Soluble saccharides and cyclitols in alfalfa (Medicago sativa L.) somatic embryos, leaflets, and mature seeds

Plant Science 109 (1995) 191-198

Soluble saccharides and cyclitols in alfalfa (Medicago sativa L.) somatic embryos, leaflets, and mature seeds Marcin Horbowiczavd, Ralph L. Obendoff a, Bryan D. McKersieC, Donald R. Viandsb ‘Seed Biology, Department of Soil, Crop and Atmospheric Sciences, 619 Braafield Hall, Cornell University, Ithaca, New York 14853-1901, USA bDepartment of Pkmt Breeding and Biometry, Cornell University Agricultural Experiment Station, Cornell University, Ithaca, New York 14853-1901, USA ‘Department of Crop Science. University of Guelph. Guelph, Ontario NlG 2WI. Canaab dResearch Institute of Vegetable Crops, Skierniewice. Poland Received 10 January 1995; revision received 5 April 1995; accepted 2 May I995

Soluble carbohydrates were identified and quantified during development, maturation and desiccation of somatic embryos of alfalfa (Medicago sativa L.) and compared to soluble carbohydrates in leaflets and mature seeds, to relate changes in soluble carbohydrates to maturation events. Somatic embryos have elevated levels of sucrose. However, in contrast to mature seeds, alfalfa somatic embryos do not accumulate Dpinitol or the galactosyl derivatives of D pinitol such as galactopinitol A, galactopinitol B and ciceritol. Lower levels of stachyose accumulate during maturation of somatic embryos, but stachyose increases to levels in mature seeds during desiccation of somatic embryos. When stachyose accumulation is limited in somatic embryos, galactinol and digalactosyl myo-inositol increase. Reducing sugars decline to low levels during desiccation of somatic embryos and sucrose: oligosaccharide ratio decreases from 2.7 to 0.9, approaching the ratio 0.2 to 0.3 in mature dry seeds. Except for the lack of pinitol and galactosyl pinitols, changes in soluble carbohydrates during the maturation and desiccation of alfalfa somatic embryos are typical of changes occurring in mature seeds that have been reported to be associated with desiccation tolerance and storability. Keywords: Sugars; Desiccation tolerance; Synthetic seeds; Artificial seeds; Somatic embryo; Embryo maturation; Stachyose; Rafftnose

1. lll~on

Somatic embryos may be used as synthetic or artificial seeds. This technology has potential as a l Corresponding author, Tel.: +l 607 2551709; Fax: +l 607 2552644.

plant-breeding tool in the development of new alfalfa (Medicago sutivu L.) cultivars [l] or as a propagation system for sterile plants or high-value plants in other species. Somatic embryos are genetically identical to the donor plant and are morphologically similar to zygotic embryos in early stages of development. When provided with

0168~9452!95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04155-N

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hf. Horbowicz et al. /Plant

the proper nutrients in the maturation media, alfalfa somatic embryos deposit storage proteins and carbohydrates and acquire desiccation tolerance [2]. After drying to 0.15 g Hz0 g-’ fresh weight, somatic embryos may be stored for several months, and then imbibed and germinated. However, the vigor of seedlings obtained from these somatic embryos remains less than that of seedlings obtained from seeds [2]. Morphological and biochemical differences between desiccated somatic embryos and seeds may explain this low vigor. Morphologically, alfalfa somatic embryos do not have fully developed cotyledons and lack an endosperm and testa. Biochemically, alfalfa seeds contain 3 major forms of storage reserves, proteins (362 mg g-’ dry mass) (31 and oil (103-109 mg g-’ dry mass) [4,5] in the cotyledons, and galactomannan (55-90 mg g-’ dry mass) [6] in the endosperm. Mature alfalfa seeds also contain 60- 105 mg g-l dry mass of soluble carbohydrates [7-91 including stachyose (32-39 mg g-’ dry mass), raBinose (1 1- 13 mg g-’ dry mass) and sucrose (12-22 mg g-’ dry mass) [8,10] in the embryo. In contrast to seeds, alfalfa somatic embryos contain qualitatively similar although fewer storage proteins 111,121. However, instead of galactomannan being the endospermic carbohydrate reserve in seeds, somatic embryos contain starch and sucrose [2] in the embryo in the absence of an endosperm. When the accumulation of storage carbohydrate and protein was manipulated during maturation of somatic embryos by changing the carbon/nitrogen balance in the maturation medium, seedling vigor was correlated positively to the quantity of storage proteins deposited in the embryo, but was independent of starch content [2]. The low vigor of desiccated somatic embryos is likely a consequence of improper maturation that may have caused the accumulation of inadequate storage reserves, poor morphological development, dehydration/rehydration damage, or insufficient desiccation tolerance. RaBinose and stachyose, galactosyl sucrose oligosaccharides, in orthodox seeds and zygotic embryos have been associated with maturation, tolerance to desiccation, and storability [9,13- 151. Similar associations during maturation of somatic

