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Fluid drilling as a delivery system for somatic embryo-derived plantlets of carrot (Daucus carota L.)
Scientia Horticulturae, 47 ( 1991 ) 209-220
209
Elsevier Science Publishers B.V., Amsterdam
Fluid drilling as a delivery system for somatic embryo-derived plantlets of carrot (Daucus carota L. )* Sherry L. Kitto, Wallace G. Pill and Donna M. Molloy Delaware Agricultural Experiment Station, Department of Plant and Soil Sciences, College of Agricultural Sciences, University of Delaware, Newark, DE 19717-1303, USA (Accepted 3 January 1991 )
ABSTRACT Kitto, S.L., Pill, W.G. and Molloy, D.M., 1991. Fluid drilling as a delivery system for somatic embryoderived plantlets of carrot ( Daucus carota L. ). Scientia Hortic., 47: 209-220. Somatic embryos and/or somatic embryo-derived plantlets (SEPs) of carrot cultivar 'Orlando Gold' were subjected to various treatments during either suspension culture or a subsequent incubation period in fluid-drilling gel. Conversion of SEPs in the glasshouse into plants containing primary leaves was greatest when SEPs were incubated for 1-2 weeks in hydroxyethyl cellulose fluid-drilling gel ( 1.67%, w / v of N-gelTM) hydrated with a solution containing Murashige and Skoog salts and vitamins and 2% ( w / v ) sucrose. Providing 3 days of chilling at 4°C then 3 days at 25°C all under light (60/~mol m -2 s-1 ) during suspension culture (at the globular to torpedo stage) led to the greatest SEP growth following fluid drilling. Inclusion of either 250 mg Truban R fungicide or 10 mg chitosan glutamate per litre of gel improved SEP conversion. Keywords: chitosan; embryogenesis; fluid drilling; polyethylene glycol. Abbreviations: MS = Murashige and Skoog salts and vitamins; PEG = polyethylene glycol; SEP = somatic embryo-derived plantlet.
INTRODUCTION
A mature true seed, essentially a young plant in an arrested state of development, is composed of an embryo (embryonic axis plus nutritive tissues) and covering structures (seed coats). Seed coats play a role in the regulation of nutrition as well as in the protection of the embryo at early stages of its *Published as Miscellaneous Paper No. 1320 of the Delaware Agricultural Experiment Station, Contribution No. 269 of the Department of Plant and Soil Sciences. Mention of trade names in this publication does not imply endorsement by the Delaware Agricultural Experiment Station of products named, nor criticism of similar ones not named.
0304-4238/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
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S.L. KITTO ET AL.
development. As seeds mature, the major function of the seed coats becomes that of protection. Many studies have shown that somatic embryos may be produced from somatic tissues or cells cultured in vitro (McWilliam et al., 1974). Further, the development of embryos produced in vivo and in vitro is similar within the same species (Janick et al., 1982). However, somatic embryos produced in vitro lack seed coats. Therefore, systems must be developed so that somatic embryos can be handled with a minimum of damage and be delivered to seedbeds in the glasshouse or field. Four handling systems to facilitate delivery of somatic embryos have been proposed: fluid drilling (Drew, 1979; Baker, 1985 ), encapsulation of somatic embryos in alginate gel (Fujii et al., 1987 ), desiccation of somatic embryos (Gray, 1989 ), or desiccation of somatic embryos encapsulated in water-soluble resin (Janick et al., 1989 ). The fluid-drillingtechnique, normally involving the transfer of germinated seeds to the seed bed in a protective carrier gel (Gray, 1981 ), provides the potential for bulk handling of many small plantlets without the need for individual handling. The present study examined techniques for sowing somatic embryo-derived plantlets (SEPs) of carrot. The five experiments presented here examined the composition of the fluid-drilling gel, the roles of light and chilling during suspension culture, embryo incubation in the fluid-drilling gel, the addition of SEP-conditioning materials to the gel and the post-fluid-drilling environment. MATERIALS AND METHODS
E x p e r i m e n t a l protocol. ~ The overall experimental protocol, shown in Fig.
1, began with callus initiation from the hypocotyl of carrot seedlings. During subsequent suspension subcultures, somatic embryos were induced and subjected to various conditioning treatments. The conditioned embryos or SEPs were then incorporated into fluid-drilling gel. The SEP-gel mixture was incubated before being fluid drilled into the seedbed. Production a n d conditioning o f SEPs. - - Callus and cell suspensions of Daucus carota L. cultivar 'Orlando Gold' were initiated and maintained as de-
scribed by Kitto and Janick ( 1985 ). Embryos were initiated from cell suspensions by inoculating 0.2 g fresh weight of cells per 25 ml of Murashige and Skoog (MS) medium (Murashige and Skoog, 1962 ) (with 2% sucrose ) and transferring to fresh medium every 3 days. After 10-14 days, torpedo embryos precociously "germinated" to produce what we have termed somatic embryo-derived plantlets (SEPs) (Fig. 2 ). SEPs (still in suspension culture) were conditioned either under cool-white fluorescent lamps (60/zmol m -2 s-l of photosynthetically active radiation at suspension culture level; 16 h
211
FLUID DRILLING AND SOMATIC EMBRYO-DERIVED CARROT PLANTLETS
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EMBRYO TRANSFERTO FLUID-DRILLINGGEL Fig. 1. Schematic representation of protocol from embryo initiation and collection to fluid drilling of the SEP-gel mixture.
Fig. 2. SEPs derived from somatic embryos incubated under lights for 6 days with MS salts and 2% sucrose (magnification X 9.5).
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S.L. KITTO ET AL.
