Factors affecting mango (Mangifera indica L.) protoplast isolation and culture

Scientia Horticulturae 130 (2011) 214–221

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Factors affecting mango (Mangifera indica L.) protoplast isolation and culture Ramezan Rezazadeh ∗ , Richard R. Williams 1 , Dion K. Harrison 2 School of Agriculture and Food Sciences, The University of Queensland, Gatton, Qld 4343, Australia

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Article history: Received 20 December 2010 Received in revised form 8 June 2011 Accepted 29 June 2011 Keywords: Alginate Kensington Pride Mangifera indica L. Mango Proembryonic masses Protoplast culture

a b s t r a c t The major factors influencing protoplast isolation and culture of mango (Mangifera indica L.) cv. Kensington Pride were investigated. The resultant protocol was used to compare plating efficiency among 4 mango cultivars. Most responses differed between proembryonic masses (PEMs) and leaf sources. Protoplast yields of 15.22 × 106 g−1 from PEMs and 8.68 × 106 g−1 from greenhouse-derived leaves were obtained in a solution of 0.7 M mannitol CPW plus 1.5% cellulase, 1% hemicellulase and 0.75% macerozyme for PEMs or 0.5 M mannitol CPW plus 1.5% cellulose, 1% hemicellulase and 1.5% macerozyme for leaves. Culture in Ca-alginate beads with initial plating densities (IPD) of 2.5 × 104 Pp mL−1 for PEMs and 2.5 × 105 for leaves gave the highest plating efficiencies (FPE). For PEMs 1 mg L−1 2,4-d and 3.5 mg L−1 kinetin gave an FPE of 2.85% whereas lower kinetin (2 mg L−1 ) plus 0.5 mg L−1 6-BAP was most effective for leaves (FPE of 2.12%). Most protoplast mortality occurred during the first week of culture and was more severe in liquid culture. In Ca-alginate beads, protoplast survival at 14 days was higher for PEMs (30%) than leaf (21%) as was the frequency of cell division (17.6% compared to 13.6%). PEMs protoplasts continued development through embryogenesis to in vitro plantlet regeneration whereas leaf protoplasts underwent cell division up to 40-cell colonies but failed to proceed further. For PEMs, polyembryonic cvs. Kensington Pride and Keow Savoey produced higher FPE (1.95%) than monoembryonic cvs. Tommy Atkins and Keitt (1.75%). There was no effect of cultivar for leaf protoplasts. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The major limitation facing the production of mango is the shortage of superior cultivars due to difficulties in conventional breeding such as long juvenility phase, a high level of heterozygosity and polyembryony in some cultivars (Iyer and Degani, 1998). Most of the current cultivars of mango (Mangifera indica L.) are selections from open-pollinated seedling populations (Litz, 2004). Protoplast-based approaches such as somatic hybridization, organelle or DNA microinjection, DNA electroporation, and microprotoplast-mediated chromosome transfer (MMCT) have the potential to overcome some of the obstacles to conventional breeding through direct transfer of genomes into the plant cells. Among these strategies, somatic hybridization has enormous potential to introduce variation in both the nuclear and cytoplasmic genome (Ishikawa et al., 2003) but this requires efficient protoplast iso-

∗ Corresponding author. Present address: Agricultural Research Center, Bandar Abbas, 7915847669 Hormozgan Province, Iran. Tel.: +098 761 4313806-8; fax: +98 761 3332496. E-mail addresses: [email protected] (R. Rezazadeh), [email protected] (R.R. Williams), [email protected] (D.K. Harrison). 1 Tel.: +61 754601305. 2 Tel.: +61 754601313. 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.06.046

