Micropropagation of Zeyheria montana Mart. (Bignoniaceae), an endangered endemic medicinal species from the Brazilian cerrado biome

Zeyheria montana Mart. has become endangered, primarily because of deforestation of its habitat, its use as a medicinal plant extract, and the strong endemism of the species. In this study, an efficient protocol was established for the micropropagation and conservation of Z. montana germplasm using isolated mature zygotic embryos as explants. Embryos germinated in vitro 4 d after isolation and inoculation on modified Murashige and Skoog (MS) medium containing 2.0 mg/l gibberellic acid (GA₃). The addition of GA₃ also improved the germination index and accelerated the process of germination. Nodal segments from seedlings were placed on modified ¼-strength MS medium containing 0.1 mg/l 6-benzyladenine and 0.5 mg/l GA₃. Nodal segments produced 7.3 shoots per explant within 60 d. Following transfer of shoots to a medium containing 1.5 mg/l indole-3-butyric acid, roots formed. All plantlets obtained were successfully acclimatized under greenhouse conditions, and approximately 68.5 acclimatized plants could be obtained per seed each year. This protocol provides a method to preserve this rare and endangered medicinal plant.     

An efficient in vitro propagation,antioxidant and antimicrobial activities of Aphyllorchis montana Rchb.f.

An in vitro plant regeneration protocol was successfully established in Aphyllorchis montana, a saprophytic achlorophyllous orchid by culturing immature seeds. Among the six basal media evaluated for seed germination, BM-1-Terrestrial Orchid medium was found to be the best followed by Knudson C medium. Half-strength BM-1-Terrestrial Orchid medium was supplemented with different growth regulators either individually or in combinations for multiplication of shoots induction. Of the five cytokinins tested, thidiazuron at 6.8 μM was found to be most effective for multiple shoot induction yielding 17.24 ± 0.27 shoots after 10 weeks of culture. Addition of low concentration of α-Naphthaleneacetic acid (1.3 μM) in MS medium supplemented with the cytokinin thidiazuron (6.8 μM) favored shoot multiplication. The addition of organic additives to the medium containing thidiazuron enhanced the number of regenerated shoots. The plantlets were acclimatized, and the survival rate was 100%. Screening of the antioxidant and antimicrobial activities and estimation of total phenolics and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolics and flavonoid content than in vitro propagated plants.     

In vitro propagation of Hedychium coronarium Koen. through axillary bud proliferation

This report describes an efficient and reproducible protocol for large-scale multiplication of Hedychium coronarium plantlets. Axillary bud explants were cultured on Murashige and Skoog medium supplemented with 3 mg/l benzylaminopurine, 3 mg/l kinetin (KIN), and 0.2 mg/l thidiazuron, yielding a maximum of 13.2 ± 0.3 number of shoots. Sub-culturing of shoots every 4 weeks on fresh multiplication medium yielded a consistent proliferation rate. Shoot clusters containing three to five shoots were successfully rooted in KIN (3 mg/l) and indole acetic acid (0.5 mg/l), yielding a maximum of 6.3 ± 0.5 number of roots. Plantlets grown in vitro were acclimatized and subsequently transferred to the field for phenotypic evaluation. Random amplified polymorphic DNA and inter-simple sequence repeat analysis has confirmed the genetic uniformity of in vitro plantlets up to 2 years. After 2 years, these plantlets were transplanted to field, and evaluation of phenotypic characteristics was done. This study is of high significance as these could be commercially utilized for large-scale production of true-to-type plantlets.     

Morphological evaluation and antioxidant activity of <Emphasis Type="Italic">in vitro</Emphasis>- and <Emphasis Type="Italic">in vivo</Emphasis>-derived <Emphasis Type="Italic">Echinacea purpurea</Emphasis> plants

An effective in vitro protocol for rapid clonal propagation of Echinacea purpurea (L.) Moench through tissue culture was described. The in vitro propagation procedure consisted of four stages: 1) an initial stage - obtaining seedlings on Murashige and Skoog (MS) basal medium with 0.1 mg L⁻¹ 6-benzylaminopurine, 0.1 mg L⁻¹ α-naphthalene acetic acid and 0.2 mg L⁻¹ gibberellic acid; 2) a propagation stage — shoot formation on MS medium supplemented with 1 mg L⁻¹ 6-benzylaminopurine alone resulted in 9.8 shoots per explant and in combination with 0.1 mg L⁻¹ α-naphthalene acetic acid resulted in 16.2 shoots per explant; 3) rooting stage — shoot rooting on half strength MS medium with 0.1 mg L⁻¹ indole-3-butyric acid resulted in 90% rooted microplants; 4) ex vitro acclimatization of plants. The mix of peat and perlite was the most suitable planting substrate for hardening and ensured high survival frequency of propagated plants. Significant higher levels were observed regarding water-soluble and lipid-soluble antioxidant capacities (expressed as equivalents of ascorbate and α-tocopherol) and total pnenols content in extracts of Echinaceae flowers derived from in vitro propagated plants and adapted to field conditions in comparison with traditionally cultivated plants.     