photoperiod) at 25/20°C (light/dark) a n d / o r by chilling at 4°C in the light or in the dark for 3-8 days. Thefluid-drilling gel. - - T h e hydroxyethyl cellulose gel, formulated specifically as a fluid-drilling gel (Banyai, 1987 ), is designated N-gelT M (Aqualon Group, Wilmington, DE). Gel was prepared by adding powder (1.67%, w/ v) to MS solution (MS; 0, 1 or 2% ( w / v ) sucrose; 0, 250 or 500 mg active ingredient (a.i.) per litre of wettable powder formulations of the fungicides Truban, Benlate or Banrot; 0, 150, 200 or 250 g polyethylene glycol 8000 (PEG) per litre, or 0, 10, 50 or 90 mg chitosan glutamate per litre (Seacure TM, Protan Co., Redmond, WA) ) that was continuously agitated with a magnetic stirrer. The gels, after autoclaving at 125 kPa and 121 °C for 15 min, were of sufficient viscosity to suspend the SEPs for at least 1 h. Water potentials for various solutions and N-gel preparations were determined by vapor pressure osmometry (Wescor Model 5500XR, Logan, UT). A 10/tl sample was added to the chamber for aqueous solutions. For the viscous N-gel preparations, a filter paper disc was dipped into the fluid and the excess material removed before placement of the disc in the sample holder. Osmometer readings (mosmol) were converted to - MPa using the formula: ~ = i m R T, where q/is osmotic potential ( - MPa), i m is osmolality of the sample ( mosmol kg- ~), R is a gas constant ( 8.31 X 10 - 6 m 3 MPa mol- ~K - ~), and T is temperature (K). S E P incubation in gel. ~ SEPs were incubated in gel contained within parafilm-sealed eight-well polystyrene tissue culture multiplates or 125 m m × 80 mm X 20 m m polystyrene boxes. A multiplate well (26 m m X 33 m m X 10 mm ) contained five SEPs and 1 ml of the appropriate gel. Multiplates were maintained for 5 days at 25 °C in the light after which SEP vigor was rated subjectively from 1 (dead) to 5 (green and healthy). SEP-gel mixtures ( 10, 15, 20, 25, or 30 ml of N-gel) were poured into polystyrene boxes to provide a gel layer 1-2 m m thick and were incubated under a 16/8 h, 25/21 °C cycle for 0, 1, 2, 3, or 4 weeks (after these periods representative SEPs were measured for length). After incubation, SEPs plus gel (adjusted to a total volume of 15, 20, 30 or 50 ml) were mixed and placed in 125 ml plastic bags (6 oz Whirlpak, NASCO, Ft. Atkinson, WI ). Post-fluid-drilling environment in the glasshouse. ~ SEPs were either transplanted manually or were fluid drilled into 10 cm plastic petri dishes or 17 cm × 12 cm × 6 cm plastic fiats. Petri dishes were placed under a 16/8 h, 25/ 20°C regime for 2 weeks when SEP length was determined. Flats contained five furrows ( 12 cm long, I cm deep ) pressed into a growth medium (ProMix BX (Pr), Premier Brands Inc., Stanford, CN; RediEarth R (Re), W.R. Grace,
FLUID DRILLING AND SOMATICEMBRYO-DERIVEDCARROT PLANTLETS
213
Fogelsville, PA; vermiculite (V); perlite (P) or 50% ( v / v ) combinations of Re or Pr with either V or P). SEPs were either sown uncovered or were covered with about 5 m m of the appropriate growth medium. Flats in the glasshouse were either placed under intermittent mist (6 s every 6 min) or were surface irrigated once daily. Flats removed from the mist were surface irrigated once daily. Flats in the laboratory were placed in clear plastic boxes under lights (60/tmol m -2 s-i, 16/8 h, 25/20°C). Percentage emergence of cotyledons (above the growth medium surface) and percentage embryo conversion (plantlets having primary leaves) were determined at various times after sowing. RESULTS
Fluid-drilling gel composition. - - While SEP vigor was greatest in gel prepared without sucrose, SEP conversion in the glasshouse 20 days after transplanting was greatest for those that had been incubated in gel prepared from 2% ( w / v ) sucrose (Table 1 ). SEPs incubated in gel containing Benlate did not survive after transplanting (data not shown). Plants converted from SEPs incubated in gel containing 2% ( w / v ) sucrose and 250 mg a.i. Truban per litre appeared normal 12 weeks after transplanting into growth medium under glasshouse conditions (observation not shown ). The total water potential of the SEPs, estimated by vapor pressure osmoTABLE 1 Vigor of SEPs of carrot cultivar 'Orlando Gold' 5 days after incorporation in N-gel containing MS medium, sucrose and two fungicides; and SEP conversion in the glasshouse 20 days after transplanting Sucrose (%, w / v ) 0 0 0 0 0
Fungicide (mg a.i.1-1) Truban Truban Banrot Banrot
SEP vigor t (mean _+ SD)
0 250 500 250 500
4.0 + 1.02 4.0+ 1.17 4.0 _ 1.05 2.9 + 0.64 4.1 _+0.78
SEP conversion2 (%) 0 0 0 0 0
1
-
0
2.7_+ 1.03
0
1 1 1 1
Truban Truban Banrot Banrot
250 500 250 500
3.2+ 1.14 3.4_+0.91 2.7_+0.75 3.6+ 1.22
0 0 0 0
2 2 2 2 2
Truban Truban Banrot Banrot
0 250 500 250 500
2.4+0.64 2.8+0.55 3.1 +0.52 2.6 + 0.64 3.0 _+0.50
7.0 12.5 4.8 7.1 4.8
~Scale from 1 (dead) to 5 (green and healthy); n = 25. 2plants with primary leaves.
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S.L. K1TTO ET AL.
metry of crushed SEPs, was - 0 . 3 9 MPa (20°C). Water potentials of 1.67% ( w / v ) N-gel prepared with MS salts and 0%, 1%, and 2% ( w / v ) sucrose were - 0 . 2 5 MPa, - 0 . 3 1 MPa, and - 0 . 3 9 MPa, respectively. Thus, the SEPs should have received a net influx of water until equilibrium was reached. Duration o f light and gel incubation periods. - - SEPs incubated in gel, compared with those not incubated in gel, were longer and had greater percentage emergence (Table 2 ). SEP length increased with up to 3 weeks incubation in gel provided that pretreatment at 25 °C in the light was for a duration of 6 days or less. SEP growth was greatest with 4 days pretreatment at 25 °C in the light followed by 3 weeks incubation in the gel. Pretreatment (25 ° C, light) for more than 6 days reduced the gel incubation period required for maximal growth. Cotyledonary emergence 1 week after fluid drilling was maximal when SEPs were incubated in gel for 1-2 weeks. A light period of more than 6 days, irrespective of the gel incubation period, greatly reduced SEP emergence. Chilling duration in the light or dark. - - Regardless of chilling duration, increasing the subsequent incubation period at 25°C in the light from 0 to 6 days increased SEP growth before fluid drilling (Fig. 3 (A)). Light or dark TABLE2 SEP length at time of fluid drilling and SEP cotyledon emergence 1 week after fluid drilling as influenced by duration of culture exposure to continuous light at 25 °C and subsequent duration of SEP incubation in 1.67% N-gel prepared with MS salts and 2% sucrose Duration of light (days)
Incubation in gel (weeks)
SEP length after incubation in gel ( m m )
SEP emergence 1 week after fluid drilling (%)
4
0
6.4 10.9 12.2 26.2 14.4
4.1 31.0 52.5 18.3 20.0
0
8.2
1
11.8
2 3 4
13.2 15.1 11.9
9.7 41.8 45.0 16.8 7.5
0 1 2 3 4
9.6 15.2 22.2 12.1 10.8
8.2 22.5 15.2 12.5 15.0
1
2 3 4 6
8
LSDo.o5
2.14
24.43
FLUID DRILLING AND SOMATIC EMBRYO-DERIVED CARROT PLANTLETS
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A
215
/
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_ --~
/"
110
I 0
I 3
I 6
Days
of
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I 0
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I 3
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dark
Fig. 3. SEP length at: (A) the time of fluid drilling; (B) 2 weeks after fluid drilling. SEPs were subjected to 0, 3, or 6 days of chilling (4°C) in the light (open symbols) or dark (closed symbols ) followed by 0 ( O, • ), 3 ( A, • ), or 6 ( [2, • ) days of light at 25 ° C before fluid drilling. Vertical bars, LSD, P < 0.05, n=25.
