Acmella oppositifolia micropropagation by single-node culture

Acmella oppositifolia plantlet formation was achieved by subculturing single-node explants on Murashige and Skoog medium without growth regulators. The explants from 1-month-old in vitro plantlets produced shoots over a 7-day culture period. From these in vitro cultured nodes readily rooted shoots elongated on auxin-free MS medium. Plants produced were easily acclimatized and subsequently flowered in a greenhouse. This species is of medicinal value in tropical America from Mexico to Colombia.     

珠美海棠试管苗的土壤支撑生根培养和带坨移栽

以粘壤土做培养基支撑物,选用只含有0．5mg·L^-1IBA和15g·L^-1蔗糖的液体培养基,珠关海棠试管苗茎段生根培养的结果显示,试管苗生根率可达到100．0％,明显高于琼脂支撑培养,根长也显著提高,并且长出正常的根毛。土支撑培养生根的珠美海棠试管苗开瓶炼苗21d后带坨移入营养钵中,移栽后不喷雾、并毋需覆盖塑膜、午后空气相对湿度控制在50％-65％,其成活率达到92．2％;在开瓶炼苗21d后,黑暗中叶片气孔的关闭率从26．7％增加到88．5％。     

The ultrastructure of micropropagated and greenhouse rose plant stomata

Stomata of leaves from in vitro grown rose plantlets remain opened in the dark. The ultrastructure of their guard cells was studied after a 7 h light and a 7 h dark period, and compared to that of functional stomata from plants which have been acclimatized to greenhouse conditions. Qualitative and quantitative observations concerning the shape of the guard cells, mitochondria, plastids and starch grains, demonstrated the similarity in guard cell ultrastructure. The peculiarity of guard cell ultrastructure of in vitro cultured plants was the inability to close in the dark; vacuolar area was 40% of the whole guard cell area during both light and dark period whereas, in guard cells from greenhouse plants, the vacuolar area was 40% of the whole guard cell area during the light and only 25% during the dark period. These results indicate that stomata from in vitro plants are duly developed and possess an ultrastructure suitable for a typical functioning. The inability to close in the dark results from atypical water relation.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

We established a plant regeneration system for Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis. Embryogenic tissues were successfully induced on three kinds of Smith media from megagametophyte explants containing pre-cotyledonary embryos of C. obtusa plus-trees. Factors affecting somatic embryo maturation were examined. The concentration of polyethylene glycol 4000 in the medium was a critical factor for embryo maturation and its effective concentration was 150 g/l. The addition of 30 g/l maltose to the medium had a positive effect on embryo maturation, but sucrose was ineffective. The mature somatic embryos germinated at a germination frequency of approximately 60%, and the presence of activated charcoal was effective in stimulating plantlet growth. The plantlets acclimatized successfully in a greenhouse. To our knowledge, this is first report describing details of a plant regeneration method for C. obtusa via somatic embryogenesis.Abbreviations ABA Abscisic acid - PEG Polyethylene glycol 4000 - SM1 Smith Standard Embryonic Tissue Capture Medium - SM2 Smith Standard Embryogenesis Medium - SM3 Smith Embryo Develop Medium     

Thidiazuron induced high frequency axillary shoot multiplication in Psoralea corylifolia

The effect of thidiazuron (TDZ) was studied on in vitro axillary shoot proliferation from nodal explant of Psoralea corylifolia - an endangered medicinal plant. Proliferation of shoots was achieved on Murashige and Skoog (MS) medium supplemented with 0.5, 1, 2, 3, 4 and 5 μM TDZ. The maximum number (13.6 ± 1.4) of shoots per explant were obtained from nodal segment cultured on 2 μM TDZ for 4 weeks and this increased to 29.7 ± 2.1 on hormone free MS medium after 8 weeks. The in vitro proliferated and elongated shoots were transferred individually on a root induction medium containing 0.5 μM indole-3-butyric acid (IBA) and within 4 weeks 4.5 ± 0.5 roots per shoot were produced. The regenerated plantlets were transferred to 1:1 soil and vermiculite mixture and acclimatized with 80 % survival rate. Fully acclimatized plants were grown in garden soil in greenhouse and their morphological and physiological parameters were comparable with seedlings.     

