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.     

Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

Breeding of disease-resistant seedless grapes using Chinese wild Vitis spp.: I. In vitro embryo rescue and plant development

Seedless grape cultivars (Vitis vinifera L.) are widely grown in Europe, America and Asia. Fungal diseases are a great threat of them. Several wild Chinese Vitis species showed high resistance to many fungal diseases. Therefore, an investigation was conducted to assess the potential to incorporate these species in a breeding project for development of the disease-resistant seedless cultivars. Hybridization was conducted using V. vinifera as female parents and the wild Chinese Vitis spp. as male parents. In-ovulo embryo rescue was used to develop hybrid plants from the seedless females. An efficient protocol is reported here for the in vitro embryo rescue and plant development from the cross Emerald Seedless × Beichun. Ovules were excised from immature fruits 7 weeks after pollination (WAP) and cultured in the double-phase ER medium supplemented with 6.0% sucrose and 0.3% activated charcoal (AC). Following 8–12 weeks of culture, embryos were removed from the ovules and transferred onto WPM supplemented with 1.0 μM 6-benzyladenine (6-BA), 2.0% sucrose and 0.2% AC and solidified with 0.6% agar. After 8 weeks of culture, the embryos germinated and subsequently grew into whole plantlets. With the optimized parameters developed in the present study, about 34.0%, 91.2% and 77.4% of embryo formation, embryo germination and plant development were obtained, respectively. When this protocol was applied to 11 other cross combinations, genotype was found to significantly influence embryo formation, embryo germination and plant development, with different frequencies of hybrid plants successfully obtained in all crosses.     

In vitro propagation of Lychnis senno Siebold et Zucc., a rare plant with potential ornamental value

Lychnis senno is a rare and valued ornamental plant. Seed propagation is not efficient because of the low germination rate. To grow commercially L. senno in China, a protocol for in vitro germination and propagation of this species was developed. Various germination rates were obtained by treating seeds with GA₃ during 1–6 months storage period. The highest germination rate reached 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4 °C. Axillary shoot proliferation was induced in the nodal segments of the seedlings on medium containing specific concentrations of BA and NAA [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497]. Maximum number of shoots was developed on a medium supplemented with 5 mg/l BA and 0.5 mg/l NAA, while the higher shoots were observed on a medium supplemented with 0.5 mg/l BA and 0.05 mg/l NAA. Rooting was induced in 91.7% of the regenerated explants on a half-strength MS medium supplemented with 0.5 mg/l NAA. The plantlets grew well and flowered after transfer to the greenhouse. The chromosome numbers of seedlings and propagated plants were also determined to be 2n = 2x = 24.     

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.     

Production of protocorm-like bodies with bioreactor and regeneration in vitro of Oncidium ‘Sugar Sweet’

To produce mass propagules of Oncidium ‘Sugar Sweet’, we tested the feasibility of producing protocorm-like bodies (PLBs) using 5 l balloon type air-lift bioreactors, and selected an optimal culture medium for shooting and rooting in vitro. The results showed that liquid bioreactor cultures were more efficient for PLBs proliferation when compared to solid- and liquid-agitated flask cultures. The maximum PLBs biomass (326.3 g per bioreactor, FW) was obtained in the immersion bioreactor culture, with the growth ratio reaching 10.9 after 50 days of culture. This was obviously higher than the ebb and flood bioreactor culture. During bioreactor culture, sucrose and electric conductivity (EC) in the culture medium were negatively correlated with PLBs growth; the highest PLBs fresh and dry biomass was obtained 40 days after culture. An inoculation density of 20 g (FW) was optimal for PLBs growth in a 3 l working volume of 5 l bioreactor. Furthermore, MS medium supplemented with 2.0 mg l⁻¹ BA for shooting and 0.5 mg l⁻¹ IBA for rooting was optimal during in vitro culture, the plantlets were successfully established in the potting substrate.     