Science IO9 (1995) 191-198

embryos have not been reported. The objectives of this study were to determine if differences in soluble carbohydrates exist between somatic embryos and mature seeds of alfalfa and to determine if these differences may be important in the maturation and desiccation of somatic embryos. 2. Methods 2.1. Tissue culture Petiole sections from young, fully expanded leaves of alfalfa (Me&ago sativu L., clones RL34 and S4-23-2) were cultured for 14 days on a modified SH medium containing K2S04, proline and thiaproline [2]. The callus was transferred to liquid B5 culture medium for 7 days. Small clusters of embryogenic cells were isolated by sieving sequentially through 50- and 200-pm nylon screens (Nitex). The 200-500~pm fraction was transferred to BOi2Y agar medium supplemented with 50 g 1-l sucrose (development medium) [2]. After 7 days on development medium, the embryos were matured in 2 steps, referred to as maturation phase I and phase II. During maturation phase I, embryos were placed for 10 days on BOi2Y medium containing 50 g sucrose l-‘, 50 mM glutamine and 25 mM potassium sulphate (Sigma Chemical Company) added prior to autoclaving [2]. Subsequently, in maturation phase II, desiccation tolerance of the somatic embryos was induced by exposure to 20 PM abscisic acid (ABA) (Sigma Chemical Company) and 50 g I-’ sucrose for 10 days. Embryos were dried to 0.15 g Hz0 g-i fresh weight overnight in a laminar flow hood and stored at a relative humidity of 43% generated in a desiccator containing a saturated solution of K&OJ at 20-25°C in darkness for at least 7 days. Germination of the dry somatic embryos was conducted as previously described [2]. 2.2. Plant tissues Alfalfa somatic embryos that were visually separated into globular, torpedo and cotyledonary stages of development (desiccated embryos of line U-23-2 stored dry for 3-6 months), leaflets from field-grown and greenhouse-grown ‘Guardsman’ alfalfa plants, and mature seeds of ‘Guardsman’ (3 replications) and ‘Rangelander’ (2 replications)

hi. Horbowicz et al. /Plant Science 109 (1995) 191-198

cultivars of alfalfa were analyzed for soluble sugar, oligosaccharide, cyclitol, and galactosyl cyclitol composition. Three replicate samples of young expanding leaf blades and fully expanded mature leaf blades were harvested on 6 October 1994 from 5week-old vegetative fall regrowth of field-grown plants at Ithaca, NY, USA. Old leaflets were harvested from greenhouse-grown plants during seed formation. Globular stage embryos (20 mg) were divided into 3 replicate samples, and torpedo stage and cotyledonary stage embryos were divided into 4 replicate samples before analysis. To test the effect of desiccation, alfalfa somatic embryos (line RL34, also known as A70 or A7034) were matured as described above. Three replicate samples were harvested after maturation phase II (after ABA treatment), but before drying, and then freeze-dried. Another 3 replicate samples, after maturation phase II, were air-dried to about 0.15 g Hz0 g-’ fresh weight in a laminar flow hood for 48 h before harvest. 2.3. Analysis of saccharides and cyclitols Tissues were extracted with ethanol:water (l:l, v/v) and heated for 45 min at 80°C to inactivate degradative enzymes. Extracts were passed through a 10 000 Mr cut-off filter and evaporated to dryness in a stream of nitrogen gas. Trimethylsilylsylderivatives were analyzed by high resolution gas chromatography using phenyl a-Dglucoside as internal standard 191. Unknown components are reported by relative retention time (Rt) to the internal standard. Saccharides and cyclitols were identified by comparison with authentic standards as available. Glucose, fructose, galactose, sorbitol, maltose, myo-inositol, sucrose, raffinose, stachyose, and phenyl a-Dglucoside (internal standard) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Galactinol was a gift from T.M. Kuo (Peoria, IL, USA); Dchiro-inositol and D-pinitol were a gift from S.J. Angyal (Kensington, NSW, Australia). O-a-D-galactopyranosyl-( l -2)-4-O-methyl-Dchiro-inositol (galactopinitol A), O-a-Dgalactopyranosyl-(l -2)-3- O-methyl-Dchiro-inositol (galactopinitol B), Dpinitol, and D-chiro-inositol were a gift from J.G. Streeter (Wooster, OH, USA); ver-

