Plant Cell, Tissue and Organ Culture63: 109–120, 2000.© 2001Kluwer Academic Publishers. Printed in the Netherlands.

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A hypocotyl-derived somatic embryogenic system inBrassica junceaCzern& Coss and its manipulation for enhanced storage lipid accumulation

Anita Kumari, G.S. Cheemaa & S.K. Munshi∗Department of Biochemistry andaBiotechnology Centre, Punjab Agricultural University, Ludhiana 141 004, India(∗requests for offprints)

Received 23 March 1999; accepted in revised form 31 October 2000

Key words:growth and development, growth regulators, soluble sugars, enzyme assays, lipid composition

Abstract

A simple and reproducible protocol for induction, growth and development of somatic embryos from hypocotylexplants of Indian mustard (Brassica junceaL. Czern & Coss) var. RLM 198 is reported. The HDSE (Hypocotyl-derived somatic embryos) were fleshy globular to torpedo structures that were maintained by regular subculturingevery three weeks. These embryos developed non-synchronously into the heart shaped-stage while some werematured into a green cotyledon-stage bearing embryos in the same medium. The HDSE accumulated as much as50.2% lipid content on a dry weight basis at 14 DAC (days after culture) using a culture medium supplementedwith 10% PEG (Polyethylene glycol 6000) in comparison to less than 15% lipid content in 2% sucrose (control) or20µM ABA (abscisic acid). An increase in total soluble sugar content was observed with 2.5% PEG and increasingPEG concentration caused a decrease in their contents in HDSE. The activities of invertase, acetyl CoA carboxylaseand 1-14C-acetate incorporation into lipids in HDSE were enhanced significantly in the culture medium containing10% PEG. The content of triacylglycerols in HDSE was maximum with 10% PEG supplemented culture medium.The wax content in HDSE increased progressively with an increase of PEG concentration in the culture medium.The ABA and PEG supplementation increased the content of membrane lipids when the data was expressed ona 100 g dry weight basis. The proportion of palmitate and erucate decreased and that of oleate, linoleate andlinolenate increased at 14 DAC in HDSE in 10% PEG supplemented culture medium. Thus, the manipulation ofculture conditions significantly altered total lipid content, membrane lipid composition and the quality of storagelipids.

Abbreviations:2,4-D – 2,4-dichlorophenoxyacetic acid; BA – benzyladenine; NAA –α-naphthaleneacetic acid;GA – gibberellic acid; ABA – abscisic acid; PEG – polyethylene glycol 6000; TAG – triacylglycerols; PL –phospholipids; GL – glycolipids; ST – sterols; SEM – scanning electron microscopy; TEM – transmission electronmicroscopy; IM – induction medium; MM – maintenance medium; DAC – days after culture; DAF – days afterflowering

Introduction

There is increased interest in the bulk production ofspecial metabolites by the use of rapidly emergingplant tissue culture techniques (Fu et al., 1999). Suchproduction systems will eliminate restrictions of sea-sons and climatic zones, to which the plants in thefield are subjected. Morphogenesis through a somaticembryogenic pattern that results in the production of

discrete bipolar entities capable of growth and devel-opment like zygotic embryos is of immense practicalimportance for its efficiency in cloning for develop-mental studies, genetic manipulations and syntheticseeds. Somatic embryos are known to accumulate stor-age reserves, e.g., proteins (Crouch and Sussex, 1981)and lipids (Avjioglu and Knox, 1989), and have beenextensively used in experiments investigating the pos-sibility of developing bioreactor systems which are

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suitable for the mass production of plant metabolites,in vitro (Preil and Beck, 1991).

In Brassicaspecies,in vitro embryogenosis hasbeen observed in different explants viz, immaturezygotic embryo (Maheswaran and Williams, 1986),hypocotyl (Dietert et al., 1982; Kirti and Chopra,1989), young leaves (Pareekh and Chandra, 1978) andprotoplasts (Kirti, 1988). Microspore-derivedembryosrepresent an array of genetically variable populations(Taylor et al., 1993). However, somatic embryos raisedfrom hypocotyls constitute a cloned genotype and,once established, requires normal tissue culture tech-niques to maintain. There is no previous report dealingwith the development of hypocotyl-derived somaticembryo inB. junceawith their capacity for the accu-mulation of storage lipids. In this laboratory, somaticembryos raised from hypocotyl explants ofB. junceaby a simple and reproducible protocol, have beenmaintained for more than a year by regular subcul-turing (Kumari et al., 1995). These somatic embryosalso accumulate lipids in lipid bodies which havewell-defined membrane proteins (Kumari, 1995).

