Asymbiotic seed germination,in vitro seedling development,and greenhouse acclimatization of the threatened terrestrial orchid <Emphasis Type="Italic">Bletia purpurea</Emphasis>

Procedures for asymbiotic seed germination and seedling acclimatization were developed for Bletia purpurea, a threatened North America native terrestrial orchid. Six asymbiotic orchid seed germination media (Knudson C, PhytoTechnology Orchid Seed Sowing Medium, Malmgren Modified Terrestrial Orchid Medium, Vacin &; Went Modified Orchid Medium, ½-strengh Murashige &; Skoog, and BM-1 Terrestrial Orchid Medium) were examined for their effectiveness in promoting seed germination and protocorm development of B. purpurea in either a 0/24 h or 16/8 h L/D photoperiod. Germination occurred regardless of medium or photoperiod treatment. However, advanced seedling development (Stage 6) only occurred on Vacin &; Went Modified Orchid Medium in the 16/8 h L/D photoperiod. Further effects of photoperiod on in vitro seedling development were also examined. Shoot length, leaf width, root number and length, and fresh weight and dry weight in the 16/8 h L/D photoperiod were all significantly different when compared to the 8/16 h and 12/12 L/D photoperiods. In vitro seedlings were readily acclimatized to greenhouse conditions. Seedlings showed high survival all potting media. Seedlings acclimatized in Fafard Mix 4 potting medium developed significantly longer roots. Corm formation occurred regardless of potting media used.     

Isolation and propagation of keratinocytes derived from Cashmere goat fetus

The study was conducted to isolate epidermal keratinocytes from Cashmere goat fetus with the aim to develop suitable conditions for keratinocyte cultivation and propagation. The methods developed for keratinocyte culture include (i) use of a feeder-layer of mitotically inactivated fibroblasts obtained from goat and mouse fetal skin, (ii) use of a substrate such as collagen IV, or (iii) without use of any substrate. Epidermal cell removal was established by enzymatically separating keratinocytes from 12 to 16 weeks aged fetal skin tissues treated with 0.125% trypsin solution overnight at 4 degrees C. The cells were maintained in all culture conditions with serum containing medium. Keratinocyte multiplication and proliferation were comparable in different culture conditions and the improved cellular attachment and growth have been obtained in cultures on feeder layers. Colony forming keratinocytes on feeder layer were heterogeneous in their growth potential. In feeder free conditions, high cellular density was required at plating for sub-cultivation as their poor attachment in culture dishes. This study reports the comparative efficacy of different culture conditions for keratinocyte isolation and in vitro propagation originating from Cashmere goat fetus.     

Progress in the biotechnology of trees

An increasing world population and rise in demand for tree products, especially wood, has increased the need to produce more timber through planting more forest with improved quality stock. Superior trees are likely to arise from several sources. Firstly, forest trees can be selected from wild populations and cloned using macropropagation techniques already being investigated for fruit tree rootstocks. Alternatively, propagation might be brought aboutin vitro through micropropagation or sustained somatic embryogenesis, with encapsulation of the somatic embryos to form artificial seeds. Tree quality could be improved through increased plant breeding and it is likely that experienced gained, to date, in the breeding of fruit species will be useful in devising strategies for forest trees. Since the development of techniques to regenerate woody plants from explant tissues, cells and protoplasts, it is now feasible to test the use of tissue culture methods to bring about improvements in tree quality. Success has already been achieved for tree species in the generation of somaclonal and protoclonal variation, the formation of haploids, triploids and polyploids, somatic hybrids and cybrids and the introduction of foreign DNA through transformation. This review summarizes the advances made so far in tree biotechnology, and suggests some of the directions that it might take in the future.     

