Indian Journal of BiotechnologyVol 2, January 2003, pp 38-47

Mutants for Biochemical and Molecular Insights into Embryogenesis in Plants**

Amita Bhattacharya, Preeti Sharma and Paramvir Singh Ahuja*Institute of Himalayan Bioresource Technology, Palampur 176061, India

Despite the advancements that have been made in understanding the intricate mechanisms of embryogenesis, alot remains elusive. However, the understanding of the mutants of different stages of embryo development viz. tsIl,gnom, mickey, lee etc. have opened up new vistas towards gaining insights into their biochemical and molecular basisof embryogenesis. Today, studies on mutants have not only played a major role in the easier manipulation of PGRsill vitro but have also led to the important discoveries of genes that are involved in the process of embryo patternformation and in elucidating the underlying genetic, molecular and physiological mechanisms of inducing polarity,cotyledon initiation and pro-vascular tissue differentiation. The present review deals with the different mutants andthe role they have played in the understanding of molecular and biochemical insights of somatic embryogenesis.

Keywords: mutants, somatic embryos, PGRs, biochemical, molecular, morphogenesis, maturation and germination

IntroductionThe unique phenomenon of producing embryos

from any vegetative cell and its ability to eventuallydevelop into a complete plant has traversed a longpath since its discovery by Reinert (1958) and Stew-ard et at (1958). As a result of this, efficient somaticembryogenesis systems have been developed in awide variety of plants (Zimmerman, 1993). Somaticembryogenesis has also contributed significantly to-wards the understanding of zygotic embryogenesis.

Embryogenesis represents the critical stage in thedevelopment of a plant at which its basic organizationand body plan have their inception and extensivework has been done on the morphological, biochemi-cal and molecular aspects (Kaplan & Cooke, 1997).However, one of the most basic question in develop-mental biology with respect to how does an undiffer-entiated mass of cells become programmed to behaveas an embryo and more importantly how the undiffer-entiated mass of cells that comprises the early embryotakes on a pattern in which different cell layers and/orregions adopt particular developmental fates, still re-mains elusive (Chasan, 1993). Despite the fact that alot needs to be understood regarding the above ques-tions, mutants have played a significant role (Kaplan& Cooke, 1997; Gallois, 2001).

Identification of mutants at different stages of de-velopment of somatic and zygotic embryos has

*Author for correspondence:TeI:01894-230411; Fax: 01894-230433E-mail: psahuja@ihbt.csir.res.in**IHBT Publication Number: 2216

opened new vistas towards gaining insights into theirbiochemical and molecular basis of embryogenesis(Krautwig & Lorz, 1996; Mordhorst et at, 1998). Thein vitro technique of 'embryo culture' has shown thatisolated zygotic embryos have the ability to completetheir development in vitro. This enabled the identifi-cation of several forms of mutants of plant growthhormones that are important for the normal develop-ment of embryos (Reinbothe et al, 1992; Liu et at,1993). These studies not only helped in easier ma-nipulation of plant growth regulators, in vitro, but alsoled to important discoveries of genes that are involvedin the process of embryogenesis and also their expres-sion (Sheridan, 1995; Heberle et al, 1997; Shen et at,2001; Magioli et al, 2001). The 'embryo culture tech-nique' when utilized for studies on mutants of themodel system 'carrot' revealed the involvement ofseveral secreted proteins during embryo development(Cordewener et at, 1991; De Jong et al, 1992). Mu-tants have also helped in dispelling the earlier beliefof maternal or post-fertilization events being the ab-solute requirement for specifying pattern and for in-ducing polarity in the plant embryos.

A conceptual model of basic pattern formation inplants was presented for the first time by Jurgens et at(1991). However, this was largely derived from thestudies on Drososphila developmental genetics. Thevery same year, the mutants of embryo developmentwere discovered in Arabidopsis thaliana. Mayer et at(1991) and Meinke (1991a; 1991b) demonstrated em-bryo pattern formation in plants to be geneticallycontrolled. Mutants with disturbed pattern formation

BHA IT ACHARY A et al: BIOCHEMICAL AND MOLECULAR INSIGHTS INTO EMBRYOGENESIS IN PLANTS 39

contributed much to the knowledge of 'control of re-gional specificity in plant embryos'. The understand-ing of the underlying molecular mechanisms of thepolarity inducing processes gained considerably fromthese phenotypic mutants.

The present article reviews the role of mutants inthe understanding of the process of embryo patternformation and also in elucidating the underlying ge-netic, molecular and physiological mechanisms ofinducing polarity, cotyledon initiation and pro-vascular tissue differentiation.

The Process of EmbryogenesisIn plants, the process of zygotic embryogenesis

commences after fertilization wherein, the singlecelled zygote undergoes gradual morphological dif-ferentiation in order to give rise to successive devel-opmental stages. The striking similarity of thesestages in most angiosperm and gymnosperm plantspecies suggests the existence of a common gene ex-pression programme for pattern formation and cellspecialization (Jurgens et al, 1991). The process ofdevelopment in somatic embryos closely resemblesthat of zygotic embryos, both morphologically as wellas temporally. In dicots, the stages of developmentinclude, the first recognizable globular stage and theoblong stage followed by the heart stage and the tor-pedo stage (Zimmerman, 1993). While the shift fromthe globular stage to the oblong stage signals the shiftfrom the isodiametric to bilaterally symmetricalgrowth and beginning of the heart stage, the globularto heart transition is clearly marked by the outgrowthof the two cotyledons, the elongation of the hypocotyland the beginning of radicle development. This tran-sition continues through the torpedo and the plantletstages (Schiavone & Cooke, 1987). Despite beingsimilar, an important difference between the somaticand zygotic embryogenesis includes the ability of thesomatic embryos to develop outside the physical con-straints and the informational context of maternal tis-sue (Zimmerman, 1993). The other important differ-ence includes the absence of endosperm and/or sus-pensor differentiation in somatic embryogenesis.

The question of whether it is the differential abilityof somatic cells or the presence of specific responsivecell type that is responsible for somatic embryogene-sis is still not defined (De long et al, 1993). The iden-tification of the original cells actually involved in thedevelopmental processes of somatic embryogenesis isdifficult and can proceed either from an asymmetrical

cell cluster, a symmetrical cell or an aberrantly shapedcell cluster. The first embryonic developmental stageor initiation of morphogenesis is intimately linked tothe development of polarity and subsequent asymmet-ric division leading to cell differentiation. Although,the first asymmetric cell division in case of zygoticembryogenesis is initiated by a gradient of lightwherein, the plane of division is generally perpen-dicular to the light axis (Dodeman et al, 1997), yet,other stresses are also known to be important.

