Evaluation of conditions for in vitro storage of commercial and minor grapevine (Vitis vinifera L.) cultivars

In vitro culture represents a tool for the ex situ conservation of a high number of sanitised plants in a reduced space. However, the culture media and/or other growing conditions need to be optimised to minimising plant growth and storage cost. Growth on MW medium was evaluated in the commercial cultivars ‘Airén’, ‘Bobal’, ‘Chardonnay’, ‘Garnacha Blanca’, ‘Moscatel de Alejandría’, ‘Moscatel de Grano Menudo’, ‘Pedro Ximénez’, ‘Pinot Blanc’, ‘Pinot Gris’, ‘Sauvignon Blanc’, and ‘Tempranillo’; the minor cultivars ‘Chelva’, ‘Valencí Negre’, ‘Valencí Blanc’, and ‘Verdil’; and the endangered cv. ‘Esclafacherre’. Different growth rates were observed among cultivars: those with faster growth need to be subcultured every 1.5–2.0 months; those with the slowest growth every 3.5–4.0 months. The effect of halving the sucrose in MW reduced the growth of the cultivars that grew faster without compromising survival. When IBA was removed from MW, growth was also reduced in some cultivars. Therefore, small modifications of the MW composition are adequate for grapevine in vitro storage under standard incubation conditions. This is an advantage with respect to the change of temperature used in other work to achieve growth reduction, and allows the use of the same chamber for different in vitro culture procedures.     

The Influence of Some Genetic and Environmental Factors on the Concentration of L-Ascorbic Acid in the Tomato Fruit

Summary

An efficient, reproducible protocol has been developed for in vitro multiplication of Sida cordifolia L. High-frequency, multiple shoots (90%) were obtained indirectly from nodal explants. Callus was induced when nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg l^–1 Kinetin (Kn). These nodal calli were then cultured in order to differentiate multiple shoots on MS medium supplemented with 0.5 mg l^–1 Kn plus 0.5 mg l^–1 naphthaleneacetic acid (NAA). Roots were induced from these multiple shoots following culture on MS medium supplemented with 0.8 mg l^–1 NAA for 4 weeks. Finally, these in vitro plantlets were hardened, acclimatised, and successfully transferred to the field.     

In vitro development and germination of immature olive embryos

Summary

In vitro culture methods were used to germinate olive embryos prior to maturation. Fruit, seed and embryo development were established with consecutive sampling from 20 to 100 days after bloom. For that same period, embryo development and germination success were determined by in vitro culture trials using one-third strength MS medium with or without the addition of zeatin. For early developmental stages, when isolation of the embryo was difficult, a cut portion of the seed containing the embryo was used for culture. The embryos cultured within the cut seed portions germinated and formed normal plantlets. Histological observations indicated a close similarity between the natural and in vitro immature embryo differentiation pattern, progressing through preglobular, globular, heart-shaped and torpedo-shaped stages. In some cases, however, the in vitro immature embryos developed or germinated abnormally. The presence of zeatin (0.25 mg l^–1) in the culture medium and the use of a cut seed-portion containing the immature embryo allowed in vitro germination sooner after bloom than previously obtained. On the contrary, zeatin was a handicap for mature olive embryo in vitro germination, which reached 100% seedling formation when no plant growth regulators were used.     

In vitro callus-induction in globe artichoke (Cynara cardunculus L. var. scolymus) as a system for the production of caffeoylquinic acids

