Metabolic profiling of ethephon-treated sweet cherry (Prunus avium L.)

Increasing costs and decreasing labor availability for sweet cherry harvest in Washington State, USA, has reinvigorated commercial and research interest of mechanized harvest. Ethephon (2-chloroethyl phosphonic acid) can be used to improve fruit abscission for mechanical harvest. Our previous work shows that 3.5 l ha⁻¹ ethephon enhances red color and reduces firmness of the cultivar ‘Bing’. In the current study, we used metabolic profiling of cultivars ‘Bing’, Chelan’, and ‘Skeena’ fruit meso and exocarp tissue to better understand underlying quality-related metabolism associated with ethephon application. Trees were treated using air-blast sprayer 13–14 days prior to harvest and fruit samples were harvested every 7–10 days starting at least 17 days prior to commercial harvest. Nearly 200 identified and partially characterized metabolites from mesocarp and exocarp tissue were characterized and evaluated. Principal component analysis models revealed changes in the metabolome associated with both natural ripening and ethephon-induced changes, including associations to key color, acid, and sugar components, such as cyanidin 3-glucoside, malic acid and sugar metabolism.     

Pre-harvest foliar application of Prohexadione-Ca and gibberellins modify canopy source-sink relations and improve quality and shelf-life of ‘Bing’ sweet cherry

This research evaluated the potential of gibberellins (GA), and Prohexadione-Ca (PCa) to affect sweet cherry (Prunus avium) fruit size and quality. The results demonstrate the ability of ostensibly counter-acting plant growth regulators to significantly improve sweet cherry cv ‘Bing’ fruit size, fruit quality and postharvest characteristics compared to the current commercial application of GA₃ alone. In 2008, we found that the combination of GA₃ or GA_4/7 (30 mg l⁻¹) with PCa (150 mg l⁻¹) applied to entire 3-year-old limbs 30 days after anthesis increased fruit size and improved fruit quality in ‘Bing’. In 2009, we investigated the effect of application timing in larger-scale field trials, comparing treatments made at 30 or 37 days after anthesis, on fruit quality, storability and sensory attributes after storage. Treatment with PCa + GA₃ or PCa + GA_4/7 delayed fruit maturity by about 7 days compared to the untreated control. Both the first and second applications of PCa + GA_4/7 resulted in 35–40% fruit being ≥10 g, compared with only 20% in the control. PCa + GA₃ treatment also showed greater potential for improving fruit storability by maintaining fruit firmness, sweetness, and consumer appeal than PCa + GA_4/7. PCa alone or in combination with GAs inhibited current shoot growth and delayed fruit coloring development. After 30 days of 4°C storage, fewer than 5% fruit from untreated trees were rated as healthy and marketable, compared to 50 and 30% fruit from PCa + GA₃ treatment applied at 30 or 37 days after anthesis, respectively. In conclusion, preharvest foliar application of PCa + GA₃ at the onset of Stage II of fruit development shows potential to affect canopy source-sink relations and improved quality and shelf life of ‘Bing’ sweet cherries.     

Response of ‘Clausellina’ Satsuma mandarin to 3,5,6-trichloro-2-pirydiloxyacetic acid and fruitlet abscission

The application of the synthetic auxin 3,5,6-trichloro-2-piridyloxyacetic acid (3,5,6-TPA) isopropyl ester at the onset of cell enlargement stage, significantly thinned fruitlets in ‘Clausellina’ Satsuma mandarin. The magnitude of the response was related to the concentration applied, increasing the percentage of abscised fruit with higher concentrations, which was up to 70% at 25 mg l⁻¹. The magnitude of the response also depended on the organ subject to treatment, abscission being greater when applied to the leaves rather than to fruit. Results suggest that a photosynthetic disorder was responsible for a reduction in fruit growth rate, triggering abscission mechanisms producing ethylene and abscission.     