Conceptually, the entire process of embryogenesisin higher plants can be summarized into three over-lapping phases during morphogenesis (i) a polar axisof the plant body is defined with specification of theshoot and root apices wherein the embryonic tissueand organ systems are formed, (ii) the second phaseof embryo maturation is characterized by accumula-tion of storage reserves and acquisition of desiccationtolerance and (iii) the final phase of desiccation anddevelopmental arrest (West & Harada, 1993).

Role of Phenotypic Mutants in Understanding theMorphological and Physiological basis of Morpho-genesis

In Arabidopsis thaliana studies on six mutants ofapical-basal pattern for mutation viz. gnom, gurke,Jackel monopteros, rootless and shoot meristemless,the three mutants of radial defects i.e. keule, knolleand raspberry and the three mutants with alteredshape i.e.fass, knopf, and mickey) have confirmed thefact that polarity of the female gamete or zygote isgenetically pre-determined (Weigel, 1993; Meinke etal, 1994; Yadegiri et al, 1994). The gnom mutants notonly fail to elongate but also produce an enlarged api-cal cell. This is due to the inclination of the plane ofdivision at an abnormal degree as a result of whichphenotypic mutants like the ball-shaped seedlingswithout root and cotyledons are produced. CI10111

mutants led to the discovery of the fact that the CI10111

gene is responsible for the early events in embryomorphogenesis (Mayer et al, 1991) and also to theconclusion that the alignment of the microtubules is ina fashion perpendicular to the axis prior to asymmet-ric division.

Cell polarity followed by asymmetric divisions areimportant for the initiation of somatic embryogenesis.In general, auxins promote asymmetric divisions inthe explant tissue to give rise to small daughter cellsfrom which the somatic embryos arise. These cellseither single (Backs-Husemann & Reinert, 1970; No-

40 INDIAN J BIOTECHNOL, JANUARY 2003

mura & Komamine, 1985) or in tight clusters aredesignated as the pro-embryonic masses or the PEMs(Halperin, 1966). PEMs generally develop on nutrientmedia containing high concentrations of auxins(Schiavone & Cooke, 1987; Michalczuk et al, 1992a,b). These PEMs are cells or cell clusters with densecytoplasm and are enriched with proteins, mRNAs,etc. They are replete with gene products necessary tocomplete the globular stage of embryogenesis (Zim-merman, 1993). The PEMs also contain many othermRN As and proteins whose continued presence gen-erally inhibits the elaboration of the embryogenic pro-cess. The removal of auxins results in inactivation ofthese genes so as to allow the embryogenic pro-gramme to proceed smoothly. However, differentfactors like the presence of other PGRs, pH shift,electrical fields (Smith & Krikorian, 1990), tempera-ture, other environmental or cellular signals (Hause etal, 1993) or nutrients in the medium may also play animportant role. Till date it has been possible only tocompare the somatic and the zygotic embryos fromthe globular stage onwards. Heterogeneity or vari-ability have been reported in the initial patterns ofboth somatic and zygotic embryogenesis (Toonen etal, 1994). However, it is still not clear whether it isthe specific responsive cell type or the differentialability of the somatic cells that become embryogenicare actually responsible for the initial steps of somaticembryogenesis (De Jong et al, 1993).

Role of Mutants in Understanding Pattern Forma-tion during Embryogenesis

The importance of heterogenous partitioning forcontrolled cell expansion and asymmetric division inembryogenesis has been indicated (De Jong et al,1993). In other words, co-ordinately regulated celldivisions are important for generating cells that willexpand, take on a pattern and finally form the plantbody. This was confirmed by the studies involving thedominant negative cdc2aAt mutant of Arabidopsisthaliana, which showed that these co-ordinated celldivisions are brought about by plant cyclin dependentkinases (CDKs) and cyclins are modulated by a vari-ety of internal and external signals (Hemerly et al,2000). Since cell division events are essential for em-bryo patterning and morphogenesis (Mansfield & Bri-arty, 1991), it is not surprising that the cdc2aAt mu-tant of Arabidopsis shows a high range of distortionson the apical-basal pattern, defective leaves and incor-rect phyllotactic pattern.

However, polarized cell expansion and divisionplane alignment are not required for spatial develop-ment (Tras et al, 1995). This was proven by the gurkeand fackel mutants with disturbed apical and basalpatterns. Generally, the first asymmetric division inzygotic embryos is followed by three crucial stagesviz. (i) the octant stage composed of two to four cells,(ii) the protoderm formation and (iii) initiation ofprimordia. The small apical cell produced after thefirst asymmetric division neither enlarges during theabove three steps nor during the subsequent periclinaldivisions when the protoderm is formed. This wasproved by the emblOl-l mutant of Arabidopsis tha-liana. While the protoderm formation prevents cellexpansion and is essential for the remaining develop-mental phases, the emblOl-l mutants of Arabidopsisshow uncontrolled cell expansion leading to the pro-duction of enlarged cells which fill the whole seed.Embryo specific mutants of Zea mays i. e. emb *-85J 8and emb*-8521 fail to form protoderm and hencecannot acquire bilateral symmetry as compared to theemb*-853 and emb*-8542 embryos that exhibit thewild type characteristics. Similarly the knolle mutantsproved that specification of radial axis is required fora normal apical and basal pattern and the Knolle geneis involved in vesicular trafficking or in membranefusion and hence in cytokinesis (Lukowitz et at,1996). About 40 genes are reported to control theformation of embryo axis pattern elements (Mayer etai,1991).

Role of Mutants in Understanding the Formation ofMeristem or Initiation of Primordia

Orientation of dividing cells and their subsequentcell morphogenesis and differentiation are tightlycontrolled (Berleth, 1998). While patterning in theembryos is not important for tissue differentiation,positional control is of considerable importance. Thiswas proven true by the raspberry mutants. Rather de-velopment of root meristem or somatic tissues or inother words, determination of cell fate requirespositional control. In this regard, the monopteros orthe gnom mutants have indicated that while themonopteros gene is responsible for the organization ofthe basal region, the gnom is epistatic to the monop-teros (Berleth & Jurgens, 1993). These studies havenow made it possible to conclude that the root devel-opment is totally dependant on organized andpositional segmentation and cell-cell interaction.