lation and culture systems. Plant regeneration has been reported from protoplasts of different sources such as leaves of axenic shoot cultures (Ortin-Parraga and Burgos, 2003) and calli (Miranda et al., 1997). For mango, there is one published report of successful plant regeneration from protoplasts derived from proembryonic masses (PEMs) of M. indica cv. Amrapali (Ara et al., 2000). We have extended this to other mango cultivars and explored glasshousegrown leaves as an alternative vegetative source, by examining osmolarity, digesting enzymes, initial plating density (IPD), culture type, plant growth regulators (PGRs) and four mango cultivars: Kensington Pride, Keow Savoey, Tommy Atkins and Keitt). 2. Materials and methods 2.1. Plant materials PEMs were induced on nucellus of immature fruits as described by DeWald et al. (1989). Slow growing PEMs turned to fast growing suspension when proliferated in liquid culture and deletion of 2,4-d from the media at the last sub-culture produced high-quality PEM free of black clumps (based on our preliminary results). Suspension cultures were passed through a 250-␮m sieve and the filtrate was centrifuged at 50 × g to pellet cell aggregates. For leaves, seedlings were grown under 40% shade and 70% humidity for two consecutive flushes. Partially expanded leaves were surface sterilized, mid ribs

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and necrotic areas removed and the remaining tissues cut into thin strips (0.2–0.5 mm). 2.2. Protoplast isolation Protoplast yield (Pp g−1 – number of protoplasts per gram of tissue) was determined using a double-chamber haemocytometer. Viability was measured by fluorescein diacetate (FDA) staining (Widholm, 1972) at isolation (0 h) and 24 h after (24 h). Cell wall regeneration was confirmed with calcofluore white (FBA) (Nagata and Takebe, 1970). Enzyme concentrations used were representative of published protocols and all enzyme solutions were adjusted to pH 5.8 and filter-sterilized with 0.22 ␮m Millipore (Millex® ) filter. Protoplasts were isolated as follows: one gram of drained PEMs pellet or fresh leaf strips were transferred to 10 mL of enzyme mixture in a 250-mL Erlenmeyer flask and incubated in darkness at 27 ◦ C on rotary shaker at 30 rpm. After 14 h (PEMs) or 16 h (leaves), suspensions were passed through a 75-␮m sterile stainless steel sieve or 40-␮m nylon sieve, respectively. The filtrate was transferred to a 15-mL falcon tube and centrifuged at 100 × g for 5 min. After discarding the supernatant, protoplast pellets were washed twice with CPW media of the corresponding osmolarity. Protoplasts were purified through density gradient centrifugation by placing 2 mL of crude protoplast suspension on the top of a 4 mL of a 21% (PEMs) or 25% (leaves) sucrose pad. After centrifugation at 80 × g for 3 min, protoplasts were collected by Pasteur pipette (from intermediate density) and re-suspended in fresh medium. 2.3. Data analysis Data from all experiments were analyzed by ANOVA using Minitab® (14) and Tukey’s test was utilized for comparison of means except comparison of culture types where Duncan’s multiple range was used. Original data were analyzed for IPE but for MPE and FPE data were first transformed to Arcsine (SQRT (P)) × 180/3.14 where P = proportion (0–30%), due to homogeneity of variances. The nominated probabilities are reported with the results. 2.4. Osmolarity and protoplast yield Mannitol and sorbitol (Fig. 1) were dissolved in CPW salts (Frearson et al., 1973), supplemented with 30 g L−1 sucrose, 0.75% cellulase R-10 (Yakult Honsha Co., Ltd.), 0.75% hemicellulase (Sigma), 1% macerozyme R-10 (Yakult Honsha Co., Ltd.). Two

Fig. 2. Effect of total enzyme concentration (%) (bottom) and three different enzymes on PEM (closed) and leaf (open) protoplast yield (Y × 106 g−1 ).

separate completely randomized experiments (CRD) were conducted with 14 osmoticum treatments (0.5, 0.6, 0.7, 0.8, 0.9 M each separately and in combinations to give equivalent total molarities) with 4 replicates. The data were combined for the analyses. 2.5. Enzyme combinations and protoplast yield and viability

Fig. 1. Effect of mannitol (), sorbitol () or total osmoticum (×) concentrations on the yield (Y × 106 g−1 ) of protoplasts from PEM (closed) and leaf (open).