Highly efficient in vitro proliferation and genetic stability analysis of micropropagated Ceropegiaevansii by RAPD and ISSR markers: A critically endangered plant of Western Ghats

Ceropegiaevansii McCann (family: Asclepiadaceae), a critically endangered plant of Western Ghats has acquired significant importance due to its medicinal implications, edible tubers, and ornamental flowers. This study deals with the optimization of axillary bud proliferation using nodal explants followed by genetic stability analysis of regenerants. Maximum number of shoots (11.6 ± 1.1) was observed on the Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (4.0 mg/l) and indole-3-acetic acid (0.3 mg/l) with 85% shoot multiplication frequency. In vitro-grown shoots were rooted best in 1/2 MS medium supplemented with indole-3-butyric acid (1.0 mg/l) with an average of 10.3 ± 0.9 roots per shoot and 92% rooting frequency. Plantlets were acclimatized best (90%) in a mixture of sterile soil, sand, and coco peat (1:2:1). Micropropagated plants were subjected to random amplified polymorphic DNA and inter simple sequence repeat markers analyses. Collectively, 759 bands were generated which were monomorphic and similar to the mother plant. Findings of this study are the first report on micropropagation and assessment of genetic stability of micropropagated plantlets in C. evansii which suggests that axillary shoot proliferation can safely be used as an effective tool for propagation and conservation of C. evansii.     

Efficient direct plant regeneration from immature leaf roll explants of sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) using polyamines and assessment of genetic fidelity by SCoT markers

A rapid and efficient method for in vitro direct plant regeneration from immature leaf roll explants of Saccharum officinarum L. (sugarcane) cv. Co 86032 was developed by the application of exogenous polyamines (PA). The effect of explant source from apical meristems and pre-culture of explants in the dark on shoot regeneration was studied. Adventitious shoot regeneration occurred on the proximal regions of immature leaf roll explants when pre-incubated in the dark for 2 wk and the regeneration response was decreased from the middle to distal end. A higher number of direct shoots (130 primary shoots explant^?1) and multiple shoots (657 secondary shoots explant^?1), were obtained with a combination of spermidine (103.27 μM), spermine (49.42 μM), and putrescine (31.04 μM) along with plant growth regulators. Shoot induction was increased up to twofold and multiplication was increased up to threefold in the medium supplemented with PA. Profuse rooting was observed in putrescine (93.12 μM), spermidine (68.84 μM), and spermine (24.71 μM), with mean number of 57 roots. A twofold increase in the number of roots was observed in medium supplemented with PA with respect to control cultures, which facilitated the successful transplantation and acclimatization process of in vitro propagated sugarcane plants. Histology and scanning electron microscopy analyses supported adventitious direct shoot regeneration from immature leaf roll explants. The genetic stability of in vitro regenerated plants was confirmed using start codon targeted polymorphism marker system.     

Micropropagation of Mandevilla moricandiana (A.DC.) Woodson

A protocol was developed for micropropagation of Mandevilla moricandiana (A.DC.) Woodson, a native plant from Brazil. Shoots, obtained from in vitro plantlets were used as source of nodal segments for shoot production from axillary buds. The nodal segments were grown on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine and/or indole-3-acetic acid to induce axillary bud elongation. After a 2-mo culture period, the medium supplemented with 1.0 mg?L^?1 6-benzyladenine gave the largest number of nodal segments per explant. The nodal segments obtained from plants developed under these conditions were grown on medium supplemented with different concentrations indole-3-acetic acid, ??-naphthaleneacetic acid, and indole-3-butyric acid. The use of the medium supplemented with indole-3-acetic acid and indole-3-buryric induced shoot elongation and shoot development, formation of basal callus, and/or indirect organogenesis of roots. Following transfer of shoots to soil, the plants with only basal callus showed 10% survival and developed roots from callus, while in vitro-rooted plants had a maximum 40% survival rate ex vitro. Regardless of the auxin added to the rooting medium, the acclimatization period allowed the plants rooted in vitro to develop their shoots fully. The protocol developed here is suitable for the production of shoots and rooted plantlets of M. moricandiana.     