Micropropagation of Allium wallichii kunth, a threatened medicinal plant of Nepal

Summary Bulbs and aerial parts of the Nepalese plant Allium wallichii are widely used for medicinal purposes and as a spice. Due to overharvesting the natural populations of the species have been increasingly reduced and the domestication of the species should be considered. For the purpose of the production of plantlets suitable for field culture, a micropropagation procedure based on multiple shoot culture has been established. Multiplication factors of 4.6 on average were possible on MS medium supplemented with 20 μM zeatin. After rooting on MS medium with 10 μM indolebutyric acid, plantlets were acclimatized to greenhouse conditions and transferred to the field with good success. Part of the PhD thesis of P. R. Malla.     

Plant regeneration of Agave tequilana by indirect organogenesis

Summary Indirect organogenesis was developed in Agave tequilana. Leaf segments and meristematic tissue from the central head (‘pi?a’) were evaluated as explant sources. A minimal-sized explant with high bud-forming capacity (19.5 BFC) was obtained through a cross section of meristematic tissue from in vitro plantlets. In callus culture, the best growth response was due to naphthalene acetic-acid (NAA) presenting a contrasting response compared to 2,4-dichlorophenoxyacetic acid (2,4-D). Regeneration from meristem segments and callus was obtained using 1.1 μM 2,4-D and 44 μM 6-benzylaminopurine (BA). The regeneration capacity of callus was maintained for 3 mo. Shoots regenerated were rooted in a hormone-free MSI medium and acclimatized in a greenhouse with a 100% survival.     

Effects of sucrose concentration,supporting material and number of air exchanges of the vessel on the growth of in vitro coffee plantlets

Growth of coffee (Coffea arabusta) plantlets cultured in vitroas affected by sugar, types of supporting material and number of air exchanges of the vessel was investigated. Single node cuttings of in vitro coffee plantlets were cultured on half strength MS medium with or without 20 g l⁻¹ sucrose. Two types of supporting material, agar and Florialite, and two levels of air exchange expressed by number of air exchanges per vessel, 0.2 and 2.3 h⁻¹, were studied. At the end of a 40-day culture period, fresh weight, shoot length, root length and leaf area of plantlets when cultured on Florialite soaked in sugar-free medium and under the higher number of air exchanges were greater than those in sugar containing medium. Callus was observed at the shoot base of plantlets grown on agar medium containing sucrose. Photosynthetic ability of coffee plantlets in vitro was also significantly increased when grown on sugar-free medium with the high number of air exchanges and Florialite as a supporting material. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of kiwifruit using non-axenic shoot tips

Kiwifruit (Actinidia chinensis Planch.) shoot tips were subjected to a standard surface sterilization procedure and cultured on a Murashige and Skoog basal medium in the presence of two surviving bacterial contaminants. The fresh weight increase of the cultures and the number of shoots produced were greater in liquid medium than in medium solidified with 0.4 or 0.8% agar. A greater number of shoots was obtained with 125 ml than with 50, 250, or 500 ml Erlenmeyer flasks. A concentration of 2 mgl^-1 N⁶-benzylaminopurine (BAP) gave a greater increase in fresh weight than either 0 or 4 mgl^-1.Shoots cut from proliferating cultures were dipped in 0.05% indolebutyric acid (IBA) and rooted directly in a peat: vermiculite: perlite mix. Over 93 % of 907 plantlets produced were successfully acclimatized. The productivity of the method was comparable to that reported for the axenic culture of meristems. The contaminants which survived the initial surface sterilization procedure thus presented no major obstacle to the in vitro propagation of kiwifruit.     

