Somatic embryogenesis in angiosperm cell tissue and organ cultures

25 Pages·2009·1.71 MB·English

lndian Proe. .dacA .ieS (Plant rS Vol. ,69 .oN ,4 October ,6891 .pp 247-27l.
(cid:14)9 Printed ni India.
Somatic embryogenesis in angiosperm celi tissue and organ cuitures*
N S RANGASWAMY
Department of Botany, ytisrevinU of Delhi Delhi, 011 ,700 India
SM deviecer 82 tsuguA 6891
A~traet. survey A of literature swohs that somatic sisenegoyrbme stricto sensu has been
deveihca ni only 201 ,seieeps tneuqesbus plantlet formation ni 15 of these seiceps and
lautneve propagation ni stilt fewer of urreht and The zygotic contrast sornatie between
sisenegoyrbme si rather The marked. potential for somatic sisenegoyrbme si genotype-
.cificeps Ultrastructural, histological dna lacimehcoib studies ale limited. Published
ecnedive swohs that auxins tceffa both transc¡ dna lanoitalsnart involved events ni
eht induction of somatie ;sisenegoyrbme ,ssetehtenon eht action primary of auxin si not
inhibitors Growth known. gnirrucco ni gnipoleved sdees Ÿ)alp a tnacifingis tole ni citamos
.sisenegoyrbme Employing yllacigolotsih simpler tissue systems sueh su nucellus and
endosperm sa explants primary si Some advocated. snoitatimil ni eht gnitsixe studies dna
prospects certain era .deifitnedi
.sdrowyeK Somatic sisenegoyrbme usnes stricto; citogyz ;sis~negoyrbme ;seilonehp .epytoneg
1. Introduetion
In the short period of a quarter century foltowing the reports by Reinert (1958) and
then by Steward et al (1958) of the formation of plantlets in carrot tissue cultures
through struetures simulating zygotic embryos, somatic embryogenesis has acquired
unlimited importance in ag¡ sylviculture, horticulture and in fact in some
industries which are dependent on continual supply of basic plant material of elite
quality. Not surprisingly therefore the topie has been discussed in over 25 specific
reviews and other publications in rather quick successiorg to cite a few: Reinert
(1963), Maheshwari and Rangaswamy (1965), Steward et al (1966), Halperin (1970),
Johri (1971), Vasil and Vasil (t972), Street and Withers (1974), Street (1979), Sharp
et al (1980), Evans et al (t981), Ammirato (1983), Raghavan (1983) and Lutz et al
(1985). No apology is needed for another review of the rapidly mounting literature.
Somatic embryogenesis is the development of embryos from somatic tissues as well
as from situations which do not involve directly gametes, haploid eells, of
gametophytes. Of whatever o¡ ah embryo is ab inirio a bipolarized entity
bounded by euticle. In embryogenesis in ovulo the zygote enjoys a definitive position
at the micropylar pole in the developing seed. This reference location enables an easy
recognition of bipola¡ exomorphicaUy in the developing embryo and ah easy
deciphe¡ of the morphogenetic events during embryogenesis. But in tissue cultures
the mere appearance of globular bodies without their further development into any
bipolarized stage of embryogenesis cannot be considered by any known crite¡ of
morphogenesis or embryology as the manifestation of somatic embryogenesis. Also,
erutavetiL* ended survey rebmeceD 5891
247
248 N S Rangaswamy
to be of practical value somatic embryogenesis should culminate in the formation of
plantlets and their successful propagation. This review therefore critically examines
the contemporary iiterature (up to Dec. 1985) to discover in which and how many
species somatic embryogenesis sensu stricto has been achieved and, in which of them
plantlets have been obtained in vitro and established in nature and draws certain
inferences to project the prospects.
2. Somatic embryogenesis versus zygotic embryogenesis
Somafic embryogenesis sensu stricto is the development of embryos of at least two
distinct stages that ate not necessarily successive. In final analysis the end product of
somatic embryogenesis si essentially similar to that of zygotic embryogenesis.
