In vitro propagation of garlic by shoot proliferation

Shoot buds (5–8 mm long), excised from dormant cloves of the New Zealand commercial garlic (Allium sativum L.) and a virus-free French cultivar ‘Rose-de-Kakylis’, proliferated both axillary and adventitious shoots on B-5 basal medium supplemented with 0.5 mg l^?1 isopentenyladenine (2-ip) and 0.1 mg l^?1 naphthaleneacetic acid (NAA). An 8-fold increase in shoot number occurred every 6 weeks. Shoots were readily rooted in B-5 + 0.01 mg l^?1 2-ip + 0.2 mg l^?1 NAA and transferred to pots, where about 70% of the shoots formed established plants. The plants raised by this shoot-proliferation method retained the diploid condition of the parents.     

Aseptic micropropagation of Rhododendron P.J.M. hybrids

Summary

Shoot tips established in vitro better than single nodes. Five-cm shoot tips gave better Stage I results than 2-cm shoot tips. The best microshoot proliferation rates were obtained with isopentenyladenine (2iP) at 5 and 10 mg l^?1; tetrahydropyranyl-benzyladenine was ineffective. Microshoots rooted well, regardless of the level of 2iP in the Stage II medium. Stem-cutting-macropropagated plants generally were more variable in height, had less basal branching and were more erect than plants from microculture.     

Callus and axillary-bud culture of Vitis vinifera ‘Sylvaner Riesling’

Healthy growth of serially subcultured callus of the grape Vitis vinifera cultivar ‘Sylvaner’ was obtained by incubation at 30° C in continuous light in a defined culture medium containing 2% w/v sucrose, 1.0 mg l^?1 1-naphthaleneacetic acid (NAA) and 0.2 mg l^?1 kinetin (K). Organogenesis was not induced in this callus by alteration in the absolute or relative levels of NAA and K.Continued shoot initiation was obtained by culture of axillary buds in a medium containing 10^?5 M Benzyladenine (BA). Plantlets could be generated from these shoot buds by transfer to media containing 10^?7 M BA or lacking a cytokinin.     

In vitro propagation of ‘Troyer’ citrange from epicotyl segments

Isolated epicotyl, root meristem and root segment tissues of ‘Troyer’ citrange [Poncirus trifoliata (L.) Rat. × Citrus sinensis (L.) Osbeck] were established in continuous culture to compare their regeneration potential. Callus was obtained from these explants on a Murashige—Skoog (MS) medium containing NAA (10 mg l^?1) and BAP (0.1–10 mg l^?1). Formation of shoots from root segments was direct without callus formation on MS medium containing BAP (10 mg l^?1) and NAA (1 mg l^?1). Shoot formation from epicotyl callus occurred on MS medium containing 0.25 mg l^?1 BAP and 0.1 mg l^?1 NAA. Formation of shoots from epicotyl segments occurred on MS medium containing BAP (0.5 mg l^?1) and NAA (0.1–1.0 mg l^?1), while rooting of regenerated shoots occurred in treatments containing 2.0 mg l^?1 NAA alone. This system provides a rapid method for propagation of ‘Troyer’ citrange.     

The influence of naphthaleneacetic acid on the growth of in vitro cultivated seedlings of Bromeliaceae

The role of NAA during in vitro seed germination and subsequent growth was examined in three Bromeliaceae: Guzmania minor var. ‘Vella’ Mez, Guzmania lingulata var. ‘Splendens’ Mez and Vriesea splendens var. ‘Fire’ Lem. NAA incorporation in the medium resulted in a strong stimulation of root and shoot growth, with an optimum response at 0.5–0.8 mg l^?1. It is hypothesized that the usual slow growth of bromeliad seedlings in soil is primarily due to poor rooting, which is the result of auxin deficiency.     

