Meristem culture and micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of oleoresin

Summary

Meristems of ginger with or without leaf primordia were induced to form shoots on three-quarter strength Murashige-Skoog’s (MS) medium containing sucrose 6%, coconut milk (CM) 20%, ascorbic acid (AA) 100 mg l^?1, glutamine (GL) 400 mg l^?1, activated charcoal (AC) 250 mg l^?1, 6-benzylaminopurine (BAP) 0.5 mg l^?1, indolebutyric acid (IBA) 0.4 mg l^?1 and agar 0.8%. Meristem-derived shoots exhibited consistent multiplication on three-quarter strength MS medium containing sucrose (3%), AA (100 mg l^?1), AC (100 mg l^?1), BAP (4–5 mg l^?1) and agar (0.8%). Liquid media (agitated or static) were less effective than a solid (agar-gelled) medium for micropropagation. Kinetin and naphthalene acetic acid (NAA) incorporated at various levels (0.01–0.8 mg l^?1) with or without added BAP and IBA neither improved plantlet formation nor enhanced shoot multiplication. The in vitro plants were successfully established in vivo and the rhizome yield was comparable with that of plants grown by conventional methods.     

Comparison of shoot induction ability of different explants in herbaceous peony (Paeonia lactiflora Pall.)

Shoot induction ability of explants of herbaceous peony was investigated in semisolid MS medium containing BA, TDZ and GA₃. Callus was readily induced from stem without node and petiole explants within 2 days of culture but failed to generate shoots. Adventitious shoots were successfully produced from meristematic regions only: bud eyes on nodal stem sections, and junctions of petioles and petiolules. No shoots were induced from internode sections, petiole without junctions, or leaf sections. Nodal sections were the most efficient explants. There were up to 20 shoots in one explant generated within 20 days of culture. TDZ was more effective than BA to induce shoots. The 100% shoot induction rate was obtained in medium containing 0.1–3 mg L⁻¹ of TDZ. However, higher concentrations of TDZ inhibited shoot elongation and only large leaf clusters were produced. Combinations of BA and TDZ failed to increase shoot induction rates but caused shoots shorter. The 2–60-min pretreatment of explants with 20 mg L⁻¹ TDZ solution was very effective to induce adventitious shoots directly, but both shoot number and shoot length tended to decrease as treatment time increased. GA₃ was beneficial for shoot and stem elongation.     

Clonal propagation of Poncirus trifoliate through culture of shoot primordia

Summary

In Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.     

The effect of light,darkness and temperature on micropropagation of the pear rootstock BP10030

There was no effect of irradiance level on surviving percentages of shoot tip explants of the pear rootstock BP10030, but low irradiance stimulated the initial growth of the explant. Irradiance had a strong effect on shoot multiplication. With an increase in photosynthetic photon flux (PPF) from 10 to 80 μmol m^?2s^?1, shoot number and length and shoot fresh and dry weights increased. The greatest number of shoots and the longest ones were obtained with a 16 h photoperiod, while the highest fresh and dry weight of shoots were produced with a 24 h photoperiod. Rooting percentage and the number of roots were markedly promoted under 80 μmolm^?2s^?1 PPF. Photoperiods of 8, 16 and 24 h produced similar effects on rooting percentages and the numbers of roots. Four to seven days of darkness were the optimum for rooting. Rooting percentage and the number of roots increased with increased temperature during darkness between 5 and 25°C. A further increase in dark temperature up to 30°C reduced rooting percentage and root number.     

Effect of gibberellic acid (GA3) on elongation and rooting of ‘St. Julien A’ rootstock in vitro

‘St. Julien A’ (Prunus instititia L.) rootstock was induced to proliferate shoots on a modified half-strength Murashige and Skoog (MS) medium. Cultures treated with 12.5 mg l^?1 gibberellic acid (GA₃) produced elongated shoots suitable for rooting. Elongated shoots were placed in media with indolebutyric acid (IBA) or indole-3-acetic acid (IAA) with or without a 16-day dark incubation. Light (16-h photoperiod) inhibited rooting. IAA (4 mg l^?1) was ineffective in promoting rooting. Rooting was best when shoots were incubated in the dark with IBA (4 mg l^?1). GA₃ was deleterious to shoots, causing chlorosis and apical die-back. Light regime interacted with auxin treatments in affecting shoot condition. Shoot condition was better on shoots treated with IBA and dark-incubated; while those treated with IAA were better when light-incubated.     