In vitro propagation of Phalaenopsis via culture of cytokinin-induced nodes

A new procedure for in vitro propagation of orchids belonging to the genus Phalaenopsis was developed. In contrast to commonly employed propagation methods that make use of leaf, root, or shoot tip tissues, we have used elongated stems of 6-benzyladenine-induced young seedlings as starting material for propagation. The elongated stem consisted of several nodes of which top nodes were used for cyclic propagation of new explants and the middle nodes for producing shoots or multiple adventitious buds. The whole procedure of proliferation could be completed within 7 months, and about 2,300 plantlets were produced from a single induced stem in a single year. This method may be used for propagation of seedlings in the case of lack of seeds in orchid breeding or for propagation of vegetative buds developed on flower stalks of rare orchid varieties when available flower stalks are limited. It may also have great potential for the propagation of wild threatened orchid species.Abbreviations PLB(s) protocorm-like body(ies) - zeatin 6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine - 2ip 6-(,-dimethylallylamino)purine - kinetin 6-furfurylaminopurine - BA 6-benzyladenine     

Rapid in vitro propagation,conservation and analysis of genetic stability of Viola pilosa

A protocol for in vitro propagation was developed for Viola pilosa, a plant of immense medicinal value. To start with in vitro propagation, the sterilized explants (buds) were cultured on MS basal medium supplemented with various concentrations of growth regulators. One of the medium compositions MS basal + 0.5 mg/l BA + 0.5 mg/l TDZ + 0.5 mg/l GA₃ gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoots on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found to be the best medium for further in vitro shoot multiplication. 100 % root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA. In vitro formed plantlets were hardened and transferred to soil with 83 % survival. Additionally, conservation of in vitro multiplying shoots was also attempted using two different approaches namely slowing down the growth at low temperature and cryopreservation following vitrification. At low temperature retrieval rate was better at 10 °C than at 4 °C after conservation of in vitro multiplying shoots. In cryopreservation–vitrification studies, the vitrified shoot buds gave maximum retrieval of 41.66 % when they were precooled at 4 °C, while only 16.66 % vitrified shoots were retrieved from those precooled at 10 °C. Genetic stability of the in vitro grown plants was analysed by RAPD and ISSR markers which indicated no somaclonal variation among in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetical effects.     

In vitro propagation of Litsea cubeba (Lours.) Pers., a multipurpose tree

 A rapid clonal propagation system has been developed for Litsea cubeba. Following investigation of a range of cytokinins and a variety of explant sources (shoot tip, node, leaf and petiole) it was established that 6-benzyladenine with shoot tip explants gave optimal multiple-shoot induction. In vitro rooting on growth-regulator-free medium was possible and over 100 plantlets were successfully weaned to the glasshouse. Received: 11 July 1996 / Revision received: 24 December 1996 / Accepted: 22 March 1999     

Adventitious root induction in Ophiorrhiza prostrata: a tool for the production of camptothecin (an anticancer drug) and rapid propagation

Roots of Ophiorrhiza prostrata D. Don serve as a rich source of camptothecin (CPT), an anticancer drug. Because of the large-scale collection of its roots, the plant has become a threatened species. The present study accomplishes the induction of adventitious roots as a means for the production of CPT as well as for the large-scale propagation of this anticancer drug plant using leaf and internode explants. The biomass yield and CPT content of adventitious roots induced from different explants were compared to roots developed on ex vitro rooted stem cuttings. Adventitious roots were produced on half-strength Murashige and Skoog (MS) medium supplemented with 10.74 μM α-naphthaleneacetic acid and 2.32 μM kinetin at mean fresh weights of 0.753, 0.739 and 0.748 g roots from leaf, internode and shoot, respectively. CPT yield from in vitro derived roots after 50, 80 and 120 days of incubation (0.028, 0.06 and 0.1% dry weight, respectively) was not significantly different from those harvested at the same age from ex vitro rooted (0.03, 0.06 and 0.13%, respectively) stem cuttings. CPT from subcultured roots derived from solid (0.08%) medium was lower than from suspension culture medium (0.12%). Subsequent cultures of the adventitious roots showed a stable production of CPT (0.16%). The yield of CPT from 360-day-old plant-derived roots was 0.19%. Elicitation using methyl jasmonate and acetyl salicylic acid exhibited no enhancement in CPT yield. In vitro propagation through direct shoot regeneration was achieved from the adventitious roots upon transfer to MS medium with 8.87 μM N ⁶-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) with a mean of 21.2 shoots per culture in 50 days. The shoots upon subculture on medium having the same level of BA and IBA underwent rapid proliferation. The shoots transferred to field conditions after in vitro rooting exhibited 95% survival. Adventitious root induction, from leaf and internode explants, enables the feasible production of CPT as well as the large-scale rapid propagation of this species which can safeguard it from extinction.     