However, the gamut of morphogenetic events du¡ zygotic embryogenesis,
particularly cell division and cell destiny, follow a predictably high degree of
precision which has provided the basis for both classhŸ and the laws of
embryogeny. With the acquisition of radial and then bilateral symmetry the young
embryo becomes morphogenetically stable. Cell vacuolation does not occur before
the embryo reaches the heart-shaped stage. All the cells derived from the zygote
together constitute the embryo; by the time the embryo becomes fully differentiated,
only its suspensor which represents a numericaily stabilized population of ceUs might
be 'wasted' through autolysis. Contradistinctly embryogenesis in Uec suspension and
tissue cultures is highly plastic and greatly influenced by the milieu and the gradients
attained in the culture medium as well as by the site of initiation of morphogenesis,
i.e., whether embryogenesis begins in a cell aggregate or in an individual ceU. In
either case the laws of embryogeny are not invariably followed. For example, in
suspension cultures of carrot, the so-called model system, the planes of cell divisions
in early somatic embryogenesis are extremely irregular and precocious vacuolation
of eells occurs (Hatperin 1966); often a large number of embryos rail to acquire
bilateral symmetry (McWilliam et al 1974). ~rI somatic proembryos which develop
through regular cell divisions, the cell lineage corresponds to the Onagrad type, but
that in the zygotic embryo corresponds to the Solanad type of embryogeny
(McWilliam et al 1974). In fact there has been no instance of a cell in exclusive
isolation that has faithfully duplicated in vitro the embryologic saga of that unique ab
initio bipolar cell, the zygote. Whether free cells in suspension cultures follow Errera's
laws si also debatable (Steward 1958; McWilliam et al 1974). And although a callus
may be derived from a cell, not all its derivatives participate in eonstituting a somatic
embryo. Thus, in somatic embryogenesis, save the rare instances of epidermal origin
of embryos, a greater amount of cellular material is wasted; such a callous
comportment si seld6m known of a zygote and weakens the key note of biological
evolution namely, 'maximal efficiency with minimal material'. Be as it may, cells in
exile are cetls in revolt. But this has its bonus; callus-mediated somatic
embryogenesis si ideal for selecting desirable variants.
3. Somatie embryogenesis sensu stricto
It is regrettable that without adequate ontogenetic studies to confirm ab initio
bipotarity, any globular body and forked body formed in tissue cuttures have been
Somatic embryogenesis in angiosperm cell tissue and organ cultures 249
described respectively as globular and dicotyledonous somatic embryos. More often
than not, young shoot buds differentiating in tissue masses simulate embryos of
cotyledonary stage. Some workers on monoeot species have reported heart-shaped
and torpedo stage somatic embryos and grass embryo structures in non-
graminaceous species! If verbal claims and claims based whoUy on exomorphic
semblance of structures to embryos and retrieval of information merely from titles of
research papers are accredited, the number of angiosperm species in which somatic
embryogenesis has been reported will be over 200, but on the basis of unequivocally
recognizable ab initio bipolarity of the observed entity and of unmistakable
demonstration of at least one other stage of e mbryo besides the globular 'body', the
number of species in which somatic embryogenesis has been achieved todate
(literature survey ended December 1985) is only 201 (table .)1
4. Factors of somatic embryogenesis
From time to time nearly 50 substances belonging to some 01 classes of organic and
inorganic compounds, naturally occurring plant juices, plant extracts and some
physical faetors have been reported to influence somatie embryogenesis, but the
cause and effect relation has not be.en unequivocally established for any, except
perhaps auxins. Reports about some factors are diamet¡ conflicting and about
some others meagre of solitary. So, what makes a ceil competent for embryogenesis
is still empirical. What complicates the problem and hence challenging is that factors
which govern plant cell, tissue and organ differentiation are the same as tbose that
govem dedifferentiation or disdifferentiation. Of the several ehemical factors, the
involvement of auxins has been extensively studied.