Exogenous gibberellic acid increases the fruit weight of ‘Comte de Paris’ pineapple by enlarging flesh cells without negative effects on fruit quality

In mainland China, the most popular pineapple cultivar is ‘Comte de Paris’. Gibberellic acids have been widely applied to enhance fruit growth in various species. To evaluate the effect of gibberellic acid (GA₃) on ‘Comte de Paris’ pineapple production and quality, pineapple fruits were sprayed with GA₃ at concentrations of 5, 20, 50, or 100 mg l⁻¹ at both 0 and 15 days after flowering (DAF). Fruits were sampled every 15 days from 0 to 60 DAF (maturation) for flow cytometric analysis and histological observation. The results showed that the treatments with the three highest concentrations of GA₃ significantly increased fruit weight, and the most effective concentration was 50 mg l⁻¹ GA₃, which increased the flesh weight by 20.3% compared to the control. Although treatment with GA₃ had little effect on the total soluble solids and fruit juice pH, it increased the vitamin C content. Although flow cytometric analysis showed that the 50 mg l⁻¹ GA₃ treatment had only a slight impact on the number of S phase cells, histological observations indicated that the increase of fruit volume and flesh weight under this GA₃ treatment was not due to the increase of cell number but a result of the increase of cell area in the fruit flesh.     

Ethephon and Aventrol as tools to enhance spring rape productivity

Ethephon and Aventrol were used as tools to provoke the processes taking part in the formation of rape seed yield and quality. Investigations on spring rape (Brassica napus L.) cultivars ‘Terra’ and ‘Landmark’ were carried out from 2008–2010. Ethephon (10 mM) and Aventrol (1 l/ha — pinolene 960 g/l) were used on different plant growth stages: BBCH-62–64 and BBCH-72–74, BBCH-80–82, respectively. Impact of ethephon manifested itself as activation of ethylene evocation by siliqua and a slight activation of growth of siliqua dehiscence zone. Siliqua cell plasmalemma and tonoplast H⁺-ATPases activation under the influence of ethephon occurred but did not lead to the destruction of transmembrane electrochemical potential. Extra seed yield and crude fat yield increased; tendency towards a lowering of the saturated/unsaturated fatty acid ratio was observed. Under the influence of Aventrol the dehiscence zone of siliqua was more closed when compared to the control and the ethephon treated variants, seed loss was significantly lowered and transmembrane cation transport was not damaged. The seed yield increased, and this was due to the accumulation of extra crude fat. Aventrol did not change the fatty acid content in rape seed oil. The positive impacts of ethephon and Aventrol for spring rape seed yield formation and possible mechanisms are discussed.     

The influence of plant growth regulators and storage on root induction and growth in <Emphasis Type="Italic">Pelargonium zonale</Emphasis> cuttings

The effects of post harvest application of ethylene, abscisic acid (ABA), indole-3-butyric acid (IBA) treatments or dark storage on root induction and continued growth of regenerated roots in Pelargonium cuttings were investigated using hydroponics in the greenhouse. Ethylene markedly increased rooting percentage in ‘Greco’ and ‘Surfing’, reduced the number of roots per cutting in ‘Surfing’ and had no effect on the total root lengths in the two cultivars. Ethylene treatment reduced fresh root mass in ‘Surfing’, increased dry root mass and reduced root water content in both cultivars. ABA (50 μM) enhanced rooting percentage in ‘Greco’, reduced the number of roots per cutting, reduced total root lengths and fresh root mass in both cultivars. ABA increased dry root mass and reduced root water content in ‘Surfing’ but this effect was not apparent in ‘Greco’. Storing cuttings in the dark for 4 days had no effect on rooting percentage and number of roots per cutting in ‘Greco’ and ‘Surfing’. However, dark storage reduced total root lengths in ‘Surfing’ and reduced fresh root mass in ‘Greco’. Dark storage had no effect on dry root mass and water content in both cultivars. Applying 4 μl l⁻¹ IBA in the rooting solution induced maximum (100%) root induction in ‘Surfing’. However, IBA reduced the number of roots per cutting in ‘Greco’, reduced total root lengths and fresh root mass in the two cultivars. IBA treatment profoundly increased and reduced dry root mass and root water content, respectively, in ‘Greco’ and ‘Surfing’. The enhanced root induction observed after IBA and ABA applications could be ascribed to their influence on ethylene biosynthesis, since ethylene treatment increased rooting percentage in both cultivars. However, high ABA (100 μM) and IBA (12 μl l⁻¹) levels or dark storage reduced the ability of induced roots to continue growth. We attribute our results to plant stress-response mechanism and ethylene appears to play an important role in the process of root initiation and root growth in Pelargonium cuttings.     