For protoplast isolation, enzymes (Fig. 2) were dissolved in CPW with 0.7 M (PEMs) or 0.5 M (leaves) mannitol plus 30 g L−1 sucrose. Two separate 3 × 2 × 3 (cellulose:hemicellulase:macerozyme) CRD factorial experiments were replicated 4 times. Protoplast yield and viability were determined at 0 h and 24 h.

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2.6. Other procedures affecting protoplast isolation Other procedures tested to improve the isolation of protoplasts were shaker speed, pre-plasmolysis duration and the inclusion of PVP in the medium. A range of conditions were tested but only the key results are reported below. 2.7. Culture of isolated protoplasts The first media dilution was performed 10 days after protoplast isolation (based on preliminary experiments where cell wall regeneration was completed between 7 and 10 days, and cell division commenced 48–96 h later). Initial plating efficiency (IPE) was defined as the percentage of cells undergoing one division after 2 weeks; intermediate plating efficiency (MPE) the percentage of microcolonies of 2–10 cells after another week; final plating efficiency (FPE) the percentage of colonies of more than 10 cells after 4 weeks. The basic protoplast culture medium (extrapolated from various published protocols) consisted of B5 major salts (Gamborg et al., 1968) with 4 mM CaCl2 and without (NH4 )2 SO4 ; MS minor salts (Murashige and Skooge, 1962) plus 20 mg L−1 thiamine–HCl, 10 mg L−1 pyridoxine, 2 mg L−1 nicotinic acid, 5 mg L−1 pantothenic acid, 2% coconut water, 30 mg L−1 ascorbic acid, 1.5 g L−1 lglutamine, 100 mg L−1 myo-inositol, 500 mg L−1 proline, 30 g L−1 sucrose plus osmotica. The pH of was adjusted to 5.8 and the media filter sterilized. Cultures were kept under darkness at 25 ± 2 ◦ C. 2.8. Comparison of IPD, culture type and PGRs on protoplast regeneration 2.8.1. Liquid culture Protoplasts were plated at the required initial plating density (IPD) in 3 mL of culture media as a shallow layer in 65 mm × 10 mm Petri dishes sealed by parafilm. For medium dilution, 500 ␮L was replaced with fresh mannitol-free medium after cell wall formation and every 4 weeks thereafter until cell colony formation. Plating efficiency was observed under an inverted microscope (400×), 10 fields per Petri dish. 2.8.2. Ca-alginate bead culture Sodium alginate (Sigma) was dissolved in osmoticum at a concentration of 4% (w/v) (twice the final concentration) by stirring for 4 h then filter sterilized. Protoplasts were re-suspended at twice the desired final IPD in calcium-free culture medium then mixed with alginate solution by gently swirling the tubes. The mixture was then dropped (40-␮L droplets) into medium containing the same composition minus Na-alginate and plus 50 mM CaCl2 . After 1 h, the solidified Ca-alginate beads were washed twice × 10 min with the culture medium and fifty beads were suspended in 3 mL of culture medium then incubated at 30 rpm. Dilution was carried out by replacing 15% of the solution phase with mannitol-free medium. The plating efficiency was determined by observing 5 droplets each in 5 cross-sections. Cell colonies were released after depolymerization of Ca-alginate beads in 20 mM sodium citrate solution according to Scheurich et al. (1980). 2.8.3. Agarose-bead culture Low melting agarose (Sigma) at 1.2% (w/v) in osmoticum was autoclaved and cooled to 40 ◦ C in a water bath. Suspensions of protoplasts in culture media (double strength) were mixed with the same volume of agarose solution at 40 ◦ C and 40 droplets (50 ␮L) of solution were placed in each Petri dish. The droplets in each Petri dish were submerged in 3 mL culture media. Dilution was carried