Sucrose, abscisic acid and polyethylene glycol6000 are substances that are known to regulate thematuration of somatic embryos (Avjioglu and Knox,1989; Attree et al., 1991) and also to improve stor-age reserve accumulation in various systems, viz.,Theobroma cacao(Pence et al., 1981),Daucus carota(Dutta and Appelqvist, 1989),Apium graveolens(Kimand Janick, 1991) andBrassica napus(Avjioglu andKnox, 1989). The aim of the present study is to report

(i) the induction, maintenance and development ofsomatic embryogenic system capable of storagelipid accumulation,

(ii) their characterization by (a) scanning electronmicroscopy (SEM), (b) transmission electronmicroscopy (TEM), (c) plant conversion capab-ility and biochemical profile and

(iii) to study the effect of sucrose, ABA and PEG onthe accumulation of storage lipids in the HDSEof B. juncea.

Materials and methods

Materials

B. junceaL. var. RLM 198 seeds were procured fromOilseed Section of the Department of Plant Breeding,Punjab Agricultural University, Ludhiana. All chem-icals used were of analytical grade. Sodium acetate

Table 1. Response ofB. junceaexplants to different induc-tion media after eight weeks of culture

Medium∗ Explant % response± S.E.

Callus Somatic

embryogenesis

IM1 Hypocotyl – 17± 1.2

Cotyledon – 20± 0.8

IM2 Hypocotyl – 72± 1.5

Cotyledon – 60± 1.6

IM3 Hypocotyl 5 + 0.8 –

Cotyledon 4 + 0.7 –

IM4 Hypocotyl 28± 1.9 –

Cotyledon 20± 1.8 –

∗The culture medium contained (µg ml−1), IM1 = 2,4-D(0.25), BA (0.50) and NAA (0.50); IM2 = 2,4-D (0.50) andBA (1.0); IM3 = 2,4-D (0.50) and BA (0.50); IM4 = BA(2.0) in half strength MS medium with 2% sucrose solution;S.E. – Standard error.

(1-14C) of high specific activity was procured fromBhabha Atomic Research Centre, Mumbai.

Somatic embryo induction, growth andcharacterization

Mature seeds were thoroughly washed with tap waterfor 20 min and surface sterilized with 0.1% mer-curic chloride for 5 min followed by 3–4 washingswith sterile distilled water in the laminar flow cabinet.The seeds were inoculated in jars containing solidifiedhalf-strength MS (Murashige and Skoog, 1962) me-dium with 1% sucrose, pH 5.8. These jars were keptin the dark for 36 h at 25± 1◦C and then illuminatedwith white fluorescent tube lights, at 21µmol m−2

s−1 in a 16 h photoperiod. One-centimeter hypocotyland cotyledon explants from 7-day-old seedlings wereplanted on modified MS medium (Edamine not used;sucrose 2% instead of 1%; 0.70% agar; pH 5.8) sup-plemented with 0.25 to 1.0µg ml−1 2,4-D, 0.1 to 0.5µg ml−1 NAA, 0.20 to 2.0µg ml−1 BA, 0.1 to 1.0µgml−1 GA3, 1.0 µg ml−1 IAA (indolacetic acid), 1.0µg ml−1 Kinetin singly and in combination. Initially50 explants, in triplicate, were used for each inductionmedium. The induction medium (IM2), half-strengthMS medium containing 0.5µg ml−1 2,4-D, and 1.0µg ml−1 BA, with 2% sucrose displayed efficiencymuch better than other induction media (Table 1). Forlater experiments on time course response and bio-

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chemical characterization, a minimum of 100 explantsper replication were used in each group or treatment.The organized structures formed on the induction me-dium, were separated from the explant and transferredto fresh medium (1M2). This medium showed max-imum dry matter and lipid accumulation comparedto other combinations and was used for later exper-iments. The somatic embryos were maintained andmultiplied through regular subculturing on fresh me-dium every 3 weeks. The cultures were examined atweekly intervals. There were three replications andduplicate observations in each replication. The HDSEwere characterized by– scanning electron microscopy (SEM) by the

method used by Maheswaran and Williams (1985),– transmission electron microscopy (TEM) by using

the method of Fowke (1984),– conversion to plantlet to document their entity as

embryos, and– biochemical analysis (Munshi et al., 1990).

