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.     

Effect of organic and inorganic fertilizers applied during successive crop seasons on growth and nitrate accumulation in lettuce

A romaine-type lettuce (Lactuca sativa L.) cv. Corsica was cultivated during three successive crop seasons (late-spring, late-autumn and late-winter) in the same soil of an experimental greenhouse in S.W. Peloponnese, Greece. Seven long-term fertilization treatments were tested for their effect on plant growth and nitrate concentration in the external lettuce leaves. Treatments included: three different doses of organic fertilization (composted sheep manure) applied at the start of each crop season, three different doses of inorganic N fertilization applied via fertigation during each crop season, and a control treatment in which no fertilizer was applied. A drip irrigation system was used to water all plants. The highest nitrate levels were observed in the medium and maximum inorganic fertilization treatments (572–664 mg kg⁻¹) in all crop seasons. They were significantly higher compared to the respective organic fertilization treatments (253–435 mg kg⁻¹) and all other fertilization treatments (148–435 mg kg⁻¹). Crop season affected lettuce growth more than nitrate accumulation in the lettuce leaves: lettuce biomass production was the smallest and most uniform in the late-autumn season and did not respond to the fertilization treatments tested (ranging from 409 to 439 g plant⁻¹), while in the late-spring season biomass production was the highest and most variable (561–841 g plant⁻¹), it correlated with nitrate concentration in the leaves and in the medium and maximum inorganic fertilizer doses it significantly exceeded production from all other fertilization treatments (827–841 g plant⁻¹). Following the three crop seasons the residual availability of N, P and K was clearly enhanced in the soil receiving the organic compared to the inorganic fertilization. Nitrate concentration in lettuce leaves was far below the upper limits set by the European Commission in all fertilization treatments throughout the three crop seasons, a result attributed mainly to the sufficient level of light intensity and duration throughout the year in Southern Greece.     

Rapid seedlings regeneration from seeds and vegetative propagation with sucker and rhizome of Eremospatha macrocarpa (Mann & Wendl.) Wendl and Laccosperma secundiflorum (P. Beauv.) Kuntze

To develop efficient seedling production methods for Laccosperma secundiflorum and Eremospatha macrocarpa, a study was conducted to examine regeneration using offsets combined with several physical and chemical treatments of seeds. Offsets categorized into small, medium and large diameters, were planted in three conditions: shaded and open nursery, and greenhouse. We tested sucker from E. macrocarpa, and sucker and rhizome from L. secundiflorum. For both species, high viability percentage (ranging from 55% to 100%) were observed for small and medium suckers planted in shaded nursery and greenhouse, against less than 49% for sucker planted in open nursery. The mean seedling emergence times were estimated to 84, 77 and 75 days after planting (DAP) for small, medium and large sucker of L. secundiflorum, respectively under open nursery condition, and 76, 75, 95 DAP for small, medium and large suckers of the same species, respectively in shaded condition. Greenhouse has a significant positive effect on E. macrocarpa seedlings emergence time. For this species, the mean seedling emergence times were estimated to 43 DAP for small sucker and 76, 93 DAP for medium and large suckers. No seedling was obtained from rhizome planted in all the growing conditions tested. Concerning seed dormancy breaking, germination percentages and rates were determined for 13 treatments. The best treatments were pre-soaking unscarified seeds for 4 days in 1.01 g l⁻¹ and 0.10 g l⁻¹ KNO₃, with 79% and 68% of germination, respectively and in 3.46 × 10⁻³ g l⁻¹ GA₃ for 68% of germination. These methods are suggested to improve germination of L. secundiflorum seeds. Successful and recommended methods for E. macrocarpa are pre-soaking scarified seeds in 3.46 × 10⁻³ g l⁻¹ and 3.46 × 10⁻⁴ g l⁻¹ GA₃, 96% and 94% of germination, respectively. Dormancy, probably a combination of mechanical and chemical dormancy, is present in the two species.     

Seed inoculation with Azospirillum mitigates NaCl effects on lettuce

Data on the growth-promoting effects of Azospirillum on lettuce exposed to either normal or saline conditions, is scarce. Lactuca sativa L., cv Mantecosa seeds were colonized with A. brasilense Sp245 cells during imbibition. Germination percentages were determined after 7 d treatments with 0, 30, 50 or 80 mol m⁻³ NaCl. In another experiment, seeds germinated in Hoagland were irrigated for 30 d with 0, 30, 50 or 80 mol m⁻³ NaCl supplemented media. Vegetative growth proceeded in a growth chamber with a 13–11 h day–night cycle. Buffer-imbibed seeds were considered non-inoculated controls. Plant samples were taken at 0, 14, 20, and 30 d after the onset of NaCl treatments and dissected in aerial and root portions. The weights of both tissues were measured. Azospirillum-inoculated seeds had significantly higher germination percentages than controls in all treatments. Inoculated dried seeds stored up to 30 d maintained such characteristic in most of the treatments, particularly at 80 mol m⁻³ NaCl. Plants grown from inoculated seeds and irrigated with saline media displayed higher total fresh and dry weights and biomass partition to the aerial portion, than non-inoculated controls. Azospirillum-inoculated lettuce seeds had better germination and vegetative growth than non-inoculated controls after being exposed to NaCl.     