Cultural conditions affect somatic embryogenesis in Catharanthus roseus L. (G.) Don

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.     

Epidermal transpiration, ultrastructural characteristics and net photosynthesis of white spruce somatic seedlings in response to in vitro acclimatization

Mortality of transplanted somatic seedlings at the stage of acclimatization is often high and likely due to rapid change in environmental conditions. To investigate the potential of in vitro acclimatization of somatic seedlings before soil transfer, somatic seedlings of white spruce ( Picea glauca [Moench] Voss) were germinated on a liquid medium supplemented with sucrose. After 6 weeks in germination, sucrose was omitted from the medium for a supplementary 6 weeks at which time somatic seedlings were acclimatized in vitro in their germination tubes before transfer to soil. In vitro acclimatization of somatic seedlings was realized by transferring the test tubes containing the germinated somatic seedlings to the greenhouse for 9 days. During this period, the culture tube lids of acclimatized somatic seedlings were lifted progressively increasing air exchange between the tube and the greenhouse whereas, for non-acclimatized somatic seedlings the culture tubes were maintained closed during in vitro acclimatization. In vitro acclimatized somatic seedlings had higher asymptotic net photosynthesis ( P _n) at light saturation than non-acclimatized seedlings (6 versus 4.5 µmol m⁻² s⁻¹). At the end of the in vitro acclimatization period, a lower rate of epidermal transpiration was also observed for acclimatized somatic seedlings (3.85 versus 4.75% h⁻¹). Microscopic observations showed that starch granules were more abundant in needles of acclimatized somatic seedlings than in non-acclimatized somatic seedlings, probably as a result of their greater photosynthetic capacity. Needles from acclimatized somatic seedlings also showed more epicuticular wax projections than needles from non-acclimatized somatic seedlings. These structural changes may help somatic seedlings to restrict epidermal water loss and stomatal aperture.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Cistus clusii</Emphasis> Dunal,an endangered plant in Italy

Shoot tips and nodes from a genotype of Cistus clusii were cultured on a medium containing Murashige and Skoog macronutrients, Nitsch and Nitsch micronutrients, sucrose, iron, thiamine, myoinositol, and agar. This establishment medium, enriched with growth regulators and the biocide substances Plant Preservative Mixture and Thiabendazole lactate, improved explant survival by 14–16% and reduced contamination late in culture. For the proliferation stage, the explants rapidly formed axillary buds on a culture medium containing 6-benzylaminopurine (0.5 mg l⁻¹). The best response for rooting was obtained on a culture medium with a 0.1 mg l⁻¹ indolebutyric acid supplement. Rooted plantlets were acclimatized to greenhouse conditions and then transferred to the field in order to evaluate their phenotypic homogeneity. Karyotyping showed that the in vitro propagated plantlets have the same chromosome numbers as the mother plants. The success of this work indicates that micropropagation can be a useful tool for the conservation of C. clusii Dunal, an endangered plant in Italy.     

Somatic embryogenesis and plant regeneration from callus culture of Acacia catechu-a multipurpose leguminous tree

Plant regeneration via somatic embryogenesis was achieved from callus derived from immature cotyledons of Acacia catechu Willd. on Woody Plant Medium (WPM) supplemented with 13.9 M kinetin and 2.7 M 1-naphthaleneacetic acid. The addition of 0.9–3.5 mM L-proline to the medium influenced development of somatic embryos and also promoted secondary somatic embryogenesis. The light-green somatic embryos germinated on half-strength MS medium supplemented with 2% (w/v) sucrose. Somatic embryos germinated into plantlets that were acclimatized in the greenhouse and subsequently transferred to the field.     

