Plant biotechnology

1. ud formation
the type of organogenesis: 1% agar supported only flower regeneration; shoot bud
increases in frequency
with decreasing agar
concentration,
while only shoot budsaa
in liquid medium. In some species, light seems to have an inhibitory effect,andmo
quality of light may be important.
The optimuim
temperialure
for shoot regeneration h
with the plant species.
are formey
effect, and even the
ation may Vary
Shoot bud regeneration is markedly improved by exposure of explants to a weaka
current. For example, application of IuA electric current to tobacco callus mainto
shoot regeneration
medium inereased shoot bud regeneration
five-fold. The shoot hisOn
5.5.5. Electrical Stimulation
appeared in the most negative region of the callus irrespective of the polarity of
derived
thec from
Similarly. exposure to an electric current induced shoot buds in wheat calli derived6
mature zygotic embryos; these calli regenerated only roots without electric stimulation
5.6. SOMATIC EMBRYOGENESIS
A somatic embryo (SE) is an embryo derived from a somatic cell, other than zygote, usial
on culture in vitro. Somatic embryogenesis may be defined as the process of development f
a bipolar structure like zygotic embryo from a nonzygotic somatic cell; the SE does not ha
vascular connections with the tissue from which it develops. In contrast, embryosdevelopin
from zygotes are called zygotic embryos or often simply embryos, while those derived from
pollen are known as pollen embryos or androgenic embryos. Somatic embryogenesis was
reported in 1968 independently by Steward and coworkers, and by Reinert in cell suspensions
of carrot (Daucus carota). By 1978, somatic embryogenesis was reported from 80 species
belonging to 33 families; the list has expanded considerably since then (over 100 speciesby
1993).
5.7. DEVELOPMENTAL PATTERN OF SEs
SEs generally originate from single cells, which divide to form a group of meristematic cells.
Usually, this multicellular group becomes isolated by breaking cytoplasmic connectionswit
the other cells around it and subsequently by cutinization of the outer walls of this
differentiating cell mass. The cells of meristematic mass continue to divide to give rise lo
globular (round ball-shaped), heart-shaped, torpedo and cotyledonary stages(Fig 3.4
general, the essential features of SE development, especially after the globular stage.
comparable to those of zygotic embryos. Somatic embryogenesis may occurdirectly fromi
explant without an intervening callus phase (direct somatic embryogenesis) or a callusp
may precede SE regeneration (indirect somatic embryogenesis; Fig. 5.5). In prac
however, it is very difficult to differentiate these two events. Direct SE regeneration1S
nost
likely to oceur from ovules, zygotic and somatic embryos and young seedlings.
Somatic embryos are bipolar structures in that they have a radicle and a plumule
radicular end is always oriented toward the centre of callus or cell mass, while theplun
The
end always sticks out from the cell mass (Fig. 5.4). In contrast, a shoot bud is
monothey
does not have a radicular end (Table 5.1). In many SEs, radicle is suppressed so
l
often do not produce roots; in such cases, roots have to be regeneratedfromu

2. Regeneration andSomatic Embryogenesis 119
T A B L E
c5.1. A comparison between shoot buds and somatlc embryos
Shoot bud
C h a r a c t e r i s t i c
Many cells, usually superficial
Unipolar, only the shoot pole present Bipolar; both shoot and root poles
Somatic embryo
Origin Single cell, usually superficial
Polarity
Present; vascular strands connected Absent; there is
present
Vascular c o n n e c t i o n
with those present in callus/explant connection with callus/explant
vascular
no
with c a l l u s / e x p l a n t
Separation from
callus/explant
Not casily separated unless cut off Easily separated since the radicular
cnd is cutinized.