The influence of desiccation and rehydration on the survival of polyembryonic seed of Citrus suhuiensis cv. limau madu

A study was carried out to investigate the influence of desiccation and freezing followed by various presowing rehydration procedures on the desiccation sensitivity of the seed of Citrus suhuiensis cv. limau madu. The freshly harvested seeds of limau madu were desiccated under a broad range of relative humidity (RH) to various equilibrium water contents (g H₂O g⁻¹ dw). The desiccated and desiccated–frozen seeds were either directly sown under germination conditions or subjected to presowing rehydration procedures: seed preheating, prehumidification and osmoconditioning. The hydrated and desiccated seeds were sown in controlled germination conditions and the survival was evaluated 4–6 weeks after sowing. The results showed that desiccation progressively reduced the percentage of normal seedling of the seeds of limau madu and the viability is almost lost at water contents below 0.08 g H₂O g⁻¹ dw. The estimated desiccation sensitivity was substantially high (WC₅₀ = 0.143 g H₂O g⁻¹ dw) when the desiccated seeds were rapidly rehydrated (uncontrolled rehydration). In contrast, seed prehumidification, preheating and osmoconditioning (controlled rehydration procedures) markedly enhanced normal seedling percentages decreasing the estimated values of WC₅₀ (between 0.08 and 0.127 g H₂O g⁻¹ dw). While the rapidly rehydrated desiccated–frozen seeds were almost killed at water content of 0.15 g H₂O g⁻¹ dw, prehumidification and preheating have noticeably increased percentage of frozen seeds survival at the same water content. However, at water content of 0.21 g H₂O g⁻¹ dw preheating significantly (P < 0.05) increased percentage of normal seedling of the frozen seeds. Seed desiccation markedly reduced the percentages of germinated seeds with multiple seedlings. Seed controlled rehydration remarkably increased the survival of polyembryos. The beneficial effect of seed controlled rehydration on the survival of the desiccated seeds was pronounced at medium water contents (0.08–0.25 g H₂O g⁻¹ dw).     

Effects of the commercial product based on Trichoderma harzianum on plant,bulb and yield characteristics of onion

Effects of the commercial product TrichoFlow WP™ (Agrimm Technologies Ltd., New Zealand), based on the fungus Trichoderma harzianum, on quality characteristics and yield of bulb onion was investigated. Bulb sets of the local cultivar Kantartopu was planted in soil with in and between row distances of 0.15 m and 0.40 m, respectively. The product, at considerably high dosages of 5 g m⁻², 10 g m⁻² and 15 g m⁻², was mixed with water and sprinkled once to the plots at planting. Analyses of data at harvest did not show statistical significance for Trichoderma effect on total bulb yield, bulb diameter, leaf length, number of shoot apex, %titratable acidity, number of internal (fleshy) leaves, number of external (papery) leaves, %soluble solids and %bulbs with diameters of 20–39 mm, 40–69 mm and ≥70 mm. The yields obtained from the plots treated with the dosages of 5 g m⁻², 10 g m⁻² and 15 g m⁻² and the control plots were 1063.7 kg da⁻¹, 1051.0 kg da⁻¹, 1066.5 kg da⁻¹ and 985.0 kg da⁻¹, respectively. Our results showed that high dosages of the Trichoderma product were not effective in enhancing onion bulb and yield characteristics under the given conditions.     

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.     

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

Development of a plant regeneration system from seed-derived calluses of centipedegrass [Eremochloa ophiuroides (Munro.) Hack]

The objective of this study is to establish plant regeneration system with the seed of the new Chinese selection “E-126”of centipedegrass [Eremochloa ophiuroides (Munro.) Hack] as explant. In present study, the following results were obtained: (1) The medium formulation most suitable for calluses induction was identified to be MS with 1.0 mg l⁻¹ 2,4-D + 30 g l⁻¹ mannitol + 50 ml l⁻¹ coconut milk and the ratio of calluses induction was 96.0%, including 5.2% of yellow granule calluses induction. The above medium formulation was adopted for subculture. (2) The rate of shoot regeneration from yellow granular calluses was 98.0% by MS optimum medium formulation with 2.0 mg l⁻¹ KT + 50 ml l⁻¹ coconut milk. The differentiated rate retained as high as 88.0% even after 5 times of subculture and 18.6% after 15 times of subculture. The optimum medium formulation for shoot growth was identified to be MS medium plus 2.0 mg l⁻¹ BAP, 0.8 mg l⁻¹ NAA and 50 ml l⁻¹ coconut milk. (3) The optimum medium for shoot rooting was identified to be MS medium with 0.6 mg l⁻¹ NAA + 50 ml l⁻¹ coconut milk, and the rooting rate to be 98.0%. The survival rate of transplanted plantlets from tubes to basin with soil was 92.0%. In conclusion, the plant regeneration system was successfully developed in this study, which may provide basic reference for screening of somaclonal variants and genetic transformation of centipedegrass.     

