Plant Cell Reports
P l a n t Cell R e p o r t s (1993) 1 2 : 6 1 - 6 5
9 Springer-Verlag 1993
Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon Michael E. Compton and D.J. Gray Central F l o r i d a R e s e a r c h a n d E d u c a t i o n Center, University o f Florida, Institute o f F o o d a n d Agricultural Sciences, 5336 U n i v e r s i t y Ave., Leesburg, F l o r i d a 34748, U S A Received M a y 28, 1992/Revised version received O c t o b e r 16, 1992 - C o m m u n i c a t e d by J. J. F i n e r
Abstract. Cotyledon explants from immature embryos of five watermelon [Citrullus l a n a t u s (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40/JM), 0.5/JM of either BA or TDZ, and 7 g-11 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon explants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGP, combination for somatic embryogenesis was 10/~M 2,4-D and 0.5/JM TDZ~ Somatic embryogenesis was greatest (30%) when cotyledon explants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit. Key Words: C i t r u l l u s l a n a t u s - cucurbits - tissue culture Abbreviations: PGR: plant growth regulator, BA: benzyladenine, TDZ: thidiazuron, 2,4-D: 2,4dichlorophenoxyaceticacid, NAA: a-naphthaleneacetic acid, 2,4,5-T: 2,4,5-trichlorophenoxyaceticacid. Introduction
Watermelon plants have been obtained from micropropagated shoot tips (Anghel and Rosu, 1985; Barnes, 1979) and from adventitious shoots regenerated from seedling cotyledons (Anghel and Rosu, 1985; Compton and Gray, 1993; Dong et al., 1991; Srivastava et al., 1989). However, to our knowledge, watermelon plants have not yet been obtained from somatic embryos. Somatic embryogenesis has been documented for several Correspondence to: M . E . C o m p t o n
cucurbits, including cucumber (Chee, 1990a; Malepszy et al., 1982), muskmelon (Gray et al., 1993; Homma et al., 1991; Kageyama et al., 1990; Oridate et al., 1992), pumpkin (Jelaska, 1972, 1974; Juretir and Jelaska, 1991) and summer squash (Chee, 1991, 1992). The development of an efficient somatic embryogenie system may be useful for studies on genetic transformationand/orsyntheticseed. Genetic transformation systems utilizing somatic embryogenesis have been developed for cucumber (Chee, 1990b; Chee and Slightom, 1991; Trulson and Shahin, 1986b) and could aid in the development of virus resistant watermelon. Syntheticseed technologyis currentlybeing investigated for cucumber (Ladyman and Girard, 1992) and could be used to clonaUy propagate superior triploid and tetraploid watermelon genotypes. In this report we describe a protocol for obtaining watermelon plants from somatic embryos and discuss the effects of 2,4-D, TDZ, BA, genotype and explant age on somatic embryogenesis.
Materials and Methods Watermelon plants were grown in the field and fruit were harvested 14 days after self-pollination, unless stated otherwise. Immature seeds were surface-sterilized in 50% bleach (2.5 % NaOCI) plus 2 drops (100 ml-t) Triton X-100 for 15 rain, rinsed six times with sterile-distilled water and the seed coat removed (any seeds that floated to the surface were discarded). Cotyledons were excised from the embryo axis, cut in half longitudinally, and cultured abaxial side down in 100 x 15 mm potri plates that contained 25 ml o f a modified Murashige and Skoog (MS; 1962) medium [MS salts, B5 vitamins (Gamborg et al., 196g), 30 g1-1 sucrose, I g'l -I myo-inositol, 7 g l a T C agar (JRH Biosciences, Lenexa, KS)] containing test concentrations of PGRs. The pH of all media was adjusted to 5.4 prior to the addition of agar and autoclaving at 121C and 98 kPa for 20 rain. Exp~ants were incubated in the dark for three weeks before transfer to MS medium without PGRs and 16h photoperlod [3050 /zmol'm'2"s a 0aPF) from cool-white fluorescent lamps] at 25C. A minimum of eight plates were cultured per treatment with six or nine
62 explants per plate. Data recorded included the number of explants with embryos and the number of embryos per explant (data taken eight weeks after culture initiation), the number of germinated embryos (four weeks after transfer to germination medium) and the number of plants transferred to soil (two weeks after acclimatization). Statistical analysis was conducted using the GLM procedure of the Statistical Analysis System (SAS Institute, Inc., 1988). Percent data were analyzed using the Catmod procedure for categorical data (SAS Institute, Inc., 1988). Data sets with a large number of zero's were transformed using the square root transformation [(y+0.5)'~; Zar, 1984] prior to analysis.