1.4 Role of auxins
Auxin transport in immature seed embryos is strictly basipetal. (Fry and
Wangermann 1976) and during embryogenesis physiologic polarity (auxin transport
and auxin-cytokinin ratio) precedes morphologic polarity (Fry and Wangermann
1976; Przybyllok and Nagl 1977). From these facts of zygotic embryogenesis it may
be extrapolated that a polarized distribution of endogenous auxin in tissue culture
may well be a prerequisite for induction of embryogenesis in the tissue. However,
high levels of tryptophan and IAA suppress somatic embryogenesis (Sung 1979) and
exogenous supply of auxin would disturb the auxin polarity in the tissue (Fujimura
and Komamine 1979). In some instances exogenous horrnone can increase and in
other instances decrease the endogenous hormone level. Auxin concentrations which
elicit growth in cultured explants are not the same as those that induce
embryogenesis in them (Sondahl and Sharp 1977). The growth period when auxin
application is given is critical for polyembryonic effect in vivo (Ferguson et al 1979).
Likewise, the hormone regimc employed in vitro to induce proliferation of '.he
explant and eventual embryogenesis in it is critical. It si commonplace that a transfer
of callus culture from a high auxin medium to a medium containing a weak auxin, of
a low or zero level of auxih would induce embryogenesis in the callus; interestingly
this is true of callus formed from pollen grains as well (Raghavan and Nagmani
1983). Embryogenic callus grown in dark conjugates indote-3-acetic acid (IAA) with
250 N S Rangaswamy
Talde .1 Established instances of somatic embryogenesis sensu stricto and propagation
therefrom in angiosperms*.
Details of
Family and spr Embryogenesis Propagation Referente**
.1 Aaar
Mangifera indica M g h el Litz et al (1982)
Z Araliaceae
Panax yinseng M g h c Pt Chang and Hsing (1980)
3. Aquifoliaceae
Ilex aqui'otium M h t m Hu et al (1978)
4. Asr
Asclepias curassavica M g h t Prabhudesai and Narayanaswamy
(1974)
Cynanchum M g h H g Har and Hausner (1976)
vincetoxicum
Hoya carnosa M g t Maraffa et al (1981)
Pergularia minor M g rh ti Pt Prabhudesai and Narayanaswamy
(1974)
Tylophora indica M g h H h Pt Rao et al (1970)
.5 Betulaeeae
Coryllus avellana (H g h/t c) Pt P› et al (1983)
.6 Ca¡
Carica papaya M g (h) c Pt so ~-~ Litz and Conover (1983)
Carica stipulata M h t Litz and Conover (1980)
.7 Convolvulaceae
Cuscuta reflexa M c H g c Maheshwari and Baldev (1961)
lpomoea batatas M g h t Ptsofl Liu and Cantliffe (1984)
.8 Crassulaceae
Kalanchoe pinnata M g h Wadhi and Mohan Rara (1964)
.9 Cruciferae
Brassica campestris" M g h t c H g t 1P Bhattacharya and Sen (1980)
Brassica napus a M 0a) e/t c Pt Eso li Lola and Ingrato (1982)
ssp. areCoelo (H g c)
Brassica oleracea M (g h t) c Pt Singh aad Chandra (I985)
var. botrytis H g h
.01 Cucurbitaceae
Cucurbita pepo M (h t c) m Pt so fl Jelaska (1974)
I .1 Euphorbiaceae
Manihot esculenta M ti e Stamp and Henshaw (1982)
1Z Gramineae
Dactylis glomeraŸ SEM m Conger et al (1983)
H g (se cp)
Echinochloa crusgalli H and SEM sc Pt Wang and Yan (1984)
cp cr SEM se cp
Hordeum vulgare M g SEM g Kott and Ka.sha (I984)
se cp
Lolium multiflorum- M se cp cr Pi Dale et al (1981)
Oryza sativa" M m H se Pt Chen et al (1985)
cp cr
Panictan maximum M and H se cp Pt Lu and Vasi! (1982)
SEM sc cp cr
Pennisetum M g (cp cr) Pi so Vasil and Vasil (1982)
americanum SEM g se cp
H sc cp cr
Penniseturn purpureum SEM sc cp cr ~P so Wang and Vasi! (1982)
Somatic embryogenesis in angiosperm cell tissue and organ cultures 251
Table I. (Contd.)