The HDSE were subjected to different treatmentsby transferring to fresh medium containing differentsucrose concentrations at 2, 5, 10, 15 or 20% (w/v)or abscisic acid at 5, 10, 15, or 20µM or polyethyl-ene glycol 6000 at 2.5, 5.0, 7.5 or 10% all with basalmedium containing 2% sucrose. Each treatment had 3replications of 50 tubes each with duplicate samplingper replication.

Sampling

Samples were drawn for the biochemical analysis ofsomatic embryos and the callus along with zygoticembryos (seeds) from the field at 15 DAF (days afterflowering). The criteria for determining the age of theseed was similar as reported earlier (Munshi et al.,1990). In addition, samples of somatic embryos andcallus were taken at 7, 14, 21 and 28 DAC (days afterculture). The embryos were carefully taken out of testtubes so that they were free from the medium andplaced on filter paper on ice.

For dry matter determination, a weighed quantity(100–150 mg) of tissue was placed in the oven at 60◦C for 48 h. After drying, the tissue was immedi-ately placed in a desiccator until final weighing. Forlipid analysis, the weighed quantity (200–300 mg) wasboiled in 2 ml isopropanol for 10 min and homogen-ized in 20 volumes of chloroform:methanol (2:1, v/v)mixture and stored at low temperature. For sugar ana-lysis, a known quantity (200–300 mg) of tissue wasboiled in 80% ethanol and stored at low temperature.

For protein estimation, the embryonic mass (approx100 mg) was put in 0.1 N NaOH and stored.

Analytical methods

The tissue homogenate in chloroform: methanol (2:1)was subjected to lipid extraction and Folch washing(Folch et al., 1957). A suitable aliquot of lipid ex-tract in chloroform was evaporated to determine totallipid content. For the determination of triacylglycerolcontent, the lipids were separated by thin layer chro-matography using silica gel G. The TLC plates weredeveloped, after having applied the sample and tri-olein as standard, in known quantity as fine spots, ina solvent system containing petroleum ether-diethylether-acetic acid (80:20:1, v/v/v) by ascending chro-matography. The triacylglycerol (TAG) spot was visu-alized with iodine vapours and scraped off separatelyinto glass column with glass wool bed at its base. TAGwas eluted with a solvent system containing hexane-diethyl ether (95:5, v/v) and measured gravimetric-ally. The content of phospholipids and glycolipidswas estimated by the method followed by Munshiet al. (1990). The sterols content was estimated byZak (1957). The wax was extracted by immersinga known amount of tissue in redistilled chloroformfor 15 seconds and after filtering and evaporating theextract, the content was determined colorimetrically(Ebercon et al., 1977).

From the ethanol preserved samples, the sugarswere extracted and the aqueous extract was depro-teinated (AOAC, 1965) followed by the estimation oftotal soluble sugars by the method of Dubois et al.(1956). The sugar free residue after ethanol extractionwas air dried and starch was extracted with 52% per-chloric acid and determined by the method of Clegg(1956). The storage proteins were extracted by themethod used by Crouch (1982) and estimated by themethod of Lowry et al. (1951). The analysis of datawas performed according to factorial experiment inrandomized block design.

Results and discussion

The hypocotyl explants cultured on four different in-duction media showed the best somatic embryogenicresponse in 1M2 medium. The response was 72%with the hypocotyl explants as compared to 60% withcotyledon explants in 1M2 medium after 8 weeks ofculture (Table 1). The apical end of the hypocotyl

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Figure 1. Induction of somatic embryos from hypocotyl ofBrassica junceaL. (A) Embryonic mass (EM) on hypocotyl (H) (× 20) (B) Globular(g), heart (h) and Torpedo (t) shaped embryo clusters (× 20), (C) Well-developed embryo with secondary somatic embryos (SSE) derivedfrom green cotyledon of primary embryo (× 20); (D) Plantlet formation from somatic embryo on basal medium (× 0.5); (E) SEM of somaticembryos (× 56), C- cotyledonary end and R-radicular end; (F) TEM of somatic embryos (× 4900), L-lipid bodies, S-starch granules, N-nucleus,Nu-nucleolus, and arrows indicate lipid body proteins.