during chilling for up to 6 days initially had no significant effect on SEP length. However, SEP length 2 weeks after fluid drilling increased dramatically when they were chilled for 3 days in the light prior to 3 days gel incubation in the light (Fig. 3 (B) ). Post-fluid-drilling environment in the glasshouse. - - The 9.1% emergence of SEPs that were chilled (3 days at 4°C in the dark then 3 days at 25 °C in the light) was significantly lower than the 15.4% emergence of SEPs that were not chilled (6 days at 25 °C in the light), although growth medium interacted significantly with mist vs. no mist to influence percentage emergence by 5 weeks after fluid drilling (Table 3 ). Generally, Pr+ P ( 1 : 1, v / v ) or Re gave the greatest percentage emergence from fluid-drilled SEPs (Table 3). Non-chilled SEPs that were fluid drilled into Re and provided with intermittent mist gave the greatest percentage emergence (57.3%). Chilled SEPs given the same post-fluid-drilling environment had only 4.8% emergence. Gel incubation volume and SEP conditioning additives in the gel. - - Increasing the gel volume to more than 15 ml per 10 ml of SEP suspension reduced the SEP conversion rate (Table 4 ). Decreasing the water potential from - 0 . 3 9 MPa (0% PEG) to - 1 . 9 9 MPa (25% PEG) in the gel during the 1 week incubation period prior to fluid drilling did not increase SEP conversion (Table 4 ). The addition of 0.01 g of chitosan glutamate per litre of gel during the
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S.L.~TTO ETAL.
TABLE 3 Percentage cotyledon emergence of SEPs 5 weeks after fluid drilling as influenced by growth medium and irrigation. Before fluid drilling, the SEPs were given either 6 days of light at 25°C (non-chilled SEPs) or 3 days at 4°C then 3 days light at 25°C (chilled SEPs) Growth medium Pr
PrV
PrP
Re
ReV
ReP
Mist Non-chilled SEPs Chilled SEPs
10.1 5.0
1.5 0
5.7 16.7
57.3 4.8
0 0
8.5 7.8
No mist Non-chilled SEPs Chilled SEPs
18.8 4.5
8.0 14.6
21.4 24.2
25.3 25.1
16.9 5.0
10.9 0
LSDo.o~ = 21.34 (3-way interaction ) ~Growth medium: Pr, Promix BX; PrV, Pr plus vermiculite (V, 1: 1, v / v ) ; PrP, Pr plus perlite (P, 1 : 1, v / v ) ; Re, RediEarth; ReV, Re plus V ( 1 : 1, v / v ) ; ReP, Re plus perlite ( 1 : 1, v / v ) .
TABLE 4 Percentage conversion of somatic embryos conditioned for 1 week under light with PEG or chitosan in different gel volumes then fluid drilled septically onto RediEarth and placed under lights in the laboratory Gel amendment
0 0 0 0 0 15% PEG 20% PEG 25% PEG 0.01 g Chitosan 4 0.05 g Chitosan 0.09 g Chitosan
LSDo.o5
Gel volume ~
SEP length after incubation in gel 2 ( m m )
SEP conversion 3 4 weeks
7 weeks
10 15 20 25 30
21.4 23.1 23.6 23.3 20.7
0 3 1 1 1
1 10 5 3 1
30 30 30
14.8 14.1
0 0 0
0 0 0
30 30 30
18.7 13.3 14.0
5 0 1
6 0 0
2.69
JVolume of gel per 10 ml of SEP. 2Initial SEP length, 9.8 + 2.3 mm. 3Number of plants with primary leaves. 4Water-soluble chitosan glutamate (Seacure).
FLUID DRILLING AND SOMATIC EMBRYO-DERIVED CARROT PLANTLETS
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incubation period before fluid drilling led to a relatively high SEP conversion 4 weeks after fluid drilling, a conversion rate that was sustained until 7 weeks after fluid drilling (Table 4). DISCUSSION
One objective of this research was to develop a system for maturing embryos into autotrophic SEPs; therefore, somatic embryos were treated either with light a n d / o r chilling during development in suspension culture or during gel incubation. SEPs kept in the light for 4-6 days and incubated for up to 2 weeks in gel had the greatest vigor (Table 2 ). The reduction in vigor associated with SEPs kept for more than 6 days in the light and/or more than 2 weeks of gel incubation may have reflected inadequate storage reserves to support basic physiological functions (e.g. respiration). Reduced vigor and slower development of somatic embryos has been associated with low levels of stored reserves (McKersie et al., 1989 ). The beneficial effect of chilling SEPs may be likened to that of short-term low-temperature osmotic priming of seeds. The osmotic potential of the suspension culture medium ( - 0.39 MPa) may have been sufficiently negative to cause events associated with osmotic seed priming, such as increased activity of esterases and phosphatases (Khan et al., 1978) which, by decreasing the SEP solute potential, would lead to increased water influx and SEP growth upon exposure to warmer conditions. Chilling of imbibed vegetable seeds has increased the germination rate (Finch-Savage and Cox, 1982). The greater SEP length 2 weeks after fluid drilling that resulted from light rather than dark during chilling, may reflect a promotive effect of the light on SEP autotrophy. This growth differential due to irradiation during chilling reflected a delayed response, as at the end of chilling, light had minimal influence on SEP length. Although SEP length was greatest when 6 days of chilling was followed by 6 days of incubation in the light at 25 °C, these SEPs were too large to fluid drill (Fig. 3 ). Three days at 4 ° C in the light followed by 3 days at 25°C in the light led to the greatest gain in SEP length 2 weeks after the SEPs had been fluid drilled. The SEPs of this treatment ( 18 mm in length), could be fluid drilled, but from a practical standpoint, SEPs should be about 1 cm long to be fluid drilled. This decrease in length might be accomplished by beginning the entire chilling/post-chilling treatment earlier during suspension culture. From our results, sucrose and chitosan appeared to be beneficial during SEP incubation in the fluid-drilling gel. Sucrose plays two roles in vitro: as a nutritional carbon source and as an osmotic agent. Lack of SEP survival with 0 or 1% sucrose (Table 1 ) may be attributed to an insufficient supply of carbon and energy during the period when SEPs become autotrophic. Drew ( 1979 ) attempted to assist the survival of somatic carrot embryos before the
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S.L. KITTO ET AL
