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102
CHAPTER 3
SUSPENSION CULTURE, SOMATIC
EMBRYOGENESIS, PREPARATION AND
GERMINATION OF SYNTHETIC SEEDS
103
Introduction
Techniques of micropropagation or in vitro cultivation have emerged as
alternatives for species that do not have the property of producing viable seeds, that is,
species that cannot germinate and develop adequately in their natural environment
(González Paneque et al., 2004). The application of plant tissue culture technique is
one of the most frequently used strategies for commercial micropropagation of plants.
Somatic embryogenesis is the most widely adopted regeneration system for many
plant species (Malabadi et al., 2004; Malabadi & van Staden, 2006; Aronen et al.,
2007; Park et al., 2009; Malabadi & Teixeira da Silva, 2011; Malabadi et al., 2011).
The first description of in vitro somatic embryo production were carried out
independently while working with carrot (Steward et al., 1958; Reinert, 1959).
Recently it is noticed that the pioneer role of Harry Waris, working with Oenanthe
aquatic (Umbelliferae) (Krikorian & Kaarina Simola, 1999). The early history of
somatic embryogenesis has been reviewed elsewhere (Raghavan, 1986; Halperin,
1995). The application of biotechnology in plant breeding requires efficient in vitro
regeneration procedures. Somatic embryogenesis is a desirable method of plant
regeneration (Williams & Maheswaran, 1986). Redenbaugh et al., (1984) for the first
time developed a technique for hydrogel encapsulation of individual somatic embryos
of alfalfa, subsequently Fujii et al., (1987) immersed somatic embryos in a protective
matrix constituting an artificial or synthetic seed, providing a convenient method for
the propagation by cloning of elite plant varieties or species that are difficult to
propagate in their natural environment. Plant regeneration via somatic embryos start
with one or only a few cells, this type of regeneration is important for plant
multiplication, mass production and plant biotechnology such as clonal propagation
and especially genetic transformation (Gordon-Kamm et al., 1990).
Finding the right conditions to induce somatic embryos in different species
and cultivars is yet, for the greater part, based on trial and error experiments
(Jacobsen, 1991; Henry et al., 1994) analysing the effect of different culture
conditions and media and modifying especially the type and levels of plant growth
regulators. Somatic embryos can be encapsulated in various gelling systems to form
artificial seeds which are easily stored and transported to long distances (Ghosh &
Sen, 1994). The use of somatic embryos as artificial seeds is becoming more feasible
104
as the advances in tissue culture technology define the conditions for induction and
development of somatic embryos in an increasing number of plant species (Jain et al.,
2011). Due to the presence of well-developed root and shoot primordia, somatic
embryos germinate easily to produce plantlets without an additional step of rooting
(Laux & Jurgens, 1997). They have also been used in commercial plant production
and for the multiplication of parental genotypes in large-scale hybrid seed production
(Bajaj, 1995; Cyr, 2000). The success of this technique depends on the development
of a series of processes that influence the genotype of the mother or donating explant
and the concentration of exogenous plant growth regulators, which in adequate
combinations would allow for obtaining a determinant embryogenic response for the
production of somatic embryos (Guerra et al., 2001). In this pathway, cells or callus
cultures on solid media or in suspension cultures form embryo-like structures called
somatic embryos, which on germination produce complete plants. The primary
somatic embryos are also capable of producing more embryos through secondary
somatic embryogenesis.
Although, somatic embryogenesis has been demonstrated in a very large
number of plants and trees, the use of somatic embryos in large-scale commercial
production has been restricted to only a few plants, such as carrot, date palm, and a
few forest trees. Somatic embryos are produced as adventitious structures directly on
explants of zygotic embryos, from callus and suspension cultures. Somatic embryos
and synthetic seeds hold potential for large-scale clonal propagation of superior
genotypes of heterogeneous plants (Redenbaugh, 1993; Mamiya & Sakamoto, 2001).