Effect of PGRs and genotype on somatic embryogenesis. Cotyledon explants of 'Allsweet', 'Crimson Sweet', FLAS87-gate, 'Jubilee H', and 'Minilee' were prepared as above and incubated on an MS medium (as above) containing 2,4-D (Eastman Kodak, Rochester, N.Y.) at three concentrations (10, 20 or 40 taM) and 0.5 paM of either BA (Sigma Chem. Co., St. Louis, MO) or TDZ (Nor-Am Chem. Co., Wilmington, DE). Control medium was an MS medium without PGRs. Effect of embryo age on somatic embryogenesis. Zygotic embryos of 'Minilee' were removed from fruit harvested at two day intervals beginning eight days after pollination and continuing for 28 days. Cotyledon explants were incubated on MS medium containing 10 p.M 2,4-D and 0.5/aM TDZ. Somatic embryogenesis from seed-propagated plants vs plants derived from somatic embryos. 'Minilee' cotyledon explants were excised from 14-day-old zygotic embryos collected from plants propagated from seed or from plants derived from somatic embryos. Cotyledon explants were incubated on MS medium containing 10 p2vl 2,4=D and 0.5/aM TDZ.
Results and Discussion
Description of cultures. Cotyledon explants were initially 2 x 4 mm in size and white but enlarged approximately 4x and became yellowish-white after two days of culture. Callus formation was evident after two weeks. Two types of calli were observed; one being white and very friable whereas the other was yellowish-white and more compact. No somatic embryos were observed during the three weeks of dark incubation. Pigmented (yellowish-white) explants greened within 24h of transfer to medium without PGRs and 16h photoperiod. Somatic embryos, some with well developed cotyledons (Fig. 1A) and apical domes (Fig. 1B), were observed three to four weeks later. A majority (> 70%) of the embryos were fused (Fig. 1C) and over 85% germinated precociously (i.e., underwent shoot elongation prior tocotyledon development; Fig. 1D). Somatic embryos formed either directly on the explant or developed from callus. Of the embryos that originated from callus, approximately 90% originated from yellowish-white, compact callus and 10% from white, friable callus. Somatic embryos germinated on a modified MS medium without PGRs (Fig. 1E). Fused embryos were separated manually before transfer to germination medium. Germinated embryos, approximately 2 cm tall with well-developed shoots and roots, were transferred
Fig. I. Development of watermelon somatic embryos on modified MS medium with 10 paM 2,4-D and 0.5 p.M TDZ. (A) Watermelon somatic embryos possessing well developed cotyledons. 03) Apical meristem (+-) on a watermelon somatic embryo with a normal hypoeotyl and cotyledons. (C) Two watermelon somatic embryos fused at the hypoeotyl. (D) Precocious watermelon somatic embryo with poorly formed cotyledons. (E) Watermelon somatic embryo germinated on MS medium without PGRs; bar = 1 ram. CF) Watermelon somatic embryoderived plants transferred to a Magenta box containing autoclaved Pro Mix; bar = 1 cm.
to Magenta GA-7 vessels (Magenta Corp., Chicago, IL) containing 1 PreMix BX (Premier Brands, Inc., New Rochelle, N.Y.): 1 coarse vermiculite (Fig. IF) and incubated under the same conditions as the tissue cultures. Plants were acclimatized to ambient humidity levels by gradually removing the lid of the GA-7 vessels over a period of three weeks. Acclimatized plants were transplanted to 10 cm plastic pots filled with PreMix and moved to the greenhouse before being transplanted to the field. Plants were obtained from both normal and precociously-germinating embryos. Somatic embryo-derived plants formed fertile male and female
63 flowers that produced normal fruit. No difference in the number of fruit per plant or the number of seed per fruit was observed among seed propagated and somatic embryo-derived plants (data not shown).
Table 2.