Summary

Globe artichoke (Cynara cardunculus L. var. scolymus) provides a rich dietary source of bio-active compounds derived from phenylpropanoid metabolism, notably caffeoylquinic acids (CQAs) and flavonoids. Micropropagation techniques have been established for this species, but in vitro cultures have not yet been extended to generate an efficient system for the induction of callus tissue. In this study, we compared more than 100 combinations of media supplements (e.g., phytohormones, absorbers of polyphenols, and inhibitors of polyphenol oxidase), along with various light regimes, and three different genotypes of globe artichoke to define the optimal conditions for callus induction from leaf explants. This led to the elaboration of an in vitro culture protocol which resulted in a high frequency of callus induction after just 1 week in culture. The procedure used leaf explants from virus-free, meristem culturederived plantlets. Quantitative HPLC analysis revealed that, as in globe artichoke leaves, the predominant phenolic esters present in callus were mono- and di-caffeoylquinic acids (diCQA). The concentration of diCQA was three- to five-fold higher in calli than in leaves. The exposure of calli to UV-C light further enhanced the levels of CQAs. In vitro callus culture combined with UV-C irradiation may thus represent a viable production system for diCQA that is suitable for the synthesis of pharmacologically-active compounds.     

The Use of Thermotherapy and in vitro Meristem Culture to Produce Virus-Free ‘Chancellor’ Grapevines

ABSTRACT

Grapevines (Vitisspp.) are very susceptible to virus diseases. Virus infection reduces fruit yield and quality. The objective of this work was to determine the usefulness of thermotherapy (37.2°C) and in vitromeristem culture to obtain virus-free grapevine plants cv. ‘Chancellor’. Grapevine leafroll-associated virus 1,3(GLRaV-1, 3) infected grapevines were multiplied in vitrofrom two infected mother-plants in half strength Murashige and Skoog medium (1/2MS) supplemented with 0.5 mg/L of BA and the in vitroplants were initially tested by ELISA to confirm their virus status; subsequently, 96 infected in vitroplants were propagated on 1/2MS medium with BA and subjected to 0, 7, 10, 12, 14, 16, 18, 20 or 22 days of exposure at 37.2°C. Afterward, the apical meristems from the plants surviving the thermotherapy treatment were excised and transferred to fresh 1/2MS medium with 0.5 mg/L of BA and grown in a culture room until they developed into entire plants. Control plants and all the plants that survived thermotherapy were assessed for their virus status using both ELISA and RT-PCR. After 20 days of exposure at 37.2°C, 100% of the plants submitted to thermotherapy were found to be virus-free by RT-PCR and ELISA tests. Plants derived from meristems with two or three primordial leaves remained virus infected. However, when meristem culture was combined with thermotherapy (12 or more days of heat treatment), all the meristem-derived plants were virus-free.     

Direct somatic embryogenesis from explants obtained from in vitro germinated embryonic axes of Camellia sinensis (L.) O. Kuntze

Summary

Studies on direct somatic embryogenesis in several types of explant from in vitro plantlets of tea cultivar TRI 2025 were undertaken to select those most suitable to induce cotyledonary-type somatic embryos. Mature zygotic embryonic axes were surface-sterilised and cultured on MS medium without growth regulators containing 0.6% (w/v) agar. Results showed that 65% of embryonic axes that converted into plantlets at the fifth week of culture had succulent leaves. Several types of explant (normal leaves, large and small succulent leaves, hypocotyl segments and root tips) were isolated from in vitro plantlets at the fifth week and cultured on half-strength MS medium containing 2 mg l^–1 6-benzylaminopurine (BAP) and 0.2 mg l^–1 naphthalene acetic acid (NAA). Morphological and histological observations on somatic embryogenesis were made. The results indicated that somatic embryos were produced at high frequency (25 – 50%) directly from the surface of hypocotyl segments (HS) and large succulent leaves (LSL) after 6 weeks of culture. Efficient somatic embryogenesis was induced in small succulent leaves (SSL) after 16 weeks. Most somatic embryos originated directly from the cortical tissues of HS or the upper epidermal layers of SSL or LSL. HS and SSL from in vitro plantlets gave the highest production of typical, firm somatic embryos for use in tea improvement programmes and for in vitro conservation of tea germplasm.     

Improved efficiency of in vitro propagation of Camellia reticulata cv. Captain Rawes

Effects of various factors were studied on the in vitro multiplication and rooting of a clone of Camellia reticulata cv. Captain Rawes established in vitro from shoots of an adult tree. Multiplication rates were increased by repeated harvesting of cultures in which shoots were subcultured in a horizontal rather than a vertical position. Best rooting responses were obtained with 175.3 mM sucrose in the rooting medium using 8 week old shoots cultured in 500 ml glass jars; these shoots contained more anthocyanin than those from test-tube cultures. The orientation and repeated harvesting of the mother shoots had no effect on rooting response.     