establishment of autotrophism by "stocking" the suspension culture with elevated sucrose levels but this greatly reduced culture growth. If the effect of sucrose was solely nutritional then perhaps much of the sucrose requirement could be replaced by carbon dioxide enrichment and high levels of photosynthetically active radiation to induce autotrophy as shown with carnation plantlets in solidified gel culture (Kozai, 1989 ). Carbohydrates as osmotic agents have been linked to the acquisition of desiccation tolerance by embryos (Kitto and Janick, 1985 ) and to elevated endogenous proline levels in wheat callus cultures (Gabor et al., 1986 ). Thus, a higher rate of SEP conversion following conditioning in N-gel containing 2% sucrose than in 0 or 1% sucrose (Table 1 ) may reflect osmotically induced stress tolerance associated with elevated endogenous proline. The beneficial effect of 0.01 g chitosan per litre (Table 4) may be associated with stress (defense?) related plasma membrane effects as chitosan is recognized by plants as an indicator of an invasive organism (Kendra and Hadwiger, 1984). Exposure to PEG-induced water stress in the present study may have been too short and too late in SEP ontogeny as the osmotic adjustment that occurred in stress-tolerant carrot cells (Fallon and Phillips, 1989) was developed over long periods (eight subcultures ). Increasing the gel volume above 15 ml per 10 ml of SEP suspension reduced the SEP conversion rate (Table 4), a response that may be attributed to hypoxia resulting from reduced gaseous exchange. SEPs can be likened to pregerminated seeds. While the synchrony of seedling emergence is enhanced by increasing the percentage of seeds which are germinated at the time of fluid drilling (Pill and Finch-Savage, 1988 ), SEPs do not have to "germinate" but they must be able to continue growth following fluid drilling. As with pregerminated seeds, the seed-bed conditions must be conducive to SEP emergence because the option for not "germinating" has been removed. SEP conversion into plants with primary leaves 4 and 7 weeks after fluid drilling occurred under septic laboratory conditions (Table 4), confirming that the SEPs were capable of conversion but that some factor (s) in the glasshouse environment prevented SEP conversion (but did not prevent SEP emergence). While SEP conversion occurred under glasshouse conditions in early February (Table 1 ), lack of SEP conversion from March to August (Tables 2 and 3, Fig. 3 ) may be associated with higher average temperatures and reduced water availability between irrigations. Fujii et al. (1989) suggested that soil surface drying associated with some watering regimes (e.g. intermittent mist) caused reduced embryo conversion which could be alleviated with a humidity tent system. Presumably Pr + P ( 1 : 1, v/v ) or Re possessed suitable aeration and water retention (Table 3 ). When they were provided with
FLUID DRILLING AND SOMATIC EMBRYO-DERIVED CARROT PLANTLETS
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daily surface irrigation, these media gave from 20 to 25% emergence from chilled or non-chilled SEPs. Results of the experiments reported here show that SEPs were capable of conversion under both septic laboratory and glasshouse conditions following fluid drilling. Light or chilling during suspension culture followed by a lighted incubation period in fluid-drilling gel of reduced water potential that contained sucrose and fungicide or chitosan favored SEP conversion. ACKNOWLEDGMENTS
Research supported in part by a Delaware Research Partnership Grant which was funded jointly by the State of Delaware and the Aqualon Group, Wilmington, DE, USA.
REFERENCES Baker, C.M., 1985. Synchronization and fluid sowing of carrot, Daucus carota somatic embryos. M.S. Thesis, University of Florida, Gainesville, FL, 98 pp. Banyai, B.E., 1987. N-gelT M polymers for agricultural fluid-drilling. Acta Hortic., 198:111-120. Drew, R.L.K., 1979. The development of carrot (Daucus carota L.) embryoids (derived from cell suspension culture) into plantlets on a sugar-free basal medium. Hortic. Res., 19: 7984. Fallon, K.M. and Phillips, R., 1989. Responses to water stress in adapted and unadapted carrot cell suspension cultures. J. Exp. Bot., 40:681-687. Finch-Savage, W.E. and Cox, C.J., 1982. A cold-treatment technique to improve the germination of vegetable seeds prior to fluid drilling. Scientia Hortic., 16:301-311. Fujii, J.A., Slade, D.T., Redenbaugh, K. and Walker, K.A., 1987. Artificial seeds for plant propagation. Trends in Biotech., 5: 335-339. Fujii, J.A., Slade, D.T. and Redenbaugh, K., 1989. Maturation and greenhouse planting of alfalfa artificial seeds. In Vitro Cell. Dev. Biol., 25:1179-1182. Gabor, G., Simon-Sarkadi, L., Bekes, F. and Erdei, L., 1986. Genotype specific changes in amino acid and polyamine of wheat tissue culture induced by osmotic stress. In: J. Semal (Editor), Somaclonal Variations and Crop Improvement. Advances in Agricultural Biotechnology. Martinus Nijhoff, Hingham, MA, pp. 170-176. Gray, D., 1981. Fluid drilling of vegetable seeds. Hortic. Rev., 3: 1-27. Gray, D.J., 1989. Effects of dehydration and exogenous growth regulators on dormancy, quiescence and germination of grape somatic embryos. In Vitro Cell Dev. Biol., 25:1173-1178. Janick, J., Kitto, S.L. and Kim, Y.-H., 1989. Production of synthetic seed by desiccation and encapsulation. In Vitro Cell. Dev. Biol., 25:1167-1172. Janick, J., Wright, D.C. and Hasegawa, P.M., 1982. In vitro production of cacao seed lipids. J. Am. Soc. Hortic. Sci., 107: 919-922. Kendra, D.F. and Hadwiger, L.A., 1984. Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Exp. Mycol., 8: 276-281. Khan, A.A., Kar-Ling, T., Knypl, J.S., Borkowska, B. and Powell, L.E., 1978. Osmotic conditioning of seeds: Physiological and biochemical changes. Acta Hortic., 83: 267-278.
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Kitto, S.L. and Janick, J., 1985. Hardening treatments increase survival of synthetically coated asexual embryos of carrot. J. Am. Soc. Hortic. Sci., 110: 283-286. Kozai, T., 1989. Autotrophic (sugar-free) micropropagation for a significant reduction of production costs. Chronica Hortic., 29: 19-20. McKersie, B.D., Senaratna, T., Bowley, S.R., Brown, D.C.W., Krochko, J.E. and Bewley, J.D., 1989. Application of artificial seed technology in the production of hybrid alfalfa (Medicago sativa L.). In Vitro Cell. Dev. Biol., 25:1183-1188. McWilliam, A.A., Smith, S.M. and Street, H.E., 1974. The origin and development of embryoids in suspension cultures of carrot (Daucus carota L. ). Ann. Bot., 38: 243-250. Murashige, T. and Skoog, F.S., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Pill, W.G. and Finch-Savage, W.E., 1988. Effects of combining priming and plant growth regulator treatments on the synchronization of carrot seed germination. Ann. Appl. Biol., 113: 383-389.