Antioxidant response of bitter gourd (Momordica charantia L.) seedlings to interactive effect of dimethoate and UV-B irradiation

Bitter gourd (Momordica charantia L.) seedlings treated with elevated concentrations of dimethoate (100 and 200 ppm) and fixed ultraviolet-B (0.4 Wm⁻²/30 min) irradiation showed stunted growth and less photosynthetic pigments chlorophylls (Chl) content. The synergistic effects of both the stresses were more pronounced than the individual effect. However, dimethoate at low concentration (50 ppm) stimulated growth and pigmentation but with UV-B it showed slight inhibition. Reactive oxygen species (ROS) accumulated considerably in leaves due to UV-B and high concentrations of dimethoate. Combined exposure further increased the ROS leading to lipid peroxidation and electrolyte leakage. Both the stresses alone and together also caused the increase activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). High concentration of dimethoate and UV-B accelerated the accumulation of ROS particularly H₂O₂ in leaves, causing heavy damage to photosynthetic pigments and growth of bitter gourd seedlings. Simultaneous exposure of UV-B and dimethoate inhibit the growth, photosynthetic pigment and enhanced the accumulation of ROS more severely than the individual exposure. Interestingly, low concentration (50 ppm) of dimethoate significantly reduced the effects of UV-B. The results suggested synergistic effect of dimethoate and UV-B on plant growth as a function of decreased photosynthetic pigments despite increase in the activities of the antioxidant enzyme.     

Transient expression of an active human interferon-beta in lettuce

Using Agrobacterium mediated transient expression method, plant bivalent expression vector pBI121 containing GUS as a report gene was transformed into lettuce (Lactuca sativa). Through designed orthogonal analysis, intact lettuce leaves infiltrated with 200 μM acetosyringone and 0.8 OD₆₀₀ bacterial suspensions under vacuum for 30 min, then co-cultured at 24 °C for 6 ds produced a maximum GUS protein of 2.5% TSP with 21.39 nmol mg⁻¹ min⁻¹ MU activity, which was 19 times of the control (1.31 nmol mg⁻¹ min⁻¹ MU). Employed these optimized conditions HuIFN-beta was expressed in lettuce leaves. Western blot and antivirus bioactivity analyses confirmed the HuIFN-beta achieved by agrobacterium infiltration had a high biological activity (3.1 × 10⁴ IU/mL). To our knowledge, it is the first detailed orthogonal optimizing study of Agrobacterium mediated transient expression and the first report on the production of the biologically active therapeutic proteins produced by Agrobacterium mediated transient expression in lettuce. In summary, transient expression by Agrobacterium vacuum infiltration can be adopted as an efficient, inexpensive and small-scaled plant expression system for therapeutic protein production.     

Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L.

An efficient method of repetitive somatic embryogenesis and plant regeneration was established in Coriandrum sativum L. Embryogenic callus was induced from cotyledon and hypocotyl segments on Murashige and Skoog (MS) medium with 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), upon subculturing on medium having same level of 2,4-D at an interval of 3 weeks developed somatic embryos, which progressed to cotyledonary stage through early developmental stages of somatic embryogenesis. The transfer of somatic embryos at an early cotyledonary and cotyledonary stage in clumps in succession to fresh 4.52 μM 2,4-D supplemented medium developed embryos in a cyclic manner. Upon transferal to embryogenic clumps (cotyledonary embryos) to modified MS medium (4 g l⁻¹ KNO₃, 0.29 g l⁻¹ NH₄NO₃, 3 mg l⁻¹ thiamine HCl, 0.5 mg l⁻¹ pyridoxine HCl, and 5 mg l⁻¹ nicotinic acid), the embryos irrespective of the cycles underwent maturation and germination. Germinating embryos transferred to half-strength MS medium favored healthy growth of plantlets. The system of recurrent somatic embryogenesis in coriander offers a system for genes transfer and also scale-up production of modified plants.     

Factors affecting haploid induction through in vitro gynogenesis in summer squash (Cucurbita pepo L.)