MERISTEMATIC
CELL
CUTICLE
INITIATION OF SOMATIC
EMBRYO (SE) DEVELOPMENT
EARLY PROEMBRYO
HEART-SHAPED SE GLOBULAR SE
CALLUS / EXPLANT
CALLUS / EXPLANT
cOTYLEDONARY STAGE SE
TORPEDO-SHAPED SE
Fig. 5.4. Development of a somatic embryo from a single superficial cell of the explant.
ced by germinating SEs. SEs often show abnormal developmental features, e.8., three
OTe cotyledons, bell-shaped cotyledon, larger size, etc.; these problems are often
Come by the presence of ABA or a suitable concentration of mannitol. In some species.
dlooking somatic embryos are formed, but they fail to germinate; at least some ofthe
n o tgerminate in most oftheplant species.
SEs do not

3. The SEs regenerating from explant or callus are termed as
primary somatic emh.
nating SEs
from cells
many cases, SEs regenerate from the tissues of other SEs or the parts of germinat
Such SEs are called secondary somatic embryos. Ordinarily, SEs originate from5
surface of callus or explant, e.g., from epidermal cells of Ranunculus stem. S a
5.8. FACTORS AFFECTING SOMATIC EMBRYOGENESIS
Somatic embryogenesis is influenced by several factors, e.g., ()GRs, (2) nitrogensou
type of explant, (4) explant genotype and (5) other factors. itrogen source, (3
5.8.1. Growth Regulators
omatic
xplant,
In most species an auxin (generally, 2,4-D at 0.5-5 mg/L) is essential for sOn
embryogenesis. The auxin causes dedifferentiation of a proportion of cells of the evnl
which begin to divide. In carrot, these small, compact cells divide
asymmetrically, and th.
daughter cells stick together to produce cell masses called proembryogenic masse
embryogenic clumps (ECs). In the presence of auxin, the ECs grow and break up into smal.
cell masses, which again produce ECs. But when the auxin is either removed or redueed
(0.01-0.1 mg/L) and cell density is lowered, each EC gives rise to few to several SEs:ea
SE is believed to develop from a single superficial cell. The ability to regenerate SEs,ie
totipotency, is acquired by cells during dedifferentiation in response to high auxin treatment
but the mechanism is not well known. High auxin prevents its own polar transport. Auxins
promote hypermethylation of DNA, which may have a role in totipotency acquisition.
Some glycoproteins produced by totipotent cell
masses are secreted into the medium; when these
proteins are added into the culture medium they
speed up the process of acquisition of totipotency. A
class of proteins, called arabinogalactan proteins
(AGPs; 90% carbohydrates with a protein backbone),
is involved in SE regeneration. AGPs are a
heterogeneous group of proteoglycans found in
plasma membranes, cell walls and plant secretions.
The type of AGPs expressed in cells changes during
root differentiation and flowering, and such changes
may presage developmental processes. AGPs may
function as markers for cell position during
morphogenesis, and they may induce differentiation
processes. When extracellular AGPs secreted into the
culture medium by embryogenic suspension cultures
of carrot and some other plant species were isolated
and added into less embryogenic suspension cultures, Fig. 5.5. Somatic embryos (SEs
the embryogenic potential of the latter was markedly
increased. Thus AGPs seem to play an important role
during the early stages of somatic embryogenesis,
e.g.. transition from globular to heart-shapedstage.
d e v e l
rom
ing trom callus produced
young (14d old) ZYgotic
embry
of wheat (Triticum aesv
cultured on MS me
medium
s t i v u n
containing 1 mgL-12,4-D

4. hoot
Regeneration
andSomatic
Embryogenesis
S h o
In m
ess: (i) SE induc
SE induction occurs on a
high auxin (upto 40-60 mg/L 2,4-D) medium, andu
oroce nment is achieved on a low auxin or
GR-free medium. In the SE ind
any species like carrot, coffee, alfalfa, etc. somatic embryogenesis is a two
step
121
SEdevelo
esp PEMs). In different cell lines of carrot, PEMs develop to different stages
/L 2,4-D) medium, and (ii)
induction phase,
proembryogenic
stages (from
to the PEM
do not synthesize auxin. The globular stage SEs, however, become sensitive
SE lls dedifferentiate, become
totipotent and, in many species, form
proembryog
e x p l a n t
masses (PE
stage. Th
only to globular stage SEs) on the induction medium before reverting to the PEM
The cell masses from the PEM stage to the
globular stage appear to be
insensiuve
auxin and they do to be insensitive to
to,
and begin to synthesize their own auxin. Cells can be maintained in embryogenic stage on
o induction medium for
prolonged periods (over 10 years in carrot). But in most crops, the
hrvogenic potential of cultures declines with time, and is eventuallylost.