Effect of PGRs and genotype on somatic embryogenesis. A significant interaction was observed between 2,4-D concentration and cytokinin type. The percentage of explants that produced embryos was greatest when MS medium contained 10 pM 2,4-D and 0.5 prn TDZ, and poorest on MS medium with 40/JM 2,4-D and 0.5 pM BA (Table 1). The number of embryos per responding explant was greater at 10 pM 2,4-D, regardless of the cytokinin type, than at 20 pM 2,4-D plus TDZ or 40 pM 2,4-D plus BA. Explants incubated on medium without PGRs failed to produce somatic embryos. The PGR combination from which watermelon somatic embryos were derived also influenced embryo germination. The percent germination was greatest for embryos derived from medium with TDZ regardless of the 2,4-D concentration and for embryos formed on medium with 20 pM 2,4-D and BA (Table 1). Embryo germination was poorest on medium supplemented with 10 pM 2,4-D plus BA. Cytokinin type and auxin concentration did not affect the percentage of plants surviving in soil. Other cucurbit somatic embryogenic systems generally employ 2,4-D, 2,4,5-T or NAA concentrations ranging from 1.8 to 22.7 pM combined with 0 to 4.4 pM BA (Chee 1990a, 1991, 1992; Homma et al., 1991; Jelaska, 1972, 1974; Jureti6 and Jelaska, 1991; Kageyama et al., 1991; Malepszy and Nadolska-Orczyk 1983; Oridate and Oosawa, 1986). Work by Gray et al. (1993) demonstrated that somatic embryogenesis in muskmelon was improved by
Allsweet FLAS87-gate Crimson Sweet Minilee Jubilee II
Table 1.
2,4oD (tiM) 0 10 10 20 20 40 40
Effect of 2,4-D concentration and cytokinin type on somatic embryo production and somatic embryo germination, a Cytokinin (0.5 pM) NONE BA TDZ BA TDZ BA TDZ
Explants with embryos (%)
Number of embryosb
Germinated embryos(%)~
0 2.9 13.7 3.6 5.4 0.6 3.6
0 4.0 3.3 2.7 2.1 2.0 2.5
0 12.5 39.1 43.3 35.1 0 46.7
+_ 1.3 _+ 2.7 _+ 1.4 _+ 1.7 _+ 1.4
_ 1.1 _+ 0.6 +_ 0.7 +_ 0.5 _ 0.8
_+ _ _+ _
9.7 8.1 20.2 14.2
_+ 19.1
aData for 'Allsweet', 'Crimson Sweet', FLAS87-gate and 'Minilee' were combined (nonsignificant 3-way interaction). There were 168 explants p e r treatment. +_ values represent standard error of the mean. bNumber of embryos per responding e~plant. cEmbryos were germinated on MS medium without plant growth regulators.
Effect of genotype on somatic embryo production and somatic embryo germination."
Genotype
Nb 336 336 168 336 168
E.xplants with embryos (%) 7.1 6.5 3.5 3.3 0
+ _ _
1.4 1.3 1.4 0.9
Germinated embryos (%)c 33.2 46.1 13.8 42.0 0
+_ _ _ _
9.2 9.5 8.0 19.1
*Data were average across all 2,4-1) concentrations and cytokinin types. _ values represent standard error of the mean. bNumber of explants per genotype. CEmbryos were germinated on MS medium without plant growth regulators.
substituting TDZ for BA which agrees with the results obtained for watermelon somatic embryogenesis. A significant difference was observed in the ability of cotyledonexplants fromdifferentwatermelongenotypes to undergo somatic embryogenesis. The percent of explants that produced embryos was greatest for 'Allsweet' and FLAS87-gate, and intermediate for 'Crimson Sweet' and 'Minilee' (Table 2). 'Jubilee II' explants failed to produce somatic embryos. The percentage of somatic embryos that germinated was greater for 'Allsweet', FLAS87-gate and 'Minilee' than for 'Crimson Sweet'. The number of embryos per responding explant was similar for all the genotypes tested. Similar variation in the ability of different genotypes to undergo somatic embryogenesis has been reported for other cucurbits (Gray et al., 1993; Oridate et al., 1992; Trutson and Shahin, 1986a). Effect of embryo age on somatic embryogenesis. In the previous experiments, embryo explants were established from fruit harvested 14 days after pollination. In order to optimize the embryogenic response, explants were established from zygotic embryos removed from fruit harvested at two day intervals beginning eight days after pollination and continuing for 28 days. The percentage of 'Minilee' explants that produced embryos was greatest when cotyledon explants were established from 18-day-old embryos (Fig. 2). The percentage of explants that formed embryos was significantly less when explants were established from 22-, 20-, 14-, 28-, 24-, 16-, and 26-day-old embryos. Explants from 8-, 10- and 12-day-old embryos failed to produce somatic embryos. Other cucurbit systems utilize cotyledon explants from mature seed for somatic embryogenesis (Chee, 1990, 1991, 1992; Gray et al., 1993; Jelaska, 1974; Malepszy and Nadolska-Orczyk 1983; Kageyama et al., 1991; Oridate et al., 1992). We have investigated using cotyledon explanlts from mature seeds; however, these explants callused profusely regardless of the 2,4-D concentration and cytokinin type, and failed to form
64 5O
Table 3.