Effect of 1-methylcyclopropene on postharvest physiological changes of ‘Zaohong’ plum

Plum is a highly perishable fruit and postharvest fruit softening limits its shelf life. The aim of this work was to study the specific effects of 1-methylcyclopropene (1-MCP) treatment on physiological changes in ‘Zaohong’ plums. Plums were treated with 500 nL L⁻¹ 1-MCP at 20°C for 18 h followed by 20°C storage. The results showed that 1-MCP treatment significantly reduced endogenous ethylene production and the activities of ethylene biosynthetic enzymes’ (1-aminocyclopropane-1-carboxylic acid synthase, ACS and 1-aminocyclopropane-1-carboxylic acid oxidase, ACO) in plum fruit during storage when compared with untreated fruit. Although 1-MCP treatment inhibited ethylene production and 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation, it did not inhibit the accumulation of N-malonyl-ACC (MACC). Higher firmness was also found in 1-MCP-treated plums than in controls. During storage, superoxide anion (O₂^−·) and hydrogen peroxide (H₂O₂) levels decreased in 1-MCP-treated fruit. 1-MCP treatment also regulated superoxide dismutase (SOD) and catalase (CAT) activities during storage. Xylanase activity was upregulated while activities of polygalacturonase (PG), pectin methyl esterase (PME) and cellulase enzymes in the fruit were downregulated by 1-MCP treatment. In conclusion, 1-MCP might be a potent compound for extending both storage period and shelf life of ‘Zaohong’ plums by suppressing ethylene biosynthesis, regulating cell wall degradation enzymes and reducing fruit softening.     

Effects of exogenous B supply on growth, B accumulation and distribution of two navel orange cultivars

To investigate the effects of boron (B) on growth, B concentration and distribution of two navel orange cultivars, ‘Newhall’ (Citrus sinensis Osbeck) and ‘Skagg’s Bonanza’ (Citrus sinensis Osbeck) grafted on the rootstock trifoliate orange [Poncirus trifoliata (L.) Raf.], B at five levels was exogenously supplied to 1-year-old grafted plants of both cultivars under greenhouse conditions. Plants were grown in sand:perlite (1:1, v/v) medium and were irrigated every 2 days with half-strength Hoagland’s No. 2 nutrient solutions containing different B, 0.01, 0.05, 0.10, 0.25 and 2.50 mg l⁻¹ (0.25 and 2.50 mg l⁻¹ were considered as control and excess B treatment, respectively, and the other three B levels were considered as low B treatments). After treatments for 183 days, leaves (from basal, middle, upper parts of the shoots), stem of scion, stem of rootstock and root were separately sampled. Our results showed that plant growth (plant height, root volume and dry weights of various parts) was inhibited in response to low or excess B supplies in both cultivars. It was found that B concentrations in the upper leaves of both cultivars were substantially higher than those in the basal leaves when low concentrations (≤0.05 mg l⁻¹) of exogenous B were applied, suggesting that B was preferentially translocated to the upper-younger leaves to support their growth. Analysis of B distribution in different parts indicated that translocation of B from the root to the scion’s shoots (stems and leaves of scion) may be restricted upon exposure to low B conditions. When B was inadequately supplied, growth of ‘Skagg’s Bonanza’ was better than ‘Newhall’, implying that the former cultivar was more tolerant to low B status, which may be due to the higher efficiency of B translocation from the root to the scion’s shoots. However, when the plants were treated with excess B (2.50 mg l⁻¹), both cultivars showed a similar degree of B toxicity. The probability of scion–rootstock interactions in relation to the differential responses of growth and different efficiency of B translocation involved in the two orange cultivars following the long-term low B stress were discussed.     

Regeneration of peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L. Batsch) cultivars and <Emphasis Type="Italic">Prunus persica</Emphasis> × <Emphasis Type="Italic">Prunus dulcis</Emphasis> rootstocks via organogenesis

Somatic peach plants were regenerated from callus derived from the base of stem explants of the scion cultivars ‘UFO-3’, ‘Maruja’, ‘Flariba’ and ‘Alice Bigi’, and the peach × almond rootstocks ‘Garnem’ and ‘GF677’. A protocol for organogenic plant regeneration was developed using three culture media containing different concentrations of 6-benzyladenine (BA) and indolebutyric acid to produce organogenic calli. Shoots were obtained from sliced calli after their transfer to a differentiation culture medium containing 2 mg l⁻¹ BA and 1 mg l⁻¹ α-naphthalene acetic acid. Using this procedure, up to 29 regenerated plants per callus were obtained. The highest regeneration rate was obtained with the peach × almond rootstocks. This work provides an effective protocol that could be utilized for peach transformation research.     

