary An efficient, rapid and large-scale propagation of the woody, aromatic and medicinal shrub, Holarrhena antidysenterica, through in vitro culture of nodal segments with axillary buds, is described. N⁶-benzyladenine used at 15 μM was the most effective in inducing bud break and growth, and also in initiating multiple shoot proliferation at the rate of 43 microshoots per nodal explant with axillary buds, after 30 d of eulture. By repeated subculturing of nodal explants with axillary buds, a high-frequency multiplication rate was established. Efficient rooting was achieved with 35 μM indole-3-butyric acid which was the most effective in inducing roots, as 80% of the microshoots produced roots. Plantlets went through a bardening phase in a controlled plant growth chamber, prior to ex vitro transfer Micropropagated plants established in garden soil were uniform and identical to donor plants with respect to growth characteristics and vegetative morphology.