Restoration of rooting competence in a mature plant of Garcinia indica through serial shoot tip grafting in vitro

Apical and axillary buds from a high yielding, early fruiting elite tree (more than 20 years old) were cultured in woody plant medium (WPM) supplemented with 0.9 μM N⁶-benzyl adenine (BA). Multiple shoots were obtained on WPM basal medium containing 8.9 μM BA and 0.5 μM thidiazuron (TDZ). Elongation of axillary shoots was obtained in half-strength WPM medium supplemented with 0.4 μM BA. For root initiation, the elongated shoots were transferred to half strength WPM basal medium containing 2.5–245 μM indole-3-butyric acid (IBA) or 2.7–268.5 μM α-naphthaleneacetic acid (NAA) or the shoots were subjected to 2.5–53.9 mM IBA, 2.7–59.1 mM NAA dip for (30 s–30 min) and then transferred to half strength WPM basal medium. However, rooting was never achieved even after 2 months of culture.     

High frequency plantlet regeneration from callus and artificial seed production of rock plant Pogonatherum paniceum (Lam.) Hack. (Poaceae)

Pogonatherum paniceum (Lam.) Hack. is a rock plant with good potential for vegetative recovery on naked lands. A high frequency in vitro regeneration system was developed for P. paniceum. Calli were induced from explants of mature seeds, seedlings, young leaves, and stem segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 2.0 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.2 mg L⁻¹ 6-benzylaminopurine (BAP). High induction rates (59.57%) and regeneration rates (100%) were obtained from mature seed explants; calli were sub-cultured for over 2 years and still retained a high regenerative capacity. One seed explant resulted in 69,997 plants in 1 year. Shoot buds derived from calli were used for encapsulation in liquid MS medium containing 3% sucrose and two different alginate matrices (3% sodium alginate (w/v) + MS medium containing 3% sucrose and 3% sodium alginate + 1% activated carbon (w/v) + MS medium containing 3% sucrose) with a 20-min exposure to 2% CaCl₂ and 0.3% bavistin (w/v). The capsule with 3.0% sodium alginate (w/v) and 1% activated carbon (w/v) showed a higher conversion rate (61.58%) and stronger plantlets under non-aseptic conditions. These systems are useful for the rapid clonal propagation and dissemination of artificial seed material of P. paniceum for eco-recovery.     

Interspecific hybridization in Lonicera caerulea and Lonicera gracilipes: The occurrence of green/albino plants by reciprocal crossing

Lonicera caerulea L. var. emphyllocalyx (Maxim.) Nakai is a berry crop cultivated in cold regions. So far, commercial cultivars have been mainly introduced from selection of wild plants. Therefore, fruit traits and other agricultural characteristics have been limited. In this study, interspecific crosses between L. caerulea var. emphyllocalyx and Lonicera gracilipes var. glabra Miquel were examined to increase genetic variability of L. caerulea var. emphyllocalyx. Seedlings were obtained from reciprocal crosses between L. caerulea var. emphyllocalyx and L. gracilipes var. glabra. The hybrid nature of seedlings was confirmed with random amplified polymorphic DNA analysis. Viable green plants were obtained efficiently from L. gracilipes var. glabra × L. caerulea var. emphyllocalyx. In contrast, all plants produced from L. caerulea var. emphyllocalyx × L. gracilipes var. glabra were albino. These albino plants were very weak and only survived in culture condition. The chlorophyll deficiency was unilaterally observed, suggesting the occurrence of nuclear–cytoplasmic incompatibility. Viable F₁ hybrids obtained from L. gracilipes var. glabra × L. caerulea var. emphyllocalyx are amphidiploid (2n = 4x = 36) as showing same to both parents. The hybrid plants are expected to increase the variability of fruit traits, and may have heat tolerance from L. gracilipes var. glabra.     

Adventitious shoot regeneration from hypocotyl slices of mature apricot (Prunus armeniaca L.) seeds: A feasible alternative for apricot genetic engineering