Improved shoot regeneration from nodules of Charybdis numidica in a temporary immersion system

Summary

A procedure for improved shoot regeneration from meristematic nodules of Charybdis numidica in a temporary immersion culture system was developed by optimizing immersion frequencies, volume of the nutrient medium, and alternating application of growth regulators. Modified liquid MS medium with 3% sucrose, 20 μM BAP and 5 μM NAA (shoot induction medium) was used to induce microshoot formation on 15 g of nodules in 1000 ml bottles. Volumes of medium (250 or 500 ml) and immersion frequency (5 min every 12 or 24 h) did not significantly influence shoot regeneration rates. Shoot induction medium additionally supplemented with 5 μM paclobutrazol in most cases led to less shoots but this effect was not significant, either. Microshoots formed under these conditions were severely hyperhydrated. Nearly complete elimination of hyperhydricity and enhanced formation of properly elongated shoots were achieved by running a shoot induction step with induction medium containing paclobutrazol followed by an elongation step with a medium supplemented only with 5 μM gibberellic acid GA₃. This two-step procedure yielded about 900 healthy shoots per bottle after a two-month cultivation period. Root induction was performed ex vitro during acclimatization and the plantlets could be established in the greenhouse with good success.     

Changes in the competence of Quercus robur ‘Fastigiata’ to grow in vitro as affected by seedling rootstocks and differential pruning

Three physiologically distinct forms of the same genotype of Quercus robur ‘Fastigiata’, all derived from adult budwood, were identified following the growth of severely-pruned hedge plants grafted onto juvenile seedling rootstocks. Only explants from the form displaying the juvenile-like characteristic of over-winter leaf retention were capable of establishing sustainable cultures in vitro, although growth was not as rapid as those from a two year old seedling source. Pruning to release correlative inhibition of buds low on the scion framework produced vigorous shoot growth, but explants from these failed to show sustained growth in vitro. Withholding light from stockplants reduced excessive phenolic oxidation in explants taken from flushing unripe shoots, however, despite a beneficial effect on culture initiation, later growth was dependent upon the juvenile- or adult-like physiology of the original explant. A new medium was devised for this subject, which in addition to promoting growth generally, also stimulated the flushing of shoot apices. The relevance to in vitro culture of expression of juvenile habit in the stockplant is discussed.     

Plum Rootstock Trials at East Malling

Summary

Dormant axillary buds excised from crowns of pineapple (Ananas comosus L., Merr.) cultured on growth regulator free Nitsch medium sprouted after 8–10 d. Sprouted buds produced multiple shoots (7–10 shoots per bud) upon transfer to solidified Murashige and Skoog medium supplemented with 9.67 μM NAA, 9.84 μM IBA and 9.29 μM KIN. Each isolated shoot upon subculture to liquid medium of the same composition further proliferated to form more multiple shoots (60–65 shoots) and were maintained on a gyratory shaker (90–100 rpm). In vitro grown shoots were rooted on White medium supplemented with 0.54 μM NAA and 1.97 μM IBA. In vitro plantlets were established in cups with soilrite and hardened for four weeks. Phenotypic variants such as albinos, white streaked shoots and shoots with elongated internodes were observed in in vitro cultures. Approximately 520 in vitro produced plantlets were established in the field and these plants exhibit somaclonal variation. Thirty-eight plants were found to be yellowish, spineless with anthocyanin streaks and three were anthocyanin rich, spined plants.     