An assessment of the in-vitro multiplication rates of fourteen black currant cultivars

Summary

The in-vitro multiplication rates of fourteen black currant (Ribes nigrum) cultivars from a range of geographical origins were compared. Shoot tips and axillary buds were cultured for 21 days in a medium supplemented with 2.2 mg l^?1 6-benzylaminopurine. The highest shoot multiplication rate achieved was by cv. Ben Lomond, where the mean rate was 3.53 shoots from a single initial expiant. Mean rates between 1.50 and 3.33 were obtained from other cultivars. The results are discussed in relation to the parentage of each cultivar.     

Cryopreservation of in vitro almond shoot tips by vitrification

Summary

Shoot tips of two almond scion cultivars, ‘Ne Plus Ultra’ and ‘Nonpareil 15-1’, and one almond/peach hybrid rootstock were successfully cryopreserved using a one-step vitrification technique. Three week old in vitro cultures were cold-hardened at 4°C on the multiplication medium (Murashige and Skoog for ‘Ne Plus Ultra’ and the hybrid rootstock; Almehdi and Parfitt for ‘Nonpareil 15-1’) for three weeks. Shoot tips, 2–2.5 mm long, were excised and precultured for 1 d at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond scion cultivars and 60 min for the hybrid rootstock, and then stored under liquid nitrogen (LN) for at least 3 d. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄NO₃ or (NH₄)₂SO₄. Shoot regeneration was usually observed within 2–3 weeks. Survival after LN, recorded as the percentage of shoot tips that produced at least one new shoot four weeks after thawing, was 87.5, 60.0 and 72.5% for ‘Ne Plus Ultra’, ‘Nonpareil 15–1’ and the hybrid rootstock respectively. The one-step vitrification method is a promising simple technique for cryopreserving almond scion and rootstock shoot tips from in vitro cultures.     

Shoot production from onion callus tissue cultures

Shoots have been produced on callus derived from onion set and from seedling radicle tissue. Whilst callus of set origin responded optimally to medium containing the cytokinin 6-(3-methyl-2-buten-1-ylamino)-purine (2iP) at 2.00 mg l^?1 and naphthaleneacetic acid (NAA) at 0.06 mg l^?1, seedling radicle callus showed a range of response. The medium used for callus initiation, the age of callus, and the provision of a dark period following inoculation on to the organogenesis medium have been shown to be critical for shoot formation. A limited number of embryoids have been produced from cultures; their occurrence was usually associated with an increase in shoot numbers for the generative callus tissue. Meristemoid areas have been observed in light micrographs of callus cultured upon organogenesis media.     

Rejuvenation influences indirect organogenesis from leaf explants of Pomegranate (Punica granatum L.) ‘Kandhari Kabuli’

Sequential subculturing leads to a gradual physiological change in cells that may be termed ‘rejuvenation’. The effect of repetitive subculturing on callus induction and shoot regeneration from leaf explants of Punica granatum L. ‘Kandhari Kabuli’ were investigated. Surface-sterilised leaves were cultured on 1.0× Murashige and Skoog (MS) medium supplemented with 4.0 mg l^–1 α-naphthaleneacetic acid (NAA) and 2.0 mg l^–1 6-benzyladenine (BA) for callus induction. Shoots were regenerated from callus on 1.0× MS medium supplemented with 1.5 mg l^–1 BA, 0.5 mg l^–1 kinetin, and 0.25 mg l^–1 NAA. Subculturing of callus onto fresh medium maintained the rate of shoot formation and substantially increased the production of shoot buds up to the second subculture. Following further subculture passages, a lower shoot regeneration potential from callus was observed. A maximum shoot bud induction from callus of 63.9% was observed at the second subculture passage. The rate of multiplication of in vitro shoots increased until the fourth subculture, then became constant. Similarly, in vitro rooting of micro-shoots increased up to the third subculture, followed by a decline during further subculturing.     