Rapid in vitro propagation of Dendrobium hybrids through direct shoot formation from foliar explants,and protocorm-like bodies

In vitro propagation protocol for Dendrobium hybrids Sonia 17 and 28, two highly priced commercial cut flower cultivars through direct organogenesis from in vitro derived foliar explants was established. Rapid clonal propagation was achieved by subsequent induction of protocorm-like bodies (PLBs) and its conversion to shoots. No significant differences were observed in the induction of direct shoots, shoot multiplication, PLBs formation and subsequent shoot development and rooting of shoots between the two cultivars. Leaf explants from flower stalk node derived shoots cultured on half-strength Murashige and Skoog (MS) medium supplemented with 44.4 μM N⁶-benzyladenine (BA) developed more than seven shoots per explant. The isolated shoots transferred onto the same medium induced more than eight PLBs from the base within 60 days, which upon transferral to fresh medium having the same level of BA facilitated rapid proliferation. More than 200 PLBs were yielded from fifth subculture. Half-strength MS medium containing 6.97 μM kinetin (Kn) facilitated conversion of more than 90% PLBs to shoots. PLBs exhibited proliferation without decline up to the 15th subculture. Half-strength MS medium with 2 g l⁻¹ activated charcoal was the best for in vitro rooting. Plantlets of the hybrids exhibited more than 80% ex vitro establishment.     

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.     

Progress towards clonal propagation of Camellia japonica cv. Alba Plena by tissue culture techniques

Summary

Optimal rooting conditions have been determined for shoot cultures of Camellia japonica cv. Alba Plena derived from a 50 year old tree: dipping the base of shoots in 1 g l^?l solution for 15 min, followed by 12 days’ darkness, induced 87% rooting in shoots cultured with Woody Plant Medium (WPM) macronutrients. Halving the auxin concentration or exposure time considerably reduced this rate, and root formation was severely inhibited if an initial dark period for the entire shoot was not used. The type of support (agar or paper bridges) did not significantly affect the rooting percentage or number of roots per rooted shoot, but liquid media induced greater root elongation. No significant differences were observed between the use of WPM and a modified Heller’s medium as regards the rooting percentages achieved, but the number of roots formed was considerably greater with WPM, as was the survival rate after transfer to soil (70% for transfer as against 35% with the modified Heller’s medium). Best survival rates were achieved when shoots were transferred to the acclimatization soil 12 days after auxin treatment, i.e. immediately after the dark period.     

Castanea sativa plantlets proliferated from axillary buds cultivated in vitro

Chestnut plants were proliferated in vitro from axillary buds of juvenile shoots. N6-Benzyl-aminopurine (BAP) at 0.1?0.5 mg l^?1 was optimal for shoot multiplication. The important role played by the macronutrient formula on shoot multiplication, and especially on the rooting-stage, is emphasized. The MS ($\text{1}\text{2}$ NO₃) macronutrients gave the best rooting percentage as well as the highest number of roots per rooted shoot. In these experiments, shoots remained in the 3 mg l^?1 indole-3-butyric acid (IBA) medium for 12 days, after which they were transferred to an auxin-free medium where roots developed fully. Optimum rooting was achieved by immersing the 1 cm basal end of shoots in concentrated IBA solutions (0.5?1 mg ml^?1) for periods ranging from 2 to 15 min.     