The induction of
embryogenic state must involve the down-regulation of the current
nonembryogenic gene programme. This may be brought about DNA hypermethylation, which
c induced by auxins like 2,4-D. Auxins may affect somatic embryogenesis through
acidification of cytoplasm and/or cell wall. For example, wounded ZEs (zygotic embryos) of
Carrot form embryogenic cultures on GR-free medium; these cultures can be maintained in
the embryogenic state on a GR-free medium at pH 4.0 having NH as the sole source of
nitrogen. The new generation of GRs, e.g., oligosaccharides, jasmonates, polyamines and
brassinosteroids also induce somatic embryogenesis in some species.
The embryogenic cultures are maintained and proliferated in a medium similar to that
used for SE induction. Low pH of the medium is essential for maintaining the embryogenic
potential of the culture. Carrot PEMs cultured on a GR-free medium buffered at pH 5.8
promptly develop into SEs, while those cultured at pH 4.0 remain in the embryogenic state.
2,4-D is particularly effective for the establishment and maintenance of embryogenic cultures.
When ECs are transferred from induction medium to an appropriate medium, SE
differentiation proceeds from globular, heart-shaped, torpedo to cotyledonary stages; this is
called SE development phase. Clearly in such species, GR requirements for thetwo phases
are drastically different. In most cases, SEs begin to germinate immediately after the
coyledonary stage; this is called SE conversion. But often the plantlets so obtained are rather
weak. It is, therefore, desirable to subject SEs to a maturation phase following their
evelopment; in this phase the SEs usually do not grow but undergo biochemical changes to
ECome more sturdy and hardy. SE maturation is achieved by culturing than on a high sucrose
upo 6% or even 40%) medium or in the presence of a suitable concentration (0.2-0.4 mg/L)
ABA, or by subjecting them to partial desiccation (usually, achieved by enclosing SEs in
e , Sealed and empty Petri dishes). In most species, SE maturation improves their
steril
conversion, often by several-fold.
nere is some evidence that the physical factors in the culture environnment play an
tant role in the induction and development of somatic embryos and their conversion. In
and carrot, a period of starvation of embryogenic
cultures increased embryo development
lac veSion. Culture of the embryogenic calli on half-strength MS medium or medium
and conversion.
medium or medium
afaesucrose, or under conditions of reduced humidity (69.3%) increased SE production by
lackin
O 3.4 4.5. But starvation oftheembryogenic calli for5 days by culturing themin
12-well
production by
culturing them in
a
factor of 3.4 to 4
"plates without nutrient medium enhanced embryo produetion by 20-fold; it also

5. Plant Tissue Cultureand Plant Biotechnology
122
improved SE maturation and germination. In case of wheat, somatic
embrogenesis
promoted by 40 mM NaCI and KCI, but this effect was genotype-dependent.
Was
In some species, e.g., Cicerarietinum, wheat, etc., SE induction and development
take place on the same high auxin (5.5 mg/L 2,4-D in C. arietinum) medium, althoughth
frequency of mature embryos is rather low. In some species, SEs are produced in response to
a cytokinin, e.g., BAP induces SEs in hypocotyls of young zygotic embryos of Trifolium sn
pea, etc. But SEs are produced on immature cotyledons of these explants when 2,4-D is used
in the medium. It seems that cytokinins are effective in SE regeneration from embryogenic
cells of young zygotie embryos, while auxins are effective on differentiated cells of both
embryos and somatic tissues. Many workers have used combinations of auxins and cytokinins
for SE regeneration in different species, but the role of cytokinin in these systems is not
known.
may
In some species, SEs regenerate superficially from cotyledons and hypocotyls of
developing SEs and even from germinating SEs and plantlets; this is termed as secondary
embryogenesis or recurrent embryogenesis. In alfalfa, recurrent cycles of somatic
embryogenesis occur in GR-free medium, each SE giving rise to about 30 SEs. More often,
recurrent embryogenesis is initiated by maintaining the SEs on a lower level of auxin than
that used for their induction. In some other cases, the same auxin levels are used both for SE
induction and recurrent embryogenesis.