out by replacing 500 ␮L of liquid phase with media free of mannitol. The plating efficiency was determined by observing 5 droplets each in 5 cross-sections. 2.8.4. Semisolid culture Gelrite (Sigma) at 3.4 g L−1 was melted and mixed with the same volume of double strength culture media at 40–50 ◦ C. Two mL of the mixture was poured into each Petri dish. Protoplast suspension was layered and mixed with the top layer of this semisolid gel. Cultures were maintained stationary in darkness. For dilution, the gel was bathed twice for 30 min with filter-sterilized liquid medium with ¾ initial concentration of mannitol. The plating efficiency was measured by observing 10 fields for each replication using an inverted microscope (400×). Protoplasts were cultured at different IPD (Figs. 4 and 6) and cytokinin combinations (Fig. 5) in each of the four culture types. PEMs and leaf were tested in separate CRD factorial experiments replicated 5 times. 2.9. Protoplast development in response to culture type In two separate CRD experiments with 6 replicates, protoplasts were cultured in each of the four culture types; PEMs protoplasts at an IPD of 2.5 × 104 Pp mL−1 in culture medium containing 1 mg L−1 2,4-d and 3.5 mg L−1 kinetin; leaf protoplasts at an IDP of 2.5 × 105 Pp mL−1 in medium containing 0.5 mg L−1 6-BAP, 1.0 mg L−1 2,4-d and 2 mg L−1 kinetin. Survival rate and percentages of divided cells were recorded after 14 days. 2.10. Comparing the protoplast plating efficiency of 4 cultivars In separate CRD experiments with 10 replicates, differences in protoplast plating efficiency of cvs. Kensington Pride, Keow Savoey, Tommy Atkins and Keitt were compared using Ca-alginate beads. PEMs protoplasts were cultured at 2.5 × 104 Pp mL−1 in a medium containing 1 mg L−1 2,4-d and 3.5 mg L−1 kinetin. Leaf protoplasts were cultured at 2.5 × 105 Pp mL−1 in medium containing 0.5 mg L−1 6-BAP, 1.0 mg L−1 2,4-d and 2 mg L−1 kinetin. 3. Results 3.1. Osmolarity and protoplast yield The yield of protoplasts was dependent on the total concentration of osmoticum irrespective of whether it was mannitol, sorbitol or a mixture (Fig. 1) but sorbitol or the mixture (data not shown) released fewer protoplasts than mannitol alone. The response was much less for leaf protoplasts compared to PEMs (Fig. 1). The highest protoplast yield was at 0.7 M osmoticum with manitol producing 13.2 × 10−6 Pp g−1 from PEMs (Fig. 3e–i). The highest yield for leaf protoplasts was 5 × 10−6 Pp g−1 with 0.5 M mannitol (62% of that for mannitol). The osmolarity of the enzyme solution with 0.5 M and 0.7 M mannitol was ∼800 and ∼1100 mmol kg−1 , respectively. Compared with leaves, PEMs produced far less debris after digestion. The protoplast diameter ranged from 16 to 26 ␮m for PEMs compared to 11–15 ␮m for leaf protoplasts (Fig. 3b and h). 3.2. Enzyme combinations and protoplast yield and viability Cellulase, hemicellulase and macerozyme interacted significantly (P < 0.001) to effect protoplast yield. Overall, protoplast yield from PEMs or leaf was proportional to the total enzyme concentration above a minimum of 2% (Fig. 2). On the basis of overall responses within this range, hemicellulase had the greatest effect

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Fig. 3. Response surface graphs illustrating interaction among enzymes for PEM protoplasts. For each graph in the left column the third factor is the low level and shows a similar interaction between each enzyme pair. In the right column the third factor is at the low level; hemicellulose is additive at high cellulose and macerozyme. Derived from Design Expert® Software.