Influence of BAP Concentrations and Nutrient Medium Composition on <Emphasis Type="Italic">In Vitro</Emphasis> Regeneration of ‘Öküzgözü’ and ‘Boğazkere’ (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) Cultivars

This study has been conducted with the aim to determine the type of nutrient medium that can be used in micropropagation studies for ‘Öküzgözü’ and ‘Bo?azkere’ and to specify BAP concentrations. In the study where ejectors with a length of 0.7–0.8?cm that are obtained with single-node culture are used, it was focused on four different nutrient media such as MS, DKW, QL and WPM and on six different concentrations such as 0.2–0.4–0.6–0.8–1.0–1.5 mg l^?1 BAP. Single-node suspension explants which will be used in initiating the culture, are taken into culture in MS nutrient medium and the nutrient medium is supported with 30?g l^?1 sucrose, 6?g l^?1 agar and 1?mg l^?1 BAP. In the trial environment, parameters such as number of shoots, shoot length (cm), number of nodes and callus ratio have been investigated. For both grape varieties, the best outcome was obtained with MS nutrient medium with respect to number of shoots, shoot length, and number of nodes. These values were found as 4.66, 1.24 and 6.39 for ‘Öküzgözü’ variety respectively, whereas they are determined as 6.28, 1.15 and 6.81 for ‘Bo?azkere’ variety respectively. In both grape varieties in DKW nutrient medium, starting from the 2nd week of culture, obscuration began to appear on the shoots and after this stage no other development has taken place.     

核桃试管嫩茎生根的形态结构及激素调控研究

 以核桃品种‘新早丰’试管嫩茎为试材, 对其诱导生根过程中的形态结构及相关的生长素(IAA) 和脱落酸(ABA) 变化进行了研究。证实诱导生根过程中核桃嫩茎不定根原基发生于形成层, 特别是髓射线正对的形成层部分; 根原基起始分化期为诱导第6 天左右, 伸长期是第10天; 如果12 d之后仍放在诱导培养基中, 生根率下降, 并且出现茎基愈伤化、茎尖变黑和叶片脱落等现象; 若生根诱导10 d后转入无植物生长调节剂培养基, 培养5 d左右可见根尖突出表皮, 根系发育正常; 同时与不定根形态发生相应的内源IAA和ABA的变化是根原基的发生期和伸长期, 内源IAA出现高峰, 内源ABA呈上升趋势,IAA /ABA值在根原基的发生前为最大, 随后降低。本研究不仅从形态结构证实了二步生根法的合理性, 而且从生理学角度阐述了不定根发生的IAA /ABA调控机制。     

Influence of medium composition and vessel ventilation on in vitro propagation of Phillyrea latifolia L.

Micropropagation of Phillyrea latifolia L. a wild species present in Mediterranean coastal areas having drought and salt tolerance was performed using explants from adult plants. Shoots were induced from nodal explants on the Rugini’s initial medium (IM). Then these were proliferated on either Rugini olive medium (OM) or Linsmaier and Skoog (LS) medium, each supplemented with 2.22 μM 6-benzylaminopurine (BA) or 4.56 μM zeatin (Z). Rooting (66.1±11%) was induced on shoots grown in perlite soaked with half-strength Rugini olive proliferation medium (OMr) containing 2.69 μM α-naphthaleneacetic acid (NAA) and 160 mg l⁻¹ putrescine. Both shoot multiplication and rooting were performed using Magenta^® GA-7 (Sigma) vessels either non-permeable or permeable to gas exchanges. Contamination (about 40%) was observed during the first five passages notwithstanding the addition of cefotaxime to the culture medium, but a high proliferation rate (90%) of explants provided enough healthy plant material. The highest shoot proliferation was observed on LS medium and zeatin whereas the presence of the ventilated filters reduced fresh weight of explants growing on LS media and did not affect shoot growth on OM media. During rooting, the use of ventilated vessels in comparison with the closed ones enhanced development of roots, and doubled the dry weight of plantlets. The vessel ventilation combined with the artificial substrate (perlite) was beneficial for in vitro acclimatization of rooted Phillyrea plantlets.     

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.     

Propagating palms in vitro with special emphasis on the date palm (Phoenix dactylifera L.)