Adventitious shoot regeneration from hypocotyl slices of mature apricot seeds has been achieved with regeneration percentages of 31.7%, 44.4%, and 46.9% for the cultivars ‘Canino’, ‘Dorada’, and ‘Moniqui’, respectively. Regeneration was significantly affected by the parental origin of the explants (P < 0.05) but not by thidiazuron or 3-indolebutyric acid for any of the three cultivars, at the levels tested. None of the other factors studied (basal medium, 2,4 dichlorophenoxy-acetic acid pulses, dark incubation period, or addition of silver thiosulfate) affected significantly shoot regeneration percentage from ‘Canino’ hypocotyl sections. The effect of paromomycin on regeneration was genotype-dependent and different dose–response curves were obtained for each cultivar. While 40 μM paromomycin completely inhibited regeneration from ‘Canino’ sections, some buds were obtained from ‘Dorada’ and ‘Moniqui’ explants. The two aminoglycoside antibiotics tested, kanamycin and paromomycin, showed differing toxicity on ‘Canino’. A lower concentration of kanamycin (20 μM) than of paromomycin inhibited totally adventitious regeneration from ‘Canino’ explants. Agrobacterium-mediated transformation experiments, with the non-oncogenic strain AGL1 harboring the binary plasmid p35SGUSINT, were performed and GUS assays were carried out after four weeks to determinate stable transformation events. The utilization of paromomycin (10 μM) as the selective agent increased significantly both the number of explants that presented at least one transformation event (P < 0.05) and the number of large area or calli expressing the gus gene (P < 0.001), compared with the addition of kanamycin (10 μM). Moreover, when 10 μM paromomycin was added to the medium some massively transformed explants were observed and a chimerical bud was regenerated.     

Evaluation of ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid for tolerance to Xanthomonas axonopodis pv. citri and Xylella fastidiosa

Asiatic citrus canker (ACC), caused by Xanthomonas axonopodis Starr & Garces pv. citri (Hasse) Vauterin et al., and citrus variegated chlorosis (CVC), caused by Xylella fastidiosa Wells et al., are considered the main diseases affecting sweet orange scion varieties in Brazil. Among commercial varieties, mandarins and tangerines are recognized as tolerant to these pathogens. We report herein the production of ‘Hamlin’ sweet orange (Citrus sinensis L. Osbeck) + ‘Montenegrina’ mandarin (Citrus deliciosa Ten.) allotetraploid somatic hybrid plants by protoplast fusion with improved disease tolerance that could be used as a donor of resistance genes in interploid hybridisation. Somatic hybridisation was confirmed by leaf morphology, flow cytometry and RAPD analyses. The somatic hybrid was propagated by grafting and cultivated in a screenhouse for tolerance assays. For X. axonopodis pv. citri assays, buds were collected from both ‘Hamlin’ sweet orange and the somatic hybrid and grafted onto ‘Cleopatra’ mandarin (Citrus reshni hort. ex Tanaka). As a negative control, buds from ‘Mexerica Tardia’ mandarin (C. deliciosa) were collected and grafted onto ‘Cleopatra’ mandarin. Two-month old plants with at least one young vegetative flush were individually spray-inoculated with a 10⁶ CFU mL⁻¹X. axonopodis pv. citri suspension and incubated in a growth chamber, at 27 °C, under 16-h photoperiod. The somatic hybrid showed a statistically significant reduction in susceptibility to ACC 30 days after inoculation. Compared to ‘Hamlin’ sweet orange, disease severity was reduced by 70%, with similar tolerance to that of the mandarin negative control. For X. fastidiosa assays, buds were collected from the somatic hybrid and its parental plants and grafted onto ‘Rangpur’ lime (Citrus limonia Osbeck). The developed plants were needle-inoculated with a X. fastidiosa suspension (8.7 × 10¹⁰ CFU mL⁻¹) into the new growth flush stem. Bacterial population was quantified both at 4 (at the inoculation point) and 8 months (50 cm above the inoculation point) after inoculation. The first evaluation detected X. fastidiosa in 63% of ‘Hamlin’ sweet orange and ‘Hamlin’ + ‘Montenegrina’ mandarin samples. In the second evaluation, X. fastidiosa was detected in 47.4% of ‘Hamlin’ sweet orange and 10.5% of ‘Hamlin’ + ‘Montenegrina’ somatic hybrid samples, suggesting that bacterial movement was restricted in the somatic hybrid. X. fastidiosa was not detected in both evaluations in samples collected from leaves of ‘Montenegrina’ mandarin. These results indicate that the ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid has potential for improved disease tolerance that should enhance its value regarding future use in citrus breeding programs.     