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Fluid drilling as a delivery system for somatic embryo-derived plantlets of carrot (Daucus carota L. )* Sherry L. Kitto, Wallace G. Pill and Donna M. Molloy Delaware Agricultural Experiment Station, Department of Plant and Soil Sciences, College of Agricultural Sciences, University of Delaware, Newark, DE 19717-1303, USA (Accepted 3 January 1991 )
ABSTRACT Kitto, S.L., Pill, W.G. and Molloy, D.M., 1991. Fluid drilling as a delivery system for somatic embryoderived plantlets of carrot ( Daucus carota L. ). Scientia Hortic., 47: 209-220. Somatic embryos and/or somatic embryo-derived plantlets (SEPs) of carrot cultivar 'Orlando Gold' were subjected to various treatments during either suspension culture or a subsequent incubation period in fluid-drilling gel. Conversion of SEPs in the glasshouse into plants containing primary leaves was greatest when SEPs were incubated for 1-2 weeks in hydroxyethyl cellulose fluid-drilling gel ( 1.67%, w / v of N-gelTM) hydrated with a solution containing Murashige and Skoog salts and vitamins and 2% ( w / v ) sucrose. Providing 3 days of chilling at 4°C then 3 days at 25°C all under light (60/~mol m -2 s-1 ) during suspension culture (at the globular to torpedo stage) led to the greatest SEP growth following fluid drilling. Inclusion of either 250 mg Truban R fungicide or 10 mg chitosan glutamate per litre of gel improved SEP conversion. Keywords: chitosan; embryogenesis; fluid drilling; polyethylene glycol. Abbreviations: MS = Murashige and Skoog salts and vitamins; PEG = polyethylene glycol; SEP = somatic embryo-derived plantlet.
INTRODUCTION
A mature true seed, essentially a young plant in an arrested state of development, is composed of an embryo (embryonic axis plus nutritive tissues) and covering structures (seed coats). Seed coats play a role in the regulation of nutrition as well as in the protection of the embryo at early stages of its *Published as Miscellaneous Paper No. 1320 of the Delaware Agricultural Experiment Station, Contribution No. 269 of the Department of Plant and Soil Sciences. Mention of trade names in this publication does not imply endorsement by the Delaware Agricultural Experiment Station of products named, nor criticism of similar ones not named.
0304-4238/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
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S.L. KITTO ET AL.
development. As seeds mature, the major function of the seed coats becomes that of protection. Many studies have shown that somatic embryos may be produced from somatic tissues or cells cultured in vitro (McWilliam et al., 1974). Further, the development of embryos produced in vivo and in vitro is similar within the same species (Janick et al., 1982). However, somatic embryos produced in vitro lack seed coats. Therefore, systems must be developed so that somatic embryos can be handled with a minimum of damage and be delivered to seedbeds in the glasshouse or field. Four handling systems to facilitate delivery of somatic embryos have been proposed: fluid drilling (Drew, 1979; Baker, 1985 ), encapsulation of somatic embryos in alginate gel (Fujii et al., 1987 ), desiccation of somatic embryos (Gray, 1989 ), or desiccation of somatic embryos encapsulated in water-soluble resin (Janick et al., 1989 ). The fluid-drillingtechnique, normally involving the transfer of germinated seeds to the seed bed in a protective carrier gel (Gray, 1981 ), provides the potential for bulk handling of many small plantlets without the need for individual handling. The present study examined techniques for sowing somatic embryo-derived plantlets (SEPs) of carrot. The five experiments presented here examined the composition of the fluid-drilling gel, the roles of light and chilling during suspension culture, embryo incubation in the fluid-drilling gel, the addition of SEP-conditioning materials to the gel and the post-fluid-drilling environment. MATERIALS AND METHODS
E x p e r i m e n t a l protocol. ~ The overall experimental protocol, shown in Fig.
1, began with callus initiation from the hypocotyl of carrot seedlings. During subsequent suspension subcultures, somatic embryos were induced and subjected to various conditioning treatments. The conditioned embryos or SEPs were then incorporated into fluid-drilling gel. The SEP-gel mixture was incubated before being fluid drilled into the seedbed. Production a n d conditioning o f SEPs. - - Callus and cell suspensions of Daucus carota L. cultivar 'Orlando Gold' were initiated and maintained as de-
scribed by Kitto and Janick ( 1985 ). Embryos were initiated from cell suspensions by inoculating 0.2 g fresh weight of cells per 25 ml of Murashige and Skoog (MS) medium (Murashige and Skoog, 1962 ) (with 2% sucrose ) and transferring to fresh medium every 3 days. After 10-14 days, torpedo embryos precociously "germinated" to produce what we have termed somatic embryo-derived plantlets (SEPs) (Fig. 2 ). SEPs (still in suspension culture) were conditioned either under cool-white fluorescent lamps (60/zmol m -2 s-l of photosynthetically active radiation at suspension culture level; 16 h
211
FLUID DRILLING AND SOMATIC EMBRYO-DERIVED CARROT PLANTLETS
CARROTSEEDLING ~ HYPOCOTYL/~ ~ 2-WEEK
EXPLANT
TRANSFER CALLUSINITIATION
........
s MA,NTENANCE ~
~
3 (For2Weeks) SFERS
~
,RDUDT,ON
EMBRYOCOLLECTION
~ ~ l l b
S
r ~
SEED \,IP z~ ~. ~-,'7 "~_~ '~r ~,~
v~:~ %
~
CARROTS GROWN FROM FLUIDDRILLED SEPs
CELL SUSPENSION
on i ionmg Treatments (e.g osmotica, chilling)
~1~
~
EMBRYO/ SEP-GEL INCUBATION TREATMENT %
t
~.~ ....
~ EXTRUSIONOFTHE SEP-GEL MIXTUREINTOA GLASSHOUSEFLAT
EMBRYO TRANSFERTO FLUID-DRILLINGGEL Fig. 1. Schematic representation of protocol from embryo initiation and collection to fluid drilling of the SEP-gel mixture.
Fig. 2. SEPs derived from somatic embryos incubated under lights for 6 days with MS salts and 2% sucrose (magnification X 9.5).
212
S.L. KITTO ET AL.