Community dynamics of evergreen broadleaf forests in southwestern Japan. II. Species composition and density of seeds buried in the soil of a climax evergreen oak forest

The species composition and density of seeds in the soil of a climax evergeen oak forest in the Kasugayama Forest Reserve, Nara, Japan were studied by directly extracting seeds from soil samples and using soil samples in planting boxes as the basis of germination tests. A total of 33 species were identified from all seeds collected in 6 stands: 11 evergreen broadleaf, 15 deciduous broadleaf, 2 coniferous, 2 liana, 1 herb, and 2 grass species. Species producing sap fruits and dry fruits accounted for 60% and 40% of the total number of species, respectively. The species composition of all the seeds was independent of the species composition of the forest vegetation. The mean density of the seeds was 22,134 seeds/m²·5 cm.Eurya japonica, of which seeds were found in all soil samples, was the most abundant species, followed byCryptomeria japonica, Ilex micrococca, andBoehmeria longispica. Pioneer species such asMallotus japonicus, Zanthoxylum ailanthoides, andAralia elata were found in all soil samples in spite of the paucity of adult trees in the forest. Seeds of evergreen oaks were relatively aboundant but no viable seeds were found. ViableE. japonica, I. micrococca, Symplocos prunifolia, andB. longispica seeds were abundant.     

Pseudocopulation of an orchid by male ants: a test of two hypotheses accounting for the rarity of ant pollination

Summary The orchid Leporella fimbriata is pollinated by pseudocopulation with winged males of the ant Myrmecia urens. This recently studied interaction provides a unique opportunity to examine the two current hypotheses concerning the apparent rarity of ant pollination systems worldwide. The first hypothesis requires a series of specialized growth forms and floral characteristics regarded as adaptations to ant pollination. L. fimbriata does not possess them. The second considers the pollenicidal effects of secretions from the metapleural gland of ants. These glands are absent in M. urens males and it may be that the occurrence of ant pollination requires the absence of metapleural glands in the vector.     

An efficient system for the production of clonal plantlets of the medicinally important aromatic plant: Salvia africana-lutea L.

An in vitro cultivation protocol was developed for S. africana-lutea a species threatened by over collection due to its importance as an aromatic medicinal plant in the Western Cape of South Africa. Adventitious shoot induction was most successful using hypocotyls as explants for propagation on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 4.4 μM BA only; 2.7 μM NAA and 4.4 μM BA; or 2.9 μM IAA and 9.3 μM kinetin respectively. For continuous subculture, IAA and BA (μM) at a ratio of 2.9:4.4 or 2.9:8.9 had the best regeneration potential producing approximately three plantlets per nodal explant. Plantlets had 4–5 nodes that could be utilized for the following subculture phase to induce axillary shoots. The tissue culture of S. africana-lutea not only favoured rapid multiplication but was also characterized by seasonal in vitro flowering that was in synchrony with that of plants growing in the wild. This propagation regime has the capacity for producing 2000–3000 plants from one shoot after 3 four-week long subculture cycles, making it highly attractive for implementation as an in vitro conservation strategy. The micropropagated plants were easily acclimatized (88%) within a month after rooting in vitro and planted ex vitro in a sand:soil:peat moss:vermiculite (1:1:1:1; v/v) mixture.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

In vitro propagation of green-foliaged Dracaena fragrans Ker.

Callus tissue was induced in young stem segments cultured on MS based media supplemented with 0.25–0.5 mg l^-1 2, 4-D. Shoots were differentiated on media containing 0.5–1.0 mg l^-1 BA and 0.5–2.0 mg l^-1 IBA or 0.1–0.2 mg l^-1 NAA. The same media were suitable for shoot multiplication. Shoot elongation and rooting were strongly inhibited by BA and stimulated by auxins IBA and NAA. Medium containing 0.5 mg l^-1 IBA was optimal for rooting. Root elongation was stimulated by light and inhibited in darkness. Transfer of rooted plantlets to outdoor conditions was feasible and special hardening procedures were not required. Among more than 5000 plants produced by this procedure only 9 off-type plants with variegated leaves were found.     