Explants with embryos (%)
Effect of explant source on somatic embryo production and somatic embryo germination."
40Explant source b
2O
0
, 8
1()
12
14 16 18 20 22 D a y s after pollination
24
26
28
Fig. 2. Effect of embryo age on somatic embryogenesis of 'Minilee'. Embryos were removed from fruit harvested at two day intervals beginning eight days after pollination and continuing for 28 days. Cotyledon explants were incubated on MS medium with 10 # M 2,4D and 0.5 g M TDZ~ Vertical bars represent standard error of the mean.
somatic embryos (data not shown).
Somatic embryogenesis from seed-propagated plants vs plants derived from somatic embryos. A significant interaction was observed between the genotype and explant source (seed-propagated vs somatic embryoderived plants). The percentage of cotyledon explants that produced embryos was more than doubled when 'Crimson Sweet' explants were established from zygotic embryos of somatic embryo-derived plants compared to seed-derived plants (Table 3). The percentage of embryos that germinated was improved when explants were prepared from cotyledons of somatic embryoderived plants compared to seed-derived plants. The number of embryos per responding explant was increased 1.6x when FLAS87-gate explants were collected from somatic embryo-derived plants compared to seed-derived plants; however, the percentage of explants with embryos and the percentage of germinating embryos was not altered. Using immature zygotic embryos from an embryogenic plant failed to significantly improve somatic embryogenesis for 'Minilee'. In conclusion, cotyledons from immature zygotic embryos provide the best explant source for somatic embryogenesis in watermelon. Somatic embryos develop rapidly and plants can be transferred to the field as early as 12 weeks after culture initiation. Plants obtained from somatic embryos appeared normal and true-to-type. Only one polyploid (4n) plant was identified. These features make somatic embryogenesis a promising method for obtaining genetically transformed watermelon plants, or clonally propagating triploid or tetraploid genotypes once the response level is improved.
Crimson Sweet Seed SE-1 SE-2 FLAS87-gate Seed SE-1 Minilee Seed SE-1
Nc
F_xplants with Number of Germinated embryos (%) embryo# embryos (%y
48 162 162
6.3 + 3.5 14.2 • 2.7 14.2 • 2.7
4.3 • 2.8 5.4 • 1.2 4.3 • 0.9
6.7 38.7 • 31.1 •
96 162
12.5 • 3.4 12.9 • 2.4
2.3 • 0.4 3.8 • 0.6
45.8 • 11.2 34.7 • 6.7
96 162
5.2 • 2.3 9.3 • 2.3
2.8 • 0.6 2.2 • 0.4
42.0 • 19.1 24.4 • 9.3
6.8 7.8
"Explants were incubated on MS medium plus 10 pM 2,4-d and 0.5 g M TDZ. + values represent standard error of the mean. bExplants were cotyledons from 14-day-old immature seeds collected from plants propagated from seed (seed) or from plants derived from somatic embryos (SE). cNumber of explants per treatment. dNumber of embryos per responding explant. cEmbryos were germinated on MS medium without plant growth regulators.
Acknowledgerrurnts. This work was supported by a grant from the State of Florida High Technology and Industry Council Applied Research Grants Program. Seed was provided by Dr. Gary W. Elmstrom, University of Florida, CFREC/IFAS, Leesburg, F L This is Florida Agricultural Experiment Station Journal Series No. R02350.
References
Anghel I, Rosu A (1985) Rev. Roum. Biol.-Biol. V6g6t. 30:43-55. Barnes LR (1979) Scientia Horticulturae 11:223-227. Chee PP (1990a) HortScience 25:792-793. Chee PP (1990b) Plant Cell Rep. 9:245-248. Chee PP (1991) Plant Cell Rep. 9:620-622. Chee PP (1992) HortScience 27:59-60. Chee PP, Slightom JL (1991) J. Amer. Soc. Hort. Sci. 116:1098-1102. Compton ME, Gray DJ (1993) J. Amer. Soc. Hort. Sci. (In Press). Dong J-Z, Jia S-R (1991) Plant Cell Rep. 9:559-562. Gamborg OL, Miller RA, Ojima K (1968) Exp. Cell Res. 50:151-158. Gray D J, McColley DW, Compton ME (1993) J. Amer. Soc. Hort. Sci. (In Press). Homma Y, Sugiyama K, Oosawa K (1991) Japan J. Breed. 41:543-551. Jelaska S (1972) Planta 103:278-280. Jelaska S (1974) Physiol. Plant. 31:257-261. Jureti~ B, Jelaska S (1991) Plant Cell Rep. 9:623-626. Kageyama K, Yabe K, Miya]ima S (1990) Plant Tissue Cult. Lett. 7:193-198.