In vitro propagation of the neotropical giant bamboo, Guadua angustifolia Kunth, through axillary shoot proliferation

Guadua angustifolia Kunth was successfully propagated in vitro from axillary buds. Culture initiation, bud sprouting, shoot and plant multiplication, rooting and acclimatization, were evaluated. Best results were obtained using explants from greenhouse-cultivated plants, following a disinfection procedure that comprised the sequential use of an alkaline detergent, a mixture of the fungicide Benomyl and the bactericide Agri-mycin, followed by immersion in sodium hypochlorite (1.5% w/v) for 10 min, and culturing on Murashige and Skoog medium containing 2 ml l^?1 of Plant Preservative Mixture^®. Highest bud sprouting in original explants was observed when 3 mg l^?1 N⁶-benzylaminopurine (BAP) was incorporated into the culture medium. Production of lateral shoots in in vitro growing plants increased with BAP concentration in culture medium, up to 5 mg l^?1, the highest concentration assessed. After six subcultures, clumps of 8–12 axes were obtained, and their division in groups of 3–5 axes allowed multiplication of the plants. Rooting occurred in vitro spontaneously in 100% of the explants that produced lateral shoots. Successful acclimatization of well-rooted clumps of 5–6 axes was achieved in the greenhouse under mist watering in a mixture of soil, sand and rice hulls (1:1:1).     

Anatomy of normal and hyperhydric leaves and shoots of in vitro grown Simmondsia chinesis (link) schn

Summary The anatomy of normal and hyperhydric in vitro shoots and leaves from micropropagated simmondsia chinensis (Link.) Schn. (jojoba) was compared with that of seedlings (control plants). In vitro normal plantlets displayed good development and survived during the acclimatization stage. In vitro hyperhydric plantlets presented numerous anatomical defects, such as hypertrophy of the mesophyll and of the stem cortex, malformed non-functional stomata, epidermal discontinuity, and xylem hypolignification; they did not survice acclimatization. The study of the anatomical features of in vitro jojoba shoots and leaves allowed determination of the structural condition of the plantlets and prediction of which plantlet would survive the critical acclimatization stage.     

Optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of shoot tip explants of green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100?% shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5?+?2.46 µM IBA and about 90?% of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301?+?AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23?°C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T₀ plants. Stable GUS activity was detected in flowers of T₀ plants and leaves of T₁ plants. PCR analysis of T₁ progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.     

Cell division study in ‘pollen mother cells’ and ‘pollen grains’ of in vivo and ex vitro plants in Drimiopsis botryoides

The various normal and abnormal stages of meiosis and pollen mitosis of Drimiopsis botryoides are described, and a comparison between naturally propagated in vivo and tissue culture derived ex vitro plants in respect to their cytological behaviour presented. We also describe the floral morphology and investigate the relationship between the floral developmental stages and the progression of microgametogenesis. In total, 33 bivalents are observed in diakinesis, which indicate the diploid number 2n = 66 and this number is cross-checked by a haploid set of n = 33 chromosomes in pollen mitosis. Only 6.8% and 4.9% meiotic abnormalities were recorded on in vivo and ex vitro plants, respectively, which led to the formation of non-viable pollen. Finally, the microspores have to develop into tri-cellular male gametophyte. Only 0.2% pollen grains are found with a micro-nucleus. Though the higher pollen viability was recorded on both in vivo (89.3 ± 4.1%) and ex vitro (92.1 ± 4.6%) plants, but surprisingly the pollen germination rate is extremely low with 13.6 ± 1.74% and 21.3 ± 1.55%, respectively. The present study obviously enriches the cytological database of D. botryoides and may help future research on androgenesis and genetic improvement.     

Rapidin Vitro propagation and enhanced stevioside accumulation inStevia rebaudiana Bert

A very rapid and efficient regeneration method has been established using mature expiants ofStevia rebaudiana Bert. Adventitious shoots were induced from nodal expiants of field-grown plants on four basal media supplemented with various combinations of auxins and cytokinins. The best performance (23.4 ± 2.1 shoots per expiant) was obtained on a Murashige and Skoog (MS) medium containing 2 mg L^-1 IAA and 0.5 mg L^-1 kinetin. Roots were then produced when thesein vitro- regenerated shoots were transferred to an MS medium supplemented with 3% sucrose and 2 mg L^-1 IBA. When acclimatized to soil, the rooted plants had a 98.4% survival rate. Following transplantation in the field, stevioside contents were similar between the regenerated plants (10.68 mg g^-1 dry weight) and the mother plants (12.01 mg g^-1 dw).     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