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Table 2. Time course response ofB. junceahypocotyl to IM2 medium for somatic em-bryogenesis

Week after culture % response± S.E.

4 15.5± 1.8

5 37.5± 2.3

6 59.0± 3.0

8 77.0± 3.1

IM2 – As in Table 1; S.E. – Standard error.

explant was more responsive to somatic embryogen-esis as compared to basal end (Figure 1A), thoughin certain cases, embryos were visible in the middleof the explant also. The responding explants formedembryogenic masses associated with scanty yellowishcallus (embryogenic) distinct from non-embryogeniccallus (Figure 1A). The embryogenic patterns display-ing organized structures were evident after 4 weeksof culture in 1M2 and the progressive response upto 8 weeks was reproducible in a separate set ofexperiment (Table 2). The embryos were fleshy glob-ular structures that developed into torpedo and heart(Figure 1B) shaped structures. Some of the embryosdeveloped cotyledons (Figure 1C) but most of themcarried cotyledonary notches which could not growinto well-developed cotyledons. Embryo developmentwas non-synchronous and at any stage of time globularto early cotyledonary embryos along with the maturestages were observed as inB. napus(Loh and Ingram,1983).

The identity of somatic embryos, after examina-tion through a stereomicroscope, was confirmed bytransferring them to a conversion medium. The well-formed and mature embryos showed greening andconverted to plantlets (Figure 1D). The purpose ofplantlet development was simply to establish the ger-mination potential of the developed embryos. TheSEM studies showed the developing embryos as dis-tinct bipolar structures with radicular and cotyledon-ary poles, in clustered orientation, at different stages ofgrowth (Figure 1E). The ultrastructural visualizationby TEM (Figure 1F) revealed that the cells containedplastids with small and large starch grains and numer-ous lipid bodies in cytoplasm, which also containedlarge nuclei with prominent nucleoli. The dividingcells were clearly visible with well defined septae.The lipid bodies had well defined membrane havingelectron dense proteins.

The fact that apical end of the explant respon-ded more efficiently to form embryos than the basalend, is similar to the observation recorded inB. napus(Tykarska, 1976) and other plants (Reiser and Fischer,1993). This could be related to the intrinsic polarityof the explant (Zimmerman, 1993). Even though 0.5µg ml−1 2,4-D and 1.0µg ml−1 BA were provided inthe culture medium, the endogenous level of growthhormones is likely to be the determining factor in theinduction of somatic embryogenesis and subsequentlyelaboration of proper morphogenesis in embryo de-velopment. The same medium (1M2) was used forsubculturing to produce secondary embryos and theirmaintenance. The higher dry matter and lipid contentin somatic embryos maintained on 1M2 suggested theimportance of 2,4-D in the culture medium though themedium also contained BA. This is in sharp contrast toremoval of auxin in carrot cultures for somatic embryodevelopment (Zimmerman, 1993). This suggests thatthe withdrawal of auxins is not conducive toBrassicajunceafor the growth of somatic embryos from hypo-cotyl explants as reported earlier by Kirti and Chopra(1989).

The somatic embryos were further confirmed bytheir biochemical profiles of storage product accu-mulation. The somatic embryos contained storagelipids like triacylglycerols, sterols and partial gly-cerides, visualized by TLC, storage proteins and starch(Table 3) as accumulated by zygotic embryos in theseeds (Munshi et al., 1990). However, the quantity wasless than the zygotic (seed) embryo of equivalent agegrowing in the field but significantly higher in com-parison to non-embryogenic callus. The accumulationof TAG in HDSE was lower than zygotic (seed) em-bryos (Table 3). The proportion of palmitate (16:0) andstearate (18:0) was higher and that of oleate (18:1),linoleate (18:2), linolenate (18:3) and erucate (22:1)lower in HDSE as compared to zygotic (seed) embryos(Table 3). This trend is similar to that reported in so-matic embryos ofBrassica napus(Avjioglu and Knox,1989; Taylor et al., 1992). The observation that thesomatic embryos convert to the plantlet stage may beattributed to the use of storage reserves like lipids andstarch, as normally occurring in zygotic counterparts(Munshi et al., 1990). The somatic embryos culturedfor 7 to 28 days accumulated dry matter, lipids andwax content (Table 4) higher than non-embryogeniccallus; the maximum lipid content was observed at14 DAC. Somatic embryos accumulated wax on theirouter surface which is similar to surface wax in leaves(Hall and Jones, 1961).