65 Kageyama K, Yabe K, Miyajima S (1991) Japan. J. Breed. 41:273-278. Ladyman JAR, Girard B (1992) HortScience 27:164165. Malepszy S, Nadolska-Orczyk A (1983) Z. Pflanzenphysiol. 111:273-276. Malepszy S, Niemirowicz-Szczytt K, Wiszniewska J (1982) Acta Biologica 10:218-220. Murashige T, Skoog F (1962) Physiol. Plant. 15:473497. Oridate T, Oosawa K (1986) Japan. J. Breed. 36:424428.
Oridate T, Atsumi H, Ito S, Araki H (1992) Plant Cell Tissue Organ Cult. 29:27-30. SAS Institute, Inc. (1988) Release 6.03. SAS Institute, Inc., Cary, N.C. Srivastava DR, Andrianov VM, Piruzian ES (1989) Plant Cell Rep. 8:300-302. Trulson AJ, Shahin EA (1986a) Plant Sci. 47:35-43. Trulson AJ, ShahLn EA (1986b) Theor. Appl. Genet. 73:11-15. Zar JH (1984) Biostatistical analysis. Second edition. Prentice-Hall, Inc., Englewood Cliffs, N.J.
P l a n t Cell R e p o r t s (1993) 1 2 : 6 1 - 6 5
9 Springer-Verlag 1993
Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon Michael E. Compton and D.J. Gray Central F l o r i d a R e s e a r c h a n d E d u c a t i o n Center, University o f Florida, Institute o f F o o d a n d Agricultural Sciences, 5336 U n i v e r s i t y Ave., Leesburg, F l o r i d a 34748, U S A Received M a y 28, 1992/Revised version received O c t o b e r 16, 1992 - C o m m u n i c a t e d by J. J. F i n e r
Abstract. Cotyledon explants from immature embryos of five watermelon [Citrullus l a n a t u s (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40/JM), 0.5/JM of either BA or TDZ, and 7 g-11 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon explants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGP, combination for somatic embryogenesis was 10/~M 2,4-D and 0.5/JM TDZ~ Somatic embryogenesis was greatest (30%) when cotyledon explants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit. Key Words: C i t r u l l u s l a n a t u s - cucurbits - tissue culture Abbreviations: PGR: plant growth regulator, BA: benzyladenine, TDZ: thidiazuron, 2,4-D: 2,4dichlorophenoxyaceticacid, NAA: a-naphthaleneacetic acid, 2,4,5-T: 2,4,5-trichlorophenoxyaceticacid. Introduction
Watermelon plants have been obtained from micropropagated shoot tips (Anghel and Rosu, 1985; Barnes, 1979) and from adventitious shoots regenerated from seedling cotyledons (Anghel and Rosu, 1985; Compton and Gray, 1993; Dong et al., 1991; Srivastava et al., 1989). However, to our knowledge, watermelon plants have not yet been obtained from somatic embryos. Somatic embryogenesis has been documented for several Correspondence to: M . E . C o m p t o n
cucurbits, including cucumber (Chee, 1990a; Malepszy et al., 1982), muskmelon (Gray et al., 1993; Homma et al., 1991; Kageyama et al., 1990; Oridate et al., 1992), pumpkin (Jelaska, 1972, 1974; Juretir and Jelaska, 1991) and summer squash (Chee, 1991, 1992). The development of an efficient somatic embryogenie system may be useful for studies on genetic transformationand/orsyntheticseed. Genetic transformation systems utilizing somatic embryogenesis have been developed for cucumber (Chee, 1990b; Chee and Slightom, 1991; Trulson and Shahin, 1986b) and could aid in the development of virus resistant watermelon. Syntheticseed technologyis currentlybeing investigated for cucumber (Ladyman and Girard, 1992) and could be used to clonaUy propagate superior triploid and tetraploid watermelon genotypes. In this report we describe a protocol for obtaining watermelon plants from somatic embryos and discuss the effects of 2,4-D, TDZ, BA, genotype and explant age on somatic embryogenesis.