photoperiod) at 25/20°C (light/dark) a n d / o r by chilling at 4°C in the light or in the dark for 3-8 days. Thefluid-drilling gel. - - T h e hydroxyethyl cellulose gel, formulated specifically as a fluid-drilling gel (Banyai, 1987 ), is designated N-gelT M (Aqualon Group, Wilmington, DE). Gel was prepared by adding powder (1.67%, w/ v) to MS solution (MS; 0, 1 or 2% ( w / v ) sucrose; 0, 250 or 500 mg active ingredient (a.i.) per litre of wettable powder formulations of the fungicides Truban, Benlate or Banrot; 0, 150, 200 or 250 g polyethylene glycol 8000 (PEG) per litre, or 0, 10, 50 or 90 mg chitosan glutamate per litre (Seacure TM, Protan Co., Redmond, WA) ) that was continuously agitated with a magnetic stirrer. The gels, after autoclaving at 125 kPa and 121 °C for 15 min, were of sufficient viscosity to suspend the SEPs for at least 1 h. Water potentials for various solutions and N-gel preparations were determined by vapor pressure osmometry (Wescor Model 5500XR, Logan, UT). A 10/tl sample was added to the chamber for aqueous solutions. For the viscous N-gel preparations, a filter paper disc was dipped into the fluid and the excess material removed before placement of the disc in the sample holder. Osmometer readings (mosmol) were converted to - MPa using the formula: ~ = i m R T, where q/is osmotic potential ( - MPa), i m is osmolality of the sample ( mosmol kg- ~), R is a gas constant ( 8.31 X 10 - 6 m 3 MPa mol- ~K - ~), and T is temperature (K). S E P incubation in gel. ~ SEPs were incubated in gel contained within parafilm-sealed eight-well polystyrene tissue culture multiplates or 125 m m × 80 mm X 20 m m polystyrene boxes. A multiplate well (26 m m X 33 m m X 10 mm ) contained five SEPs and 1 ml of the appropriate gel. Multiplates were maintained for 5 days at 25 °C in the light after which SEP vigor was rated subjectively from 1 (dead) to 5 (green and healthy). SEP-gel mixtures ( 10, 15, 20, 25, or 30 ml of N-gel) were poured into polystyrene boxes to provide a gel layer 1-2 m m thick and were incubated under a 16/8 h, 25/21 °C cycle for 0, 1, 2, 3, or 4 weeks (after these periods representative SEPs were measured for length). After incubation, SEPs plus gel (adjusted to a total volume of 15, 20, 30 or 50 ml) were mixed and placed in 125 ml plastic bags (6 oz Whirlpak, NASCO, Ft. Atkinson, WI ). Post-fluid-drilling environment in the glasshouse. ~ SEPs were either transplanted manually or were fluid drilled into 10 cm plastic petri dishes or 17 cm × 12 cm × 6 cm plastic fiats. Petri dishes were placed under a 16/8 h, 25/ 20°C regime for 2 weeks when SEP length was determined. Flats contained five furrows ( 12 cm long, I cm deep ) pressed into a growth medium (ProMix BX (Pr), Premier Brands Inc., Stanford, CN; RediEarth R (Re), W.R. Grace,
FLUID DRILLING AND SOMATICEMBRYO-DERIVEDCARROT PLANTLETS
213
Fogelsville, PA; vermiculite (V); perlite (P) or 50% ( v / v ) combinations of Re or Pr with either V or P). SEPs were either sown uncovered or were covered with about 5 m m of the appropriate growth medium. Flats in the glasshouse were either placed under intermittent mist (6 s every 6 min) or were surface irrigated once daily. Flats removed from the mist were surface irrigated once daily. Flats in the laboratory were placed in clear plastic boxes under lights (60/tmol m -2 s-i, 16/8 h, 25/20°C). Percentage emergence of cotyledons (above the growth medium surface) and percentage embryo conversion (plantlets having primary leaves) were determined at various times after sowing. RESULTS
Fluid-drilling gel composition. - - While SEP vigor was greatest in gel prepared without sucrose, SEP conversion in the glasshouse 20 days after transplanting was greatest for those that had been incubated in gel prepared from 2% ( w / v ) sucrose (Table 1 ). SEPs incubated in gel containing Benlate did not survive after transplanting (data not shown). Plants converted from SEPs incubated in gel containing 2% ( w / v ) sucrose and 250 mg a.i. Truban per litre appeared normal 12 weeks after transplanting into growth medium under glasshouse conditions (observation not shown ). The total water potential of the SEPs, estimated by vapor pressure osmoTABLE 1 Vigor of SEPs of carrot cultivar 'Orlando Gold' 5 days after incorporation in N-gel containing MS medium, sucrose and two fungicides; and SEP conversion in the glasshouse 20 days after transplanting Sucrose (%, w / v ) 0 0 0 0 0
Fungicide (mg a.i.1-1) Truban Truban Banrot Banrot
SEP vigor t (mean _+ SD)
0 250 500 250 500
4.0 + 1.02 4.0+ 1.17 4.0 _ 1.05 2.9 + 0.64 4.1 _+0.78
SEP conversion2 (%) 0 0 0 0 0
1
-
0
2.7_+ 1.03
0
1 1 1 1
Truban Truban Banrot Banrot
250 500 250 500
3.2+ 1.14 3.4_+0.91 2.7_+0.75 3.6+ 1.22
0 0 0 0
2 2 2 2 2
Truban Truban Banrot Banrot
0 250 500 250 500
2.4+0.64 2.8+0.55 3.1 +0.52 2.6 + 0.64 3.0 _+0.50
7.0 12.5 4.8 7.1 4.8
~Scale from 1 (dead) to 5 (green and healthy); n = 25. 2plants with primary leaves.
214
S.L. K1TTO ET AL.
metry of crushed SEPs, was - 0 . 3 9 MPa (20°C). Water potentials of 1.67% ( w / v ) N-gel prepared with MS salts and 0%, 1%, and 2% ( w / v ) sucrose were - 0 . 2 5 MPa, - 0 . 3 1 MPa, and - 0 . 3 9 MPa, respectively. Thus, the SEPs should have received a net influx of water until equilibrium was reached. Duration o f light and gel incubation periods. - - SEPs incubated in gel, compared with those not incubated in gel, were longer and had greater percentage emergence (Table 2 ). SEP length increased with up to 3 weeks incubation in gel provided that pretreatment at 25 °C in the light was for a duration of 6 days or less. SEP growth was greatest with 4 days pretreatment at 25 °C in the light followed by 3 weeks incubation in the gel. Pretreatment (25 ° C, light) for more than 6 days reduced the gel incubation period required for maximal growth. Cotyledonary emergence 1 week after fluid drilling was maximal when SEPs were incubated in gel for 1-2 weeks. A light period of more than 6 days, irrespective of the gel incubation period, greatly reduced SEP emergence. Chilling duration in the light or dark. - - Regardless of chilling duration, increasing the subsequent incubation period at 25°C in the light from 0 to 6 days increased SEP growth before fluid drilling (Fig. 3 (A)). Light or dark TABLE2 SEP length at time of fluid drilling and SEP cotyledon emergence 1 week after fluid drilling as influenced by duration of culture exposure to continuous light at 25 °C and subsequent duration of SEP incubation in 1.67% N-gel prepared with MS salts and 2% sucrose Duration of light (days)
Incubation in gel (weeks)
SEP length after incubation in gel ( m m )
SEP emergence 1 week after fluid drilling (%)
4
0
6.4 10.9 12.2 26.2 14.4
4.1 31.0 52.5 18.3 20.0
0
8.2
1
11.8
2 3 4
13.2 15.1 11.9
9.7 41.8 45.0 16.8 7.5
0 1 2 3 4
9.6 15.2 22.2 12.1 10.8
8.2 22.5 15.2 12.5 15.0
1
2 3 4 6
8
LSDo.o5
2.14
24.43
FLUID DRILLING AND SOMATIC EMBRYO-DERIVED CARROT PLANTLETS
60
A
215
/
50
E
==
40 /
//
•
30
/
2O
_ --~
/"
110
I 0
I 3
I 6
Days
of
chilling
I 0
in light
I 3
or
I 6
dark
Fig. 3. SEP length at: (A) the time of fluid drilling; (B) 2 weeks after fluid drilling. SEPs were subjected to 0, 3, or 6 days of chilling (4°C) in the light (open symbols) or dark (closed symbols ) followed by 0 ( O, • ), 3 ( A, • ), or 6 ( [2, • ) days of light at 25 ° C before fluid drilling. Vertical bars, LSD, P < 0.05, n=25.