Suspension cultures of plant cells are becoming increasingly important as
experimental material for investigations concerning plant growth and secondary
metabolism. The induction of somatic embryos by suspension culture may constitute a
viable means of rapid clonal propagation. The use of somatic embryos for clonal
propagation and artificial seed production, and their cryopreservation in germplasm
banks, would be beneficial for the medicinal important species. Somatic
embryogenesis of plants in suspension is a potentially useful system for the rapid
propagation of plant material (Vasil, 1984; Osuga & Komamine, 1994). An
embryogenic cell suspension consists of a heterogeneous population composed of
smaller and larger cells either single or cluster of different sizes. Even in cultures with
a high level of embryogenic capacity only a small proportion of the cells will be able
105
to undergo embryogenesis. However, it is still difficult to predict which of these cells
will develop into somatic embryos. A closer examination of the surface of an
embryogenic callus suggests that embryogenic cells are characterized by having
denser cytoplasm than surrounding cells (Christopher et al., 2003).
In suspension, proembryogenic cell clusters form and can be separated from
the single cells and the larger clumps of callus by sequentially sieving through nylon
membranes of 500 and 224 mm pore size. The suspension, however, cannot be
maintained in this embryogenic state. If the 224-500 mm embryogenic fraction is
placed back into suspension, the single cell population increases, the embryogenic cell
clusters disperse and they become a progressively smaller proportion of the culture
(Lai & McKersie, 1994). Identifying techniques to stabilize these embryogenic
cultures is critical for the scale-up of this technology. The organic and inorganic
components of the liquid suspension culture medium have been precisely defined,
also physical factors including inoculants density (weight of callus added to 40 ml of
liquid suspension) are also critical and show precise optimal values (McKersie &
Brown, 1996). Redenbaugh et al., (1984) developed a technique for hydrogel
encapsulation of individual somatic embryos of alfalfa. Since then encapsulation in
hydrogel remains to be the most studied method of artificial seed production
(Redenbaugh & Walker, 1990; McKersie & Brown, 1996). A number of substances
like potassium alginate, sodium alginate, carrageenan, agar, gelrite, sodium pectate,
etc. have been tested as hydrogels but sodium alginate gel is the most popular
(McKers & Bowley, 1993). However, Molle et al., (1993) found that for the
production of synthetic seeds of carrot, 1% sodium alginate solution, 50 mM Ca2+ and
20–30 min were satisfactory for proper hardening of calcium alginate capsules. They
have suggested the use of a dual nozzle pipette in which the embryos flow through the
inner pipette and the alginate solution through the outer pipette. As a result, the
embryos are positioned in the centre of the beads for better protection. The technology
of hydrogel encapsulation is also favoured for synthetic seed production from these
micropropagules (Redenbaugh & Walker, 1990; Redenbaugh, 1993; Ara et al., 1999).
Synthetic seeds can be produced either as coated or non-coated, desiccated
somatic embryos or as embryos encapsulated in hydrated gel (Redenbaugh, 1993).
Successful utilization of synthetic seeds as propagules of choice requires an efficient
and reproducible production system and a high percentage of post-planting conversion
106
into vigorous plants. Artificial coats and gel capsules containing nutrients, pesticides
and beneficial organisms have long been thought as substitutes for seed coat and
endosperm (Bajaj, 1995). However, this technology is still in the developmental stage,
and currently cannot compete with the other methods of commercial plant
propagation (Cyr, 2000).
Artificial seed production is a potential technique for plant multiplication and
preservation, especially as it has been considered to be promising for propagation of
non-seed producing plants, transgenic plants and other plants that need to keep
superior traits by means of asexual propagation (Saiprasad, 2001). Plant artificial seed
in a narrow sense, means the beads formed by encapsulating somatic embryo with
coating materials. Its effect varied with different species, coating materials,
maintained solutions and its concentration and condition (Nhut et al., 2005).
Nowadays, it is widely used in Paulownia elongata, Chrysanthemum morifolium,
Cymbidium spp respectively (Ipekci & Gozukirmizi, 2003; Halmagyi et al., 2004;
Nhut et al., 2005). Currently, systems of artificial seed production have progressed
substantially in this area, the most advanced being in seeding under ex vitro or field
conditions, obtaining high percentages of conversion to plants (Nieves et al., 2003).