193

bascose, Dchiro-inositol, and Dpinitol were a gift from P. Wtirsch (Lausanne, Switzerland); verbascose and Dchiro-inositol were a gift from P. Adams and R.G. Jensen (Tucson, AZ, USA); and Dononitol, sequoyitol and L-(+)-bomesitol were a gift from F.A. Loewus (Pullman, WA, USA). To facilitate identification of unknowns, the temperature program was changed to an initial temperature of 14O“C(ISO’C), adjusted to 180°C (200°C) at l.S”C/min (3”C/min), adjusted to 325°C at 7”C/min, and held at 325°C for 30 min. This program clearly separated Dpinitol, Dononitol, sequoyitol, D-chiro-inositol, L-(+)-bornesitol, myo-inositol, glucose and fructose. Peaks identified as a-galactosides disappeared after incubation of extracts with green coffee bean a-galactosidase (EC 3.2.1.22) (Boehringer Mannheim Corporation, Indianapolis, IN, USA), and compounds were hydrolyzed to their monomeric components with 2 N trifluoroacetic acid. Peaks identified as Dpinitol, D-chiro-inositol, and myo-inositol were verified by GC-MS. Amounts of unknown carbohydrates were estimated by calculation with the nearest known standard. Quantities are expressed as mean f S.E. of the mean for 2-4 replications. 3. Rear& and diseuasIon 3.1. Leaves Alfalfa leaflets contained free cyclitols including large quantities of Dpinitol (3-O-methyl-Dchiroinositol) and lesser quantities of Dononitol (4-Omethyl-myo-inositol) and ntyo-inositol (Table 1). Sequoyitol (5-O-methyl-Dmyo-inositol) and D(-)bomesitol (l-O-methyl-Dmyo-inositol), if present, were below the level of detection in extracts of leaBet tissues. Sucrose was the major sugar (30-36% of total). Trehalose was not detected in alfalfa leaflets. Glucose, fructose, maltose, galactinol and ratEnose were detected in field-grown leaflets, but these compounds were reduced to trace amounts in old leaves from greenhouse-grown plants (Table 1). Presence of galactinol and rafllnose in leaves from the cool field environment is consistent with the stimulation of galactinol synthase by cool temperature [ 161. D pinitol increased with increased age of leaflets and comprised 20-53% of total soluble carbohydrates.

194

M. Horbowicz et al. /Plant Science 109 (1995) 191-198

Table 1 Sugars and cyclitols in alfalfa (Medicago sativa L. cv. ‘Guardsman’) leaflets from field- and greenhouse-grown plants Sugar or cyclitol

Field-grown alfalfa

Greenhouse alfalfa

Young leaf

Mature leaf (% of total)

Fructose GlucoSe o-pinitol n-ononitol myo-inositol Sucrose Maltose Galactinol Ratlinose Unknowns