Materials and Methods Watermelon plants were grown in the field and fruit were harvested 14 days after self-pollination, unless stated otherwise. Immature seeds were surface-sterilized in 50% bleach (2.5 % NaOCI) plus 2 drops (100 ml-t) Triton X-100 for 15 rain, rinsed six times with sterile-distilled water and the seed coat removed (any seeds that floated to the surface were discarded). Cotyledons were excised from the embryo axis, cut in half longitudinally, and cultured abaxial side down in 100 x 15 mm potri plates that contained 25 ml o f a modified Murashige and Skoog (MS; 1962) medium [MS salts, B5 vitamins (Gamborg et al., 196g), 30 g1-1 sucrose, I g'l -I myo-inositol, 7 g l a T C agar (JRH Biosciences, Lenexa, KS)] containing test concentrations of PGRs. The pH of all media was adjusted to 5.4 prior to the addition of agar and autoclaving at 121C and 98 kPa for 20 rain. Exp~ants were incubated in the dark for three weeks before transfer to MS medium without PGRs and 16h photoperlod [3050 /zmol'm'2"s a 0aPF) from cool-white fluorescent lamps] at 25C. A minimum of eight plates were cultured per treatment with six or nine
62 explants per plate. Data recorded included the number of explants with embryos and the number of embryos per explant (data taken eight weeks after culture initiation), the number of germinated embryos (four weeks after transfer to germination medium) and the number of plants transferred to soil (two weeks after acclimatization). Statistical analysis was conducted using the GLM procedure of the Statistical Analysis System (SAS Institute, Inc., 1988). Percent data were analyzed using the Catmod procedure for categorical data (SAS Institute, Inc., 1988). Data sets with a large number of zero's were transformed using the square root transformation [(y+0.5)'~; Zar, 1984] prior to analysis.
Effect of PGRs and genotype on somatic embryogenesis. Cotyledon explants of 'Allsweet', 'Crimson Sweet', FLAS87-gate, 'Jubilee H', and 'Minilee' were prepared as above and incubated on an MS medium (as above) containing 2,4-D (Eastman Kodak, Rochester, N.Y.) at three concentrations (10, 20 or 40 taM) and 0.5 paM of either BA (Sigma Chem. Co., St. Louis, MO) or TDZ (Nor-Am Chem. Co., Wilmington, DE). Control medium was an MS medium without PGRs. Effect of embryo age on somatic embryogenesis. Zygotic embryos of 'Minilee' were removed from fruit harvested at two day intervals beginning eight days after pollination and continuing for 28 days. Cotyledon explants were incubated on MS medium containing 10 p.M 2,4-D and 0.5/aM TDZ. Somatic embryogenesis from seed-propagated plants vs plants derived from somatic embryos. 'Minilee' cotyledon explants were excised from 14-day-old zygotic embryos collected from plants propagated from seed or from plants derived from somatic embryos. Cotyledon explants were incubated on MS medium containing 10 p2vl 2,4=D and 0.5/aM TDZ.
Results and Discussion
Description of cultures. Cotyledon explants were initially 2 x 4 mm in size and white but enlarged approximately 4x and became yellowish-white after two days of culture. Callus formation was evident after two weeks. Two types of calli were observed; one being white and very friable whereas the other was yellowish-white and more compact. No somatic embryos were observed during the three weeks of dark incubation. Pigmented (yellowish-white) explants greened within 24h of transfer to medium without PGRs and 16h photoperiod. Somatic embryos, some with well developed cotyledons (Fig. 1A) and apical domes (Fig. 1B), were observed three to four weeks later. A majority (> 70%) of the embryos were fused (Fig. 1C) and over 85% germinated precociously (i.e., underwent shoot elongation prior tocotyledon development; Fig. 1D). Somatic embryos formed either directly on the explant or developed from callus. Of the embryos that originated from callus, approximately 90% originated from yellowish-white, compact callus and 10% from white, friable callus. Somatic embryos germinated on a modified MS medium without PGRs (Fig. 1E). Fused embryos were separated manually before transfer to germination medium. Germinated embryos, approximately 2 cm tall with well-developed shoots and roots, were transferred
Fig. I. Development of watermelon somatic embryos on modified MS medium with 10 paM 2,4-D and 0.5 p.M TDZ. (A) Watermelon somatic embryos possessing well developed cotyledons. 03) Apical meristem (+-) on a watermelon somatic embryo with a normal hypoeotyl and cotyledons. (C) Two watermelon somatic embryos fused at the hypoeotyl. (D) Precocious watermelon somatic embryo with poorly formed cotyledons. (E) Watermelon somatic embryo germinated on MS medium without PGRs; bar = 1 ram. CF) Watermelon somatic embryoderived plants transferred to a Magenta box containing autoclaved Pro Mix; bar = 1 cm.
to Magenta GA-7 vessels (Magenta Corp., Chicago, IL) containing 1 PreMix BX (Premier Brands, Inc., New Rochelle, N.Y.): 1 coarse vermiculite (Fig. IF) and incubated under the same conditions as the tissue cultures. Plants were acclimatized to ambient humidity levels by gradually removing the lid of the GA-7 vessels over a period of three weeks. Acclimatized plants were transplanted to 10 cm plastic pots filled with PreMix and moved to the greenhouse before being transplanted to the field. Plants were obtained from both normal and precociously-germinating embryos. Somatic embryo-derived plants formed fertile male and female
63 flowers that produced normal fruit. No difference in the number of fruit per plant or the number of seed per fruit was observed among seed propagated and somatic embryo-derived plants (data not shown).