Haploid production using in vitro ovule cultures has long been recognized as an important tool to produce haploid and homozygous double-haploid plants for genetic studies and plant breeding programs. In the present study, four experiments were carried out to study the influence of genotype, position of female flowers on plant stem, temperature and sucrose concentration on the in vitro gynogenesis induction of squash. (1) Ovules of 12 genotypes were excised from female flowers, 1 day before anthesis, and cultured onto MS medium containing 3% sucrose and 1 mg l⁻¹ from each of kinetin and 2,4-D (2,4- dichlorophenoxy acetic acid). Differences in response among genotypes were demonstrated. Raad F₁ showed the highest percentage of responding ovules and number of plantlets per dish with 48.8% and 15 plants, respectively. The results revealed that genotype is a key factor influencing the in vitro gynogenesis in squash. (2) Ovules were excised from first, second and third female flower of two hybrids (Giad and Raad) and cultured onto the mentioned above medium. The highest percentage of responding ovules and number of plantlets per dish were obtained from ovules excised from the second female flower on the plant stem. (3) Effect of temperature (4 and 32 °C) for 0, 4, 7 and 12 days on the ovule culture of Queen F₁ was studied. Ovules incubated at 4 or 32 °C for 4 days produced a better embryogenic response. (4) Three sucrose concentrations (30, 60 and 90 g l⁻¹) were tested with the ovule cultures of the local cultivar (Eskandrani). Differences among sucrose concentrations were statistically significant and ovules cultured on the MS medium containing 30 g l⁻¹ produced the best result. MS medium containing 90 g l⁻¹ did not produce gynogenic ovules.     

Effect of genotype,explant size,position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture

An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l⁻¹ thidiazuron (TDZ), 0.8 mg l⁻¹ adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 2.0 mg l⁻¹ kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l⁻¹ IBA. All plantlets acclimatized well in the greenhouse.     

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%).     

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.     

Callus induction and plant regeneration from embryonic axes of Kosteletzkya virginica

Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L⁻¹ indole-3-acetic acid (IAA), 0.3 mg L⁻¹ kinetin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L⁻¹ IAA, 0.5 mg L⁻¹ zeatin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (3) 96.67% rooting on MS medium containing 30 g L⁻¹ sucrose and 8 g L⁻¹ agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l⁻¹) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l⁻¹) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l⁻¹ ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l⁻¹) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.     

In vitro germination of walnut (Juglans regia L.) embryos

The present studies were undertaken with a view to standardize the medium and culture conditions for embryo culture of five cultivars of walnut viz., ACO 38853, Netar Akhrot, Gobind, Solding Selection and Blackmore. Embryos from mature fruits were aseptically excised and cultured on MS medium supplemented with different combinations of BAP, kinetin and GA₃. Best performing medium was MS with 0.5 mg l⁻¹ kinetin, 0.5 mg l⁻¹ BAP and 2 mg l⁻¹ GA₃ yielding 66.6% germination in Netar Akhrot after 12 days of culturing. Percent germination of excised embryos was higher when GA₃ and cold treatments were simultaneously applied as compared to those when applied separately. Netar Akhrot was found to be the best responding cultivar, which had a range of 25–66.6% embryo germination under different culture conditions. Plantlets with shoots and roots have been obtained in Netar Akhrot and ACO38853 and are transferred to soil after hardening.     

Foliar application of chlorocholine chloride improves leaf mineral nutrition,antioxidant enzyme activity,and tuber yield of potato (Solanum tuberosum L.)

In southern China, potatoes (Solanum tuberosum L.) are grown in the late season and the crops are often subjected to low temperature stress particularly during the tuber bulking stage. Exogenous chlorocholine chloride (CCC) treatment has been found to improve crop performance under suboptimal growth conditions; however, the physiological mechanisms underlying the beneficial effects have not been fully understood. The objective of this study was to investigate the effects of CCC treatment on mineral nutrition, antioxidant enzyme system, and tuber yield of potato (cv. Zhongshu 3) under field conditions. The plants were foliar sprayed twice with 1.5, 2.0 and 2.5 g l⁻¹ CCC at 24 and 28 days after emergence (DAE), respectively; and plants without CCC treatment were serviced as control. Leaf samples were collected on 56 DAE for determination of mineral nutrition contents and antioxidant enzyme activity. Results showed that 1.5 and 2.0 g l⁻¹ CCC treatments significantly increased the contents of P, K, Ca, Mg, Fe, Mn, Zn and Cu in potato leaves. These treatments also increased superoxide dismutase (SOD), peroxidases (POD) and catalase (CAT) activities in the leaves. A positive linear relationship was found between SOD activity and the content of Fe + Mn + Zn + Cu. Tuber yield was significantly increased by CCC treatment. It is concluded that treatment with certain concentration of CCC (e.g. 1.5–2.0 g l⁻¹) improves mineral nutrition and SOD, POD and CAT activities in potato leaves; which might have contributed to the higher tuber yield of the crop grown under suboptimal conditions.