Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.     

Asymbiotic seed germination,mass propagation and seedling development of Vanda coerulea Griff ex.Lindl. (Blue Vanda): An in vitro protocol for an endangered orchid

The application of modern biotechnology for conservation of any endangered species requires an efficient in vitro regeneration protocol. In this study a reliable protocol was developed for in vitro seed germination, protocorm multiplication and subsequent plantlet regeneration of Vanda coerulea, an endangered orchid species. Among the four basal media evaluated for asymbiotic seed germination, Phytamax was found to be the best followed by Murashige and Skoog (MS). Phytamax was also found good for protocorm development. For protocorm like body (PLB) regeneration, protocorms were then further cultured on Phytamax media fortified with different phytohormones either individually or in combinations. The frequency of protocorm like body (PLB) regeneration significantly relied on kinds and concentrations of plant growth regulators used. A combination of 1-naphthaleneacetic acid (NAA) (5.36 μM) and 6-benzyle amino purine (BAP) (3.80 μM) was found to be suitable for maximum PLB regeneration. Healthy plantlets were induced from PLBs when cultured on same basal medium supplemented with activated charcoal (AC – 3.0 g/l). Plantlets with well developed leaves and roots were transplanted to pots filled with a mixture of charcoal, brick pieces and sphagnum moss and transferred to the greenhouse. This protocol will enable mass propagation and conservation of this exquisite orchid.     

In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl.

The effects of sodium hypochlorite treatment and growth medium on in vitro seed germination and seedling growth of the leopard orchid (Ansellia africana Lindl.) were investigated. Forty minutes treatment of seeds with 1.75% sodium hypochlorite stimulated germination (70.6%) and seedling development when measured at Stage 5 (emergence of first leaf) on P668 medium after 8 weeks of culture. The growth medium played a significant role in determining the germination response of A. africana seeds. Dark pretreatment of seed cultures significantly enhanced the germination percentage and the growth of rhizoids on the protocorms. Leaf growth in terms of length was substantially enhanced on P668 medium. It was significantly reduced in modified Knudson C medium after 16 weeks of culture. Seedlings developed on P668 medium showed a significantly better growth performance in relation to leaf length, leaf number, root length, fresh and dry weights per plant in vermiculite after 12 weeks of ex vitro growth in a mist house with 90% relative humidity. Further studies developing a suitable method for in vitro symbiotic germination could assist in reintroduction of this threatened orchid species into the wild.     

High frequency multiplication of Phalaenopsis gigantea using trimmed bases protocorms technique

This study was conducted to determine the effects of coconut water (CW) and activated charcoal (AC) on multiplication of Phalaenopsis gigantea protocorms. The protocorms used for this study were obtained by germinating seeds in vitro. Protocorms with trimmed and untrimmed bases were cultured on XER basal medium containing 0, 10, 15 or 20% (v/v) CW; and 0, 1, 2 or 2.5 g AC l⁻¹. Trimmed protocorms exhibited the highest percentage of proliferation on a medium containing 15% (v/v) CW and 2.5 g AC l⁻¹ (56.82 ± 38.86%) with an average of 4.24 ± 2.89 protocorms formed per protocorm. Untrimmed protocorms cultured on a medium containing 20% (v/v) CW without AC produced the highest percentage of new protocorms (6.93 ± 6.28%) with an average of 0.72 ± 0.57 per protocorm. When CW was added to a medium singly, 10% (v/v) CW induced a higher degree of proliferation on trimmed protocorms (5.68 ± 10.14%) with an average 0.50 ± 0.84 new protocorms per protocorm. Untrimmed protocorms proliferate to a much lower extent (2.57 ± 2.74%) with an average of 0.72 ± 0.57 protocorms per protocorm when cultured on a similar medium. A high concentration of CW enhanced proliferation on untrimmed protocorms, but increased mortality of trimmed protocorms. The addition of CW with AC to media increased protocorm proliferation and survival of both trimmed and untrimmed protocorms. When cultured on all media, trimmed protocorms produced a higher number of new protocorms (an average 0.5–7.0) as compared to untrimmed protocorms (0.3–1.9). Comparative studies showed that trimmed protocorms produced up to 10 times more new protocorms than untrimmed ones. Altogether this study showed that trimmed protocorms cultured on a medium containing CW and AC can be used for high-frequency multiplication of P. gigantea seedlings.     