In vitro establishment of Picea pungens f. glauca and P. sitchensis seedling rootstocks with an assessment of their suitabilities for micrografting with scions of various Picea species

A successful technique was developed for micrograftirig resting-bud and growing-tip scions of three Picea spp. (P. abies (L.) Karst, P. pungens f. glauca (Regel) Beissn. (and its clone ‘Koster’) and P. sitchensis Bong) on P. pungens f. glauca and P. sitchensis rootstocks. In vitro rootstock production under both sterile and non-sterile conditions and micrografting using two types of graft (apical and side) are described. Both types of graft were equally successful although apical grafts were preferred since the additional operation of removing at four to five weeks the epicotyl above the graft following union was unnecessary. Culture medium strength was found to affect micrografting success rates: 1/3- strength B5 medium with 2% (w/v) sucrose giving the best results. Antioxidant additives applied to the micrograft unions were found to reduce significantly union success rates whilst the physiological age of scions used had no effect on graft union successes. A modified micrografting method was developed from which preliminary results indicated that high success rates are possible. The method is suitable for attempts to rejuvenate mature bud materials of these important ornamental and forest conifers by serial grafting.     

Influence of the antimitotic agents colchicine and oryzalin on in vitro regeneration and chromosome doubling of diploid bananas (Musa spp.)

Summary

In vitro cultures of the four diploid banana cultivars, Sannachenkadali (AA), Anaikomban (AA), Kunnan (AB) and Thattillakunnan (AB) were treated with two antimitotic agents, colchicine (C₂₂H₂₅NO₆) and oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide) to induce ploidy alterations, particularly the induction of tetraploids, which would serve as useful breeding material for creating newer, improved triploids. Both antimitotic agents, but particularly colchicine, had a negative effect on the in vitro regeneration of the four cultivars. This was reflected in terms of delay in regeneration, reduced multiple shoot regeneration rates, regeneration of smaller microshoots with lower fresh weights, and reduced response to rhizogenesis. Colchicine, as expected, had a negative effect on the number of multiple shoots regenerated. However, oryzalin at lower concentrations (10 mM and 20 mM) resulted in the regeneration of more microshoots per culture than the untreated control. The chromosome doubling capacity of colchicine was equal to that of oryzalin only at 125–200 times higher concentrations. The cultivars exhibited a genome-related response.     

In vitro propagation of ‘Arka Ravi’ chrysanthemum on growth regulator-free medium harbouring endophytic bacteria

Summary

To develop a micropropagation protocol for the elite chrysanthemum cultivar, ‘Arka Ravi’, in vitro cultures were established using surface-sterilised nodal microcuttings (1.0 – 1.2 cm) on semi-solid MS medium. Microbial contamination was observed in 22% of cultures during the initiation phase. Cultures that were devoid of obvious contamination were transferred to culture bottles containing MS medium supplemented with 30 g l^–1 sucrose, 2.5 g l^–1 Phytagel^® and either benzyl adenine (BA) or kinetin (KIN) supplied at 0, 1, 5, 10, or 20 µM, and were monitored, over eight in vitro passages, for their growth and microbial association. Shoot-tip and nodal microcuttings yielded a single shoot, coupled with rooting, in medium devoid of BA or KIN which was the best medium for continuous micropropagation. Rooting was inhibited with increasing concentrations of BA or KIN, and one or more shorter shoots with condensed internodes were induced, resulting in low rates of propagation. Culture indexing (i.e., testing the medium and tissue from visibly clean cultures using enriched bacteriological media) revealed quiescent endophytic bacteria associated with 80 – 100% of such cultures. Three distinct colony morphotypes were isolated and were identified as Ralstonia, Enterobacter, and Methylobacterium spp., based on their 16S rRNA gene sequences. These endophytes did not interfere with normal micropropagation, but tended to grow actively and outgrow older cultures, especially at higher cytokinin concentrations. Stable micropropagation of ‘Arka Ravi’ chrysanthemum for ≥ 2 years, with their resident endophytic bacteria in a covert form, was achieved on basal MS medium with > 90% shoot growth and rooting, a four-to-five-fold propagation rate at each 2 – 3 week sub-culture cycle, and with > 90% establishment of rooted plantlets ex vitro. These results suggest that in vitro cultures of chrysanthemum often harbour endophytes with no obvious indications of their presence or with possible hidden effects during micropropagation.     