5.8.2. Nitrogen Source
The form of nitrogen has a marked effect on somatic embryogenesis. In carrot, NH, has a
promotive effect on SE regeneration. In fact, induction of SEs in carrot occurs only when
about 5 m mol/kg of cell fresh weight NH is present in the cells. This level of endogenous
NH is reached with only 2.5 m mol/L ofexogenous level of NH4, while 60 m mol/L NO;
is needed for the same. Therefore, the presence of a low level of NH, (in carrot 10 m mol/L IS
optimal) in combination with NOg is required for SE regeneration. In carrot, NH; is essentia
during SE induction, while SE development occurs on a medium containing NOG as the sole
nitrogen source. But in case of alfalfa, there is an absolute requirement for NH during
induction as well as differentiation of SEs; 5 mM NH; is optimum for SE induction, and1
20 mM is optimum for SE differentiation. In species like orchardgrass and alfalfa, C, H or d
ccmbination of certain amino acids promotes SE development and germinability of the SEs
5.8.3. Genotype of Explant
Explant genotype has a marked intuence on SE regeneration, and in many casesit n
determine whether or not SE regeneration will occur. For example in the case of species l
carrot and alfalfa, almost any and every explant shows embryogenic potentia But in many
other species, embryogenic potential is confined to embryonal or highly juvenil
Juvenile tissues
cereals like wheat are good examples, where immature ZEs have to be used for a consiste
and high frequency response. Strong genotypie effects have been shown in many specl
e.g., alfalfa, wheat, maize, rice, chickpea, etc. Irn case of wheat, chromosome 4B is importa
in regeneration, a major gene anteeng regeneration is located on the long arm
chromosome 2D, minor genes are present On the long arm of chromosome 2A and short a

6. 9osenerationand SomaticEmbryogenesis
Shool
of
2B,
and a regulato
e n e r a t i o
ation ability is mainly additive and highly heritable in maize, rice and wheat, but in
dominance seems to be more
important. In the cases of wheat, rice
atory gene is situated on the long arm of chromo:
123
nosome 2B. Variation for
cytoplasmha
sociated with mitochondrial genome.
barley
sm has a strong influence on
regeneration. In wheat, this effect appears to be
and maize,
byt
genoty
the GR regime during culture. It may, therefore, be postulated that at least a part of the
atvpic effect on regeneration may be concerned with endogenous GR levels and/or
In groundnut,
In groundnut, the relative regeneration potential of different cultivars is greatly influenced
sensitivity to exogenous GRs. In case of alfalfa, regeneration apacity is governed by two
jominant genes. In addition, recurrent
selection successfully improved regeneration of the
hybrid produced by crossing two poorly regenerating parents, viz.. Du Puits (10%
egeneration) and Sarnac (14% regeneration). The selected line, "Regan-s', showed 67%
regeneration, and is tetraploid (4x). Similarly, a diploid (2r) line of alfalfa, called "HG2, was
developed by chromosonme manipulation; the line HG2 shows 96% regeneration.
5.8.4. Explant
The type of explant has a strong influence on embryogenesis. Immature ZEs have been found
to be best explant for embryogenesis, e.g., in cereals, legumes, conifers, etc. In case of wheat,
the optimum stage of ZE development is 11-14 days after anthesis. But in few species like
alfalfa and carrot, almost all explants show embryogenesis.
5.8.5. Electrical Stimulation
Exposure of explants/cells to a mild electrical field may promote shoot/SE regeneration. In
case of alfalfa mesophyll protoplasts, exposure to an electric field of O.02 V DC for 20 hr
considerably promoted the embryogenic response. This promotive effect seems to be due to
changes in cell polarity effected by the organisation of microtubules. The electrical
Stumulation induces asymmetric first division coupled with a relatively short period of cell
expansion; these effects may be important in embryogenesis.