Shoot apical meristems are a group of indetermi-nate stem cells that are organized during embryogene-

BHA ITACHARY A et al: BIOCHEMICAL AND MOLECULAR INSIGHTS INTO EMBRYOGENESIS IN PLANTS 41

sis. Each of the mutants pt and clv (clavata) of Arabi-dopsis thaliana embryos indicated that their defectsare first detected at early heart stage and broad shootapical meristems (SAM) are produced. Double mu-tants i.e. pt clv showed additive effects with enlargedSAMs which later produced short fascinated inflores-cence stems and also had a additive effect on thenumber of rosette leaves. These mutants indicated thatthe CL V and PT genes act in an independent manneron pathways that control SAMs (Mordhorst et al,1998). A recessive mutant of maize, abphyll, indi-cated that defects in them first appeared during em-bryogenesis and played a vital role in regulating mor-phogenesis and in determining the future phyllotaxyof shoots. The embryo mutants of maize, emb*-853and emb*-8542 are important in overcoming the mu-tants defective in development of functional shootapical meristems and leaf primordia.

Role of Mutants in Understanding the HormonalRegulation of Embryogenesis

Mutants have shown that development entails a se-ries of interacting processes where the alteration ofone factor triggers successive abnormal events (Goe-

·bel-Tourand et al, 1993). Generally, these interactingprocesses are governed by plant growth regulatorswhich act as signaling molecules during embryo-genensis. Mutants with a disturbed balance of auxinsand cytokinins have perturbed spatial patterns likemultiple or fused cotyledons, shoot-meristemless em-bryos, etc. While the shoot-meristemless mutants ei-ther completely lack or have only partially organizedapical meristems that do not interfere with the devel-opment of other parts of the embryo (Barton & Po-ethig, 1993; Long et al, 1996), the pin1 mutants ofArabidopsis thaliana have fused cotyledons due to theinhibition of polar transport of auxins (Okada et al,1991; Liu et al, 1993; Cooke et al, 1993). Liu and co-workers used excised zygotic embryos of Brassicajuncea to show that polar transport of auxin is essen-tial for the establishment of bilateral symmetry duringembryogenesis. Using a wide array of inhibitors ofpolar transport of auxins on globular embryos theyshowed that cotyledon formation became decoupledfrom the development of bilateral symmetry whereininstead of two cotyledons forming on opposite sidesof the embryo, a ring of cotyledon tissue emerged toencircle the embryo. In Arabidopsis, embryos homo-zygous for the mutant pin1 also develop a collar likeor fused cotyledon. This was further elucidated by the

use of an auxin antagonist like 2-p-clorophenoxy iso-butyric acid or CIPB that does not have any effect onpolar auxin transport. Based on the fact that the use ofCIPB did not produce any aberrant structures, Liu etal (1993) and Cooke et al (1993) postulated that theauxin polar transport was an absolute requirement forthe establishment of bilateral symmetry in globularembryos. The activity of polar transport of auxin de-termines where the two cotyledons will be formed atthe side towards which the embryo accumulatesmaximum auxin becomes the future site of cotyledonformation. The cells at these sites not only amplifiedtheir own levels of polar auxin transport but also in-hibited the polar transport of auxin to all the neigh-bouring cells except the most distant one (Chasan,1993).

The mickey mutants of Arabidopsis (Mayer et aI,1991) indicated that an optimal balance of hormoneswas required for normal vasculature and subsequentmeristem development but any disturbance in thisbalance resulted in abnormalities as is evident in theirphenotypic mutants. The shoot-meristemless mutantsdisplayed reduced meristems, abnormal fuzzy vascu-lar strands and/or hypertrophic cell distortion (Goe-bel-Tourand et al, 1993). A similar response was ob-served in species like soybean (Barwale et al, 1986),alfalfa (Dos Santos et al, 1983) and Vitis longii (Gray& Mortensen, 1987).

Role of Mutants in understanding the UnderlyingBiochemical Basis of Embryogenesis

Mutants have shown that the process of embryo-genesis is characterized by well marked stages, ofwhich, the transition from the globular to heart shapeis the most crucial. Recent studies with mutants haveindicated that certain cell wall proteins either promoteor inhibit the process of somatic embryogenesis (Cor-dewener et al, 1991; De long et al, 1992; Kreuger &Van Holst, 1993; De long et aZ, 1993). Giuliano et at(1984) produced a chemical mutant that was sensitiveto temperature. This temperature sensitive ts11 mutantshowed a transitional arrest from the globular to theheart stage. Aberrant protoderm formation in thesemutants was actually responsible for this develop-mental arrest at non-permissible temperatures. How-ever, the addition of a mixture of proteins secreted bythe embryogenic wild type cell lines of carrot wascapable of overcoming this developmental arrest (LoSchiavo et al, 1990). These proteins were later foundto be glycosylated extra-cellular proteins that were

42 INDIAN J BIOTECHNOL, JANUARY 2003

involved in different stages of development. Whilethe extra-cellular protein 3 (EP3), an acidic endo-chitinase of 32 kDa was responsible for the rescue ofts11 embryos, (De long et al, 1992), the EPI proteinwas found to be secreted only by the non-embryogenic cells (Van Engelen et al, 1991). In thesame year, another extra-cellular protein (EP2) wasidentified to be involved in lipid transfer and wassynthesized only by embryogenic cells and somaticembryos (Sterk et al, 1991). De long et al, (1992)showed that the EP3 proteins could be substituted bythe N-acetylglucosamine containing lipooligosaccha-rides or nodulation factors or Nodklv-VrAc CI8:4) ofRhizobium leguminosarum bacterial variety vieiae.

Maturation and Germination

The stages of somatic and zygotic embryo devel-opment are similar in all aspects, except for the factthat the somatic embryos do not become dormant.Genetic studies based on mutants have revealed theinfluence of late embryogenesis specific genes in-volved in maturation. The leafy cotyledon or lee mu-tants of Arabidopsis cause defects in the differentia-tion process of cotyledons and in the maturation spe-cific events such as storage reserve accumulation,desiccation tolerance and maintenance of quiescence(Meinke et al, 1994). While the wild type LECl genefunctions in both regions of the embryo axis and lackstorage organelles, the Lee mutations result in thetransition of cotyledons of embryos and seedlings intoleaf like structures, characterized by trichomes, sto-mata and mesophyll cell differentiation and absenceof protein and lipid storage bodies. Therefore, the leemutant embryos germinate precociously, implyingthat embryonic and post-germinative programmesoccur simultaneously.