Shoot production from cultured Allium porrum tissues

Large numbers of shoots have been obtained from the excised basal regions of leek plants, cultured on BDS medium. Shoots could be induced optimally upon media within the range of 6.0–8.0 mg 1^?1 6-(3-methyl-2-buten-1-ylamino)-purine (2iP) and 1.0–2.0 mg 1^?1 napthaleneacetic acid (NAA). Sub-culture of induced shoots onto fresh media, from the above hormone ranges, resulted in further multiplication. Sub-culture, onto fresh medium from the above hormone ranges, of the callus-like region around the bases of induced shoots and adjacent tissue, could also result in shoot production. Internally developing shoot primordia occurred in close proximity to meristematic regions, whereas superficially developing primordia did not show distinct affinities, although there was evidence of localised pockets of meristematic cells amidst senescing explant material at the surface of the culture. Such regions may represent early stages in the formation of the superficial primordia. The production of large numbers of leek shoots in vitro is mentioned in relation to present techniques of micro-propagation and cryopreservation.     

Shedding of the upper parts of grapevine shoots

Shedding of the upper parts of unlignified grapevine (Vitis vinifera L.) canes is frequent during autumn. Stress conditions during the growing-season lead to similar shedding of shoot tips. The separation process occurs above a node in the boundary area between the distal part of the diaphragm and the pith.Ethephon (2-chloroethyl-phosphonic acid) sprays, at 750 or 1500 mg l^?1, induced separation of the shoot tips of ‘Perlette’ and ‘Queen of the Vineyards’. Ethylene (C₂H₄) 200 vpm and ethephon at 10, 100 and 1000 mg l^?1 enhanced separation in shoot explants, whereas 2,4,5-TP (2,4,5-trichlorophenoxy-propionic acid) at concentrations above 1 mg l^?1 arrested it.This phenomenon may be regarded as a shoot abscission process which may represent adaptation of self-pruning of the grapevine.     

美国芦荟茎尖和茎段离体培养植株再生研究

以美国库拉索芦荟（Aloe veraL.）茎尖和幼茎段为外植体,MS培养基配以不同浓度的NAA和6-BA进行离体组织培养。结果表明,以MS＋6-BA3 mg·L^-1＋NAA 0.5 mg·L^-1培养基对芽的诱导效果最佳,芽的诱导分化率最高,且试管苗健壮;增殖培养基以MS＋6-BA 2 mg·L^-1＋NAA 0.2 mg·L^-1效果最佳,有利于芽苗增殖;生根培养基用1/2 MS＋IBA 0.5 mg·L^-1＋1.5%蔗糖效果最好;芦荟外植体用0.1%升汞消毒时间以10 min为宜。通过研究采用一些措施有效地控制玻璃化苗的发生。     

Evaluation,tissue culture propagation,and dissemination of ‘Saba’ and ‘Pelipita’ plantains in Costa Rica

Fruit characteristics of the disease-resistant bluggoe-type (ABB) cooking bananas (Musa × paradisiaca L.) ‘Saba’ and ‘Pelipita’ were compared with those of the susceptible ‘Currare’ (‘Horn’). Yields of ‘Saba’ and ‘Pelipita’ were similar to those of ‘Currare’; however, ‘Saba’ and ‘Pelipita’ yielded fewer hands with a greater number of small fruit when compared with ‘Currare’. Shoot-tip cultures of both clones were readily initiated on a modified Murashige and Skoog (MS) basal medium supplemented with N⁶-benzyladenine (BA) in combination with indole-3-acetic acid (IAA). Propagation cultures were initiated by splitting shoot tips along their longitudinal axis and re-culturing the individual pieces to basal medium supplemented with 5 mg l^?1 BA. Transfer of axillary shoots to hormone-free medium resulted in rapid and extensive root formation. Plantlet survival after transfer to methyl bromide-treated soil exceeded 90%. Establishment in the field was achieved following procedures normally used for vegetative propagation of this crop. Trial plantings of in vitro propagated ‘Saba’ and ‘Pelipita’ were established in various provinces of Costa Rica for grower evaluation and for future comparison of growth and reproductive development with plants of these and other cultivars propagated from corms.     