Details of
Family and species Embryogenesis Propagation Reference**
Saccharum officinarum M and SEM Pt so Ho and Vasil )3891(
gsccp (H gsc
cr)
Secale cereale a M sc cp Pt so fl La er al )4891(
SEM sc
Setaria italica M gl SEM Pt Xu et al )4891{
sc cp cr
Sorghum arundinaceum SEM g sc cp Pt so Boyes and Vasit 1,4891(
Sorgham bicolor M and H )g( Pt so se Wernicke et al (I982)
sc cp cr
Triticum aestivum ~ M and H sc Magnusson and Bornman (1985)
cp cr
Zea mays ~ (M g sc cp cr (Pt) so se Armstrong and Green (1985)
SEM sc cp)
.31 Hammamelidaceae
Liquidambar M t c Pt Sommer and Brown (1980)
styraciflua
.41 Iridaceae
Iris. sp. M g r H g m Pt Reuther )7791(
.51 Juglandaceae
Juglans regia M g c H c Pi so Tulecke and Mcgranahan (1985)
.61 Lauraceae
Sass~ifras randaiense M gc Pt so t-~ Chen and Wang )5891(
.71 Leguminosae
Albiz~.ia lebbeck M g h t Gharyal and Maheshwari (1981)
Cyamopsis M )g( h t c Pt McHughen and Swartz )4891(
tetragonoloba
Glycine max M (g h) c H c Pt Lippmann and Lippmann (1984)
Glycine soja M g h Gamborg et al )3891(
Medicago sativa H g h c Dos Santos et al )3891(
SEM g c
(M ghc)
Trifolium ar(cid:127)ense M Ir el Bhojwani er al )4891(
Trifolium repens Mhtc Maheswaran and Williams (1984)
Vigna aconitifolia Mhc Bhargava and Chandra (1983)
.81 Liliaceae
Alliton sativum M sc ep r Abo )7791(
Asparagus silanic~JJo M e H g c Pt Reuther )7791(
Beltevalia romana M e c Lupi et al )5891(
.91 Loranthaceae
Dendrophthoe falcata M g c Johri and Bajaj )3691(
Nuytsia floribunda M t c Nag and Johri )6791(
Scngrula pulverulenta M g h Johri and Bhojwani (1970)
Taxillus vestitus M t c Nag and Johri )6791(
.02 Malvaceae
Gossypium M g t c Finer and Smith (1984)
klotzschianum
Hibiscus acetosella M g hi t e Reynolds and Blackmon (1983)
Sida rhombifolia M g t Rangaswamy et al )0891(
.12 Musaceae
Musa sp. M g c Cronaucr and Krikorian (1983)
.22 Palmae
Elaeis guineensis M le mi Nwankwo and Krikorian (1983)
252 N S Rangaswamy
Table I. (Contd.)