Effects of development,temperature, and calcium hypochlorite treatment on in vitro germinability of Phalaenopsis seeds

There are no standardized procedures for sanitizing orchid seeds for propagation by tissue culture and there is insufficient information about the optimum stage of orchid seed development for best germination. Phalaenopsis amabilis flowers were hand-pollinated and fruits harvested 90, 105, and 120 d after pollination (DAP) for seed developmental analysis. Embryo cell number per seed was counted after staining with 4′-6-diamidino-2-phenylindole and viewing through a confocal microscope. Germination percentage and cell number per embryo increased from 14 to 61% and 41 to 66%, respectively, during fruit development from 90 to 120 DAP. Seeds from mature, browning (∼140 DAP) Phalaenopsis Sogo Lit-Angel and Phalaenopsis spp. breeding line 9450 seed pods failed to germinate until frozen at −196, −80, or −18 °C and thawed or chilled at 4 °C for 10 d. Germinability in 140 DAP seeds was correlated with cracked testa after freezing and thawing. P. amabilis seeds were treated with 0, 5, 10, or 15% calcium hypochlorite (CH) for 5, 10, or 15 min. Ninety six percent of untreated seeds from 90 DAP fruit produced protocorms within 40 d after sowing (DAS). Exposing seeds to 5% CH for 10 or 15 min decreased germination to 85 and 73%, respectively. Exposure to 10 or 15% CH for 5, 10, or 15 min produced seed germination percentages of less than 40%. Protocorms developed root hairs and shoot primordia by 50 DAS and an average of one leaf and root by 85 DAS after treatment with either 0 or 5% CH. Higher concentrations delayed or inhibited protocorm development. Green fruits 120 DAP produced the highest percentage of protocorms, while ∼140 DAP seeds from browning fruit were dormant but cold treatments increased germination.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

Mass propagation of Satyrium nepalense D.Don.—A medicinal orchid via seed culture

An in vitro plant regeneration protocol was successfully established in Satyrium nepalense, a terrestrial orchid by culturing immature seeds from unripe fruits. Seeds were germinated on Murashige and Skoog, Knudson C and Knudson C modified Morel medium. The germination of the seeds and development of protocorm was highest in MS medium (86.7%) followed by Knudson C modified Morel medium (74%) and Knudson C medium (61.2%). Among the cytokinins tried for multiple shoot induction from the protocorm, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) was found to be superior. Indole-3-butyric acid (IBA) was effective for inducing healthy roots. Well-developed plantlets were hardened in vermicompost (leaf litter + cow dung 1:1), sand and coconut husk (1:1:1).     

Gibberellin producing Neosartorya sp. CC8 reprograms Chinese cabbage to higher growth

Gibberellins production by soil fungi received little attention, although substantial work has been carried out on other growth promoting aspects of soil borne fungi. We investigated gibberellins production and growth promoting capacity of a novel fungal strain of Neosartorya, which was isolated from the roots of Chinese cabbage (Bassica rapa L. ssp. pekinensis). Fungal culture filtrates (CF) obtained from pure cultures of 16 endophytic fungi were bioassayed on Waito-C, in order to investigate plant growth promoting capacity of these fungi. The fungal isolate CC-8 induced maximum shoot length of Waito-C (13.0 cm) as compared to control treatments. In a separate experiment, the CF of fungus CC-8 significantly promoted plant length and biomass of Chinese cabbage. The fungal CF also increased endogenous bioactive GA₁ and GA₄ contents of Chinese cabbage. Gibberellin analysis of CF of CC-8 showed presence of both physiologically active and non active gibberellins in higher concentrations (GA₁, 1.42 ng/ml; GA₃, 5.93 ng/ml; GA₄, 11.36 ng/ml; GA₇, 3.25 ng/ml; GA₉, 0.79 ng/ml; GA₁₅, 1.18 ng/ml). The culture filtrate of CC-8 produced higher amounts of GA₃, GA₄, GA₇ and GA₉ than wild type Fusarium fujikuroi, a well known gibberellins producing fungus. The fungal isolate CC-8 was later identified as a new strain of Neosartorya species on the basis of traditional and advance molecular techniques.     