Table 2.
Effect of PGRs and genotype on somatic embryogenesis. A significant interaction was observed between 2,4-D concentration and cytokinin type. The percentage of explants that produced embryos was greatest when MS medium contained 10 pM 2,4-D and 0.5 prn TDZ, and poorest on MS medium with 40/JM 2,4-D and 0.5 pM BA (Table 1). The number of embryos per responding explant was greater at 10 pM 2,4-D, regardless of the cytokinin type, than at 20 pM 2,4-D plus TDZ or 40 pM 2,4-D plus BA. Explants incubated on medium without PGRs failed to produce somatic embryos. The PGR combination from which watermelon somatic embryos were derived also influenced embryo germination. The percent germination was greatest for embryos derived from medium with TDZ regardless of the 2,4-D concentration and for embryos formed on medium with 20 pM 2,4-D and BA (Table 1). Embryo germination was poorest on medium supplemented with 10 pM 2,4-D plus BA. Cytokinin type and auxin concentration did not affect the percentage of plants surviving in soil. Other cucurbit somatic embryogenic systems generally employ 2,4-D, 2,4,5-T or NAA concentrations ranging from 1.8 to 22.7 pM combined with 0 to 4.4 pM BA (Chee 1990a, 1991, 1992; Homma et al., 1991; Jelaska, 1972, 1974; Jureti6 and Jelaska, 1991; Kageyama et al., 1991; Malepszy and Nadolska-Orczyk 1983; Oridate and Oosawa, 1986). Work by Gray et al. (1993) demonstrated that somatic embryogenesis in muskmelon was improved by
Allsweet FLAS87-gate Crimson Sweet Minilee Jubilee II
Table 1.
2,4oD (tiM) 0 10 10 20 20 40 40
Effect of 2,4-D concentration and cytokinin type on somatic embryo production and somatic embryo germination, a Cytokinin (0.5 pM) NONE BA TDZ BA TDZ BA TDZ
Explants with embryos (%)
Number of embryosb
Germinated embryos(%)~
0 2.9 13.7 3.6 5.4 0.6 3.6
0 4.0 3.3 2.7 2.1 2.0 2.5
0 12.5 39.1 43.3 35.1 0 46.7
+_ 1.3 _+ 2.7 _+ 1.4 _+ 1.7 _+ 1.4
_ 1.1 _+ 0.6 +_ 0.7 +_ 0.5 _ 0.8
_+ _ _+ _
9.7 8.1 20.2 14.2
_+ 19.1
aData for 'Allsweet', 'Crimson Sweet', FLAS87-gate and 'Minilee' were combined (nonsignificant 3-way interaction). There were 168 explants p e r treatment. +_ values represent standard error of the mean. bNumber of embryos per responding e~plant. cEmbryos were germinated on MS medium without plant growth regulators.
Effect of genotype on somatic embryo production and somatic embryo germination."
Genotype
Nb 336 336 168 336 168
E.xplants with embryos (%) 7.1 6.5 3.5 3.3 0
+ _ _
1.4 1.3 1.4 0.9
Germinated embryos (%)c 33.2 46.1 13.8 42.0 0
+_ _ _ _
9.2 9.5 8.0 19.1
*Data were average across all 2,4-1) concentrations and cytokinin types. _ values represent standard error of the mean. bNumber of explants per genotype. CEmbryos were germinated on MS medium without plant growth regulators.