Corm induction and multiplication of Amorphophallus albus in vitro

Summary

Amorphophallus albus, belonging to the family Araceae, has attracted widespread attention due to its considerable economic and medicinal importance. The natural propagation coefficient of A. albus is very low, which limits application of this crop. In vitro corms can be used for propagation of A. albus and have been proved to be superior over in vitro plantlets. To optimise procedures for in vitro corm production and multiplication, the effects of phytohormones, sucrose concentrations and incubation conditions with desirable phytohormone combinations for callus induction, corm formation and corm growth of A. albus were investigated. The results showed that calli were induced at high frequency from petiole segments on Murashige and Skoog medium supplemented with 1.0 mg l^–1 naphthaleneacetic acid (NAA) and 1.0 mg l^–1 6-benzyladenine (BA). Compact nodular calli were desirable for corm formation, and optimum corm formation was obtained in the presence of 0.5 mg l^–1 NAA and 2.0 mg l^–1 BA. With this auxin and cytokinin combination, an increase in sucrose concentration from 2% to 6% (w/v) significantly increased the corm formation rate and favoured corm growth, but negative effects occurred at higher sucrose concentrations. By incubating over a range of temperatures from 19°C – 28°C, 22°C produced the largest numbers of corms and highest mean fresh weight of each corm. Short-day (8 h) or long-day (16 h) photoperiods did not affect corm formation and growth significantly, except that corm weight fell under long-day conditions. The multiplication rate of in vitro corms was enhanced by apical meristem wounding. It was possible to store in vitro produced corms at 4°C for as long as 90 d to overcome apical dormancy and accelerate sprouting after planting into soil. This work has established an efficient protocol for multiplication of A. albus through an in vitro corm system.     

In vitro propagation of GF677 hybrid rootstock (Prunus persica × Prunus amygdalus) from mature cotyledons

Adventitious shoot regeneration from mature cotyledons of GF677 rootstock (Prunus persica × Prunus amygdalus) was achieved in vitro. Thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-yl-urea; TDZ) at 32 µM gave the highest percentage of cotyledons forming adventitious shoots (68.8%) and the highest number of shoots per cotyledon (4.8) on Quoirin and Lepoivre (QL) medium. On QL medium containing 32 µM TDZ, exposure of the proximal segments of cotyledons to darkness at the start of culture increased the percentage of cotyledons forming adventitious shoots (62.5%) when compared with those kept under light conditions (15%). A combination of 0.72 µM gibberellic acid and dark treatment resulted in at least 2.7-fold more elongated shoots than non-treated shoots. The highest rooting percentage (100%) occurred on 0.5× Murashige and Skoog medium supplemented with Gamborg (B5) vitamins and 2 mg l^?1 indole-3-butyric acid. Rooted plantlets were acclimatised under greenhouse conditions with a 70% survival rate.     

Determination of essential oil contents and micropropagation of Gaultheria fragrantissima,an endangered woody aromatic plant of India

Summary

Leaves of mature of Gaultheria fragrantissima Wall. plants (Indian wintergreen) were collected from various locations in the Eastern Himalayan region on the Indo-China border and were analysed by steam distillation and gas chromatography to identify an elite line that contained 1.79% (v/v) essential oils, 98% of which was methyl salicylate. Subsequently, a highly reproducible micropropagation protocol using adult shoot tips from this elite genotype was developed in order to conserve this highly-valued, endangered, woody oil-bearing aromatic shrub in India. Among several plant growth regulator (PGR) combinations tested for in vitro multiplication, up to 35 shoots per explant could be induced within 14 weeks of culture on woody plant medium (WPM) fortified only with 0.22 mg l^–1 thidiazuron (TDZ). These shoots could elongate on WPM containing 1.0 mg l^–1 kinetin. Rooted plantlets were acclimatised ex vivo, with 70% success. Random amplified polymorphic DNA (RAPD) analysis indicated the genetic uniformity of both the micropropagated plantlets and the donor plants. This is the first report on in vitro micropropagation of G. fragrantissima.     