6.67 3.81 13.75 2.80 6.89 24.13 0.72

0.34 f 0.10 1.21 f 0.17 6.81 f 0.96

I.1 0.5 1.8 10.0

Total

67.05 f 6.93

100.0

f 1.32 ZIZ0.73 * 0.55 l 0.10 f 1.29 f 2.85 f 0.13

9.9 5.7 20.5 4.2 10.3 36.0

(mg/g dry mass) 13.89 f 7.22 f 19.69 f 2.82 f 6.52 zt 29.18 f 1.87 f 0.72 f 1.89 zt 8.37 f

-

Old leaf (% of total)

(% of total)

1.78 0.89 3.05 0.39 0.66 7.00 0.25 0.11 0.16 0.58

15.1 7.8 21.4 3.0 7.1 31.6 2.0 0.8 2.1 9.1

0.47 0.40 28.84 1.30 4.28 16.11 0 0 0 2.66

0.9 0.7 53.4 2.4 7.9 29.8 0 0 0 4.9

92.20 zt 9.67

100.0

54.06

100.0

-

Values are mean f SE. of the mean for 3 replications of tield-grown leaves. A single pooled sample of greenhouse leaves was analyzed.

Young leaflets contained 16 unknowns, mostly in trace amounts, but together accounting for 10% of the total soluble carbohydrates. The number of these unknowns decreased with leaf age. One prominent unknown appeared between glucose (peak 1) and ononitol. Between citrate and fructose, 2 peaks of unknowns were observed in trace amounts. The position of the 2 peaks suggests that their identity may be a di-O-methyl-rnyo-inositol such as dambonito1 (1,3-di-0-methyl-myo-inositol) that is synthesized from myo-inositol with D(-)-bornesitol as the precursor [ 171 or liriodendritol (l&h-Omethyl-D-myo-inositol) with Dononitol as the precursor (181. Vegetative tissues of alfalfa are reported to contain sucrose, glucose, fructose, trehalose, Dpinitol, Pononitol, and myo-inositol [19-221. It has been deduced that Dpinitol, D-ononitol, and myoinositol are translocated in the phloem because the honeydew of pea aphids (Acyrthosiphon pisum) which feed on alfalfa leaf phloem contains relatively large quantities of myo-inositol and Dpinitol and small amounts of Dononitol [21]. However, no evidence suggests that cyclitols substitute for sucrose as the major transported carbohydrate. Based largely on work with crimson clover (Trifo-

lium incarnatum L.) leaflets, early work identified

sequoyitol as the precursor in biosynthesis of Dpinitol [23]. Later, D-ononitol was identified as the preferred precursor to Dpinitol in leaves of several legumes including alfalfa, crimson clover, and Ononis spinosa [24]. Perhaps both pathways are active in transformation of myo-inositol into D-pinitol, depending upon the species or the conditions. 3.2. Seea5 Mature seeds of alfalfa had no detectable fructose or glucose but stored mostly (83-86%) sucrose and galactosyl sucrose oligosaccharides as soluble components (Table 2). ‘Guardsman’ seeds contained about 55% of the soluble components as stachyose, 10% as raffmose, 6% as ciceritol, 3% as verbascose, and 14% as sucrose. myo-Inositol and ~pinitol were present as free cyclitols and also as galactosyl derivatives. The galactosyl derivatives of myo-inositol included galactinol (O-o-Bgalactopyranosyl-( 1- 3)-D-myo-inositol; also known as O-~~galactopyranosyl-(l - Q-L-myo-inositol), and digalactosyl myo-inositol (Rt = 2.68) (O-~-Dgalactopyranosyl-( 1 - 6)-O-a-Dgalactopyranosyl(1 - 3)-D-myo-inositol) [25,26]. Higher oligomers,

IU. Horbowicz et al. /Plant Science 109 (1995) 191-198

trigalactosyl and tetragalactosyl derivatives of myoinositol, may also occur in some seeds [26]. Mature alfalfa seeds contained the galactopinitol A series of @nitol, galactopinitol A (O-cr-~galactopyranosyl-( l -2)-4-O-methyl-D&‘ro-inositol), ciceritol (O-o-~galaetopyranosyl-( 1- 6)-O-cr-Dgalactopyrnosyl-( 1- 2)-4-O-methyl-D&r&nositol) [271, and trigalactopinitol A (probably the unknown with