Biocontrol of mildew with Bacillus subtilis in bitter gourd (Momordica charantia L.) seeds during germination

The bitter gourd seed has a thick, hard seed coat. Mildew often occurs during germination and causes uneven and low rates of seed germination. However, the problems caused by mildew can be overcome by treating seeds with hot water, by soaking in water, or by using microorganisms. Seeds of the ‘Ching Pi’ bitter gourd were treated in a water bath at 60 °C for 10 min and then soaked in tap water at 25 °C for 24 h. The resulting germination percentage was 86.7%, and the resulting percentage of mildewed seeds was 10%. The biocontrol potential of three commercially available Bacillus subtilis solutions was examined. For seeds primed with Huodijun B. subtilis solution, the germination percentage was 73.3% and the mildewed percentage 6%. In dual cultures, the antibiotic content in the Huodijun B. subtilis solution was significantly greater than in Yunghsing and Huolibao, the other B. subtilis solutions examined. B. subtilis effectively inhibited the growth of Rhizoctonia solani mycelium and caused abnormal mycelial growth.     

Phloroglucinol enhances recovery and survival of cryopreserved Dendrobium nobile protocorms

The aim of the present study was to evaluate the effect of phloroglucinol in the recovery and survival of cryopreserved Dendrobium nobile protocorms. The exposure of protocorms to 2 M glycerol osmoprotective solution for 20 min followed by 10 min in PVS2 vitrification solution with 1% phloroglucinol resulted in the highest protocorm recovery and survival (68%). A positive effect of phloroglucinol was observed when combined with glycerol and PVS2. Phloroglucinol added at 1% provided an increase of over 100% in protocorm recovery and survival as compared to the same treatment without phloroglucinol. However, when sucrose was added to treatments, a negative effect was observed with a reduction in survival by 90%. Protocorms that survived cryopreservation were successfully regenerated into plants and acclimatized with 100% survival in greenhouse. Survival of cryopreserved D. nobile protocorms was a determining factor for seedling survival and growth into normal and fully functional plants. This study demonstrated an efficient procedure for cryopreservation and subsequent recovery and survival of cryopreserved D. nobile protocorms using phloroglucinol as a cryoprotectant additive.     

Methods to break seed dormancy in Cyclocarya paliurus (Batal)Iljinskaja

Cyclocarya paliurus is native to China and is the sole species in its genus. However, the seeds remain deeply dormant for 2 years in their natural environment. We tested different pretreatments of chemical scarification and exogenous application of gibberellic acid (GA₃) for efficacy in breaking dormancy and speeding germination. In contrast to scarified seeds, non-scarified seed did not germinate, indicating that C. paliurus seeds have hard, impermeable seed coat dormancy. Exogenous application of GA₃ significantly enhanced germination of scarified seeds. Compared with seeds stratified in sand with water, the germination of seeds stratified in sand moistened with 400 ppm GA₃ for 60 days was significantly increased and germination rate was over 90% after 120 days. Analysis of variance indicated that both GA₃ concentration and stratification medium had significant effects on seed germination and final germination percentage. Germination was higher for longer stratification periods, but no significant difference in germination was observed after 90 days. These results suggested that C. paliurus seeds exhibit both exogenous and endogenous dormancy. A combination of chemical scarification and exogenous application of GA₃ alleviated seed dormancy in a relatively short period of time.     

Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids

Paphiopedilum armeniacum, Paphiopedilum micranthum and Paphiopedilum delenatii are endangered orchid species. These three Paphiopedilum species and their hybrids are difficult to distinguish morphologically. In this study, rDNA-ITS (internal transcribed spacer) sequences were used to design species-specific SCAR (sequence characterized amplified regions) markers to distinguish P. armeniacum, P. micranthum, P. delenatii and their respective hybrids. The developed markers efficiently amplified 600 bp DNA product for P. armeniacum and its hybrids (SCAR-600armF/Pap-ITS2R), 300 bp product for P. delenatii and its hybrids (SCAR-300delF/Pap-ITS2R) and 700 bp product for P. micranthum and its hybrids (SCAR-700micF/Pap-ITS2R). The effectiveness of designed species-specific markers was also confirmed by using multiplex polymerase chain reaction amplification with a combination of developed three SCAR markers.     

Seed-coat anatomy and proanthocyanidins contribute to the dormancy of Rubus seed

Rubus seed has a deep double dormancy that restricts germination due to seed coat structure and chemical composition. Improved germination of diverse Rubus species required for breeding improved blackberry and raspberry cultivars is partly dependent on the seed coat structure. This study evaluated the seed coat structure of three species with thin (R. hoffmeisterianus Kunth & C. D. Bouché), medium (R. occidentalis L.) and thick (R. caesius L.) seed coats. The three species exhibited distinctive seed-coat cell composition. The very thin testa (0.086 mm) of R. hoffmeisterianus had little exotesta (surface) reticulation; with the meso- and endotesta composed of sclereids of homogenous shape and size. R. occidentalis had a thick testa (0.175 mm) and a highly reticulate exotesta; the meso- and endotesta were composed of several diverse types of sclereids. R. caesius had the thickest seed coat (0.185 mm) but only moderate exotesta reticulation; the meso- and endotesta were composed of large, irregular, loosely arranged sclereids. R. occidentalis, a medium size seed, was the most heavily lignified with seed-coat thickness similar to R. caesius, the largest seed. Proanthocyanidins (PAs) from dry seed of six Rubus species were extracted and quantified by high performance liquid chromatography. R. hoffmeisterianus, a thin only slightly hard seed, had half the PA (0.45 μg/seed) of R. occidentalis with a thick, extremely-hard seed coat and diverse sclereids (1.07 μg/seed). PA content and sclereid composition both appear contribute to seed coat hardness and resulting seed dormancy. The effectiveness of sulfuric acid for Rubus seed scarification is likely due to degradation of PAs in the testa.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Development of seedless and Mal Secco tolerant mutant lemons through budwood irradiation

Mal secco (caused by Phoma tracheiphila (Petri) Kantsch. and Gik.) is the most destructive fungal disease of lemon plantations worldwide and seedless lemons would be preferred by most consumers. Five dosage levels, 0, 3, 5, 7, and 9 kiloradian (krad), of cobalt (⁶⁰Co) gamma irradiation were applied to budstick of ‘Kutdiken’ lemon (Citrus limon (L.) Burm. f.) clone KT-2A. Mutations were stabilized in three vegetative generations. Three hundred fifty-eight and 478 M1V3 (mutation one and vegetation three) plants were evaluated for seed number and mal secco tolerance in the field and the greenhouse, respectively. LD₅₀ was around 5 krad gamma irradiation for ‘Kutdiken’ lemon. The seed number varied from 0 to 34 per fruit. The level of mal secco tolerance also varied significantly among the plants from 1.0 (no symptom) to 4.3 (high level of disease occurance). The stable seedless and mal secco tolerant plants were obtained from 5 and 7 krad irradiation: the three mutants from 5 krad irradiation gave more lemon-like fruits, while 7 krad irradiation caused altered tree morphology and early maturation of fruits. This study shows considerable potential for lemon cultivar improvement aiming to obtain seedless and mal secco tolerant lemons.     