Details of
Family and species Embryogcnesis Propagation Reference**
Phoenix dactylifera M (g)e Pt Tisserat and de Mason )0891(
Hgm
.32 Papaveraceae
Eschscholzia M g( )c Pt Kavathekar et al )7791(
californica
Macleaya cordata M g h t c Kohlenbach )6691(
Papaver orientale M g t Pt Schuchmann and Wellmann
)3891(
Papaver somniferum M g c H( and Pt Schuchmann and Wr
MES )c )3891(
.42 Ranunculaceae
Ni#ella damascena Mght Raman and Greyson )4791(
Nigella sativa MghtHgh Ptfl Banerjee and Gupta )6791(
Ranunculus sceleratus M( and H g h ~c Pt Konar and Nataraja )5691(
Thalictrum urbaini M g t c Yang and Chang )0891(
.52 Rosaceae
Malus pumila Mghc Mehra and Sachdeva )4891(
~rIMIlIS 19(cid:1)ISO'tUC Mhtc Du na,7r )5891(
P yrus malus Mgh Mu et al )7791(
.62 Rubiaceae
Coffea arabica Mgtc Pt S6ndahl and Sharp )7791(
Coffea canephora MgtcHgc Pierson et al )3891(
MES g h t
.72 Rutaceae
Citrus grandis Migo He Pt Rangan et al )8691(
riC limon M cm Rangart et al )8691(
Citrus microcarpa Mghc Pt Rangaswamy )1691(
Citrus paradisi Mgh Koehba et al )2791(
Citrus reticulata Mgh Pt Sabharwal )3691(
Citrus sinensis MgheHg Pt Button et al )4791(
.82 Santalaceae
Exocarpus bidwillii M )gI c Johri )5691(
Santalum arubla Mgc Pt Bapat and oaR )9791(
.92 eaecagarfixaS
Ribes rubrum Mht Zatyk£ et al )5791(
.03 Scrophulariaceae
Di#italis lanata Mghc Tewes et al )2891(
Paulownia tomentosa Mgc Hh R adojevi6 19791(
.13 Solanaceae
Atropa belladonna ~ MgHghc Konar et al )2791(
H yoscyamus ni *req McHghc Pt os )it( Cheng and Raghavan )5891(
L ycopersicon Mgc Zapata and Sink )1891(
munaivurep
Petunia hybrida Mgh Pt Rao et al )3791(
Petunia inflata Hgc Handro et al )2791(
Solanum metongena Mghtc PI gm Gleddie et al )3891(
.23 Sterculiaceae
Theobroma cacao Hghc Kononowicz et al }4891(
.33 Theaceae
Camellia chrysantha MandHghc Pt Chengji and Hanxing )5891(
.43 Umbelliferae
Ammi majus Mghc Grewal te al )6791(
Anethum sneloe~arg MtcHgc Pt lagheS )8791(
Somatic embryoyenesis in angiosperm cell tissue and oryan cultures 253
Table I. (Contd.)
Details of
Family and species Embryogenesis Propagation Reference**
Apium oraveolens M g h e H g h Dunstan et al (I )289
(SEM g h t)
Carum carvi M g( h )t c Ammirato )4791(
Coriandrum sativum M h t e Pt Steward et al )0791(
Daucus earota M g t c H g c Pt so lf se Halperin and Wetherell )4691(
FI
Foeniculum vul#are M Ir el Maheshwari and Gupta )5691(
Petroselinum horte~e M g h c Pt Vasil and Hildebrandt )6691(
Hghc
Pimpinella anisum M g h t Kudielka and Theimer )3891(
Sium sa~e M g h t c Pt Steward et al 0970}
.53 Vitaceae
Vitis vinifera M g h t c Pt Rajasekaran et al )2891(
SEM c
*lrrespective of rnorphologic category of the vegetative part or somatic explant;inoculurn used, only the
publications that conlain convincing illustrative account of at least two stages of somatic embryos aro
tabulated. Verbal claims as well as reports that depict the ormation of merely globular 'bodies" or
embryoids are discounted.
No claim for ah exhaustivo tabutation is made, especially because of non-availability of "endemic' reports
and of publieations that have limited global circulation ta general botanical public.
*"To keep the list of references to a reasonable minimum, only the earliest work. irresper of the
explant used, that describes one or more aspects (M, morphologic; H, histologic; SEM, seanning electron
microscopio) of somatic embryogenesis in greater detail and/or the formation of plantlets through somatic
embryogenesis and their performance is cited.
Abbreviations used: c, Cotyledonary stage; cp. coleoptile; cr, eoleorhiza; ,e elongate embryo stage; epi,
epiblast; g, globular embryo; ,h heart-shaped embryo; m, mature embryo; psc, post-scutellum stage; rad,
radicle; ,cs scutellum; t, torpedo stage. F t, plantlet produeed Ft; li, plantlet flowered; Ir, plantlet set fruit;
,gm plantlet matured in green house; Pt, plamlet; se, plantlet set seed; so, plantlet transferred to soil; so~- ,~
a wef plantlets survived in soil.