195

Rt = 3.00) (~-~~actopyran~yl~l-6~~-~ galactopyranosyl-(l -6)-O-o-D-galactopyranosyl(1.2)4&nethyl-D-c/r%nositol). Gall?etopinitol B (O-o-~galactopyranosyl-( l -2)-3-O-methyl-Dchiro-inositol) represents a positional isomer. Sequoyitol and Dononitol were not detected in mature seeds. Unknowns (Rt = 1.94 and 2.60) may be galactosyl sequoyitol and digalactosyl sequoyitol or

Table 2 Concentration of soluble saccharides and cyclitols in dry alfalfa seeds (cv. ‘Guardsman’ and cv. ‘Rangelander’) and dry somatic embryos of alfalfa (Medicago sativa L., line N-23-2) Saccharide or cyclitol

Rta (ratio)

Alfalfa seed ‘Guardsman’

Fructose Glucose D-pinitol Unknownb myo-inositol Sucrose Galactopinitol A Galactopinitol B Unknown Galactinol Digalactosyl glycerolc RafEnose Ciceritol Unknown Digalactosy1 myoinositold Stachyose UnknownC Unknown’ Verbascose Total Sucrose:oligosaccharide, ratio Sucrose:non-sucrose, ratio

0

Alfalfa somatic embryos ‘Rangelander’

0

Globular stage (mg/g dry mass)

Torpedo stage

0.72 f 0.23

0.71 zt 0.29

Cotyledon stage

0.31;0.32 0.323 0.40;0.55 0.34 0.47 0.71 1.72 1.85 1.93 1.94 2.06 2.18 2.37 2.55 2.60 2.68

0.71 f 0.09 0 1.69 zt 0.12 12.57 f 0.39 1.36 f 0.15 0.23 zk 0.03 Trace 1.69 f 0.14 0.80 f 0.12 8.85 zt 0.20 5.57 f 0.24 1.21 f 0.09 0.90 l 0.20

0.39 f 0.02 0 1.23 f 0.12 11.65zt 0.43 1.13 l 0.01 0 0 1.27 zt 0.30 0.57 l 0.01 5.48 l 0.03 2.74 f 0.36 0 0.31 f 0.10

2.88 3.08 3.16 3.62

48.77 f 7.48 0.66 l 0.05 0 2.57 f 0.50

30.71 f 1.83 Trace 0 Trace

9.35 + 0.51 0 0 Trace

12.92 f 1.48 Trace 0.28 + 0.16 0.62 f 0.36

15.75 f 1.99 Trace 0.28 zt 0.16 0.80 f 0.53

87.83 + 8.69

55.30

112.62 f 1.79

146.08 f 8.65

135.50 f 5.98

0

0

0.40 l 0.09

l

2.20

0

0.32 f 2.17 l 76.91 f 0 0 0.31 zt 15.19 f 0.13 l 6.08 f 0 0 1.03 l

0.07 0.12 5.82

0.16 2.56 0.07 2.35

0.11

0.45 l 0 0.39 f 1.42 zt 103.28 f 0 0 Trace 14.52 f Trace 1.96 zt 0 0 9.42 f

0.14 0.14 0.06 5.89

0.85 0.33

0.65

0.44

l

0.26 f 0 0.95 * 1.38 f 95.11 l 0 0 0 10.25 f 0.07 l 2.13 l 0 0 8.07 zt

0.20 0.10 0.18 0.19 3.11

0.86 0.05 0.34

1.11

0.18 f 0.01

0.32 f 0.03

5.47

1.48

6.91 f 0.68

5.32 f 0.60

0.17 * 0.01

0.21 f 0.04

2.33 zt 0.59

2.17 f 0.20

2.40 zt 0.18

l

Values are mean f S.E. of the mean for 2 replications of ‘Rangelander’, 3 replications of ‘Guardsman’ and globular stage, and 4 replications of torpedo and cotyledon stages. aRetention time relative to the internal standard, phenyl o-nglucoside. bA peak with similar retention time was also present in young expanding leaflets. Woposed identification; is widespread among seeds of many species. dProposed identification; digalactosyl myo-inositol is galactosyl galactinol. ‘Proposed identification: trigalactopinitol A. ‘Proposed identification: trigalactosyl myo-inositol.