Chemical compositions and insecticidal effects of essential oils isolated from Achillea gypsicola, Satureja hortensis, Origanum acutidens and Hypericum scabrum against broadbean weevil (Bruchus dentipes)

The essential oils of aerial parts of Achillea gypsicola Hub.-Mor., Hypericum scabrum L., Satureja hortensis L., and Origanum acutidens (Hand.-Mazz.) Letswaart were analyzed in this study by GC and GC–MS and their oils were tested for toxicity against broadbean weevil (Bruchus dentipes). A. gypsicola oil contained camphor (40.17%), 1,8-cineole (22.01%), piperitone (11.29%), borneol (9.50%) and α-terpineol (1.56%) as major components. A total of 74 components were identified by GC–MS in H. scabrum oil, including α-pinene (9.26%), terpinen-4-ol (5.12%), camphor (5.94%), δ-cadinene (4.52%), pulegone (4.45%), γ-muurolene (4.12%), pinocarvone (3.97%) and β-caryophyllene (3.42%) as predominant components. The essential oils of O. acutidens and S. hortensis were characterized by high contents of carvacrol (86.99% and 55.74%), γ-terpinene (0.71% and 20.94%), p-cymene (1.95% and 12.30%), α-terpinene (0.13% and 2.04%) and β-caryophyllene (1.30% and 1.08%). All of the essential oils were toxic to adults of B. dentipes and insect mortality increased with increasing concentration of each oil. The oils (20 μl dose) brought about 100% mortality in 36 h. Although desirable insecticidal activities against the pest were achieved with the oils from all four plant species, S. hortensis and O. acutidens oils were more effective, particularly after 6 h of treatment. The current results concluded that the essential oils, in particular O. acutidens and S. hortensis oils, may be used as potential botanical insecticides against B. dentipes.     

Transgenic orchids

Breeding orchids through traditional means is a lengthy process. Therefore, it would be advantageous if transgenic technologies could be applied for orchids to improve important traits such as novel flower colour, fragrance and shape, cut-flower longevity and flowering control, abiotic stress tolerance and resistance to pests and diseases. Even though there are several genetic transformation techniques available to orchid breeders, only two (Agrobacterium-mediated transformation and particle bombardment) have been successfully and consistently used thus far. This review aims to capture the full range of studies conducted on orchid transformation with a view of providing new perspectives for future molecular breeding programmes.     

Interspecific variations in seed germination of Corylopsis

This study was initiated to investigate the differences in germination percentages and rates between Corylopsis coreana Uyeki and Corylopsis sinensis var. calvescens Rehder & E.H. Wilson following a warm stratification (WS) and cold stratification (CS), and to study the effect of different WS temperatures interacting with different durations of CS. Warm stratification at 10 °C, 15 °C, 20 °C, and 25 °C was given for 1 month (1 M 10 °C, 15 °C, 20 °C, and 25 °C WS) followed by 0 M, 1 M, 2 M, and 3 M of CS at 5 °C (0 M, 1 M, 2 M, 3 M CS) and seeds were germinated in an air conditioned greenhouse maintained at 18.5 °C/18 °C. On average, less than 1% of C. coreana seeds germinated when sown without any WS and CS or with 1 M 15 °C, 20 °C, and 25 °C WS without CS treatment. However, 26% C. coreana seeds germinated after 1 M 10 °C WS without any CS treatment. Germination was not affected by WS temperatures when followed by 2 M 5 °C CS. It is concluded that C. coreana exhibited low seed germination at 10 °C and that this temperature could be considered the upper limit of CS for C. coreana. Only 2 M CS was required for more than 90% seeds to germinate. However, C. sinensis var. calvescens required longer than 3 M CS for more than 29% seeds to germinate. This clearly shows that there is an interspecific variation in optimum dormancy-breaking requirements.