The fusea mutants of Arabidopsis, which accumu-late anthocyanins in their cotyledons during late em-bryogenesis and fail to germinate, showed the genesrequired for post-embryonic development are activein late embryogenesis (Goldberg et al, 1994). Gener-ally, the fusea mutants show normal embryogenesiswith exception to jus3 mutants which exhibit leafylike phenotype. The lethal nature also indicated thatFUSCA genes are essential for critical developmentalprocesses wherein, the accumulation of anthocyaninwas a secondary effect (Castle & Meinke, 1994).

_Some regulatory genes that prepare the embryos forsubsequent germination.

Molecular Insights

Sung and his group (1984) and Goldberg et al(1989) considered somatic embryogenesis to be anexcellent model system for studying gene regulationduring embryogenesis. Fujimura & Komamine,(1980) indicated that active RNA synthesis and sub-stantial changes in gene expression at transcriptionallevels dictate the embryo development process. Sincethen several laboratories have been searching forgenes that were regulated during somatic embryo-genesis (Zimmerman et al, 1992). Based on theirwork on protein gel analysis Sung and Okimoto(1983) showed that the embryogenic process may notinvolve the mRNAs. Therefore, the search for 'em-bryo enhanced genes' began. Approaches like cDNAlibraries and screening of the libraries by raising anti-sera against embryo proteins or radiolabelling led tosignificant successes ( Choi et al, 1987: Sterk et al,1991; Ulrich et al, 1990; Aleith & Richter, 1990:Thomas, 1993).

A number of 'embryo enhanced genes' have beenisolated whose functions are still unknown. However,some genes like DC8, DC3, EMB-1 are now knownto belong to the family of proteins called the "LateEmbryogenesis Abundant" proteins (Galau et aI,1986; Dure et al, 1989). These genes are generallyABA inducible and are thought to contribute to desic-cation tolerance. A second group of embryo abundantgenes have been isolated for the EP2 proteins (Sterket at, 1991) The EP2 gene is expressed in the proto-derm cells of both somatic and zygotic embryos and istransiently expressed in epidermal cells of leaf pri-mordia and is considered to be a marker for proto-derm development in early embryos.

Apuya & Zimmerman, (1992) isolated two othergenes, the ATP-2 gene and the translation elongationfactor EF-la and found that they were specificallyexpressed in globular embryos of carrot. Also thestudies on mutants of Arabidopsis like extracotyledon2 or xtc2 accumulation of MADS protein domainAGAMOUS 15 in the embryonic tissues of dandelion,Arabidopsis, oil seed rape and alfalfa indicate thatthese genes were active during early embryogenesis(Perry et al, 1999).

Genes responsible for embryo development like theembryo specific PEll and those responsible for thetransition from globular to heart stage (Li & Thomas,1998), the meristem formation i. e. the knl for shootmeristem and ZnHox for shoot and root meristems

BHA ITACHARY A et al: BIOCHEMICAL AND MOLECULAR INSIGHTS INTO EMBRYOGENESIS IN PLANTS 43

have been isolated from mutants like the t1 of Arabi-dopsis (Sheridan, 1995).

genesis in the plant species is listed in Table l. Al-though, the major focus has been on the production oflarge number of plants for either biotechnological im-provement or for mass propagation of importantplants using somatic embryogenesis, no work has yetbeen done on the mutants of somatic embryogenesis.

The Indian ScenarioIn India, considerable work has been done in the

recent past on different aspects of somatic embryo-

Plant species

Tablel-Work done by Indian workers on somatic embryogenesis in the different plant species in the last ten years

Explants used Mutant if any References

TreesSantalum indicum

Acacia nitoticaTermalia arjunaPinus roxburgiiGloriosa superbaHardwickia banata

Oil seedsBrassica junceaSunflowerBrassica junceaBrassica nigraArchis hypogeasafflower

Medicinal plantsAsparagus cooperiPodophyllum hexandrum

Hyoscyamus muticusAconitum heterophyllumSolanum sarrachoidesPsoralea corylifolia

CerealsMilletTriticum aestivum

Oryza sativaO. sativa

PulsesChick peaChick peaPigeon peaChick peaVigna radiata

Commercial cropsBambooBambooHevia brasiliensisAnacardium occidentale

Camellia sinensisC. sinensisC. sinensis

Hypocotyls Bapat & Rao, 1984

EndospermLeavesImmaure megagametophytesShoot apicesSemi mature zygotic embryos

Garg et al, 1996Kumari et al, 1998Mathur et al, 2000Jadhav & Hegde, 2001Chand & Singh, 2001

Mesophyll ProtoplastsImmature zygotic embryosProtoplast derived hypocotylsHypocotylsImmature leafletsCotyledons

Kirti & Chopra, 1987Sujatha & Prabakaran. 200 IKirti & Chopra, 1990Narasimhulu et al, 1992Venkatachalam, et al, 1999Mandai et al, 2001

Spear callusZygotic embryos

Ghosh & Sen, 199 IArumugam & Bhojwani,1990Giri & Ahuja, 1990Giri et ai, 1993.Banerjee et ai, 1994.Rout & Das. 2001

protoplastsLeaves and petiolesLeavesLeaves and stem

ProtoplastsScutellar callus of immature zygoticembryoscallusMature embryos

Nayak & Sen, 1991Nambisan & Chopra, 1992

Basu et al, 1997Shankdher et al, 2001

Cotyledons and immature embryo axesEmbryo axesCotyledon and leaf explantsLeavesImmature cotyledons

Sagare et al, 1993Suhasini et al, 1994Sreenivasu et al, 1997Kumar et al, 1994Girija et ai, 2000

SeedsNodal segmentsImmature inflorescenceRadicle end of immature zygotic em-bryosCotyledonsCotyledonsCotyledons

Nadgauda et al, 1993Godbole, 2002Kumari et ai, 2000Cardoza et al, 2000

Jha et al, 1992Mondal et al, 2000Mondal et al, 2001

(Contd)

44 INDIAN J BIOTECHNOL, JANUARY 2003

Tablel-Work done by Indian workers on somatic embryogenesis in the different plant species in the last ten years-Contd

Plant species Explants used Mutant if any References

Camellia sinensis Cotyledons Mondal et al, 2002Coffea arabica Cultured roots and hypocotyl segments Santa et a}, 1999Cotton Hypocotyl Kumar & Pental, 1998

Fruits

Mangifera indica Protoplasts Ara et al, 2000M. indica Nucellus Ara et al, 2000

Vegetables

Egg plant Cotyledons, leaves and hypocotyls Sharma et al, 1995Tomato Leaves and shoots Chandel & Katiyar, 2000

ConclusionWhile most of the genes that control polarity es-

tablishment, tissue differentiation and elaboration ofpatterns during embryo development have been iden-tified and characterized, the regulatory mechanismswhich co-ordinate the asymmetrical division and sub-sequent determination is still unknown. The only ex-ception is the regulatory gene, shoot-meristemless.