Coconut water and BAP successfully replaced zeatin in olive (Olea europaea L.) micropropagation

The data presented report on trials conducted during 24 months using the Portuguese olive cultivar ‘Galega vulgar’. The effectiveness of coconut water, BAP, or kinetin, as possible zeatin substitutes in olive micropropagation protocols, was investigated. In all stages of the micropropagation process, the mineral and vitamin formulation of olive medium (OM) was used. Regarding culture establishment the best results were achieved when 50 ml l⁻¹ coconut water and 2.22 μM BAP were used as medium supplements. For the in vitro multiplication stage, the highest proliferation rates with an average of 3.4 new explants on each 30 days were achieved maintaining the coconut water concentration at 50 ml l⁻¹ and increasing BAP up to 8.87 μM. The effects of IBA and activated charcoal on the in vitro root induction were also studied. Rooting rates of over 85% were obtained by basal immersion of the explants in IBA solution at 3 g l⁻¹ for 10 s, followed by inoculation in the OM culture medium, added with 2 g l⁻¹ of activated charcoal and without growth regulators. All in vitro rooted plants were transferred into Jiffy-Pots filled with vermiculite–perlite 3:1 (v/v) substrate. Those were subsequently wetted with the OM mineral solution, placed into polystyrene plates each one with 100 Jiffy-Pots capacity, which were transferred to traditional rooting mist benches, on a water-cooling equipped greenhouse. Such a simple acclimatization procedure allowed for 95% of plants survival.     

In vitro plantlets from alginate-encapsulated shoot tips of Solanum nigrum L.

Shoot tip explants obtained from in vitro proliferated shoots were encapsulated in 3% sodium alginate and 100 mM calcium chloride for the production of synthetic seed in Solanum nigrum L., a medicinally important plant. Morphogenic responses of encapsulated shoot tips to various sowing media (full or half-strength 0.8% agar-solidified or liquid MS medium or full-strength MS medium containing BAP) were evaluated in vitro. Of the six media evaluated, maximum conversion was obtained on 0.8% agar-solidified growth regulator free full-strength MS medium. The addition of MS nutrients in alginate matrix had a pronounced effect on the length of shoots that emerged from alginate beads. Encapsulated shoot tips also converted when directly sown in sterile soil moistened with liquid MS medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

High frequency early flowering from in vitro seedlings of Dendrobium nobile

Dendrobium nobile Lindl. is a popular temperate Chinese orchid commonly marketed as a traditional medicinal plant. Seedlings of Dendrobium nobile Lindl. produced floral buds (33.3–34.8%) precociously on a defined basal medium (1/2 MS) containing paclobutrazol (PP₃₃₃) at 0.5 mg L⁻¹ or thidiazuron (TDZ) at 0.1 mg L⁻¹ within 4 months of culturing. The frequency of floral buds formation can be further increased to 95.6% by growing seedlings in a PN (PP₃₃₃ 0.3 mg L⁻¹ + NAA 0.5 mg L⁻¹)-containing medium followed by transfer onto 1/2 MS medium with PP₃₃₃ and TDZ (PP₃₃₃ + TDZ). However, flower developed was deformed under 25 °C but it developed fully when grown in a lower temperature regime (23 °C/18 °C, light/dark) for 45 days. Under optimal condition, in vitro flowering was observed about 6 months after seed sowing.     

Combining ability for superoxide dismutase,peroxidase and catalase enzymes in cabbage head (Brassica oleracea var. capitata L.)

Antioxidant enzymes have been touted as beneficial for enhancing the fitness, preventing disorders, and mitigating the effects of aging and senescence. Our objective was to evaluate combining ability of superoxide dismutase, peroxidase, and catalase activity in cabbage head. Head samples were frozen immediately in liquid nitrogen and placed at −80 °C for assay. Less than unity values of σ²_gca/σ²_sca ratio for all three enzymes indicated predominance of non-additive gene action. The parents CMS-GA and Red Cabbage excelled as good general combiners for all antioxidants and indicated the value and need for multiple crossing. The crosses CMS-GA × Red Cabbage, CMS-GA × C-2, 83-2 × AC-204, 83-2 × EC-490174, 83-2 × AC-1021, Pride of Asia × C-4, and Pride of Asia × AC-1019 showed significant specific combining ability, which could be exploited through heterosis breeding. The hybrid combinations with high per se performance and favorable SCA estimate and involving at least one of the parents with high GCA estimate could be useful to increase the abundance of favorable alleles for enhancing the antioxidants in cabbage head.     