Propagation of Vaccinium in Vitro

Abstract

Micropropagation techniques are important for clonal multiplication, germplasm improvement, and gene conservation of three commercially cultivated and medicinally important Vaccinium species: cranberries, blueberries, and lingonberries. The in vitro propagation of Vaccinium species using axillary bud proliferation and adventitious shoot regeneration has been investigated in a number of previous studies. The morphogenesis seems to be highly dependent on plant growth regulators and media used for culture, which is again genotype specific. This review presents the progress in-depth of various aspects of Vaccinium propagation in vitro for its commercial production. It also discusses the issues that still need to be addressed to utilize the full potential of plant tissue culture techniques in mass propagation of Vaccinium species.     

Embryogenesis in vitro of several Citrus species and cultivars

Summary

A number of experiments were performed to obtain somatic embryos and embryogenie cell lines from several species and cultivars of citrus by in vitro ovule culture. The source of ovules (pre-anthesis and post-anthesis), incubation conditions (light and darkness) and several modifications of the culture medium were evaluated. All the species and cultivars assayed produced somatic embryos and “pseudobulbi”, but production of friable embryogenie callus was species dependent. The small proliferation of callus and globular bodies recovered from the primary cultures were suitable for recovering embryogenie callus lines by periodical subculturing into fresh medium. After several monthly subcultures discarding embryos and “pseudobulbi”, cell lines virtually devoid of organized structures were obtained from three cultivars of sweet orange, sour orange and Cleopatra mandarin. In all these species, embryogenesis was restored spontaneously and/or by substitution of sucrose by maltose or lactose in the culture medium. The embryos germinated and produced phenotypically normal plants.     

In vitro micrografting of apical and axillary buds of cacao

This study aimed to evaluate in vitro grafting of Theobroma cacao where seedlings of the UF 677 genotype were used as the rootstock and apices or axillary buds of a Trinitarian genotype were used as scion. Three methods of grafting using scions from seedlings were evaluated. Apical grafts using apex and side grafts using apex displayed better graft success (95 and 80%, respectively). However, side grafts using axillary buds reached a greater height on average and a higher number of leaves per plant (1.76 cm and 3.72, respectively). Histological studies revealed new vascular elements at the graft union area. Side grafts with axillary buds provided the highest survival rate (82%) after the acclimatization step. A shoot of at least 1 cm with two leaves is required for plant survival after transfer to ex vitro conditions. Side grafting was carried out with axillary buds from adult trees and nursery plants. Only the grafts with buds from nursery grafted plants were successful, with a rate of 26%. Overall, side grafting with axillary buds is the most appropriate method for cacao micrografting. This method can be used for clonal propagation and for the establishment of in vivo and/or in vitro cacao germplasm collection.     

Comparison of seed-derived with micropropagated male-sterile plants of Tagetes erecta L. for F1 hybrid seed production

Summary

To multiply large number of male-sterile marigold plants for F₁ hybrid seed production, an efficient protocol for in vitro cloning of field-grown differentiated male sterile plants has been developed. A comparative field performance study of tissue culture and seed-derived male sterile plants of two marigold genotypes was undertaken to test the possibility of using micropropagated plants in hybrid seed production. Tissue culture raised plants of both genotypes had superior field performance to the seed-derived counterparts. These plants were more vigorous in growth, i.e. in terms of plant height, number of secondary branches and number of leaves and plant spread, while the leaf chlorophyll contents were equal to that of seedling plants. Flowering was earlier by 2-3 weeks and the number of flowers per plant was also higher in such plants. Repeated hand pollination of sterile flowers with bagged flowers of cv. Pusa Narangi Gainda showed that seed set and bold seed yield were higher or almost comparable with the seed-derived plants. The results clearly indicate that the tissue culture can be adopted for the successful cloning of male-sterile plants, which could then be utilized for producing F₁ seeds with higher quantities of bold seeds with better storability.