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Table 3. Comparison ofB. junceahypocotyl-derived somatic embryos, raised on IM2 withnon-embryogenic callus and zygotic embryos (seeds) for certain biochemical parameters

Parameters Non-embryogenic Somatic Zygotic

callus embryos embryos

(Seeds)

Dry matter (g/100g FW) 5.39± 0.5 12.37± 0.8 14.30± 0.8

Lipids (g/100g FW) 0.39± 0.0 1.06± 0.1 1.26± 0.1

(g/100g DW) 4.64± 0.3 8.57± 0.5 18.00± 0.6

Triacylglycerols by TLC 16.3± 0.8 58.2± 1.1 88.0± 1.3

Fatty acids 16:0 ND 44.50± 0.5 16.60± 0.3

18:0 ND 10.60± 0.3 Tr

18:1 ND 11.40± 0.3 26.00± 0.3

18:2 ND 2.00± 0.0 22.90± 0.4

18:3 ND 5.50± 0.0 11.80± 0.2

22:1 ND 21.20± 0.2 27.40± 0.4

24:0 ND 1.90± 0.0 Tr

Storage proteins (g/100g FW) 0.28± 0.1 1.10± 0.1 2.00± 0.1

(g/100g DW) 5.19± 0.6 8.89± 1.1 13.99± 0.9

Starch (g/100g FW) 0.15± 0.0 1.57± 0.1 5.53± 0.5

(g/100g DW) 2.80± 0.2 13.30± 2.1 36.40± 2.5

The age of each tissue in each case was 15 DAC/DAF. Values are means± standard error.FW – Fresh weight; DW – Dry weight; IM2 – As in Table 1, ND – Not determined, Tr –Traces.

Table 4. Composition of hypocotyl-derived non-embryogenic callus and somatic embryos ofB. juncea

Days Non-embryogenic callus Somatic embryos

after

culture Dry matter Lipids Wax Dry matter Lipids Wax

% FW % FW % DW % Oil % FW % FW % DW % Oil

7 3.60 0.09 2.50 ND 6.50 0.55 8.46 3.98

14 4.80 0.14 2.91 ND 9.12 0.99 10.85 4.50

21 5.01 0.16 3.19 ND 7.73 0.81 10.34 6.80

28 5.39 0.25 4.64 ND 11.81 0.88 7.45 7.00

Critical difference (p < 0.05)

Dry matter Lipids Wax

% FW % FW % DW % Oil

Tissue (T) 0.01 0.02 0.15 0.24

Days after culture (D) 0.12 0.01 0.21 0.33

T x D 0.17 0.02 0.29 0.47

ND – Not detected.

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Figure 2. Dry matter, lipids and starch content of hypocotyl derived somatic embryos (HDSE) ofB. junceainfluenced by different concentra-tions of sucrose or ABA or PEG in the culture medium#: 2% sucrose (control); : 5% sucrose, 5µM ABA or 2.5% PEG; x: 10% sucrose, 10µM ABA or 5% PEG;1: 15% sucrose, 15µM ABA or 7.5% PEG;�: 20% sucrose, 20µM ABA or 10% PEG. Standard errors, too small forthe scale used, are not shown.

An increase in dry matter content (Figure 2) andtotal soluble sugars (Table 5) but a decrease in lip-ids, starch (Figure 2) and reducing sugars content inHDSE with increasing concentrations of sucrose in theculture medium indicated that sucrose may be takenup by the cells in proportion to its concentration inthe medium. However, its utilization was severely

affected, most likely due to the osmotic effect anddisruption of metabolic functions in the cells and theincrease in the dry matter observed could be due to thatof sucrose itself. Repression in the growth of HDSEand the effect of sucrose is consistent with the res-ults of Pence et al. (1981) and Avjioglu and Knox(1989). The improvement in the growth and develop-

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Table 5. Soluble sugar content [g (100 g dry weight)−1] influenced by different concentrations of sucrose, ABA and PEG in HDSEof B. juncea