Conservation of Vanilla species, in vitro

Vanilla (Vanilla planifolia) is a crop of great commercial importance as the source of natural vanillin, a major component of flavor industry. The primary gene pool of V. planifolia is narrow and is evidently threatened due to destruction of its natural habitats making the secondary gene pool important as a source of desirable traits especially for resistance to diseases. Many species of vanilla are considered rare and endangered hence an urgent need to conserve them, arises. Effective procedures for micropropagation and in vitro conservation by slow growth in selected species of vanilla, are described. Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, which could be stored up to 10 months with 80% germination in sterile water at 22 ± 2 °C. In vitro conservation technology of Vanilla was standardized and shoot cultures could be maintained for more than 1 year without subculture, on slow growth medium, i.e. Murashige and Skoog medium supplemented with 15 g l⁻¹ each of sucrose and mannitol in sealed culture vessels at 22 ± 2 °C. These cultures were maintained in vitro for more than 7 years with yearly subculture. The conserved material could be retrieved and multiplied normally in MS medium with 1.0 mg l⁻¹ BA and 0.5 mgl ⁻¹ IBA. The in vitro conserved plants showed good growth and developed into normal plants. This synseed and in vitro conservation system can be utilized for conservation and exchange of vanilla genetic resources.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

Effects of exogenous application of plant growth regulators on the development of ovule and subsequent embryo rescue of stenospermic grape (Vitis vinifera L.)

Seedless grapevine cultivars (Vitis vinifera L.) are widely grown in Europe, America and Asia. Abortion of zygotic embryos in seedless grapes largely limits the efficiency of breeding of seedless cultivars through genetic crossing. The present study was designed to investigate effects of exogenously applied plant growth regulators (PGRs) to the grapevines in field condition on ovule and subsequent embryo rescue of seedless grapes of small seed traces. First experiment was performed by measuring ovules weight, proportion of each category ovules in maturity and embryo development in vitro of seedless grape cv. Centennial Seedless, Thompson Seedless and Crimson Seedless sprayed by chlormequat (CCC), benzyladenine (BA), ethephon (CEPA) and putrescine (Put). The effects of different application concentration and date of CCC were further evaluated in Centennial Seedless in later experiment. The results showed that exogenous application of all PGRs did not affect the total number of ovules per berries in maturity. CCC increased the ovules weight and proportion of ovules >4 mm in length of three varieties in maturity. The effects of two application times of PGRs on weight of berries and ovules and proportion of each category ovules in maturity were not significantly different. In the proceeding of embryo rescue, CCC at 100 and 1000 mg l⁻¹, BA at 100 mg l⁻¹ and Put at 20 mg l⁻¹ increased the percentage of developed embryos of Centennial Seedless and Thompson Seedless. The results showed that the size of ovules excised for embryo rescue significantly affected embryo formation and plant regeneration. The percentage of embryos formation in ovules >4 mm in length was significantly more than in ovules 2–4 mm in length, no embryo was found in ovules <2 mm in length. Exogenous application of CCC at 100–500 mg l⁻¹ significantly increased percentage of ovules >2 mm in length by 80.0–82.7% in Centennial Seedless, therefore improving embryo formation. The statistical correlation was found between the proportion of ovules >2 mm and embryo formation (r = 0.92) in Centennial Seedless. Among the different spraying time in Centennial Seedless, CCC applied 14 days before bloom produced significantly more ovules >2 mm in length and embryos formation.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

Micropropagation of Cymbidium Sleeping Nymph through protocorm-like bodies production by thin cell layer culture

Transverse thin cell layers (tTCLs) of protocorm-like bodies of two stages of PLBs (30 d and 60 d old) of Cymbidium Sleeping Nymph were used as explants to induce PLBs by using coconut water (CW) as a natural additive. 5% (v/v) CW supplemented to KC medium induced an average of 5 PLBs per responding tTCL of 30 d old PLBs with 83% of responding tTCLs. A low percentage of responding tTCLs were observed in 60 d old PLBs’ tTCLs. Anatomical and confocal microscopic studies traced the origin of PLBs to subepidermal layers of the tTCL. A significantly high percentage of shoot regeneration was obtained from PLBs formed on 1–10% (v/v) CW from tTCLs of 30 d old PLBs in comparison to PLBs induced on control after first subculture on KC medium (without CW). The induced PLBs regenerated into plantlets with velamenous roots and these plantlets were transferred to greenhouse conditions on cocopeat:perlite (9:1) with nearly 100% survival. Post-transfer performance of the plantlets was monitored. The results suggest tTCLs as potential explants (with respect to economy of precious hybrid materials) which can overcome the slow growth of hybrid PLBs and coconut water as a single natural additive for the mass multiplication of commercially important orchids.