However, investigations on the developmental ba-sis of a number of mutant phenotypes have enabledthe identification of gene activities promoting asym-metric cell division and polarization leading to heter-ogenous partitioning of the cytoplasmic determinantsnecessary for the initiation of embryogenesis e.g.gnom.

It can thus be concluded that, mutants have nowenabled one to predict not only the direct or indirectinduction potential of somatic embryogenesis in dif-ferent plants and explants (Ogas et al, 1997; Dupire etat, 1999; Limanton et al, 2000) but also on whetherthere would be a potential for recurrent embryogene-sis (Recklinghausen et al, 2000). These studies areindeed valuable in manipulating cells and tissues ofnon-embryogenic nature to embryogenic types. Iden-tification of a super-embryogenic mutant, Jemalong2HA is specially valuable for providing insights intothe nature of totipotency in plants (Rose et al, 1999).

ReferencesAleith F & Richter G, 1990. Gene expression during induction of

somatic embryogenesis in carrot cell suspensions. Planta,183, 17-24.

Apuya & Zimmerman J L, 1992. Heat shock gene expression iscontrolled primarily at the translational level in carrot cellsand somatic embryos. Plant Cell, 4, 657-665.

Ara H, Jaiswal U & Jaiswal V S, 2000. Plant regeneration fromprotoplasts of mango (Mangifera indica L.) through somaticembryogenesis. Plant Cell Rep, 19,622-627.

Ara H, Jaiswal U & Jaiswal V S, 2000. Somatic embryogenesisand plantlet regeneration in Amrapali and Chausa cultivars ofmango (Mangifera indica L.). Curr Sci, 78, 164-169.

Arumugam N & Bhojwani S S, 1990. Somatic embryogenesis intissue cultures of Podophyllum hexandrum. Can J Bot, 68,487-491.

Backs-Husemann D & Reinert J, 1970. Embryobildung dirchisolierte Einzelzellen aus Gewenbekulturen von D. carota.Protoplasma, 70. 36-90.

Banerjee S, Ahuja P S & Pal A, 1994. Somatic embryogenesisfrom callus cultures of Solanum sarrachoides Sendt. J PlantPhysiol, 143. 750-752.

Bapat V A & Rao P S, 1984. Regulatory factors for in vitro multi-plication of sandalwood tree (Santalum album Linn.). I.Shoot bud regeneration and somatic embryogenesis in hypo-cotyl cultures. Plant Sci, 93, 19-27.

Barton M K & Poethig R S, 1993. Formation of the shoot apicalmeristem in Arabidopsis thaliana: An analysis of develop-ment in the wild type and in the shoot-meristemless mutant.Development, 119, 823-831.

Barwale U B, Kerns H R & Wildholm J M, 1986. Plant regenera-tion from callus cultures of several soybean genotypes viaembryogenesis and organogenesis. Planta, 164,473-481.

Basu S, Gangopadhyay G, Mukherjee B B & Gupta S, 1997. Plantregeneration of salt adapted callus of indica rice (var, Bas-mati 370) in saline conditions. Plant Cell Tissue Organ Cult.50, 153-159.

Berleth T & Jurgens G, 1993. The role of monopterosgene inorganizing the basal body region of the Arabidopsis embryo.Development, 118,575-587.

Berleth T, 1998. Experimental approaches to Arabidopsis em-bryogenesis. Plant Physiol Biochem, 36, 69-82.

Cardoza Vinitha, D'Souza L & Cardoza V, 2000. Direct somaticembryogenesis from immature zygotic embryos in cashewtAnacardium occidentale L.). Phytomorphology, 50, 201-204

Castle L A & Meinke D W, 1994. A FUSCA gene of Arabidop.1isencodes a novel protein essential for plant development.Plant Cell, 6, 25-41.

Chand S & Singh A J, 200 I.' Direct somatic embryogenesis fromzygotic embryos of a timber-yielding leguminous tree.Hardwickia binata Roxb. CurrSci, 80, 882-887.

Chandel G & Katiyar S K, 2000. Organogenesis and somatic em-bryogenesis in tomato (Lycopersicon esculantum Mill.), AdvPlant Sci, 13, 11-17.

BHA TT ACHARY A et al: BIOCHEMICAL AND MOLECULAR INSIGHTS INTO EMBRYOGENESIS IN PLANTS 45

Chasan R, 1993. Embryogenesis: New molecular insights. PlantCell, 5, 597-599.

Choi 1 H, Liu L S, Borkird C & Sung Z R, 1987. Cloning of genesdevelopmentally regulated during plant embryogenesis. ProcNatl Acad Sci USA, 82, 4399-4403.

Cooke T J, Racusen R H & Cohen J D, 1993. The role of auxin inplant embryogenesis. Plant Cell, 5, 1494-1495.

Cordewener 1 H G, Booij H, Van der Zandt H, Van Engelen F,van Kammen A & de Vries S C, 1991. Tunicamycin -inhibited carrot somatic embryogenesis can be restored bysecreted cationic peroxidase isoenzymes. Planta, 184, 478-486.

De long A J, Cordewener J, Lo Schiavo F, Terzi M,Vandekerckhove J, van Kammen A & de Vries S C, 1992. Acarrot somatic embryo mutant is rescued by chitinase. PlantCell, 4, 425-433.

De long A l, Schimdt E D L & de Vries S C, 1993. Early eventsin higher plant embryogenesis. Plant Mol Bioi, 22, 367-377.

Dodeman V L, Ducreux G & Kreis M, 1997. Zygotic embryo-genesis versus somatic embryogenesis. J Exp Bot, 48, 1493-1509.

Dos Santos A V P, Cutter E G & Davey M R, J983. Origin anddevelopment of somatic embryos in Medicago sativa L. (al-falfa). Protoplasma, 117, 107-115.