Treatments/ 7 DAC 14 DAC 21 DAC 28 DAC

Concentrations

Total Reducing Total Reducing Total Reducing Total Reducing

sugars sugars sugars sugars sugars sugars sugars sugars

Sucrose

2% (Control) – – 10.0 3.0 – – 7.5 2.0

5% – – 31.3 2.2 – – 30.0 11.9

10% – – 56.0 3.7 – – 38.3 6.5

15% – – 89.0 6.3 – – 54.6 4.1

20% – – 73.5 6.0 – – 85.9 3.7

ABA

Control 5.1 0.3 10.5 2.5 5.5 0.1 7.0 1.0

5 µM 6.2 0.2 18.2 6.1 4.8 0.2 5.5 3.0

10 µM 6.8 2.9 12.7 9.7 4.2 1.9 8.3 2.5

15 µM 12.6 3.4 16.1 9.4 5.7 1.5 11.8 3.3

20 µM 20.6 11.1 8.7 7.0 5.1 2.6 12.5 7.5

PEG

Control 5.1 0.3 10.3 2.5 5.5 0.1 7.0 1.0

2.5% 28.3 7.2 7.5 2.9 7.6 2.5 5.8 4.0

5.0% 20.0 7.8 7.2 4.1 10.5 3.8 4.9 2.2

7.5% 20.1 7.6 12.4 4.6 10.5 4.8 11.4 4.3

10.0% 9.9 4.6 12.5 5.5 25.2 5.6 9.2 1.4

Critical difference (p < 0.05)

Sucrose ABA PEG

Total Reducing Total Reducing Total Reducing

sugars sugars sugars sugars sugars sugars

Concentrations (C) 2.6 0.6 0.7 0.5 1.6 0.3

DAC (D) 1.3 0.4 0.7 0.5 1.4 0.2

C× D 3.2 0.9 1.5 1.1 3.2 0.5

ment of HDSE, manifested in increased size of theembryos and embryonic clusters in ABA- and PEG-supplemented culture medium, could be due to thedry matter accumulation induced by the water stressin the cell (Kermode, 1990), the conditions similar todrought which bring about desiccation for the matur-ity of embryos (Attree and Fowke, 1993). The sizeand general morphology of the embryo at 7.5 and10% PEG was conspicuous and better than at lowerconcentrations. PEG-induced uptake and utilization ofsucrose in the embryonic cells causing enhanced accu-mulation of lipids (50.2%); higher than even zygoticembryos (40 to 42%) (Munshi et al., 1990). Starch

that was highest with 2.5% PEG, showed a decreasewith increasing PEG concentration (Figure 2) sug-gesting that sucrose and starch were utilized for thebiosynthesis of lipids. An increase in the activitiesof invertase, acetyl CoA carboxylase and fatty acidsynthetase visualized by the incorporation of 1-14C-acetate into lipids (Table 6) in HDSE at 14 DAC, thephase of rapid lipid accumulation, with 10% PEG inthe culture medium, supported the view of enhancedutilization of sucrose for the biosynthesis of lipids.An observed decrease in the activities of these en-zymes from 14 to 28 DAC was possibly due to thedesiccation conditions induced by PEG. Similar phe-

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Table 6. Effect of PEG supplementation in the culture medium of HDSE ofB. junceaon the activities of invertase,acetyl CoA carboxylase and 1-14C-acetate incorporation into lipids

Treatments 14 DAC 28 DAC

Invertase (µg sucrose hydrolysed min−1 g−1 F.W.)

Control 1092± 10 1169± 12

PEG (10%) 1717± 15 2148± 17

Acetyl CoA carboxylase (DPM x 10−3 100g−1 F.W.)

Control 759± 9 70± 3

PEG (10%) 959± 9 62± 2

1-14C-acetate incorporation into lipids (DPM x 10−3 g−1F.W.)

Control 1403± 11 138± 2

PEG (10%) 2812± 17 111± 2

Critical difference (p < 0.05)

Acetyl CoA 1-14C-acetate

Invertase carboxylase incorporation

Treatments (T) 1.1 1.8 1.6

DAC (D) 0.9 0.8 0.7

T x D 1.6 2.5 2.2

Values are means± standard error.