Differential effects of abscisic acid and ethylene on the fruit maturation of <Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn.

Two litchi cultivars, a well-coloured ‘Nuomici’ and a poorly coloured ‘Feizixiao’, were used to investigate changes in endogenous abscisic acid (ABA) concentration and ethylene production during fruit maturation and to test the effects of exogenous growth regulators on litchi fruit maturation. Abscisic acid concentration in both the aril and pericarp increased with fruit maturation. Transfusion of ABA into the fruit 3 weeks before harvest accelerated, whereas transfusion of 6-benzyl aminopurine (6-BA) retarded sugar accumulation and pigmentation. The effect of 6-BA was assumed to link with the resultant decrease in ABA. In contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) concentration and ACC oxidase (ACO) activities in the aril remained relatively constant during sugar accumulation. Transfusion of aminooxyacetic acid (AOA) significantly decreased ACC concentration but had no effect on sugar accumulation in the aril. These results suggested that endogenous ABA, but not ethylene, was critical for the sugar accumulation. However, the roles of ABA and ethylene in pericarp pigmentation were rather complicated. Application of exogenous ABA promoted anthocyanin synthesis significantly, but had very little effect on chlorophyll degradation. Ethylene production in litchi fruit decreased with development, but a transient increase of endogenous ethylene production was detected just around the colour break in ‘Nuomici’. Enhanced ACO activity in the pericarp was detected during pigmentation. Ethrel at 400 mg l⁻¹ showed no effect on pericarp coloration, but accelerated chlorophyll degradation and anthocyanin synthesis at a much higher concentration (800 mg l⁻¹). Fruit dipped in ABA solution alone yielded no effect on chlorophyll degradation, but the combined use of ABA and Ethrel at 400 mg l⁻¹ enhanced both chlorophyll degradation and anthocyanin synthesis. These results indicated the possible synergistic action of ethylene and ABA during litchi fruit colouration. ABA is suggested to play a more crucial role in anthocyanin synthesis, while ethylene is more important in chlorophyll degradation. ABA can increase the sensitivity of pericarp tissue to ethylene.     

Synthetic Auxin 3,5,6-TPA Provokes <Emphasis Type="Italic">Citrus clementina</Emphasis> (Hort. <Emphasis Type="Italic">ex</Emphasis> Tan) Fruitlet Abscission by Reducing Photosynthate Availability

The aim of this study was to determine the effects of the synthetic auxin 3,5,6-trichloro-2-pirydiloxyacetic acid (3,5,6-TPA) on photosynthetic activity, photosynthate transport to the fruit, and fruitlet abscission to further explain the physiological basis of auxin-mediated citrus fruit thinning. Applying 15 mg l⁻¹ 3,5,6-TPA to trees during the fruit cell division stage significantly increased fruitlet abscission of Clementine mandarin. On treated trees, abnormal foliar development and photosynthetic damage were observed at the same time as 3,5,6-TPA reduced fruitlet growth rate. Briefly, treatment reduced chlorophyll and carotenoid concentrations and modified chlorophyll a fluorescence parameters, that is, reduced the quantum yield (ФPSII) of the noncyclic electron transport rate, diminished the capacity to reduce the quinone pool (photochemical quenching; q_p), and increased nonphotochemical quenching (q _N), thereby preventing the dissipation of excess excitation energy. In addition, the net photosynthetic flux (μmol CO₂ m⁻² s⁻¹) and leaf photosynthate content decreased in treated trees. As a result, the 3,5,6-TPA treatment significantly reduced the photosynthate accumulation in fruit from day 3 to day 8 after treatment, thus reducing fruitlet growth rate. Hence, treated fruitlets significantly increased ethylene production and abscised. Twenty days after treatment, chlorophyll a fluorescence parameters and fruitlet growth rate were reestablished. Accordingly, the thinning effect of 3,5,6-TPA may be due to a temporarily induced photosynthetic disorder that leads to reduction in photosynthate production and fruitlet uptake that temporarily slows its growth, triggering ethylene production and fruitlet abscission. Afterward, the remaining treated fruit overcame this effect, increased growth rate, and reached a larger size than control fruit.     