Dupire L, Decout E, Vasseur J & Delbreil B, 1999. Histologicaland 2-D protein pattern comparisons between a wild type anda somatic embryogenic mutant of Asparagus officinalis L.Plant Sci Limmerick, 147,9-17.

Dure III L, Crouch M, Harada J, Ho T H D, Mundy J, Quatrano RS, Thomas T & Sung T, 1989. Common amino acid sequencedomains among the LEA proteins of higher plants. Plant MolBioi, 12,475-486.

Fujimura T & Komamine A 1980. The serial observation of em-bryogenesis in a carrot cell suspension culture. New Phytol,86: 213-218.

Galau G A, Jacobsen K S & Hughes D W, 1986. Abscisic acidinduction of cloned cotton late embryogenesis abundant(Lea) mRNAs. Plant Mol Bioi, 7, 155-170.

Gallois P, 2001. Future of early embryogenesis studies in Arabi-dopsis thaliana. C. R. Academjic Science. 324, 569-573.

Garg L, Bhandari N N, Rani V & Bhojwani S S, 1996.Somaticembryogenesis and regeneration of triploid plants in en-dosperm cultures of Acacia nilotica. Plant Cell Rep, 15, 855-858.

Ghosh B & Sen S, 1991. Plant regeneration through somatic em-bryogenesis from spear callus culture of Asparagus cooperiBaker. Plant Cell Rep, 9, 667-670.

Giri A, Ahuja P S & Kumar P V A, 1993. Son-atic embryogenesisand plant generation from callus cultures of Aconitum het-eropltyllum Wall. Plant Cell Tissue Organ Cult, 32, 213-218

Giri C C & Ahuja P S, 1990. Direct somatic embryogenesis fromcultured protoplasts of Hyoscyamus muticus L. Indian J ExpBioi, 28, 249-252.

Girija S, Ganapathi A & Ananthakrishnan G, 2000. Somatic em-bryogenesis in Vigna radiata (L.) Wilczek. Indian J ExpBioi, 38, 1241-1244.

Giuliano G, Lo Schiavo F & Terzi M, 1984. Isolation and devel-opmental characterization of temperature sensiti ve carrot cellvariants. Theor Appl Genet, 67, 179-183.

Godbole S, Thakur R, Sood A, Sharma M & Ahuja P S, 2002.Somatic embryogenesis and regeneration of plantlets inbamboo (Dendrocalamus hamilioniii. Curr Sci, 83, 885-889.

Goebel-Tourand I, Mauro M C, Sossountzov L & Miginiac D A.1993. Arrest of somatic embryo development grapevine: Ahistological characterization and the effect of ABA, BAP andzeatin in stimulating plantlet development. Plant Cell TissueOrgan Cult, 33, 91-103.

Goldberg R B, Barker S J & Perez-Grau L, 1989. Regulation ofgene expression during plant embryogenesis. Cell,56, 149.

Goldberg R B, de Paiva G & Yadegiri R, 1994. Plant embryo-genesis; zygote to seed. Science, 266, 605-614.

Gray D J & Mortensen J A, 1987. Initiation and maintenance oflong term somatic embryogenesis from anthers and ovariesof Vitis longii 'Microsperma'. Plant Cell Tissue Organ Cult.9,73-80.

Halperin W, 1966. Alternative morphogenetic events in cell sus-pensions. Am J Bot, 53, 443-453.

Hause B, Hause G, Pechan P & Van Lammeren A A M. 1993.Cytoskeletal changes and induction of embryogenesis in mi-crospore and pollen cultures of Brassica napus L. Cell Bioilnt Rep, 17, 153-168.

Heberle B E & de Vries S C, 1997. Plant Embryonic Develop-ment. Biofutur, 172, 38-42.

Hemerly A S, Ferreira P C G, van Montagu M, Engler G & InzeD, 2000. Plant J, 23,123-130.

Jadhav S Y & Hegde B A, 2001. Somatic embryogenesis andplant regeneration in Gloriosa L. Indian" J Exp Bioi, 39, 943-946.

lha T B, Sumita Jha & Sen S K, 1992. Somatic embryogenesisfrom immature cotyledons of an elite Darjeeling tea clone.Plant Sci Limerick, 84, 209-213.

Jurgens G, Mayer U, Torres-Ruiz R A, Berleth T & Misera S.1991. Genetic analysis of pattern formation in the Arabidop-sis embryo. Dev Genet, 1 (suppl.), 27-38.

Kaplan D R & Cooke T J, 1997. Fundamental concepts in theembryogenesis of dicotyledons: A morphological interpreta-tion of embryo mutants. Plant Cell 9: 1903-1919.

Kirti P B & Chopra V L 1990. Rapid plant regeneration throughorganogenesis and somatic embryogenesis from culturedprotoplasts of Brassica juncea. Plant Cell Tissue Organ Cult,20,65-67.

Kirti P B & Chopra V L, 1987. A simple method of generatingsomatic embryos from rnesophyll protoplasts of Brassicajuncea (L.) Czern & Coss. Cruciferae-Newslett, No 12. 71.

Krautwig B & Lorz H, 1996. From cell to cereal plant. In vitroembryogenesis and regeneration in the Poaceae. Naturwis-senschaften, 83, 414-423.

Kreuger M & Van Holst G J, 1993. Arabinogalactan proteins areessential in somatic embryogenesis of Daucus carota L.Planta, 189, 243-248.

Kumar V D, Kirti P B, Sachan J K S, Chopra V L. 1994. Plantregeneration via somatic embryogenesis in chickpea (Cicerarietinum L.). Plant Cell Rep. 13,468-472.

Kumar S & Pental D, 1998. Regeneration of Indian cotton varietyMCU-5 through somatic embryogenesis. Curr Sci, 74, 538-540.

Kumari N, Jaiswal U & Jaiswal V S, 1998. Induction of somaticembryogenesis and plant regeneration from leaf callus ofTerminalia arjuna Bedd. Curr Sci. 75, 1052-1055.

46 INDIAN J BIOTECHNOL, JANUARY 2003

Kumari S, Sobha S, Rekha K, Jayasree R & Asokan M P, 2000.Influence of growth regulators and sucrose on somatic em-bryogenesis and plant regeneration from immature inflores-cence of Hevea brasiliensis. Indian J Nat Rubber Res, 13,19-29.