Abbreviations in brackets indicate that the information is avaitable in other publicalions. Abbreviations
in square braekets aro based on my interpretation.
aSpecies for whieh pollen embryos bayo also been reported.
aspartate at a higher rato than non-embryogenic caUus and thus achieves a lower
level of auxin conducive for embryogenesis (Epstein et al 1977). In auxin-habituated
tissues even as low as 0"01 mg/l auxin inhibited embryogenesis (Kochba and Spiegel-
Roy 1977). The raro instantes of induction of somatic embryogenesis without
exogenous auxin (Vardi et al 1975; Hu et al 1978) may represent systems that have
optimal level of endogenous auxin.
In some instances antiauxins of the phenoxyacetic acid group e.g. (2,4,5-
trichtorophenoxy acetic acid) (2,4,5-T) and (2,4,6-t¡ aeetic ac'id) (Z4,6-T)
were essential or more effective than auxins for initiating embryogenic callus
(Newcomb and Wetherell 1970; Cronauer and Krikorian 1983). Galactose and the
galactose-yielding sugars lactose and rat¡ which inhibit auxin synthesis
stimulated somatic embryogenesis even in caUus fines not responsive to any other
treatment (Kochba et al 1978b). Other inhibitors of auxin synthesis such as 5-hydroxy-
nitrobenzylbromide and 7-aza indole (Kochba and Spiegel-Roy t977) and y-irradiation
(Spiegel-Roy and Kochba 1973) which denatures auxin, also greatly stimulated
452 N S Ranoaswamy
embryogenesis. The inhibition or promotion of somatic embryogenesis by auxins,
antiauxins and auxin synthesis inhibitors is a function of age and/or stage of the
tissue culture and the residual exogenous auxin present in the culture at the time of
application of the test substance (Fujimura and Komamine .)9791
As in the most :extensively and intensively studied model system, namely carrot
and presumably in other systems as well, somatic embryogenesis si at least a 3-phased
program: )i( slow Uec division phase during which the doubling time is 85 h,
(ii) rapid cell division phase (doubling time < 7 )al during which the differentiating
structure increases in mass and (iii) organized cell division phase which leads to a
bipolarized growth pattern (Fujimura and Komamine 1982) characteristic of
embryogenesis. Each phase si the manifestation of a specific pattern of gene
activation leading to the next essential phase. In the first phase which usually lasts 3
or 4 days from culture, cell clusters in the callus become committed to differentiation;
such a commitment precedes at least one mitotic cycle. In the second phase the
culture si rather sensitive to the effects of auxins as well as cytokinins. In the third
phase of embryogenesis the culture is less sensitive to the exogenous supply of either
auxin or cytokinin (Fujimura and Komamine 1980a, .)b This explains, although
partly, the divergent reports on the effects of auxins and cytokinins, alone or in
combination, on somatic embryogenesis.
4.la Mechanism ofauxin action: Researches to explain the mechanism of role of
auxin in somatic embryogenesis are sporadic and inconclusive largely because the
cultures employed are asynchronous. In tissue cultures raised on high levels of auxin,
production of ethanol (Thomas and Murashige 1979) and ethylene (Tisserat and
Murashige 1977a; Grierson et al 1982) is high. Ethylene production can in fact serve
as ah index of auxin (2,4-D) stress in tissue cultures (Garcia and Einset 1983). High
ethylene production leads to high activity of both cellulase and pectinase resulting in
fragmentation of the callus (Wochok and Wetherell 1971). Auxin promotes calcium
ettlux (de Guzman and Fuente 1984) which could lead to cell wall loosening.