First evidence of somatic embryogenesis from needles of 1-year-old Picea abies plants

Summary Somatic embryogenesis was obtained from hypocotyls and cotyledons of one month old plantlets of Picea abies. Embryogenic yield was higher with expiants from somatic embryo-derived plantlets (80 %) than with plantlets issued from zygotic embryos (10 %). This report also describes production of embryogenic calli from needles of 14 month old somatic embryo-derived plants cultivated in greenhouse. The influence of the physiological status and genotype of the mother plant on somatic embryogenic potential is discussed.Abbreviations ABA abscisic acid - (±) ABA racemic ABA - BAP 6-benzylaminopurine - CI callus inducing culture medium - NAA 1-naphtaleneacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and cryostorage of <Emphasis Type="Italic">Syzygium francissi (Myrtaceae)</Emphasis> by the encapsulation-dehydration method

Summary An efficient procedure for the in vitro propagation and cryogenic conservation of Syzygium francissi was developed. The maximum number of shoots per explant was obtained on a Murashige and Skoog (MS) medium supplemented with 4.5 μM benzyladenine and 0.5 μM indole-3-butyric acid (IBA). The in vitro-propagated shoots produced roots when transferred to MS medium containing IBA, indold-3-acetic acid, or naphthaleneacetic acid at various concentrations. Rooted microshoots were transferred to a coco-peat, perlite, and vermiculite (1∶1∶1) mixture, and hardened off under greenhouse conditions. Ninety-five percent of rooted shoots successfully acclimatized in the greenhouse. Shoot tips excised from in vitro-grown plants were successfully cryostoraged at −196°C by the encapsulation-dehydration method. A preculture of formed beads on MS medium containing 0.75 M sucrose for 1 d, followed by 6 h dehydration (20% moisture content) led to the highest survival rate after cryostorage for 1h. This method is a promising technique for in vitro propagation and cryopreservation of shoot tips from in vitro-grown plantlets of S. francissi germplasm.     

Yellow and red pigment production by cell cultures of Carthamus tinctorius in a bioreactor

Segments taken from flower-stalk internodes of Oncidium Sweet Sugar formed somatic embryos and shoot buds directly from wound surfaces or via nodular masses proliferation within 1.5 months, when cultured on a Gelrite-gelled 1/2-MS basal medium supplemented with thidiazuron (0.1–3 mg l⁻¹) in darkness. In light, when subcultured, these nodular masses proliferated into green compact callus, and produced somatic embryos, shoot buds and/or yellowish abnormal structures spontaneously. Supplementing 0.1–1 mg l⁻¹ NAA enhanced embryo formation, but retarded proliferation of shoot buds and yellowish abnormal structures. Somatic embryos that directly formed from wound surfaces of flower stalk explants usually developed into abnormal structures, but the callus-derived embryos could germinate into PLBs and eventually developed to normal plantlets on a hormone-free basal medium for 3–4 weeks. Both the embryo-and shoot bud-derived regenerants developed into healthly plantlets when potted in sphagnum moss and acclimatized in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of water stress mediated through agar on <Emphasis Type="Italic">in vitro</Emphasis> growth of potato

With the objective to develop a practical method of screening potato for drought tolerance, shoot and root growth in plantlets raised in vitro (from nodal cuttings drawn from in vivo as well as in vitro grown plantlets) were studied in three genotypes with known root mass production under field conditions. Different levels of water stress were induced using five concentrations of agar in MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium. Water potential of various media ranged from −0.70 MPa to −0.98 MPa. Water stress in culture adversely affected plantlet growth, and the responses varied with genotype and explant source. Genotype IWA-1 was less affected than Konafubuki and Norin-1. In the experiment with explants from in vivo grown plants, the time to rooting was considerably delayed in Konafubuki and Norin-1 by an increase in agar concentration, but no such effect was observed in IWA-1. In all media, the mean number of roots and root length was greater in IWA-1 than Konafubuki and Norin-1, and the latter two genotypes were at par. At 10 gl⁻¹ agar, IWA-1 had taller plantlets, heavier foliage dry weight, root volume, as well as root dry weight than Konafubuki and Norin-1, whereas the latter two genotypes were at par for all these characteristics. This pattern was similar to the reported pattern of these genotypes for root dry weight under field conditions. However, such similarity in the in vitro and field behavior of the tested genotypes was not observed when nodal cuttings drawn from in vitro plantlets were used as explants. It is concluded that in vitro screening of potato under specific and limited water stress conditions by raising plantlets from nodal cuttings drawn from in vivo grown plants may provide a system for effectively differentiating the genotypes for their expected root mass production under field conditions.     