nomena of sucrose and starch hydrolysis seemed to bemanifested in case of ABA treatment but at a lowerrate as compared to PEG treatment and the conversionof starch to lipids was significantly less with 20µMABA. The accumulation of starch (Figure 2) and totalsoluble sugars (Table 5) from 14 to 21 DAC with 10%PEG could be due to their reduced utilization for thebiosynthesis of lipids which caused reduction in lipidcontent at 21 DAC in HDSE. An increase in dry mat-ter and lipids has been reported in somatic embryosof alfalfa (Anandrajah and McKersie, 1990), whitespruce (Attree et al., 1991) andB. napus(Finkelsteinand Crouch, 1986) on ABA and PEG supplementedcultures. Therefore, the data suggest that sucrose hy-drolysis makes available glucose initially for starchbiosynthesis and for fatty acid biosynthesis during thephase of rapid lipid accumulation (7 to 14 DAC) andthis pattern is similar to zygotic embryos (Munshi etal., 1990) and somatic embryos of other systems (Xuet al., 1990). Furthermore, the osmotically-treated so-matic embryos accumulated an enhanced level of drymatter and lipids as compared to control as a result ofincreased uptake of sucrose and a shift in the partition-ing of carbon towards fatty acid biosynthesis. It maybe noted, that somatic embryos may not experiencedesiccationper se. However, these embryos undergo

the preparation necessary for maturation which arecomparable to desiccation as in zygotic embryos andstarch acts as a protective agent against desiccation(Faure and Aarrouf, 1994).

Analysis of the composition of lipids in HDSE re-vealed an increased accumulation of wax and TAGwith period of growth in culture containing ABA andPEG in comparison to the control, and was attributableto chemical and water stress imposed on the embryos.Since the TAG was accumulated in the oil bodies, itsrelative proportion in the storage lipids was as muchas 47.1 and 57.6% with ABA and PEG, respectively(Table 7) as compared to 17% in control which is farless than the zygotic embryos (Munshi et al., 1990).However, the proportion of TAG in HDSE was signi-ficantly higher than the corresponding values reportedin microspore-derived embryos (Taylor et al., 1990)and also inB. napus(Avjioglu and Knox, 1989). Theresponse of TAG synthesis elicited by ABA and PEGwas similar to the one obtained in somatic embryos ofwhite spruce (Lawlor, 1970; Attree and Fowke, 1993),microspore-derived embryos ofB. napus(Taylor etal., 1990) and celery (Kim and Janick, 1991). Waxis present as a protective covering over the plant or-gans. The zygotic embryos inBrassicaare endowedwith a hard seed coat (testa) to sustain the post harvest

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Table 7. The proportion of various lipid classes [g (100 g lipids)−1] in HDSE ofB. junceainfluenced by differentconcentrations of ABA and PEG in the culture medium

Lipid DAF ABA ( µM) PEG (%)

class Control 5 10 15 20 Control 2.5 5.0 7.5 10.0

Wax 7 6.9 15.2 9.5 5.5 7.5 6.9 5.0 5.0 12.0 14.0

14 8.1 5.6 5.4 5.9 2.3 8.1 5.7 5.2 14.8 16.7

21 8.9 14.3 10.8 7.0 4.5 8.9 5.1 13.0 28.7 29.4

28 10.9 27.5 20.0 8.5 15.8 10.9 16.3 28.1 30.5 27.9

TAG 7 7.3 41.2 41.5 39.1 41.6 7.3 17.6 12.6 23.8 28.5

14 13.6 39.7 34.3 42.5 43.0 13.6 19.6 16.0 41.3 45.6

21 15.3 45.5 39.2 43.6 47.1 15.2 21.2 26.2 50.3 53.8

28 19.0 47.6 41.3 38.8 44.5 19.0 30.7 43.1 54.7 57.6

PL 7 6.4 10.2 5.1 7.8 6.8 6.9 5.2 2.5 2.6 0.7

14 5.6 14.2 7.5 9.8 16.4 5.6 4.4 1.4 1.4 0.8

21 4.3 5.3 9.0 7.6 9.4 4.8 2.4 1.8 0.6 1.8

28 3.4 13.6 13.1 8.6 10.6 3.4 3.0 1.5 1.0 0.5

GL 7 8.1 12.5 4.9 8.7 7.0 8.7 5.1 2.9 3.9 1.2

14 8.8 8.2 5.7 5.9 13.3 8.8 2.9 1.3 1.4 1.7

21 7.7 5.5 9.3 7.8 9.3 8.4 1.7 1.5 0.8 2.3

28 6.2 10.4 6.5 5.7 7.5 6.9 2.3 2.3 1.8 1.1

ST 7 13.5 22.1 13.4 5.0 20.3 14.5 12.2 4.6 5.5 5.4

14 11.5 27.4 14.9 10.5 20.6 11.5 5.2 4.1 3.4 2.4

21 9.1 24.5 22.7 16.0 20.2 10.1 4.3 3.1 1.9 2.8

28 8.8 35.1 14.5 7.1 21.9 9.9 5.0 3.7 2.1 1.7

Critical difference (p<0.05)