Whereas a certain degree of tissue fragmentation may be salutary for induction of
embryogenesis (Halperin and Jensen 1967), fragmentation before the callus
establishes endogenous auxin polarity (Wochok and Wetherell 1971) may be
deterrent. The converse si also true. In the absence of auxin, tissue fragmentation is
markedly decreased (Halperin and Jensen 1967). Both ethylene and etbanol mimic
the repressive effect of auxin on somatic embryogenesis (Wochok and Wetherell
;1791 Tisserat and Murashige 1977a, .)b
At the molecular level the mechanism of auxin action in somatic embryogenesis si
iess understood. Unlike tissues grown all through on auxin-containing medium (non-
embryogenic cultures), those grown initially on auxin-rich medium and subsequently
on auxin-free medium (embryogenic cultures) showed higher DNA content (Wochok
;3791 Verma and Dougall 1978; Masuda et al ,)4891 higher rates of syntheses of poly
}A( § RNA and protein (Fujimura et al 1980; Sengupta and Raghavan 1980), decrease
in replicon size of DNA (Fujimura and Komamine 1982), increase in template
activity of chromatin and non-histone proteins (evident as early as two days
following auxin omission) (Matsumoto et al 1975; Fujimura and Komamine 1982)
and variations in etectrophoretic profiles of certain isozymes, especially of peroxidase
(Wochok and Burleson 1974; Kochba et al 1977). The rates of RNA and protein
syntheses in embryogenic cells increased appreciably over those in non-embryogenic
Somatic embryogenesis in angiosperm cell tissue and organ cultures 255
cells within 2-4 h from transfer of callus culture to auxin-free medium (Sengupta and
Raghavan 1980). But the general stimulation of syntheses of RNA and protein in
plants by auxin, especially ,2 4--D, does not help explain the auxin effect in somatic
embryogenesis. Nonetheless, put together )i( the studies using cordycepin (an
inhibitor of polyadenylation) (Raghavan 1983), (ii) the studies on in vitro protein
synthesis using wheat germ system and RNA from carrot embryogenic system
(Fujimura and Komamine 1982) and (iii) the most common observation that cell
masses which differentiated on auxin-containing medium do not become
embryogenic unless transferred to ah auxin-free medium suggest that the message
component of RNA presumably remains masked (Spiegel-Roy and Kochba 1973),
that the deletion of auxin from the medium monitors molecular events which lead to
the synthesis of poly )A( + RNA in the previously auxin-conditioned cuiture and that
the induction of somatic embryogenesis si presumably controlled both at the
transcriptional (Fujimura and Komamine 1982) and translational (Raghavan 1983)
levels. In paraUel with animal systems non-histone proteins have been implicated in
the promotion of transcription. Both zygotic and somatic embryos of a species
accumulate similar storage proteins, but the latter accumulate smaller quantities and
in much ea/lier stages (Crouch 1982). UA embryo-like structures contain the
characteristic storage proteins, but not the other structures which differentiate in the
same culture, of the explants from which the cultures were derived. Likewise,
synthesis of certain lectins occurs only in the embryo and not in other parts of the
adult plant (Rouge 1974). Obviously embryogenesis would need specific messengers.
Significantly, in carrot system while auxin evoked the synthesis of embryogenic
proteins, which may be considered as developmental markers, continued presence of
auxin in the culture medium repressed their further synthesis and hence
embryogenesis (Sung and Okimoto 1981). CeU lines incapable of embryogenesis
lacked the marker proteins.
The various findings on auxin involvement in somatic embryogenesis as presented
in this mini-review are not strictly chronological developments. They have not been
based on the same experimental system or genotype. Nonetheless, it may be inferred
that auxin (especially 2, 4-D) conditions the cuiture for embryogenic competence,
but embryogenesis per se is induced in the absence of auxin. The control of synthesis
of tissue-specific proteins is an event far removed from the p¡ action of auxin.
Beeause the same hormone can evoke va¡ molecular events in different tissues
(Wareing 1971; Zeroni and Hall 1980), future research on auxin mechanism in
somatic embryogenesis must be directed to discover a primary auxin action such as
binding with a receptor or insertion into a membrane (Vanderhoef and Kosuge 1984)
and in turna control of ion fluxes. Auxins may modify the 3-dimensionat structure of
acyl lipids in membrane and thus influence the transport and/or detection at the
membrane level of chemical signals (Warren and Fowler 1979) essential for somatic
embryogenesis.