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M. Horbowicz et al. /Plant Science 109 (1995) 191-198

galaCtOSy1 OnOnitOl (O-or-D-galactopyranosyl-(1-5)-4-O-methyl-D-myo-inositol) [28] and digalactosyl ononitol, respectively. Structures of selected cyclitols and their galactosyl derivatives have been illustrated [9]. ‘Rangelander’ seeds had about 60% of the total concentration of soluble saccharides, cyclitols and galactosyl cyclitols when compared to ‘Guardsman’ seeds, but the relative proportions of individual components was similar (Table 2), except for the absence of verbascose.

3.3. Somatic embryos All stages of somatic embryos had elevated lev-

els of sucrose (Table 2) - a reflection of the high concentration of sucrose in the medium used for maturation [2]. However, all stages of somatic embryos had restricted levels of stachyose and raffmose. The elevated levels of galactinol, the galactosyl donor in the formation of stachyose from raflinose and verbascose from rafflnose, suggests an impairment to oligosaccharide biosynthesis in somatic embryos. The elevated levels of galactinol were accompanied by elevated levels of digalactosyl myo-inositol (Rt = 2.68), a higher galactosyl derivative of galactinol, and possibly small amounts of trigalactosyl myo-inositol (Rt = 3.16) in the torpedo and cotyledonary stage somatic embryos (Table 2).

Table 3 Effect of desiccation on the concentration of soluble saccharides and cyclitols in somatic embryos of alfalfa (Medicago safiva L., line RL34) after maturation phase II in standard culture but before drying and after air-drying to 0.15 g Hz0 g-’ fresh weight in a laminar flow hood for 48 h Saccharide or cyclitol

Fructose Glucose o-pinitol Unknown Unknown Unknown b myo-inositol Unknown Sucrose. Maltose Galactopinitol A Galactopinitol B Galactinol Digalactosyl glycerolC RaIlinose Ciceritol Digalactosyl myo-inositold Stachyose Verbascose Total Sucrose:oligosaccharide, ratio Sucrose:non-sucrose, ratio

Rta (ratio)

0.309; 0.317; 0.323 0.40; 0.55 0.34 0.36 0.45 0.47 0.71 0.93 1.72 1.76; 1.83 1.85

1.93 2.06 2.18 2.37 2.55 2.68 2.88 3.62

Alfalfa somatic embryos after maturation phase II Before drying (m@g dry mass)

After air drying (mg/g dry mass)

13.53 zt 0.96 4.96 zt 0.24 0 0.44 f 0.04 1.07 f 0.11 1.01 f 0.08 3.38 zt 0.17 0.49 f 0.06 84.50 f 1.46 1.31 l 0.73 0 0 5.17 l 1.17 0.40 f 0.03 14.72 f 0.20 0 0.05 * 0.03 16.57 f 0.90 0

0.42 f 0.06 0.42 + 0.09 0 0 0 0.53 * 0.21 1.29 f 0.24 0.36 + 0.04 47.98 f 5.93 0.74 f 0.11 0 0 3.43 f 0.18 0.71 l 0.04 9.18 zt 1.61 0 0.72 f 0.09 40.87 zt 1.19 1.38 f 0.16

147.59 f 0.67

108.02 l 7.64

2.70 f 0.05 1.34 * 0.06

0.93 f 0.08 0.79 zt 0.08

Values are mean f SE of the mean for 3 replicate samples. aRetention time relative to the internal standard, phenyl a-Dglucoside. bA peak with similar retention time was also found in young expanding leaflets. %oposed identification; is widespread among seeds of many species. dProposed identification; digalactosyl myo-inositol is galactosyl galactinol.