Satellite tracking reveals distinct movement patterns for Type B and Type C killer whales in the southern Ross Sea, Antarctica

During January/February 2006, we satellite-tracked two different ecotypes of killer whales (Orcinus orca) in McMurdo Sound, Ross Sea, Antarctica, using surface-mounted tags attached with sub-dermal darts. A single Type B whale (pinniped prey specialist), tracked for 27 days, traveled an average net distance of 56.8 ± 32.8 km day⁻¹, a maximum of 114 km day⁻¹, and covered an estimated area of 49,351 km². It spent several days near two large emperor penguin (Aptenodytes forsteri) colonies, a potential prey item for this form. By contrast, four Type C killer whales (fish prey specialists) tracked for 7–65 days, traveled an average net distance of 20 ± 8.3 km day⁻¹, a maximum of 56 net km day⁻¹, and covered an estimated area of only 5,223 km². These movement patterns are consistent with those of killer whale ecotypes in the eastern North Pacific where mammal-eating ‘transients’ travel widely and are less predictable in their movements, and fish-eating ‘residents’ have a more localized distribution and more predictable occurrence, at least during the summer months.     

Cherry fruit abscission: evidence for time of initiation and the involvement of ethylene

Initiation of abscission at the pedicel-fruit zone in the sour cherry (Prunus cerasus L. cv. Montmorency) occurs near the transition of Stage II to Stage III of fruit growth. The preinitiation phase is characterized by a high fruit removal force (FRF) and explants prepared from fruits during this period do not undergo abscission as indexed by a reduction in FRF. Ethylene does not cause a significant reduction in FRF either in attached fruit or in explants prepared during this period. By contrast, after initiation (Stage III of fruit growth), there is a marked decrease in FRF with fruit development, explants prepared from fruits during this period undergo abscission, and ethylene markedly promotes the loss in break-strength. Neither the rate of evolution nor the internal concentration of ethylene in the fruit were correlated with fruit abscission. Similar abscission responses, as indexed by FRF and sensitivity to ethylene, were observed in attached fruit and in detached fruit explants.     

Strapping and a synthetic cytokinin promote cell enlargement in ‘Hiratanenashi’ Japanese persimmon

The effects of N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) with and without strapping on fruit growth of ‘Hiratanenashi’ Japanese persimmon were evaluated by measuring parenchyma cell size and the number of cell layers in mesocarp throughout fruit development. Three-year-old branches were strapped with a wire 2 weeks before full bloom, and 10 mg l⁻¹ of CPPU was applied to the fruitlet at 10 days after full bloom. CPPU alone prolonged the growth period, resulting in a larger fruit diameter at harvest than the control. Strapping promoted the fruit coloration and increased final fruit diameter. CPPU plus strapping shortened the growth period compared to CPPU alone, and the mature fruit diameter was similar to CPPU alone. The increases in fruit diameter caused by CPPU and strapping were involved with the increases in length of parenchyma cell. The number of cell layers was almost similar among the treatments. These results showed that strapping promotes the rate of parenchyma cell expansion, and CPPU with and without strapping prolong the duration of cell enlargement and promote the rate of the cell expansion.     

The regulation of sweet cherry fruit abscission by polar auxin transport

Inconsistency of cropping is an important problem for UK sweet cherry production. Premature fruit abscission in Prunus can reduce yields severely, however, the environmental cues and hormonal signals that trigger abscission have not been identified. Auxin (IAA) is known to delay abscission by reducing the sensitivity of cells in the abscission zone to ethylene, a promoter of abscission. Therefore, the capacity for polar auxin transport (PAT) through sweet cherry pedicels was examined in relation to fruit abscission. Cherry ‘spurs’ (short shoots) with similar leaf areas and different fruit numbers were phloem-girdled to restrict assimilate movement. Abscission from spurs with many fruit (eight or more) occurred within 14 days of girdling, whereas abscission from spurs with few (two) fruit was minimal. The pedicels’ capacity for PAT in spurs with different fruit numbers was determined 1, 3 and 9 days after girdling (DAG). Fruit were analysed for endogenous IAA concentration 3, 5, 7 and 9 DAG. PAT inhibitors 2,3,5-triiodobenzoic acid or 1-N-naphthylphtalamic acid were applied to pedicels of fruit not expected to abscise, i.e. on spurs with few fruit. The effect of these inhibitors on fruit abscission was determined 14 DAG. The proportion of the transported [³H]-IAA was lower from the outset in pedicels from spurs with many fruit. By 9 DAG, symptoms of fruit abscission were apparent and 40% less [³H] -IAA was transported through pedicels on spurs with many fruit. Fruit endogenous IAA concentrations were similar in the two groups of spurs. Application of PAT inhibitors shortly after girdling increased fruit abscission by 30%. The results suggest that although a decline in PAT is not the only cause of fruit abscission, the maintenance of PAT contributes to fruit retention.     