Li Z & Thomas T L, 1998. PEIl, an embryo specific zinc fingerprotein gene required for heart stage embryo formation inArabidopsis. Plant Cell, 10, 383-398.

Limanton G A, Sotta B, Brown S & JuJlien M, 2000. Analysis ofhabituated embryogenic lines in Asparagus officinalis L.:Growth characteristics, hormone content and ploidy level ofcalli and regenerated plants. Plant Sci, 160, 15-26.

LiuC-m, Xu Z-h & Chua N H, 1993. Auxin polar transport isessential for the establishment of bilateral symmetry duringearly plant embryogenesis. Plant Cell, 5, 621-630.

Lo Schiavo F, Giuliano G, de Vries S C, Genga A, Bollini R, PittoL, Cozzani F, Nuti-Ronchi V & Terzi M, 1990. A carrot cellvariant temperature sensitive for somatic embryogenesis re-veals a defect in the glycosylation of extracellular proteins.Mol & Gen Genet, 223, 385-393.

Long J A, Moan E I, Medford J I& Barton M K, 1996. A memberof the KNOTTED class of homeodomain proteins encoded bythe STM gene of Arabidopsis. Nature (Lond), 379, 66-69.

Lukowitz W, Mayer U & Jurgens G, 1996. Cytokinesis in theArabidopsis embryo involves the syntaxin-related KNOLLEgene product. Cell, 84, 61-71.

Magioli C, Barroco M R, Rocha CAB, Dolores de Santigo-Fernandes L, Mansur E, Engler G, Pinheiro M & Martins G,2001. Somatic embryo formation in Arabidopsis and egg-plant is associated with expression of a glycine-rich proteingene(Atgrp-5). Plant Sci, 161, 559-567.

MandaI A K A, Gupta S D & Chatterji A K, 2001. Factors affect-ing somatic embryogenesis from cotyledonary explants ofsafflower. Bioi Plant, 44, 503-507.

Mansfield S G & Briarty L G, 1991. Embryogenesis in Arabidop-sis thaliana. II. The developing embryo. Can J Bot, 69, 461-476.

Mathur M, Arnold S von, Rajani Nadgauda, Mathur G, von-Arnold S & Nadgauda R, 2000.Studies on somatic embryo-genesis from immature zygotic embryos of chir pine (Pinusroxburghii Sarg.). Curr Sci, 79, 999-1004.

Mayer U, Torres-Ruiz R A, Berleth T, Misera S & Jurgens G,1991. Mutations affecting body organization in the Arabi-dopsis embryo. Naturei Land}, 353, 402-407.

Meinke D W, 1992. A homeotic mutant of Arabidopsis thalianawith leafy cotyledons. Science, 258, 1647-1650.

Meinke D W. 1994. Perspectives on genetic analysis of plant em-bryogenesis. Plant Cell, 3, 857-866.

Meinke D W, 1991a. Embryonic mutants of Arabidopsis thaliana.Dev Genet, 12, 382-392.

Meinke D W, 1991b. Perspectives on genetic analysis of plantembryogenesis. Plant Cell, 3, 857-866.

Meinke D W, Franzmann I H, Nickle T C & Yeung E C, 1994.Leafy cotyledon mutants of Arabidopsis. Plant Cell, 6, 1049-1064.

Michalczuk L. Cooke T J & Cohen J D, 1992a. Auxin levels atdifferent stages of carrot somatic embryogenesis. Phyto-chemistry, 31, 1097-1103.

Michalczuk L, Ribnicky D M, Cooke T J & Cohen J D, I992b.Regulation of indole-3-acetic acid biosynthetic pathways incarrot cell cultures. Plant Physiol, 100, 1346-1353.

Mondal A, Bhattacharya A, Sood A & Ahuja P S, 2000. Factorsaffecting induction and cold storage of encapsulated tea[Camellia sinensis (L.) O. Kuntze] somatic embryos. Tea.21,92-100.

Mondal A, Bhattacharya A, Sood A & Ahuja P S, 2002. Factorsaffecting germination and conversion frequency of somaticembryos of tea [Camellia sinensis (L.) O. Kuntze]. J PlantPhysiol, ( in press).

Mondal T K, Bhattacharya A & Ahuja P S, 2001. Induction ofsynchronous secondary somatic embryogenesis in Camelliasinensis (L.) O. Kuntze. J Plant Physiol, 158, 945-951.

Mordhorst A P, Voerman K J, Hartog M V, Meijer E A, van WentJ, Koornneef M & de Vries S C, 1998. Genelics,149. 549-563.

Nadgauda R S, John C K & Mascarenhas A F. 1993. Why wait solong for the bamboos to flower? BIC India Bull, 3. 14-20.

Nambisan P & Chopra V L, 1992. Somatic embryogenesis inTriticum aestivum L. Morphological observations on germi-nation. Indian J Exp Bioi, 30, 12-14.

Narasimhulu S B, Kirti P B, Prakash S & Chopra V L, 1992. So-matic embryogenesis in Brassica nigra (Koch). J Exp BOI.43, 1203-1207.

Nayak P & Sen S K, 1991. Plant regeneration through somaticembryogenesis from suspension culture-derived protopiastsof Paspalum scrobiculatum L. Plant Cell Rep, 10,362-365.

Nomura K & Komamine A, 1985. Identification and isolation ofsingle cells that produce somatic embryos at high frequencyin a carrot suspension culture. Plant Physiol, 79, 988-991.

Ogas J, Cheng J C, Sung Z R & Somerville C, 1997. Cellulardifferentiation regulated by gibberellin in the Arabidopsisthaliana pickle mutant. Science, 277, 1-94.

Okada K, Ueda J, Komaki K, Bell C J & Shimura Y, 1991. Re-quirement of the auxin polar transport system in early stagesof Arabidopsis floral bud formation. Plant Cell. 3, 677-684.

Perry S E, Lehti M D & Fernandez D E, 1999. The MADS do-main protein AGAMOUS like 15 accumulates in embryonictissues with diverse origins. Plant Physiol, 120, 121- [29.

Reinbothe C, Tewes A & Reinbothe S, 1992. Comparative mo-lecular analysis of gene expression during plant embryogene-sis: do evolutionarily conserved mechanisms control earlyplant development? Tibtech News Inj; 4, 381N-397N.

Reinert J, [958. Morphogenese und ihre Kontrolle in Gewe-bekulturen aus Carotten. Naturwissenscliaften, 45, 344-345.