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

Abstract

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.     

影响杏黄兜兰种子萌发的因素

通过在不同条件下杏黄兜兰 (Paphiopedilumarmeniacum)种子萌发的观察 ,对影响其萌发的诸因子报道如下 :1)果实的生长期与种子的萌发率有关。实验表明种子采自生长期为 6 0d的果实 ,发芽率为 3 5 % ,采自 12 0d的果实 ,发芽率为 4 0 % ,采自 180d的果实 ,发芽率为 18 3%。 2 )培养基也会影响到种子的萌发 ,种子在 1 5MS内培养 ,萌发率明显高于培养于MS ,RE和改良HyponexNo 1内的种子。 3)培养基 (1 5MS)掺入添加物也会影响到种子的萌发率 ,如掺入 10 %椰子水会促使种子的萌发率达到很高的水平 ,掺入马铃薯泥 (5 0g L)或胰化胨 (2g L) ,种子的萌发率会达到较高的水平 ,而掺入香蕉泥则会对种子的萌发率产生负面影响 ,掺入活性炭 (2g L)会促使种子萌发和幼苗发育。 4 )与固态培养基相比 ,种子在液体悬浮培养基内的萌发速度更快 ,幼苗更为整齐划一。     

Induction of tuberisation in vitro with jasmonic acid and sucrose in an Australian terrestrial orchid, Pterostylis sanguinea

Effectsof jasmonic acid (JA) and sucrose on tuber formation were studied in a commontuberous terrestrial orchid Pterostylis sanguinea D.L.Jones& M. Clements (dark banded greenhood orchid) from southwestern Australia.Seeds were germinated symbiotically in vitro on an oatmealagar (OMA) in the presence of a mycorrhizal fungus isolated from the orchidhost. Ten week-old seedlings were transferred into culture vessels containingOMA supplemented with JA (at concentrations 0, 0.1, 1, 5 or 10M), sucrose (at concentrations 0, 5, 10 or 20 gl^–1) or combinations of each. Tuber development in alltreatments occurred approximately twenty-six weeks after seed sowing. Theaddition of 5 or 10 g l^–1 sucrose with JA to themedia resulted in higher frequencies of tuber formation in comparison to thecontrol. Significantly higher proportion of seedlings produced tuber when themedium was supplemented with 5 g l^–1 sucrose incombination with 5 M JA, compared to the control.In vitro tuber formation of Pterostylissanguinea can be improved by combined addition of appropriate levelsof JA and sucrose to OMA, which will aid in rapid in vitrotuberisation of terrestrial orchids.     

In vitro propagation, restriction fragment length polymorphism, and random amplified polymorphic DNA analyses of Angelica plants

Angelica acutiloba, a medicinal plant used as a natural medicine Touki, was clonally propagated through axillary buds in vitro. No substantial differences were found in the random amplified polymorphic DNA (RAPD) pattern between the original A. acutiloba and the plant propagated in vitro, suggesting no changes in the DNA sequences and structure during in vitro propagation. The genetic similarities of several Angelica plants were investigated by restriction fragment length polymorphism (RFLP) and RAPD analyses. The RFLP and RAPD patterns of A. sinensis Diels were substantially different from those of A. acutiloba. Using ten different restriction enzymes, no RFLP was observed in the varieties of A. acutiloba. By RAPD analysis, A. acutiloba varieties can be classified into two major subgroups, i.e., A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyamae Hikino. The varieties of A. acutiloba Kitagawa in Japan and Angelica spp. in northeast China exhibited a very close genetic relationship. Received: 13 March 1998 / Revision received: 28 July 1998 / Accepted: 21 August 1998     

<Emphasis Type="Italic">Pityopus californicus</Emphasis>: structural characteristics of seed and seedling development in a myco-heterotrophic species