A two-stage solar photobioreactor for cultivation of microalgae based on solar concentrators

A novel two-stage experimental photobioreactor (PBR) with a total volume of 450 L and based uniquely on solar concentrators—linear Fresnel lenses—has been constructed and tested. Daily courses of irradiance, and also its distribution inside cultivation tubes, were studied in two unit types. The supra-high irradiance units in the ‘roof’ achieved a maximum summer value above 6 mmol photon m⁻² s⁻¹, while irradiance in the vertical-facade units was lower than ‘ambient’. In model cultivations, cultures of the cyanobacterium Arthrospira platensis were cultivated at much higher solar irradiances than those usually recorded outdoors in summer, indicating that this organism is resilient to high-irradiance (photoinhibition). Starting from a biomass density of 0.5 g L⁻¹ at optimum temperature, the cultures grew exponentially. A two-stage cultivation process of the green microalga Haematococcus pluvialis was investigated with respect to correlations between photochemical activities and astaxanthin production. The culture was first grown in low-irradiance units, and then exposed to supra-high irradiance when the rate of astaxanthin production was 30–50% higher than in the culture exposed to ‘ambient’ irradiance. Within 4 days, the astaxanthin content reached 3% of dry weight, whereas under ambient irradiance the astaxanthin content was 25% lower.     

The Effects of Gibberellin4+7 on the Vase Life and Flower Quality of Alstroemeria Cut Flowers

Leaf yellowing is a major problem in Alstroemeria and absence of leaf senescence symptoms is an important quality attribute. Two Alstroemeria cultivars ‘Yellow King’ and ‘Marina’ were sourced from a commercial farm and harvested when sepals began to reflex. Stems were re-cut under water and kept in vase solutions of gibberellin A₄₊₇ (0, 2.5, 5.0, 7.5, 10.0, 12.5 or 15.0 mg l⁻¹ [Provide^r]). Treatments and cultivars were combined in a factorial fashion and arranged in a completely randomised design. Application of GA₄₊₇ in the holding solution at 2.5–10.0 mg l⁻¹ significantly delayed the onset of leaf senescence by around 7 days and significantly increased days to 50% petal fall by ca. 2 days. Additionally, these GA₄₊₇ concentrations resulted in higher retention of leaf nitrogen, leaf chlorophyll and also increased leaf water content, while reducing leaf dry weight, all relative to untreated controls. Cultivar ‘Yellow King’ had significantly longer vase life and a better retention of leaf quality than ‘Marina’. Our results suggest that a concentration of 10 mg l⁻¹ GA₄₊₇ can be used to prolong vase life, delay leaf senescence and enhance post-harvest quality of Alstroemeria cut flowers during their transport to market.     

Highly efficient embryogenesis and plant regeneration of tall fescue (Festuca arundinacea Schreb.) from mature seed-derived calli

Summary We report a protocol for efficient plant regeneration of four tall fescue (Festuca arundinacea Schreb.) cultivars (‘Surpro’, ‘Coronado’, ‘Summer Lawn’, and ‘Fawn’) via somatic embryogenesis. Calli were initiated from mature seeds grown on modified Murashige and Skoog (MMS) medium supplemented with 7.0mgl⁻¹ (31.7μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mgl⁻¹ (0.23 μM) kinetin (Kin). Calli were maintained and proliferated by subculture at monthly intervals on MMS medium containing 4.5 mgl⁻¹ (20.4 μM) 2,4-D and 0.2mgl⁻¹ (0.9 μM) Kin. Somatic embryos (SE) were induced from seed-derived calli on SE-induction medium (MMS supplemented with 2.0 mgl⁻¹ 2,4-D and 0.2mgl⁻¹ Kin). Plantlets were regenerated from somatic embryogenic calli grown on modified SH medium supplemented with 2 mgl⁻¹ Kin. Using this optimized protocol, 78.6–82.3% of mature seeds of all four cultivars produced SE clusters, of which 93.5–95.3% regenerated into plants within 10 wk. The regenerants showed no phenotypic abnormalities.