Micropropagation and germplasm conservation of Central Park Splendor Chinese elm (<Emphasis Type="Italic">Ulmus parvifolia</Emphasis> Jacq. ‘A/Ross Central Park’) trees

Micropropagation offers opportunities to propagate, preserve and ship tree germplasm. It also reduces the risk of moving pathogens and insects with the germplasm due to built-in pathogen detection capabilities of aseptic cultures. For the past few decades, our laboratory has been involved in a project to preserve and restore a large, cold hardy, and historically important Chinese elm (Ulmus parvifolia Jacq. ‘A/Ross Central Park’) tree. Here we present three simple and efficient systems for its micropropagation, germplasm conservation and distribution: (1) in vitro plant formation from meristematic nodules (MNs), (2) plantlet generation from axillary buds, and (3) in vitro rooting of micro-cuttings from 20-years-old hedged stock plants. Newly flushed nodal segments were used as explants. WPM with 0.5 mg/l BA was found to be the best medium for meristematic shoot development and WPM supplemented with 2.0 mg/l 4-CPPU and 0.5 mg/l TDZ was best for meristematic nodule formation. Rhizogenesis of regenerants and micro-cuttings was best achieved on WPM with 1.0 mg/l NAA and 2% sucrose. Rooted plants were readily acclimatized to the greenhouse ambient environment and continued to grow well under greenhouse conditions. The survival rate of acclimatized plantlets under ex vitro conditions was 100% after 4 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 1,000 plants. Cycling of shoot explants and MNs through repetitive cultures was effective in scaling-up propagules.     

In vitro Culture and Temporary Immersion Bioreactor Production of Crescentia cujete

Crescentia cujete L. is a widely distributed medicinal tree with a diverse range of phytochemicals used as medicinal compounds. Seedlings of wild-harvested C. cujete were established in vitro and used as the starting material for the establishment of axenic cultures. Shoots were proliferated from nodal segments and were maintained over a period of more than 2 years by sequential subculture on a medium containing 1.0 μmol l⁻¹ kinetin. De novo regeneration was induced on petiole sections cultured onto a medium containing thidiazuron in combination with 2,4-dichlorophenoxyacetic acid. Axenic cultures were also used to test the efficiency of three different cultivation systems for production of biomass of C. cujete. Growth of plantlets in a temporary immersion bioreactor resulted in significant increases in biomass, leaf number, shoot height and transplant efficiency. Plantlets grown in the bioreactors were acclimatized under greenhouse conditions. Together, these experiments have established optimized parameters for propagation and growth of C. cujete plantlets in a sterile controlled environment for biochemical characterization and production of high-quality medicinal products.     

Plant regeneration from leaf explants of Rhodiola fastigiata

Summary An efficient plant regeneration protocol for rapidly propagating Rhodiola fastigiata (Hk. f. et Thoms.) S.H.FU, a traditional Chinese medicinal plant, was developed. Shoot organogenesis occurred from the leaf explants inoculated on medium with appropriate supplements of plant growth regulators. Up to 5.3 shoots formed per leaf explant cultured on a medium containing 13.32 μM 6-benzylaminopurine (BA) and 0.54 μM 1-naphthaleneacetic acid (NAA). Regenerated shoots formed complete plantlets on a medium containing 1.48 μM indole-3-butyric acid (IBA), and mature plants were established, acclimatized, and thrived in greenhouse conditions. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite Chinese medicinal plant.