on yield for PEMs (16 × 106 g−1 ) and leaf (8 × 106 g−1 ) (Fig. 2), however this effect was mainly at the highest concentrations of the other two enzymes. The interaction between the three enzymes was similar at high concentrations (Fig. 3a) whereas only hemicellulase increased yield when included at low concentrations (Fig. 3b). There was no significant effect (P < 0.001) of enzyme treatment on protoplast viability but the treatments giving the highest yields also gave the highest viabilities, initially 84–92% and 85–90% and after 24 h, 72–78% or 75–81%, for PEMs and leaf respectively. 3.3. Other procedure affecting protoplast isolation The best combinations of protoplast yield and viability for PEMs (14.04 × 106 Pp g−1 ; 91.5% viability) and leaves (7.91 × 106 Pp g−1 ; 89.9% viability) were achieved using a shaker at 30 and 45 rpm, respectively. Pre-plasmolysis for 1 h significantly increased (P < 0.05) the protoplast yield of PEMs to 15.22 × 106 Pp g−1 and leaves to 8.68 × 106 Pp g−1 without any significant changes (P < 0.05) in viability. Inclusion of 1% PVP-10 in the isolation media significantly enhanced (P < 0.001) the protoplast viability at 24 h for PEMs to 86.8% and leaves to 84.5%.

3.4. Effect of IPD, culture type and PGRs on protoplast regeneration of cv. Kensington Pride IPE, MPE and FPE for leaf and PEMs protoplasts were significantly affected by the combination of initial plating density, PGRs and culture type (P < 0.01). For leaf protoplasts, the highest percentages of IPE (13.4%), MPE (9.81%) and FPE (2.12%) were achieved with an IPD of 2.5 × 105 Pp mL−1 in Ca-alginate beads in the presence of 0.5 mg L−1 6-BAP, 2 mg L−1 kinetin and 1 mg L−1 2,4-d (Fig. 4). Overall, the highest FPE of 2.85% was obtained when PEMs protoplasts were cultured Ca-alginate beads at an IPD of 2.5 × 104 Pp mL−1 . PEMs protoplast cultures produced micro-colonies, embryos and plantlets in all treatments (Fig. 4h–p). For leaf protoplasts, the effect of the type of plating medium was proportionately similar at each stage with calcium alginate beads providing the highest plating efficiency (Fig. 6). A lower initial planting density resulted in a higher plating efficiency with both the liquid and alginate beads. The type of cytokinin in the medium followed a similar pattern between stages with the combination of 0.5BAP and 2KIN giving the highest plating efficiency (Fig. 7). Leaf protoplast cultures produced colonies of up to 40 cells (Fig. 4b–d).

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Fig. 4. Protoplast regeneration from Mango cv. Kensington Pride: (a) young leaves; (b) leaf protoplasts stained with FDA; (c) microcolonies derived from leaf protoplasts and (d) stained with FDA; (e) ovule half (arrow) in immature fruit; (f) induced PEMs on ovule half; (g) PEMs suspension culture; (h) PEMs protoplasts and (i) PEMs protoplasts stained with FDA; (j) a necrotic (arrow) and divided PEMs protoplast; (k) microcolonies of cell and PEMs regeneration; (l) microcolonies stained with FDA; (m) embryogenesis from PEMs; (n) mature embryos; (o) radicle germination of mature embryos; (p) shoot emergence on mature rooted embryos. Scale bars = 10 ␮m (b, c, d, h, i, j, and l) and =1 cm (a, e, f, g, k, m, n, o, and p).

3.5. Protoplast development in response to culture type

4. Discussion

Culture in alginate beads lead to the best subsequent development of the protoplasts and PEMs survived and developed better than leaf protoplasts. The highest survival of protoplasts after 14 days was 30% for PEMs with 5% budding and 17.6% dividing. For leaf protoplasts survival was 21% with 13.6% division.

To our knowledge this is the first report of protoplast isolation and culture from leaf of mango and PEMs of cvs. Kensington Pride, Keow Savoey, Tommy Atkins and Keitt. PEM-derived protoplasts produced embryos and plantlets. Leaf protoplasts yielded a useful number of protoplasts but they did not progress beyond cell microcolonies. PEMs and leaf protoplasts differed in their responses to the type of culture, osmolarity, enzyme combination, plating density, growth regulators and genotype.

3.6. Comparing the protoplast plating efficiency of 4 cultivars For PEMs protoplasts the polyembryonic cultivars (Kensington Pride, Keow Savoey) had significantly (P < 0.05) higher plating efficiencies at each stage compared to the monoembryonic cultivars (Tommy Atkins and Keitt) (Table 1) however there were no significant differences between cultivars for leaf protoplasts.