during chilling for up to 6 days initially had no significant effect on SEP length. However, SEP length 2 weeks after fluid drilling increased dramatically when they were chilled for 3 days in the light prior to 3 days gel incubation in the light (Fig. 3 (B) ). Post-fluid-drilling environment in the glasshouse. - - The 9.1% emergence of SEPs that were chilled (3 days at 4°C in the dark then 3 days at 25 °C in the light) was significantly lower than the 15.4% emergence of SEPs that were not chilled (6 days at 25 °C in the light), although growth medium interacted significantly with mist vs. no mist to influence percentage emergence by 5 weeks after fluid drilling (Table 3 ). Generally, Pr+ P ( 1 : 1, v / v ) or Re gave the greatest percentage emergence from fluid-drilled SEPs (Table 3). Non-chilled SEPs that were fluid drilled into Re and provided with intermittent mist gave the greatest percentage emergence (57.3%). Chilled SEPs given the same post-fluid-drilling environment had only 4.8% emergence. Gel incubation volume and SEP conditioning additives in the gel. - - Increasing the gel volume to more than 15 ml per 10 ml of SEP suspension reduced the SEP conversion rate (Table 4 ). Decreasing the water potential from - 0 . 3 9 MPa (0% PEG) to - 1 . 9 9 MPa (25% PEG) in the gel during the 1 week incubation period prior to fluid drilling did not increase SEP conversion (Table 4 ). The addition of 0.01 g of chitosan glutamate per litre of gel during the
216
S.L.~TTO ETAL.
TABLE 3 Percentage cotyledon emergence of SEPs 5 weeks after fluid drilling as influenced by growth medium and irrigation. Before fluid drilling, the SEPs were given either 6 days of light at 25°C (non-chilled SEPs) or 3 days at 4°C then 3 days light at 25°C (chilled SEPs) Growth medium Pr
PrV
PrP
Re
ReV
ReP
Mist Non-chilled SEPs Chilled SEPs
10.1 5.0
1.5 0
5.7 16.7
57.3 4.8
0 0
8.5 7.8
No mist Non-chilled SEPs Chilled SEPs
18.8 4.5
8.0 14.6
21.4 24.2
25.3 25.1
16.9 5.0
10.9 0
LSDo.o~ = 21.34 (3-way interaction ) ~Growth medium: Pr, Promix BX; PrV, Pr plus vermiculite (V, 1: 1, v / v ) ; PrP, Pr plus perlite (P, 1 : 1, v / v ) ; Re, RediEarth; ReV, Re plus V ( 1 : 1, v / v ) ; ReP, Re plus perlite ( 1 : 1, v / v ) .
TABLE 4 Percentage conversion of somatic embryos conditioned for 1 week under light with PEG or chitosan in different gel volumes then fluid drilled septically onto RediEarth and placed under lights in the laboratory Gel amendment
0 0 0 0 0 15% PEG 20% PEG 25% PEG 0.01 g Chitosan 4 0.05 g Chitosan 0.09 g Chitosan
LSDo.o5
Gel volume ~
SEP length after incubation in gel 2 ( m m )
SEP conversion 3 4 weeks
7 weeks
10 15 20 25 30
21.4 23.1 23.6 23.3 20.7
0 3 1 1 1
1 10 5 3 1
30 30 30
14.8 14.1
0 0 0
0 0 0
30 30 30
18.7 13.3 14.0
5 0 1
6 0 0
2.69
JVolume of gel per 10 ml of SEP. 2Initial SEP length, 9.8 + 2.3 mm. 3Number of plants with primary leaves. 4Water-soluble chitosan glutamate (Seacure).
FLUID DRILLING AND SOMATIC EMBRYO-DERIVED CARROT PLANTLETS
217
incubation period before fluid drilling led to a relatively high SEP conversion 4 weeks after fluid drilling, a conversion rate that was sustained until 7 weeks after fluid drilling (Table 4). DISCUSSION
One objective of this research was to develop a system for maturing embryos into autotrophic SEPs; therefore, somatic embryos were treated either with light a n d / o r chilling during development in suspension culture or during gel incubation. SEPs kept in the light for 4-6 days and incubated for up to 2 weeks in gel had the greatest vigor (Table 2 ). The reduction in vigor associated with SEPs kept for more than 6 days in the light and/or more than 2 weeks of gel incubation may have reflected inadequate storage reserves to support basic physiological functions (e.g. respiration). Reduced vigor and slower development of somatic embryos has been associated with low levels of stored reserves (McKersie et al., 1989 ). The beneficial effect of chilling SEPs may be likened to that of short-term low-temperature osmotic priming of seeds. The osmotic potential of the suspension culture medium ( - 0.39 MPa) may have been sufficiently negative to cause events associated with osmotic seed priming, such as increased activity of esterases and phosphatases (Khan et al., 1978) which, by decreasing the SEP solute potential, would lead to increased water influx and SEP growth upon exposure to warmer conditions. Chilling of imbibed vegetable seeds has increased the germination rate (Finch-Savage and Cox, 1982). The greater SEP length 2 weeks after fluid drilling that resulted from light rather than dark during chilling, may reflect a promotive effect of the light on SEP autotrophy. This growth differential due to irradiation during chilling reflected a delayed response, as at the end of chilling, light had minimal influence on SEP length. Although SEP length was greatest when 6 days of chilling was followed by 6 days of incubation in the light at 25 °C, these SEPs were too large to fluid drill (Fig. 3 ). Three days at 4 ° C in the light followed by 3 days at 25°C in the light led to the greatest gain in SEP length 2 weeks after the SEPs had been fluid drilled. The SEPs of this treatment ( 18 mm in length), could be fluid drilled, but from a practical standpoint, SEPs should be about 1 cm long to be fluid drilled. This decrease in length might be accomplished by beginning the entire chilling/post-chilling treatment earlier during suspension culture. From our results, sucrose and chitosan appeared to be beneficial during SEP incubation in the fluid-drilling gel. Sucrose plays two roles in vitro: as a nutritional carbon source and as an osmotic agent. Lack of SEP survival with 0 or 1% sucrose (Table 1 ) may be attributed to an insufficient supply of carbon and energy during the period when SEPs become autotrophic. Drew ( 1979 ) attempted to assist the survival of somatic carrot embryos before the
218
S.L. KITTO ET AL