An efficient in vitro regeneration system of fieldpea (Pisum sativum L.) via. shoot organogenesis

In-vitro regeneration in fieldpea was achieved from immature embryonic axes and cotyledonary node explants of six genotypes on modified MS media supplemented with different concentration of plant growth regulators, 6-Benzylamino purine (BAP) and Naphthalene acetic acid (NAA). The best regeneration response, leading to multiple shoot formation efficiency (22.34 shoots/explant) was observed in the medium supplemented with 1.0 mg/L BAP and 0.2 mg/L NAA and best frequency (67.55?±?4.74) was achieved on medium containing 2.0 mg/L BAP and 0.4 mg/L NAA. The shoots were subcultured on a medium supplemented with a combination of 1.0 mg/L GA₃, 2.0 mg/L BAP and 0.4 mg/L NAA, which resulted in elongation of 85 % of shoots. Rooting attempted from the elongated shoots, on half strength MS medium and supplemented with three different auxins IBA, IAA and NAA separately, exhibited similar results. Alternatively, micro-grafting of in vitro regenerated shoots onto pre-germinated root stocks raised in green house facility was attempted with high success rate (75 %). The grafted plants could be successfully hardened, fertigated with Hoagland solution and distilled water in a ratio of (1:10) for acclimatization and further development. All the genotypes tested, produced multiple shoots that could be established to mature fertile plant, hence, the medium combinations used were found to be genotype neutral.     

Micropropagation of Abelmoschus esculentus L. (Moench.) for disease free plantlets through meristem culture

Protocol was established for mass in vitro propagation of okra using meristem culture. Meristems (0.3–0.5 mm in size) were isolated from shoot tips of three-week old in vitro grown seedlings. Isolated meristems were established rapidly in MS liquid medium containing 1.0 mg/l of BAP. For shoot development from primarily established meristem, semisolid MS medium having the same concentration of BAP was found to be the most effective. Rapid shoot multiplication of mericlone was achieved from node cutting cultured in 1.0 mg/l plus 0.5 mg/l GA₃, and a maximum of nine shoots were found from each node. Effective root development from the developed plantlets was successful in 1.0 mg/l IBA. More than 75% of the micropropagated mericlones plantlets were successfully acclimatised in soil up to maturity and found to be healthy.     

Plant regeneration from leaf explants of mature sandalwood (Santalum album L.) trees under in vitro conditions

Sandalwood (Santalum album L.) is a small evergreen, hemi-parasitic tree having more than 18 woody species that are mostly distributed in South Asia, Australia, and Hawaii. Its economical importance is derived from its heartwood oil, but its difficult propagation makes conservation essential. The percentage of seed germination is poor and germination time exceeds 12 mo. Vegetative propagation can be accomplished by grafting, air layering, or with root suckers, but the production of clones is inefficient and time consuming. In this study, efficient plant regeneration was achieved via indirect organogenesis from callus cultures derived from leaf tissues of S. album. Callus induction was induced when leaf explants were cultured on woody plant media (WPM) supplemented with either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid. The highest callus frequency (100%) was obtained when leaf tissue was cultured in the medium with 0.4 mg?l^?1 TDZ. Fresh weight (141.92 mg) and dry weight (47 mg) of leaf-derived callus were highest in the medium supplemented with 0.8 mg?l^?1 TDZ. The WPM medium supplemented with 2.5 mg?l^?1 BA?+?0.4 mg?l^?1 NAA was the most effective, producing the highest number of shoot buds (24.6) per callus. The highest number of shoots per explant (20.67) and shoot length (5.17 cm) were observed in media supplemented with 5.0 mg?l^?1 BA and 3.0 mg?1^?1 Kn, respectively. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). The highest rooting percentage (91.67) and survival were achieved using WPM media with 1.5 mg?l^?1 IBA. All plantlets survived acclimatization, producing healthy plants in the greenhouse. The current investigation showed efficient in vitro regeneration capabilities of S. album from leaf explants.     