4.2 Cytokinins
Data on the nature and amounts of cytokinins used for induction of somatic
embryogenesis have been compiled (Evans et al 1981). There is no compelling
evidence fora universal requirement of cytokinins for somatic embryogenesis. But
some reports show that cytokinins promote maturation of somatic embryos
256 N S Ran~taswamy
(Fujimura and Komamine 1980a), especially cotyledon development (Ammirato and
Steward I971) and ate required for growth of somatic embryos into plantlets
(Kavathekar et al .)8791
Studies on cytokinin-auxin interaction point to a decisive effect of the hormonal
regime on explant proliferation and eventual embryogenesis. Cytokinin inhibition of
auxin effect on embryogenesis is commonly reported (Halperin 1970; Kochba and
Spiegel-Roy 1977; Fujimura and Komamine 1980a; Gleddie et al 1983). Although
cytokinin is reported to increase the rate of explant proliferation, it suppresses the
appearance of embryogenic cetls (Halperin 1970). Embryogenesis is vigorous in
suspension cultures defived from auxm-induced proliferations (Halperin .)0791 Addition
ofcytokinin within three or four days from culture promotes somatie embryogenesis
(Fujimura and Komamine 1982; Ernst and Oesterhelt 1984) as this period coincides
with the rapid cell division phase in embryogenic cultures. The presence of eytokinin
in the medium during the early phases (commitment and expression ph~ses) of
embryogenesis suppresses the expressi6n of the eommitted ,sUec whereas its addition
in the later stages does not hinder the progress of somatic embryogenesis.
The mode of action of cytokinins is not understood. Kinetin sectas to inhibit
glycolysis (MacLeod 1968) and aerobic respiration (Neumann 1968). Addition of
kinetin to caUus culture doubles its RNA content (Bryant and ap Rees 1971). Results
of experiments on 3~P-incorporation into RNA indicate that tissue cultures
contain more stable RNA in the presence of kinetin than in its absence (Sharp et al
.)089I
Bud induction in the moss Funaria by cytokinin requires calcium; it is presumed
that cytokinin effects an opening of the calcium charmels in the cell system (Saunders
and Helper 1983). Cytokinin may have a similar action in inhibiting auxin-induced
growth (Vanderhoef and Stahl 1975). Plaucibly, cytokinin-auxin interaction may
help achieve a balanced level of caleium for cytodifferentiation.
3.4 Gibberellins
Gibberellins rarely stimulate somatic embryogenesis (Kononowicz and Janick .)4891
Gibberellic acid (GAs) generally inhibits it (Halperin 1970, Fujimura and Komamine
1975; Tisserat and Murashige 1977b). But GA3 is reported to promote embryo
maturation, overcome dormancy of somatic embryos and stimulate rooting and
subsequent development of embryos into planflets (Rangaswamy 1961; Kochba et al
;4791 Kavathekar et al 1978; Lakshmi Sita et al 1979; Lu and Vasil 1981; Lu et al
.)2891 In combination with zeatin and ABA, GA3 promotes a normal development
of somatic embryos (Ammirato .)7791
Lately polar and biologically active gibberellins have been implicated in somatic
embryogenesis (Noma et al 1982). Embryogenic cultures showed rather high levels of
less polar GAs (GA4- and GAl-like GAs) and rapid metabolism of GAt into GAs
and Gibberellin (GA)-glucosides (Noma et al .)2891 Researches with anti-gibbereUins
and gibberellin antagonists have ,eb en less rewarding in our understandin~ the
mechanism of gŸ effects on somatic embryogenesis.
4.4 Abscisic acid and other growth inhibitors
That abscisic acid (ABA) promotes normal development (Norstog and Blume 1974)
and prevents precocious germination (Ihle and Dure 1972) of excised zygotic