4.1. Culture type The highest production of viable protoplasts and subsequent plant development was achieved using Ca-aginate beads rather than other liquid or semi-solid media. Similar results have been

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Table 1 Plating efficiency of four cultivars: Kensington Pride, Keow Savoey, Tommy Atkins and Keitt. Cultivar

Parameter (%) Leaf protoplast

Kensington Pride Keow Savoy Keitt Tommy Atkins

PEMs protoplast

Initial plating efficiency

Medium plating efficiency

Final plating efficiency

17.5 a* 20.5 a 11.4 b 10.7 b

6.8 a 7.1 a 3.8 b 4.0 b

2.40 a 2.95 a 1.80 b 1.70 b

Mean

2.65 a 1.75 b

Initial plating efficiency 12.90 a 12.60 a 13.70 a 11.50 a

Medium plating efficiency 9.30 a 8.40 a 7.80 a 8.60 a

Final plating efficiency 2.10 a 1.80 a 2.02 a 2.10 a

Mean

1.95 a 2.05 a

* Means with the same letters in each column are not significantly different among the investigated factors (cultivars, poly- and mono-embryonic group) according to Tukey’s test (P = 0.01).

reported for chicory (Nenz et al., 2000), sweet orange (Niedz, 1993), apple (Perales and Schieder, 1993), litchi (Yu et al., 2000) and avocado (Witjaksono et al., 1998). Mango leaf protoplasts were more sensitive to culture type than PEM protoplasts particularly for initial plating efficiency (Figs. 4 and 5). Immobilization of protoplasts in alginate (Nenz et al., 2000; Yu et al., 2000) or agarose (Sinha and Caligari, 2005) may improve protoplast isolation but the use of agarose was more difficult due to the higher temperature during solidification and perhaps reduced liquification during culture. Mechanical support of the protoplasts has been shown to have numerous benefits: reduced cell leakage by increasing the membrane stability (Schnabl et al., 1983); reduced budding (Yu et al., 2000); prevention of aggregation, reduction of diffusion of phenolics and reduced gradients in the media (Draget et al., 1988); and reduced proteolytic enzymes from dying cells (Schnabl et al., 1983). 4.2. Osmolarity The mango PEMs produced their highest yield with an osmolarity of 0.7 M compared to 0.5 M for leaf protoplasts (Fig. 1). In citrus (Grosser and Gmitter, 1990) and apple (Patat-Ochatt et al., 1988), protoplasts from callus and in vitro leaves were obtained in the same osmolarity. Differences in optimum osmolarity presumably relate to the solute content of the cells; our PEM cells were grown in nutrient-rich culture media increasing the cell solute level. Such differences in cell solute concentration may also account for the variability among cells or tissues and between batches; some protoplasts were obtained over a wide range of osmolarities. Mannitol produced a higher yield of protoplasts than sorbitol or a mixture of the two. Mannitol is an inert sugar alcohol which may be more sta-

ble in the media (Evans, 1976) while sorbitol is more readily taken up by cells (Warren, 1991). 4.3. Enzymes Leaf protoplasts required less total enzymes; they required a higher concentration of macerozyme (1.5–2%) than PEMs (1–0.75%) (Fig. 2). This may be attributed to the greater content of structural materials in leaves (evident from the greater residual debris during protoplast isolation). Plant cell walls contain pectics (Warren, 1991) and pectic polysaccharides (Carpita and Gibeut, 1993) which are degraded by macerozyme. 4.4. Plating density Protoplasts are generally cultured with an IPD range of 5 × 104 to 1 × 106 Pp mL−1 (Davey et al., 2005). In the present study with mango cv Kensington Pride, overall plating efficiency depended on the IPD with the best outcome with PEMs (19.5% cell division and 2.85% colony formation) following an IPD of 2.5 × 104 Pp mL−1 . The type of plant material is important since the best IPD for leaf protoplasts was 10-fold more at 2.5 × 105 Pp mL−1 resulting in 13.2% cell division and 2.12% colony formation. Working with mango cv. Amrapali, Ara et al. (2000) obtained 71% division at 5 × 104 Pp mL−1 IPD in liquid culture; however they made no mention of FPE. Our results compare favorably with 1.89% for pear (Ochatt and Power, 1988) and 1.7% for avocado (Witjaksono et al., 1998). Davey et al. (2005) stated that an excessively high IPD may deplete the nutrients leading to a failure to sustain cell division while low IPD causes a deficiency of growth stimulus factors for cell division.