Micrografting of almond (Prunus dulcis Mill.) cultivars “Ferragnes” and “Ferraduel”

The success of various in vitro micrografting techniques, establishment of the rootstock, size of the microscion, and the effects of culture medium on the grafted seedling development for almond cultivars “Ferragnes” and “Ferraduel” were studied. In vitro germinated wild almond seedlings developed from seeds were used as rootstocks. Shoot culture initiation was successfully achieved from the above almond cultivars by culturing mature shoot tips from forced nodal buds, about 3–5 mm, on 0.7 mg/L BA and 0.01 mg/L NAA containing a MS medium. The regenerated adventitious shoots from in vitro cultures were maintained and proliferated by sub-culturing on a fresh medium every three to 4 weeks. Regenerated shoot tips, which were micrografted onto in vitro seedlings, resulted in the restoration of shoot proliferation. The results indicated that the most successful method for the grafting of tested almond cultivars was slit micrografting. High levels of micrograft take were achieved with all ranges of scions (4–15 mm) obtained from the regenerated shoot tips. Slow growth and lack of axillary shoot development on the micrografts were noticeable when the micrografts were cultured on hormone-free germination medium. In vitro micrografted plantlets were successfully acclimatized and no problems were encountered with the establishment of micrografted plants in vivo. The developed technique has demonstrated a high potential for application in the micropropagation of almond cvs. “Ferragnes” and “Ferraduel” and thereby, represents a feasible method for the renewal of almond orchards in Turkey and elsewhere in the world.     

Rapid propagation of Cucurbita pepo L. by culture of meristem tips

A tissue culture technique has been developed for the rapid multiplication of pumpkin (Cucurbita pepo L.) clones. Meristem-tips from seedlings of cultivar ‘Cinderella’ were grown initially on MS medium containing 2.56 mg l^?1 Kinetin and 8 mg l^?1 IAA, and then transferred to experimental media. Maximum shoot proliferation occurred on MS medium containing 1 mg l^?1 BA and no auxin. Cultures were rooted after 2–3 weeks on MS medium containing 8 mg l^?1 IAA and no cytokinin.     

The effects of growth substances and photoperiod on the development of shoot apices of Vitis cultured in vitro

‘Rougeon’ grapevines were recovered from shoot apices using in vitro culture. Shoot development from apices 0.5–1 mm in length, which contained 2–4 leaf primordia was obtained with 5 × 10^?6M benzylaminopurine and 5 × 10^?7M naphthaleneacetic acid (NAA) under a 10-hour photoperiod, but not under continuous light, in a 15-hour photoperiod, or in darkness. At least one vigorous shoot was produced by 100% of the cultures.Rooting of these shoots was induced under the same photoperiod with the same basal culture media containing only 10^?7 M NAA. Roots were produced by 91% of the cultures. Zero-, 16- or 24-h photoperiods were ineffective in promoting root development.Rooted plantlets were transferred into soil with 80% of them surviving. The grapevines were allowed to reach an average length of 2 m in a growth chamber before growth conditions were modified to induce dormancy. Vines were planted in a vineyard; after 2 growing-seasons we were unable to distinguish the micropropagated vines from ‘Rougeon’ vines propagated in the standard manner, on the basis of fruit or vegetative morphology.     

马铃薯茎尖小滴玻璃化法超低温保存及其再生植株的遗传稳定性

以马铃薯试管苗为试材,对其茎尖小滴玻璃化法超低温保存的影响因素进行了研究,并对再生植株进行了遗传稳定性检测。结果表明,马铃薯茎尖依次在含有0.3 mol · L-1和0.5 mol · L-1蔗糖的液体MS培养基中预培养各1 d后,在0 ℃下PVS2处理30 min,转到铝箔条上PVS2小滴上（约15 μL）,将粘有茎尖的铝箔条在液氮里蘸一下,然后直接装入盛满液氮的冷冻管中,投入液氮至少保持1 h。室温下用含有1.2 mol · L-1蔗糖的MS液体培养基解冻并洗涤30 min后,接种到MS + 0.5 mg · L-1 Zeatin + 0.1 mg · L-1 NAA＋1.0 mg · L-1 GA3恢复培养基上,存活率和再生率最高达79.91%和62.52%。通过SSR分子标记检测,再生植株的遗传稳定性没有发生改变。     