5.8.6. Other Factors
Lertain other factors are reportea to affect SE regeneration. For example, high K* levels and
OW dissolved O, levels promote SE regeneration in some species. In some other species, e.g..
us medica, some
volatile compounds
like ethanol inhibit SE regeneration. In soybean,
Sucrose
concentrations (5 and 10 g/L) promote
SE regeneration as compared to high
entrations (20 and 30 g/L). In alfalfa, use of maltose as carbon source improves both SE
o n and maturation
(including
germination)
as compared to those on sucrose.
Oyamines seems to be needed for ZE and SE development.
The globular SEs of celery
u a 37-fold higher
polyamine
content
than the plantiets.
Putrescine appears to be the
polyaminethat
some cases,
Shows the greatest
increase, e.g.,
in celery, mango,
etc. But in sc
nond
y o g e n i c
cultures
show a higher
polyamine
content
than the embryogenic
cultures.
case of carrot
Clopment,
while a higher
DO
favours rooting.
Reduced O, in the gaseous
mixture
E S S E r e g e n e r a t i o n
in wheat. Low O, level
reduces the
amount
of2,4-D
needed forSE
SE develo
TOt,
dissolved oxygen
(DO)
below the critical level of 1.5 mg L-l is essential for

7. hology
induction and suppres precocious germination of SEs. But in alf
Ifalfa, ahighe
entration of 88% or more supports a much higher frequency of SEs than Do
concen
concentration of 18%.
does DO
5.9. MOLECULAR ASPECTS OF SOMATIC EMBRYOGENESIS
A remarkable progress has been made in the analysis of molecular events during so
ryo-specific
somat
embryogenesis, and several genes that are either specifically expressed(embryo-spee
expressed at enhanced levels (embryo-enhanced) have been cloned. But the molecular.O
events
hat the shift
somatic cell from somatic to embryogenic development many depend on a balancebet
the products of genes that govern acquisition of embryogenic competence and of those
nat lead to somatic embryogenesis are far from clear. It has been suggested tha
tween
tha
Specity the somatic development. In this section, the molecular events during the onse
of
embryogenesis, SE pattern and organ formation, and SE maturation are briefly descrihed
5.9.1. Onset of Embryogenesis
This phase consists of the transition of somatic cell to such a state that it can
develop into an
SE without any further external stimulus. Several genes have been cloned that show enhanced
expressionduring early stages of somatic embryogenesis, e.g., DC5, CEM-6, ASET-1, ASET
2, Metl, Met2 (both encode DNA methyl transferases), LECI, LEC2, SERK, etc. The genes
LECI and LEC2 (LEC, leafy cotyledon) were identified in Arabidopsis thaliana; they encode
transcription factors, and cause spontaneous SE formation in the leaves of transgenic plants
when either of them is ectopically expressed. Ectopic expression signifies the expression of a
gene in time and/or place different from its natural expression. The gene SERK (s0matit
embryogenesis receptor kinase) encodes akinase that most likely functions as areceptor for
an unknown ligand. SERK is expressed in flowers between 3 and 20 days after pollination.
and in carrot hypocotyls between 7 days after their culture in the presence of 2,4-D and upro
100-celled globular embryos. It is possible that the onset of somatic embrogenesis may
represent a signal transduction pathway beginning with the binding of a ligand (which 1sy
unknown) to a
receptor like SERK, and ending with the expression of transcription aco
like LECI and LEc2 that would activate genes responsible for embryogenic developmen
5.9.2. SE Pattern and Organ Formation
This stage signifies the start of cell division in a cell with embryogenic comperc
organisation ofthis cell mass into an SE. The cells of early zygotic embryos are
no
strong
determined in terms of the course they follow in
differentiation; as a result, they arcni
are
prone0
ogen
physiological and biochemical disturbances. The successful establishment or
c
pattern and completion of embryo differentiation requires the action of many
genes in A. thaliana. Transitions from globular to heart-shaped, and from heanes
genes,e.g
ped
to
m a y
topedo stages require activation of
specific sets of genes. The activation of thed
be initiated by the so-called "master
regulatory genes", which themselves moy
beginning of an
activating cascade of reactions.