Tissue-culture methods are described for the vegetative propagation of several palm species either through shoot tip culture or plantlet differentiation via embryogenic callus. The influence of explant size, medium composition and physical environment required for the establishment of palm shoot tips in vitro was determined. Date palm (Phoenix dactylifera L.) seedling shoot tips of various sizes were cultured in either liquid or agar modified Murashige and Skoog (MS) medium containing 0.0–1.0 mg 1^?1 α-naphthaleneacetic acid (NAA) and 0.0–15.0 mg 1^?1 benzyladenine or N⁶-(Δ²-isopentenyl) adenine (2iP) in order to enhance shoot growth and induce axillary budding. Satisfactory date palm shoot tip growth and proliferation was obtained from explants that were 3 mm in length, consisting of the apical meristem region and 2–5 adjacent leaf primordia. Optimum shoot tip development and axillary budding was obtained by initially establishing explants on an agar medium for 2 weeks, then transferring to a liquid medium. Shoot tips from several palm species were cultured on MS media containing 100 mg 1^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg 1^?1 2iP and 3 g 1^?1 activated charcoal, or on MS medium containing 1 mg 1^?1 NAA and charcoal, to determine their morphogenetic responses in vitro. Shoot tips of Metroxylon sp., Phoenix canariensis Hort. ex. Chabaud., P. dactylifera ‘Khalasa’, ‘Thoory’ and ‘Zahidi’, and P. roebelenii O'Brien planted on medium with 2,4-D and 2iP initiated callus, asexual embryos and free-living plantlets after 4–8 months in culture. Shoot tips from Erythea edulis S. Wats., P. canariensis, P. dactylifera ‘Khalasa’, Thoory' and ‘Zahidi’, Washingtonia filifera Wendl. and W. robusta Wendl. cultured on medium containing NAA developed into plantlets with well-developed leaves and adventitious roots within 2–6 months from the time of planting. In some cases, cultured date palm shoot tips gave rise to axillary buds.     

Effect of agar,galactomannan and indole-butyric acid on in vitro rooting of the pear cultivar ‘Durondeau’ and apple rootstock cultivar ‘Marubakaido’

Summary

The influence of partial substitution of agar by galactomannans in culture media supplemented with different concentrations of indole-3-butyric acid (IBA) was studied on in vitro rooting of pear (Pyrus communis L.) cultivar ‘Durondeau’ and apple rootstock (Malus prunifolia Borkh.) cultivar ‘Marubakaido’. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum) seeds. The results obtained with mixtures of agar and galactomannan (3 g l^–1 each) were compared with those from media solidified with a standard concentration of agar (6 g l^–1). The rooting of pear shoots was enhanced significantly in the presence of a mixture of agar plus cassia galactomannan compared to medium solidified with agar only. The modified media promoted a higher number of roots than the control, and increased the percentage of rooted shoots. A maximum of 84.8% rooting was obtained on half-strength MS medium (¹?2MS) supplemented with 0.49 µM IBA and solidified with a blend of agar plus cassia galactomannan. For the apple rootstock, only the number of roots per shoot was influenced significantly by the addition of galactomannan to the rooting medium. The highest number of roots per shoot was 16.67 on ¹?2MS medium gelled with a mixture of agar plus guar galactomannan supplemented with 4.90 µM IBA. The behaviour of the agar-galactomannan gel and the possibility of reduced costs when compared with systems containing only agar, suggest new biological and commercial applications for galactomannans.     

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.     

Effect of plant density and macronutrient nutrition on Delphinium shoot cultures

The effect of shoot density on the uptake of macronutrients from MS medium and growth rates of Delphinium shoot tissue cultures was determined. Multiplication rates and uptake of phosphate, nitrate and sugar per shoot increased with decreasing shoot density. Increasing the concentration of total nutrients significantly increased both fresh weight gain and multiplication rate at the high plant density (15 shoots 50 ml'1 medium) usually used for micropropagation. However, increasing the phosphate concentration (phosphate being the nutrient most rapidly depleted in the medium) resulted in higher fresh weight only, while the multiplication rates of shoots remained similar.     

In vitro vegetative multiplication of Fuchsia hybrida

Rapid development of axillary buds from shoot-tips and nodes of 18 cultivars of Fuchsia hybrida was obtained on solid Murashige and Skoog medium with BAP (1 mg l^?1 and an auxin (0.1 mg l^?1). NAA as the auxin appeared to be more active than IAA or IBA. Vegetative shoots were subsequently isolated and developed up to 15 supplementary axillary shoots on the same solid medium. Agitated and non-agitated liquid media of the same composition were less effective. One-cm long shoots could be rooted in 20 days in the presence of IBA before being transferred to soil.     

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.