M. Horbowicz et 01. / Pkmt Science 109 (1995) 191-198

Unlike alfalfa seeds, somatic embryos of line S423-2 did not accumulate tr-pinitol nor the galactosyl derivatives of Ppinitol (galactopinitol A, galactopinitol B, ciceritol, or trigalactopinitol A) (Table 2). Somatic embryos contained a new compound (Rt = 0.47) that was not found in old k&lets or seeds, but a similar peak was present in young expanding leaflets. Unknowns (with Rt = 1.94 and 2.60) in somatic embryos may be galactosyl derivatives of sequoyitol or D-ononitol. Ononitol and galactosyl ononitol have been identified in cowpea (Vigna zurg&uluru (L.) Walp.) seeds 1291. Caution should be used in identification of sequoyitol and Dononitol in somatic embryos because mannitol and sorbitol have similar retention times. Even though the culture media used in these experiments contained neither mannitol nor sorbitol, sorbitol has been shown to accumulate under certain stress conditions such as accelerated aging of seeds (P.K. Kataki, unpublished), conditions that also release glucose and fructose. 3.4. Effect of desiccation Somatic embryos of line RL34 also did not accumulate tr-pinitol or the galactosyl derivatives of Dpinitol (Table 3). The reducing sugars, fructose and glucose, declined to low levels during airdrying of the mature somatic embryos. Sucrose concentration also declined substantially during drying causing a decline in total soluble components. Stachyose concentration more than doubled during drying and small amounts of verbascose appeared. The decrease in concentration of sucrose and the increase in stachyose during drying resulted in a decrease in the sucrose:oligosaccharide ratio from 2.7 before drying to 0.9 after drying of somatic embryos (Table 3). Induction of desiccation tolerance during slow-drying of immature zygotic embryos of soybean also was associated with loss of sucrose and an increase in stachyose 1141.The decline in the sucrozoligosaccharide ratio (Table 3) was expected to be associated with the increased desiccation tolerance and storability of these somatic embryos [9,15]. 4. comelusIolls The most dramatic difference between the alfal-

197

fa somatic embryos and seeds was the absence of tr-pinitol and its derivatives. Perhaps the lack of cotyledon development or the absence of an endosperm tissue contributed to the apparent lack of these metabolites. Alternatively, the culture conditions may not have suitably mimicked the in ovule environment so that a key regulatory metabolite for this pathway was missing. Somatic embryos do not have the maternal leaf source of transported rr-pinitol. However, we have observed that in vitro-grown zygotic embryos of soybean (Glycine mm (L.) Merrill) accumulate galactosyl derivatives of tr-pinitol while maintaining a constant level of free tr-pinitol and without a maternal source of Dpinitol (M. Horbowicx and R.L. Obendorf, unpublished). Therefore, it appears more likely that one or more genes for the biosynthesis of Dpinitol were not expressed. Perhaps the gene for a myo-inositol O-methyl transferase that forms Dononitol [30], an intermediate in D pinitol biosynthesis, was not expressed in alfalfa somatic embryos. The second interesting observation concerns stachyose. Accumulation of stachyose was stimulated by desiccation of the somatic embryos with a concomitant decline in glucose, fructose, and sucrose. Stachyose accumulation also was dependent on the maturity of the somatic embryos because dried globular-stage embryos of S4-23-2 accumulated less stachyose than dried cotyledonstage embryos. Also the dried cotyledon-stage embryos of !?&23-2 accumulated less stachyose than those of RL34. Germination of this batch of S423-2 somatic embryos was 44% with root emergence and none with shoot emergence. In contrast, germination of the dried RL34 somatic embryos was 55% with an emerged radicle and 26% with a radicle and trifoliate leaf at 21 days after imbibition. AcknowIedgemeuts This work was conducted as part of Western Regional Research Project W-168 (NY-C 125423) and was supported in part by grants from Pioneer Hi-Bred International, Inc. to R.L.O. and from the Ontario Ministry of Agriculture and Food to B.D.M. We gratefully acknowledge fellowship support from The Kosciusxko Foundation to

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M. Horbowicz et al. /Plant Science 109 (1995) 191-198

M.H. We thank T.M. Kuo, S.J. Angyal, J.G. Streeter, P. Wiirsch, P. Adams, R.G. Jensen, and F.A. Loewus for supplying authentic standards, and P. Brenac for help with leaflet analysis. Technical assistance was provided by Mr. Cecilio Gregorio for the plant tissue culture experiments. Referencea

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