Several genes concerned with SE
differentiation, e.g., CUSI (a MADS-bOX
a y
E"
c o n t r o l
the
etc., and those listed in Table 5.2, have been cloned and investigated. A pro
ne)D
t o d e r n

8. oot Regeneration and
SomaticEmbryogenesis
hoo 125
rotod
ment appears to be critical for transition of globular SEs to heart-shaped SES.
devrm development is affected by carrot genes EP2, EP3 and the genes encoding AGPs
protgalactan proteins). AGPs secreted into the medium by embroygenic cultures of
araan reinitiate somatic embryogenesis in
non-embryogenic cell lines. In a
temperaturc
xpressionof
Cave mutant, Ts11, of carrot, SEs are arrested at
globular stage; this is relieved by
Sesion of EP3, which encodes an extracellular chitinase. It has been postulated that the
sens
itive mutant,
chitinases activate
exases activate AGPs by cleavage, and the cleavage products sponsor the transition of
eARIE 5.2. Some ofthegenesthat are likely to be involved in SE differentiation
TA
Gene Gene production Expression pattern/ function
Accumulates specifically at heart-shaped and
early torpedo stages; genes CHB-1 to CHB-6 of
carrot may be involved in vascular element
differentiation
CHB-2 Homoeoprotein
EP2 Lipid transfer protein (LTP) Expressed in embryogenic carrot cultures; marks
acquisition of embryogenic potential in carrot
Expression as in the case of EP2
LTP may be involved in protodermformation
Involved in normal protoderm formation; may
activate AGPs by cleavage, which liberates
lipochito-oligosaccharide-like molecules
AtLTP1 LTP
EP3
Extracellular endochitinase
TSII Extracellular endochitinase
Arabinogalactan proteins (AGPs) Initiate SE formation in nonembryogenic cell
lines of carrot; involved in transition from
globular to heart-shaped SEs; likely to be
activated by endochitinases
globular stage SEs to heart-shaped ones. It is possible that the chitinases may initiate a signal
transduction cascade needed for progression of globular SEs to heart-shaped stage through a
correct positioning of cell division planes. Further work is needed to demonstrate this
possibility and to identify the components of this cascade. Another group of proteins, called
upid transfer proteins (LTPs), is also involved in protoderm formation. For example, the
expression of genes EP2 (of carrot) and AtLTP-/ (of A. thaliana) encode LTPs, and their
CApression is highly correlated with the embryogenic potentialofcellcultures; it is likely that
Apression of EP2 marks the acquisition of, and the degree of embryogenic potential in
carrot cell cultures.
5.9.3. SE Maturation
Veral genes are expressed during the later stages of embryo development; some of such
s are listed in Table 5.3. Proteins of the LEA (late embryogenesis abundant) group are
genes
dOst studied of these proteins. These are very hydrophilic, are produced abundantly
h e later stages of SE and ZE development,
and the promoters
of their genes contain
ring the later stage
ABA
AEIy to be involved in the protection of cellular
structures during desiccation of
embryos.
are
:ponsive
elements (ABRE) so that their expression is induced by ABA. LEA proteins

9. Plant Tissue Cultureand PlantBiotechna
nology
126
TABLE 5.3. Some of the genes
expressed during
m a t u r a t i o n of SEs
Expression and ABA-responsiveness
Gene
Gene product
Expressed in SEs and ZES poorly expressed in eal
ABA inducible
calli;
DC8
LEA protein
Expression high in SEs, poor in calli; ABA inducible
Expression very low in calli; accumulates in SEs
High
accumulation in SE cotyledons
DC 3 LEA protein
EMB-1
LEA protein
PgEMB 12, LEA proteins
14. 15.