Pityopus californicus (Eastw.) H. F. Copel., a monotypic member of the Monotropoideae in the family Ericaceae, is a myco-heterotrophic species with distribution limited to the Pacific Northwest of the USA. Young embryos of P. californicus developed mycorrhizal associations in seed packets that had been buried for up to 681 days, suggesting that seeds of P. californicus may require the presence of a fungus to achieve germination. Samples of nongerminated seeds and early stages in embryo and root development were subsequently processed for light microscopy, histochemistry, and transmission electron microscopy (TEM). Nongerminated seeds possessed a thick testa, lacked a shoot and root meristem, and consisted of an embryo with large parenchymatous cells containing protein bodies and starch grains as storage reserves. In the earliest developmental stage (seed coat still attached), fungal hyphae were present on the testa surface and between the testa and embryo. This stage was followed by embryo elongation, the organization of a root apical meristem, and the development of a well-developed fungal mantle surrounding the elongated embryo. At least two morphotypes were identified based on structural characteristics of the mantle. One of these, with ascomycetous septa, had Cenococcum-like features. Late-stage embryo/early root development revealed a typical mantle and Hartig net, with fungal pegs penetrating the outer tangential walls of epidermal cells. Transfer cell-like deposits of wall material, similar to those described in Monotropa spp., enclosed fungal pegs. The development of a Hartig net and fungal pegs suggests that nutrient exchange interfaces are required for seedling development.     

Cell divisions in cotyledons after germination: localization, time course and utilization for a mutagenesis assay

Post-germinative proliferation of cells was studied in cotyledons of Nicotiana tabacum L., Petunia hybrida Vilm. and Arabidopsis thaliana (L.) Heynh. Patterns of cell divisions after germination were characterized by clonal analysis in cotyledons of N. tabacum. The fate of initial cells, which are formed by the end of embryogenesis, was quite variable: cells could undergo between one to seven, and most often, between three to five anticlinal divisions after germination. Sector shape suggested that there were more divisions in length than in width, particularly at the periphery of the cotyledon. The boundaries of clones generated by irradiation of mature seeds did not intersect the midvein, and in most cases, did not intersect lateral veins. The time course of cell divisions during post-germinative development was analyzed cytologically in cotyledons of N. tabacum and P. hybrida. No divisions were detected up to the second day after sowing (DAS), when the radicle emerged. Cotyledon cells started to divide at a rapid rate between 2 and 3 DAS, reaching a mitotic index of about 2% at 3–4 DAS. A rapid decline followed the peak, and no divisions were detected 6–7 DAS. Similarities between leaf and cotyledon development are discussed. In addition, we show that divisions in cotyledons of N. tabacum and A. thaliana chlorophyll mutants can be exploited for a quick and sensitive bioassay from which the effects of various mutagens and DNA repair genes can be assessed.     

The use of in vitro culture to improve the propagation of Rhododendron ponticum subsp. baeticum (Boiss. & Reuter)

Rhododendron ponticum subsp. baeticum is endemic in the southern region of the Iberian Peninsula. The relict populations of this species are vulnerable, due mainly to difficult conditions for the establishment of seedlings, resulting in a virtual lack of sexual recruitment. In order to preserve the surviving populations, in vitro culture methods have been applied for both the sexual and the agamic propagation of the species. The in vitro germination of seeds was high when conducted with Anderson’s medium without plant growth regulators. The self-rooted seedlings obtained were easily transplanted to outside conditions. The presence of growth regulators in the medium interfered with the development of the seedlings, causing heavy callus formation. The in vitro growth of explants took place readily in Anderson’s medium plus 0.072 mg L⁻¹ of BA and 0.036 mg L⁻¹ of NAA although the explants did not form roots. Rooting was achieved by the basal dipping of the explants in hydroalcoholic solutions of 500 mg L⁻¹ IAA during the outside transplanting process. Therefore, the combination of in vitro grown explants together with ex vitro rooting, results in a good method for the agamic propagation of Rhododendron ponticum subsp. baeticum.