Fig. 5. Effect of plating density and medium type on plating efficiency of PEM protoplasts from Mango cv. Kensington Pride. Liq – liquid; CaA – calcium alginate beads; Agr – agarose beads; SS – semi-solid; numerals – IPD (×104 Pp mL−1 ). IPE, MPE, FPE – initial, median and final protoplast efficiency, respectively. Letters indicate differences among means for CaA according to Tukey’s test.

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Fig. 6. Effect of plating density and medium type on plating efficiency (Y: %). of leaf protoplasts from Mango cv. Kensington Pride. Liq – liquid; CaA – calcium alginate beads; Agr – agarose beads; SS – semi-solid; numerals – IPD (×105 Pp mL−1 ). IPE, MPE, FPE – initial, median and final protoplast efficiency respectively.

et al., 1973; Meyer et al., 2009), apple (Patat-Ochatt et al., 1988) and red clover (Myers et al., 1989). Litz (1984) and DeWald et al. (1989) also reported differences in nucellar somatic embryogenesis between mango cultivars but Litz et al. (1998) stated that such cultivar differences were independent of them being monoor poly-embryonic. The differences between mono- and polyembryonic cultivars occurs at the IPE and particularly the MPE stages with the subsequent rate of conversion to FPE unaffected. This indicates that the genotype effect involves differences in protoplast isolation and cell division rather than the later embryogenesis. This difference is marked by the reduced initial survival of leaf protoplasts. Our findings will aid further research into the understanding and application of protoplast technologies. For example, the use of a medium that enables regeneration of PEM-derived protoplasts but not leaf protoplasts could enable these alternate sources of protoplasts to be used as regenerable and non-regenerable parents respectively, in somatic hybridization and cybridization. Future research will focus on deflasking the resulting in vitro plantlets and improving the leaf protoplast regeneration.

Acknowledgements 4.5. Growth regulators Auxin and cytokinin have both been reported necessary for protoplast regeneration in fruit trees (Ochatt, 1992; Perales and Schieder, 1993; Ortin-Parraga and Burgos, 2003). In this study, media lacking PGRs, induced initial cell division but did not support further cell division of leaf protoplasts. The combination of 0.5BAP and 2KIN provided the highest plating efficiency at each stage (Fig. 6). Kinetin increased cell division while increased 6-BAP stopped cell division (Fig. 7). 4.6. Genotype We found a marked difference between the responsiveness of protoplasts from mono- and poly-embryonic cultivars of mango. Protoplast plating efficiency of PEMs was much higher at each stage for poly-embryonic cultivars (Kensington Pride and Keow Savory) compared to the mono-embryonic cultivars (Tommy Atkins and Keitt) (Table 1). However, plating efficiency of leaf protoplasts was not cultivar dependant. Differences in protoplast regeneration have been reported among genotypes of petunia (Frearson

Fig. 7. Effect of cytokinin combination (mg L−1 ) on plating efficiency (Y: %) of leaf protoplasts from Mango cv. Kensington Pride. BAP – benzyl amno purine; KIN – kinetin. IPE, MPE, FPE – initial, median and final protoplast efficiency, respectively.

The authors would like to thank the Iranian Agricultural Research Organization and School of Agriculture and Food Sciences, The University of Queensland, Australia, for the financial support of this study. The assistance of Allan Lisle and Ross Bourne are also gratefully acknowledged.

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