Rose R J, Nolan K E & Bicego L, 1999. The development of thehighly regenerable seed line Jema!ong 2HA for transforma-tion of Medicago transcatula - implications for the regener-ability via somatic embryogenesis. J Plant Physiol, 155. 788-791.

Rout G R & Das P, 200l.Studies on in vitro somatic embryogene-sis of Psoralea corylifolia Linn. - An endangered medicinalplant. Gartenbauwissenschaft, 66, 202-206.

Sagare A P, Suhasini K & Krishnamurthy K V, 1993. Plant regen-eration via somatic embryogenesis in chickpea (Cicer arieti-num L.). Plant Cell Rep. 12, 652-655.

Santa R, Sreenath i-I, Srinivasan C S & Rama S, 1999. Morphoge-netic responses of cultured root and hypocotyl tissues ofCoffea arabica L. J Coffee Res, 27, 87-92.

Schiavone F M & Cooke T J, 1987. Unusual patterns of somaticembryogenesis in domesticated carrot: Developmental ef-fects of exogenous auxins and auxin transport inhibitors. CellDiffer, 21, 53-62.

BHA ITACHARY A et al: BIOCHEMICAL AND MOLECULAR INSIGHTS INTO EMBRYOGENESIS IN PLANTS 47

Shankhdhar D, Shankhdhar S C, Pant R C & Mani S C, 2001.Regeneration in rice (Oryza sativa L.) through somatic em-bryos. Phytomorphology, 51, 79-81.

Sharma P, Rajam M V & Sharma P, 1995 Genotype, explant andposition effects on organogenesis and somatic embryogenesisin eggplant (Solanum melongena L.). J Exp Bot, 46, 135-141.

Shen Q, Gomez C A, Zhang P, Walker-Simmons M K, Sheen J &Ho T H, 2001. Dissection of abscisic acid signal transductionpathways in barley aleurone layers. Plant Mol Bioi, 47, 437-448.

Sheridan W F, 1995. Genes and embryo morphogenesis in angio-sperms. Dev Genet, 16,291-297.

Smith D L & Krikorian A D, 1990. Somatic proembryo produc-tion from excised, wounded zygotic carrot embryos on hor-mone free medium: Evaluation of the effects of pH, ethyleneand activated charcoal. Plant Cell Rep, 9, 468-470.

Sreenivasu K, Malik S K, Kumar P A & Sharma R P, 1997. Plantregeneration via somatic embryogenesis in pigeonpea [Caja-nus cajan (L.) Millsp]. Plant Cell Rep, 17,294-297.

Sterk P, Booij H, Schellekens G A, van Kammen A & de Vries SC, 1991. Cell-specific expression of the carrot EP-2 lipidtransfer protein gene. Plant Cell, 3, 907-921.

Steward F C, Mapes M 0 & Smith J, 1958. Growth and organizeddevelopment of cultured cells II. Organization in culturesgrown from freely suspended cells. Am J Bot, 45, 705-708.

Suhasini K, Sagare A P & Krishnarnurthy K V, 1994. Direct so-matic embryogenesis from mature embryo axes in chickpea(Cicer arietinum L.). Plant Sci Limerick, 102, 189-94.

Sujatha M & Prabakaran A J, 2001. High frequency embryogene-sis in immature zygotic embryos of sunflower. Plant CellTissue Organ Cult, 65, 23-29.

Sung Z R & Okimoto R, 1983. Co-ordinated gene regulation dur-ing somatic embryogenesis in carrots. Proc Natl Acad SciUSA, 80, 2661-2665.

Sung Z R, Fienberg A, Chornewu R, Borkird C, Furner I & SmithJ, 1984. Developmental biology of embryogenesis from car-rot culture. Plant Mol Bioi Rep, 2, 3-14.

Thomas T L, 1993. Gene expression during plant embryogenesisand germination: An overview. Plant Cell, 5,1401-1410.

Toonen M A J, Hendricks T, Schimdt E D L, Verhoeven H A. vanKammen A & de Vries S C, 1994. Description of somaticembryo-forming single cells in carrot suspension culture em-ploying video cell tracking. Planta, 194, 565-572.

Tras J, Bellini C, Nacry P, Kronenberger J, Boucher D & CabocheM, 1995. Normal differential pattern in plants lacking micro-tubular pre-prophase bands. Nature (Lond), 375, 676.

Ulrich T U, Wurtele E S & Nikolau B J. 1990. Sequence of EMB-1, an mRNA accumulating specifically in embryos of carrot.Nucleic Acids Res, 18, 2826.

Van Engelen F A, Sterk P, Booij H, Cordewener J H G, Rook W.Van Kammen A & de Vries S C, 1991. Heterogenei ty andcell type-specific localization of a cell wall glycol-proteinfrom carrot suspension cells. Plant Physiol, 96, 705-712.

Venkatachalam P, Kishor P B K, Geetha N, Thangavelu M &Jayabalan N, 1999. A rapid protocol for somatic embryo-genesis from immature leaflets of groundnut (Arachis hypo-gaea L.). In Vitro Cell Dev Bioi Plant, 35, 409-412.

Vikrant & Rashid A, 2001. Direct as well as indirect somatic em-bryogenesis from immature (unemerged) inflorescence of aminor millet Paspalum scrobiculatum L. Euphytica. 12().167-172.

von Recklinghausen I R, Iwanowska A, Klef H. Mordhorst A P.Schell J H N & van Lammeren A A M, 2000. Structure anddevelopment of somatic embryos formed in Arabidopsis tho-liana pt mutant callus cultures derived from seedlings. Pro·toplasma, 211, 217-224.

Weigel D, 1993. Patterning the Arabidopsis embryo. Curr Bioi. 3.443-445.

West MAL & Harada J J, 1993. Embryogenesis in higher plants:An overview. Plant Cell, 5,1361-1369.

Yadegiri R, Paiva G R, de Laux T, Kolnow A M, Apuya N, Zim-merman J L, Fischer R L, Harada J J & Goldberg R B, 1994.Cell differentiation and morphogenesis are uncoupled inArabidopsis. Plant Cell, 6,1731-1745.

Zimmerman J H, 1993. Somatic embryogenesis: A model forearly development in higher plants. Plant Cell, 5, 1411-1423.

Zimmerman J H, Lin X & Hwang G J, 1992. The molecular basisfor somatic embryo development in carrot. Acta Hortic. 336.217-224.