Plantlet regeneration from cotyledon,cotyledon petiole,and hypocotyl explants via somatic embryogenesis pathway in roquette (Eruca sativa Mill)

Abstract

A high-efficiency plant regeneration protocol based on somatic embryo formation for Huining Roquette, an interesting ecotype of Eruca sativa Mill, was established for future transgenic applications. On Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), alone or in combination with 6-benzylaminopurine (BA) or kinetin (KT), the cotyledon explants, cotyledon petioles, and hypocotyls all produced embryogenic callus (ECs) or somatic embryos (SEs) to different extents. After transferring onto hormone-free MS medium, the ECs or SEs from the different explants and media, all of them developed shoots with a frequency of 6–48%, and then produced roots with a frequency of 2–29%. As regards the probability of shoot differentiation, cotyledon explants appeared similar to hypocotyls, but superior to cotyledon petioles; 2,4-D + KT worked more effectively than 2,4-D alone and 2,4-D + BA for callus induction and shoot differentiation. The optimal hormone combinations for plant regeneration of cotyledon, cotyledon petiole, and hypocotyl explants were 1.0 mg/l 2,4-D + 0.1 mg/l KT, 0.8 mg/l 2,4-D + 0.3 mg/l BA, and 1.0 mg/l 2,4-D + 0.3 mg/l KT, respectively. MS medium with 60–80 g/l sucrose was the most effective for improving SE maturation and germination.     

Some propagation methods for cloning holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.) plants

Holm oak (Quercus ilex L.), a typical evergreen tree of the Mediterranean area, is very important due to its ecological and economical values. Propagation of this species is extremely difficult and traditionally carried out only by seed germination. In this work, mature acorns were germinated in vitro and in peat substrate in aseptic and non-aseptic conditions. Explants from the seedlings obtained were propagated in vitro in WPM plus 4 μM BA. Plant regeneration was achieved from hypocotyls and root segments cultured in vitro on modified Gamborg medium plus 20 μM BA and 20 μM NAA. 13.8% of the hypocotyls and approximately 30% of the root segments developed both shoots and roots after 30 days of culture. Rooting of stem segments was obtained both in vitro and ex vitro by basal dipping in IBA solutions. Within ex vitro rooting, mother plant age had major influence on the percentage of rooting of the cuttings as the younger plants showed higher ability to root. In this way, Q. ilex plants could be propagated and cloned. The procedure described here would be a very useful tool for breeding programs since vegetative propagation of selected individuals can be achieved.     

In vitro propagation of Notocactus magnificus

Most commercially grown cacti can be easily propagated by seed and/or cuttings. A group of rare and endangered species does not fit into this category and is therefore a good candidate for in vitro propagation productions as a tool to overcome habitat and plant-destruction. The number of rare and endangered species of Cacti goes into about 100. Many show a low production and germination of seeds and plantlets are prone to damping-off, making the in vitro propagation a feasible alternative for the multiplication and conservation of their germplasm. The aim of the present investigation is to establish a protocol for the in vitro culture and plant regeneration of Notocactus magnificus, the blue cactus, a highly ornamental species, native to Brazil. The surface sterilization of the explants was achieved with immersion for 10 min in sodium hypochlorite solution for either seeds (0.25% v/v) or ribs segments (1% v/v). Callus formation was observed when explants were cultured on MS medium supplemented with sucrose at 2% (w/v), 2,4-dichlorophenoxyacetic acid 0.5 μM, benzylaminopurine 4.4 μM, thiamine HCl 0.4 mg l⁻¹ and i-inositol 100 mg l⁻¹. The regeneration of shoots was carried out on MS medium supplemented with either different concentrations of benzylaminopurine and 1-naphthaleneacetic acid, or kinetin and indole-3-acetic acid. The highest number of shoots occurred when MS medium was supplemented with benzylaminopurine 22.2 μM, sucrose 3% (w/v) and agar 0,6% (w/v). In vitro spontaneous rooting of shoots was observed after eight months under culture on MS medium. Only in vitro rooted shoots developed into normal plants under glasshouse culture conditions. This in vitro protocol should be useful for the conservation as well as mass propagation of Notocactus magnificus.     

Staining for viability testing,germination and maturation of Sambucus nigra L. pollen in vitro

Freshly released pollen of black elderberry (Sambucus nigra L.) was incubated under various culture conditions until germination was achieved. Optimal conditions for germination were determined and used for maturation of unicellular microspores in vitro. Staining with 5-diphenyltetrazolium bromide, propidium iodide and iodine potassium iodide was used to assess pollen viability, nuclear phase and maturation, respectively. The germination rate was highest when fresh pollen was agitated at 40 rpm in Petri dishes containing a liquid medium consisting of Brewbaker and Kwack salts, 15% (w/v) sucrose, 500 mg/l MES sodium salt, at pH 5.0; germination reached nearly 70% after only 1 h in culture. Under these conditions, and with addition of 200 mg/l glutamine, 260 mg/l cytidine and 500 mg/l uridine, uninucleate microspores developed into mature pollen at a 12% germination rate. Our report is the first demonstration of maturation of S. nigra microspores in vitro.