Nitrogen and calcium nutrition of Petunia × hybrida ‘Coral Sea’

The effects of N and Ca nutrition on plant growth and shoot elemental content of Petunia × hybrida Hort. Vilm. - Andr. ‘Coral Sea’ were evaluated. Nitrogen and Ca were applied separately or in combination in three experiments: (1) N at 0, 100, 200 or 400 mg l^?1; (2) Ca at 0, 75, 150 or 300 mg l^?1; (3) N at 0 or 100 mg l^?1 and Ca at 0 or 150 mg l^?1 combined factorially. Shoot and root dry weights, branch length and flower number were highest when plants received 100 mg l^?1 N. Plants treated with 150 mg l^?1 Ca had the highest shoot and root dry weights. Branch length was maximal at 300 mg l^?1 Ca.Nitrogen and Ca interacted to increase shoot dry weights, branch number and length, leaf area and flower number. Increasing N concentrations increased N and decreased P, Mn and Zn shoot contents. Calcium content of shoots increased while N, P and Mg decreased in response to increasing applications of Ca to petunia plants. Minimal N and Ca tissue concentrations for optimal P. × hybrida growth were 3.3 and 0.67%, respectively.     

Increased haploid production in Saintpaulia ionantha by anther culture

Anthers of Saintpaulia ionantha containing late-uninucleate stage pollen produced callus from the anther interior after 3–4 weeks culture on Murashige and Skoog medium supplemented with 1 mg l^?1 naphthylene acetic acid (NAA) and 0.5 mg l^?1 1,6-benzylaminopurine (BAP). Shoot regeneration occurred rapidly and up to 200 shoots could be recovered from callus derived from a single anther within 10 weeks. Examination of roottip mitoses from transplanted, established plants demonstrated that, with few exceptions, the plants were haploid, thus indicating that the callus was pollen derived. Exposure of buds to low temperature prior to anther excision was inhibitory to callus production. Shoot regeneration was studied by scanning-electron microscopy.     

Production of four commercially cultivated Echinacea species by different methods of in vitro regeneration

Summary

Efficient in vitro procedures for mass propagation of four commercially important Echinacea species have been deveoped. Plants of E. angustifolia, E. pallida, E. paradoxa and E. purpurea were regenerated by three methods, namely axillary bud proliferation, adventitious shoot formation and somatic embyrogenesis. Shoot tips obtained from in vitro germinated seedlings, adventitious shoots or somatic embryo-derived plantlets, when cultured on Murashige and Skoog medium enriched with 1 μM 6-benzylaminopurine, 2 μM kinetin, 0.5 μM indole-3-butyric acid and 4 mg^–1 paclobutrazol multiplied three-fold within 3–4 weeks in culture. Incorporation of paclobutrazol in the shoot multiplication medium was necessary to recover healthy and robust shoots suitable for rooting. Direct, high-frequency shoot formation on intact leaves of shoots grown on 6-benzylaminopurine and kinetin-supplemented media, an unusual and novel observation made in this study, occurred in all the species studied. Rooting of in vitro developed shoots was achieved relatively easily with Murashige and Skoog basal medium rather than with auxin-enriched media. Culturing of hypocotyl explants on medium containing 3,6-dichloro-o-anisic acid (commonly known as dicamba), or 2,4-dichlorophenoxyacetic acid, resulted in direct somatic embryogenesis in all the species examined. The presence of cytokinin was required for somatic embryo germination, but further development of germinated somatic embryos into normal plantlets occurred in Murashige and Skoog medium. We conclude that the procedures described here could be used for rapid propagation as well as genetic transformation of commerically cultivated Echinacea species.