substituting TDZ for BA which agrees with the results obtained for watermelon somatic embryogenesis. A significant difference was observed in the ability of cotyledonexplants fromdifferentwatermelongenotypes to undergo somatic embryogenesis. The percent of explants that produced embryos was greatest for 'Allsweet' and FLAS87-gate, and intermediate for 'Crimson Sweet' and 'Minilee' (Table 2). 'Jubilee II' explants failed to produce somatic embryos. The percentage of somatic embryos that germinated was greater for 'Allsweet', FLAS87-gate and 'Minilee' than for 'Crimson Sweet'. The number of embryos per responding explant was similar for all the genotypes tested. Similar variation in the ability of different genotypes to undergo somatic embryogenesis has been reported for other cucurbits (Gray et al., 1993; Oridate et al., 1992; Trutson and Shahin, 1986a). Effect of embryo age on somatic embryogenesis. In the previous experiments, embryo explants were established from fruit harvested 14 days after pollination. In order to optimize the embryogenic response, explants were established from zygotic embryos removed from fruit harvested at two day intervals beginning eight days after pollination and continuing for 28 days. The percentage of 'Minilee' explants that produced embryos was greatest when cotyledon explants were established from 18-day-old embryos (Fig. 2). The percentage of explants that formed embryos was significantly less when explants were established from 22-, 20-, 14-, 28-, 24-, 16-, and 26-day-old embryos. Explants from 8-, 10- and 12-day-old embryos failed to produce somatic embryos. Other cucurbit systems utilize cotyledon explants from mature seed for somatic embryogenesis (Chee, 1990, 1991, 1992; Gray et al., 1993; Jelaska, 1974; Malepszy and Nadolska-Orczyk 1983; Kageyama et al., 1991; Oridate et al., 1992). We have investigated using cotyledon explanlts from mature seeds; however, these explants callused profusely regardless of the 2,4-D concentration and cytokinin type, and failed to form
64 5O
Table 3.
Explants with embryos (%)
Effect of explant source on somatic embryo production and somatic embryo germination."
40Explant source b
2O
0
, 8
1()
12
14 16 18 20 22 D a y s after pollination
24
26
28
Fig. 2. Effect of embryo age on somatic embryogenesis of 'Minilee'. Embryos were removed from fruit harvested at two day intervals beginning eight days after pollination and continuing for 28 days. Cotyledon explants were incubated on MS medium with 10 # M 2,4D and 0.5 g M TDZ~ Vertical bars represent standard error of the mean.
somatic embryos (data not shown).
Somatic embryogenesis from seed-propagated plants vs plants derived from somatic embryos. A significant interaction was observed between the genotype and explant source (seed-propagated vs somatic embryoderived plants). The percentage of cotyledon explants that produced embryos was more than doubled when 'Crimson Sweet' explants were established from zygotic embryos of somatic embryo-derived plants compared to seed-derived plants (Table 3). The percentage of embryos that germinated was improved when explants were prepared from cotyledons of somatic embryoderived plants compared to seed-derived plants. The number of embryos per responding explant was increased 1.6x when FLAS87-gate explants were collected from somatic embryo-derived plants compared to seed-derived plants; however, the percentage of explants with embryos and the percentage of germinating embryos was not altered. Using immature zygotic embryos from an embryogenic plant failed to significantly improve somatic embryogenesis for 'Minilee'. In conclusion, cotyledons from immature zygotic embryos provide the best explant source for somatic embryogenesis in watermelon. Somatic embryos develop rapidly and plants can be transferred to the field as early as 12 weeks after culture initiation. Plants obtained from somatic embryos appeared normal and true-to-type. Only one polyploid (4n) plant was identified. These features make somatic embryogenesis a promising method for obtaining genetically transformed watermelon plants, or clonally propagating triploid or tetraploid genotypes once the response level is improved.
Crimson Sweet Seed SE-1 SE-2 FLAS87-gate Seed SE-1 Minilee Seed SE-1
Nc
F_xplants with Number of Germinated embryos (%) embryo# embryos (%y
48 162 162
6.3 + 3.5 14.2 • 2.7 14.2 • 2.7
4.3 • 2.8 5.4 • 1.2 4.3 • 0.9
6.7 38.7 • 31.1 •
96 162
12.5 • 3.4 12.9 • 2.4
2.3 • 0.4 3.8 • 0.6
45.8 • 11.2 34.7 • 6.7
96 162
5.2 • 2.3 9.3 • 2.3
2.8 • 0.6 2.2 • 0.4
42.0 • 19.1 24.4 • 9.3
6.8 7.8
"Explants were incubated on MS medium plus 10 pM 2,4-d and 0.5 g M TDZ. + values represent standard error of the mean. bExplants were cotyledons from 14-day-old immature seeds collected from plants propagated from seed (seed) or from plants derived from somatic embryos (SE). cNumber of explants per treatment. dNumber of embryos per responding explant. cEmbryos were germinated on MS medium without plant growth regulators.
Acknowledgerrurnts. This work was supported by a grant from the State of Florida High Technology and Industry Council Applied Research Grants Program. Seed was provided by Dr. Gary W. Elmstrom, University of Florida, CFREC/IFAS, Leesburg, F L This is Florida Agricultural Experiment Station Journal Series No. R02350.
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