Expressed in SEs at cotyledonary stage; ABA-responsive
(also modulated by osmoticum and metal ions)
Strongly induced at globular and heart stages of SES
detectable in embryogenicecalli
PM 2.1 Metallothionein
Gca 8
Globulin-like
*
LEA, late embrogenesis abundant; SEs, somatic embryos; ZEs, zygotic embryos.
Carrot gene DC8 encodes a LEA protein, which is hydrophilic. DC8 protein is produced
in only those tissues that are either derived from embryo, e.g, cotyledons, or that are capable
of giving rise to embryogenic calli. In such tissues, ABA can increase the expression of gene
DC8& by 100-fold in 24 hr, while a 50-fold increase occurs in 15 min. Many other LEA genes
are highly expressed in embryogenic cells during the somatic embryogenesis, which may be
due to the high levels of endogenous ABA detected in embroygenic cell elusters.
In addition, genes encoding low molecular weight heat shock proteins (LMWHSP) show
a shift in regulation of their expression: regulation shifts from transcription level to
translation level during early embryogenesis, then shifts back to transcription level regulation
after some time. In addition, exposure of globular stage SEs of carrot to heat shock at 37C
for 2-3 hr can permanently block their development. In case of alfalfa, a LMWHSP was
expressed transiently in developing SEs without any heat shock, the expression being
abolished in torpedo stage embryos. But the nonembryogenic suspension cultures expresSeu
this LMWHSP only in response to a heat shock. It has been suggested that these LMWHS
are expressed in response to ABA and they may protect SEs and ZEs during desiccation.
Further studies are required to clarify the roles of LMWHSP during somatic embryogenesis.
5.10. CONCLUDING REMARKS
Plant tissue culture is the basic tool in plant biotechnology since it enables regeneration
complete plantlets from both somatic and gametic cells. Regeneration occurs either as sno
or SE, and the regeneration potential is greatly affected by the physiological state o
explants. For example, regeneration is limited to embryonic tissues of most cereals, gr
f the
rain
egumes, cotton, tree species, etc. and their older tissues have so far remained recalcd
trant.
The realisation of significance of explant type and the genotype of donor plant nas
d
the
development of regeneration systems in
many such species that were earlier regahle
recalcitrant. Regeneration events have been shown to occur in experimentally sepo
steps, each step responding differently to various GRs involved in regeneration. 'Ie
GRs influencing regenerationevents has expanded remarkably over and above the clat0
auxins and cytokinins. Both shoot and, particularly, SE regeneration appear to
resp

10. shoot
Kegeneration
and SomaticEpnbryogenesis 127
chemical stresses. In addition, some proteins secreted by embryogenic
c e l l s o f c a r r o t
n e c i e s ,S Em a t u r a t i o n
ical to the
practical
v a r i o
hysicaland
iousrrot are able to inauce sE regeneration in nonembryogenic cultures. In a
s SEmaturation and conversion remain problematic and resolution of this bottleneck is
he practical utilisation of SEs. It may be expected that as our understanding of the
nts responsible for regeneration events increases, the regeneration events may
molecular
blecmore and more amenable to control. One may even hope to be able to identify and
ecohe genes that trigger various stages of regeneration, and transfer them into otherwise
i s o l a t e
citrantspecies to make them amenable to regeneration.
MODEL QUESTIONS
SECTION A
1 Explain the meaning of "regeneration'.
2 With reference to regeneration, explain the meaning of 'induction'.
3. What are tissue cultureresponse gene?
4. How do somatic embryos differ from shoot buds ?
5. Explain the role of Lea proteins in somatic embryogenesis.
SECTIONB
1. Write short notes on: (i) induction of shoot regeneration, (ii) role of growth regulators in shoot
regeneration, (iii) role of growth regulators in somatic embryogenesis, (iv) somatic
embryogenesis, (v) nmolecular aspects of somatic embryogenesis, (vi) role of explant genotype
in tissue culture response.
SECTIONC
. Discuss the various aspects of shoot regeneration from cultured plant cells and tissues.
2. Describe the various events during somatic embryogenesis.
3. Give a brief account of the molccular aspects of somatic embryogenes.