establishment of autotrophism by "stocking" the suspension culture with elevated sucrose levels but this greatly reduced culture growth. If the effect of sucrose was solely nutritional then perhaps much of the sucrose requirement could be replaced by carbon dioxide enrichment and high levels of photosynthetically active radiation to induce autotrophy as shown with carnation plantlets in solidified gel culture (Kozai, 1989 ). Carbohydrates as osmotic agents have been linked to the acquisition of desiccation tolerance by embryos (Kitto and Janick, 1985 ) and to elevated endogenous proline levels in wheat callus cultures (Gabor et al., 1986 ). Thus, a higher rate of SEP conversion following conditioning in N-gel containing 2% sucrose than in 0 or 1% sucrose (Table 1 ) may reflect osmotically induced stress tolerance associated with elevated endogenous proline. The beneficial effect of 0.01 g chitosan per litre (Table 4) may be associated with stress (defense?) related plasma membrane effects as chitosan is recognized by plants as an indicator of an invasive organism (Kendra and Hadwiger, 1984). Exposure to PEG-induced water stress in the present study may have been too short and too late in SEP ontogeny as the osmotic adjustment that occurred in stress-tolerant carrot cells (Fallon and Phillips, 1989) was developed over long periods (eight subcultures ). Increasing the gel volume above 15 ml per 10 ml of SEP suspension reduced the SEP conversion rate (Table 4), a response that may be attributed to hypoxia resulting from reduced gaseous exchange. SEPs can be likened to pregerminated seeds. While the synchrony of seedling emergence is enhanced by increasing the percentage of seeds which are germinated at the time of fluid drilling (Pill and Finch-Savage, 1988 ), SEPs do not have to "germinate" but they must be able to continue growth following fluid drilling. As with pregerminated seeds, the seed-bed conditions must be conducive to SEP emergence because the option for not "germinating" has been removed. SEP conversion into plants with primary leaves 4 and 7 weeks after fluid drilling occurred under septic laboratory conditions (Table 4), confirming that the SEPs were capable of conversion but that some factor (s) in the glasshouse environment prevented SEP conversion (but did not prevent SEP emergence). While SEP conversion occurred under glasshouse conditions in early February (Table 1 ), lack of SEP conversion from March to August (Tables 2 and 3, Fig. 3 ) may be associated with higher average temperatures and reduced water availability between irrigations. Fujii et al. (1989) suggested that soil surface drying associated with some watering regimes (e.g. intermittent mist) caused reduced embryo conversion which could be alleviated with a humidity tent system. Presumably Pr + P ( 1 : 1, v/v ) or Re possessed suitable aeration and water retention (Table 3 ). When they were provided with
FLUID DRILLING AND SOMATIC EMBRYO-DERIVED CARROT PLANTLETS
219
daily surface irrigation, these media gave from 20 to 25% emergence from chilled or non-chilled SEPs. Results of the experiments reported here show that SEPs were capable of conversion under both septic laboratory and glasshouse conditions following fluid drilling. Light or chilling during suspension culture followed by a lighted incubation period in fluid-drilling gel of reduced water potential that contained sucrose and fungicide or chitosan favored SEP conversion. ACKNOWLEDGMENTS
Research supported in part by a Delaware Research Partnership Grant which was funded jointly by the State of Delaware and the Aqualon Group, Wilmington, DE, USA.
REFERENCES Baker, C.M., 1985. Synchronization and fluid sowing of carrot, Daucus carota somatic embryos. M.S. Thesis, University of Florida, Gainesville, FL, 98 pp. Banyai, B.E., 1987. N-gelT M polymers for agricultural fluid-drilling. Acta Hortic., 198:111-120. Drew, R.L.K., 1979. The development of carrot (Daucus carota L.) embryoids (derived from cell suspension culture) into plantlets on a sugar-free basal medium. Hortic. Res., 19: 7984. Fallon, K.M. and Phillips, R., 1989. Responses to water stress in adapted and unadapted carrot cell suspension cultures. J. Exp. Bot., 40:681-687. Finch-Savage, W.E. and Cox, C.J., 1982. A cold-treatment technique to improve the germination of vegetable seeds prior to fluid drilling. Scientia Hortic., 16:301-311. Fujii, J.A., Slade, D.T., Redenbaugh, K. and Walker, K.A., 1987. Artificial seeds for plant propagation. Trends in Biotech., 5: 335-339. Fujii, J.A., Slade, D.T. and Redenbaugh, K., 1989. Maturation and greenhouse planting of alfalfa artificial seeds. In Vitro Cell. Dev. Biol., 25:1179-1182. Gabor, G., Simon-Sarkadi, L., Bekes, F. and Erdei, L., 1986. Genotype specific changes in amino acid and polyamine of wheat tissue culture induced by osmotic stress. In: J. Semal (Editor), Somaclonal Variations and Crop Improvement. Advances in Agricultural Biotechnology. Martinus Nijhoff, Hingham, MA, pp. 170-176. Gray, D., 1981. Fluid drilling of vegetable seeds. Hortic. Rev., 3: 1-27. Gray, D.J., 1989. Effects of dehydration and exogenous growth regulators on dormancy, quiescence and germination of grape somatic embryos. In Vitro Cell Dev. Biol., 25:1173-1178. Janick, J., Kitto, S.L. and Kim, Y.-H., 1989. Production of synthetic seed by desiccation and encapsulation. In Vitro Cell. Dev. Biol., 25:1167-1172. Janick, J., Wright, D.C. and Hasegawa, P.M., 1982. In vitro production of cacao seed lipids. J. Am. Soc. Hortic. Sci., 107: 919-922. Kendra, D.F. and Hadwiger, L.A., 1984. Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Exp. Mycol., 8: 276-281. Khan, A.A., Kar-Ling, T., Knypl, J.S., Borkowska, B. and Powell, L.E., 1978. Osmotic conditioning of seeds: Physiological and biochemical changes. Acta Hortic., 83: 267-278.
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Kitto, S.L. and Janick, J., 1985. Hardening treatments increase survival of synthetically coated asexual embryos of carrot. J. Am. Soc. Hortic. Sci., 110: 283-286. Kozai, T., 1989. Autotrophic (sugar-free) micropropagation for a significant reduction of production costs. Chronica Hortic., 29: 19-20. McKersie, B.D., Senaratna, T., Bowley, S.R., Brown, D.C.W., Krochko, J.E. and Bewley, J.D., 1989. Application of artificial seed technology in the production of hybrid alfalfa (Medicago sativa L.). In Vitro Cell. Dev. Biol., 25:1183-1188. McWilliam, A.A., Smith, S.M. and Street, H.E., 1974. The origin and development of embryoids in suspension cultures of carrot (Daucus carota L. ). Ann. Bot., 38: 243-250. Murashige, T. and Skoog, F.S., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Pill, W.G. and Finch-Savage, W.E., 1988. Effects of combining priming and plant growth regulator treatments on the synchronization of carrot seed germination. Ann. Appl. Biol., 113: 383-389.