Concentration (C) DAC (D) C× D C D C× D

Wax 0.5 0.4 0.9 0.1 0.1 0.2

TAG 0.4 0.3 0.8 0.3 0.3 0.7

PL 0.3 0.3 0.7 0.1 0.1 0.3

GL 0.3 0.2 0.5 0.2 0.1 0.3

ST 0.5 0.4 1.0 0.3 0.3 0.7

handling operations. In contrast, the HDSE is a tenderstructure raised in sterile environment on a fixed me-dium and requires wax and other unknown nonpolarcompounds as a protective covering to support the cel-lular contents. The wax content in HDSE registereda decrease with increasing ABA concentration (Table7). In contrast, with increasing PEG concentration inthe culture medium, an increase in wax content ofHDSE was noted. Our studies showed an increase inPL, GL and ST using ABA but a decrease using PEGin HDSE in the culture medium (Table 7). However,if the data is expressed on 100 g dry weight of HDSE,the content of these membrane lipids would increasesignificantly using both ABA and PEG because theoil content has increased (Figure 2). Therefore, anincrease in membrane lipid, i.e., PL, GL and ST in

HDSE using ABA and PEG suggested developmentalchanges in the cell for providing the machinery forTAG biosynthesis (Goldberg et al., 1981).

The comparison of fatty acid composition ofHDSE (Table 8) and the zygotic embryos (Munshi andKumari, 1994) revealed that the proportion of 16.0 in-creased and that of 18:1, 18:2, 18:3 and 22.1 decreasedin HDSE (Table 3) suggesting lower rates of fatty acidelongation and desaturation in somatic embryos thanzygotic embryos, as also shown in case of microspore-derived embryos inB. napus(Pomeroy et al., 1991).During the period from 14 to 28 DAC, a decrease in theproportion of 16.0 and 18.0 with an increase in 18:1and 24:0 (Table 8) suggested that elongation (from16:0 to 24:0) and desaturation (from 18:0 to 18:1) offatty acids were stimulated. An increase in elongase

119

Table 8. Fatty acid composition of HDSE ofB. junceainfluenced by 10% PEGsupplementation in the culture medium

Treatments g (100 g fatty acids)−1

16:0 18:0 18:1 18:2 18:3 22:0 22:1 24:0

14 DAC

Control 44.5 10.6 11.4 2.0 5.5 – 21.2 1.9

PEG 10% 36.8 7.8 16.9 4.6 7.8 7.1 14.3 –

28 DAC

Control 24.3 1.3 17.1 2.2 6.0 – 22.3 17.9

PEG 10% 24.1 5.2 19.2 3.7 6.7 – 15.6 20.4

Critical difference (p<0.05)

Treatment (T) 2.1 1.1 1.0 0.7 0.6 ND 2.0 0.5

DAC (D) 1.7 0.9 0.8 0.5 0.4 ND 1.0 0.2

T x D 2.3 1.3 1.2 0.9 0.7 ND 2.1 0.6

– : Not detected; ND : Not determined.

activity during desiccation phase has been reported inwhite spruce somatic embryos (Attree et al., 1991).However, it may also be noted that PEG decreasedthe proportion of 22:1 at both 14 and 28 DAC (Table8) as compared to control indicating reduced rate ofelongation of 18:1 to 22:1.

The data indicate that hypocotyl explants of In-dian mustard (Brassica juncea) can be a useful systemfor raising somatic embryos which could be used forstudies of storage lipid accumulation. The HDSE werecapable of accumulating storage lipids nearly as muchas zygotic (seed) embryos when the growth mediumwas supplemented with 10% PEG. There have beenenhanced utilization of soluble sugars and the concom-mitant changes in the requisite enzyme activities tosupport lipid biosynthesis. However, there is a scopefor enhancing TAG in storage lipid accumulated inHDSE comparable to zygotic embryos. Lipid com-position of HDSE can also be altered by manipulatingthe culture conditions. It is, therefore, imperative tostudy the qualitative and quantitative range of stor-age lipid accumulation on a laboratory scale beforeany bioreactor based bulk production system can beenvisaged.

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