There are several potential uses of synthetic seeds of those crop plants that are
vegetatively propagated and have long juvenile periods, e.g. citrus, grapes, mango,
etc. The planting efficiency of such crops could theoretically be increased by the use
of synthetic seeds instead of cuttings. Synthetic seeds have been found highly
advantageous for germplasm conservation in grape and other similar crops (Gray &
Purohit, 1991). Moreover, establishment of synthetic seeds have multiple advantages
including ease of handling, potential long-term storage and low cost of production and
supsequent propagation (Ghosh & Sen, 1994). Hence, synthetic seed technology
seems promising for propagating a number of plant species despite the fact that it has
limited production of viable micropropagules useful in synthetic seed production, its
development is anomalous and asynchronous (Ara et al., 2000).
Review of literature revealed that preparation of synthetic seeds is not
attempted on Trianthema decandra and this is the first report on synthetic seed
preparation in aforesaid plant. Therefore, the aim of the study is to evaluate the best
medium for the bead among different media (MS, B5 and Nitsch media) at different
107
concentrations of growth regulators, best percentage of alginate and to investigate
long-term storage with regard to viability and germination in Trianthema decandra. In
the present investigation somatic embryos isolated from suspension cultures were
used for encapsulation.
Review of Literature
The concept of synthetic seed was given by Murashige et al., (1990), but first
report on the development of synthetic seeds was published by Kitto and Janick
(1982). They reported the production of desiccated synthetic seeds by coating a
mixture of carrot somatic embryo in a water-soluble resin, polyoxyethylene glycol
(Polyox). Later, Redenbaugh et al., (1984) were successful in producing synthetic
seeds for alfalfa by encapsulating somatic embryos with alginate hydrogel. Since then
several research groups have been working on synthetic seeds with different plant
species including cereals, fruits, vegetables, ornamentals, medicinal plants, forest
trees and orchids (Mathur et al., 1989; Ganapathi et al., 1992; Corrie & Tandon,
1993; Maruyama et al., 1997; Rao et al., 1998; Mandal et al., 2000; Rout et al., 2001;
Sicurani et al., 2001; Nyende et al., 2003; Chand & Singh, 2004; Niranjan &
Sudarshana, 2005; Naik & Chand, 2006; Singh et al., 2006; Micheli et al., 2007; Rai
et al., 2008).
The main advantage of these non-embryogenic vegetative propagules would
be in those crops where somatic embryogenesis is not well established or do not
produce uniform quality embryos. In such cases synthetic seed system may be useful
for the propagation and delivery of tissue cultured plants (Rao et al., 1998). Based on
the technology established, there are two types of synthetic seeds: hydrated and
desiccated. Although, the most studied method involves the encapsulation of
propagules in hydrogel for synthetic seed production (Redenbaugh & Walker, 1990).
Due to absence of a nutritive tissue like the endosperm of the natural seed, synthetic
seeds have low conversion ability in some cases (Kumar et al., 2005). Addition of
nutrients, carbon sources, growth regulators and antimicrobial agents such as
antibiotics, fungicides etc. in the gel matrix which apparently served as a synthetic
endosperm, facilitated growth and survival of encapsulated propagules (Redenbaugh
et al., 1987; Gray, 1990; Bapat & Mhatre, 2005). Such additives should be non-toxic
to propagules and allow the development of plants without any variation (Redenbaugh
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& Ruzin, 1989; Bapat & Mhatre, 2005). Hindrance of the gel capsule for the
emergence of the root and shoot from encapsulated propagule is another mechanical
problem in encapsulation technology, although, adopting the self-breaking alginate
gel beads technology could overcome this shortcoming (Onishi et al., 1994). Calcium
alginate capsule pretreated with potassium nitrate becomes soften and allow the easily
emergence of shoot and root from alginate beads (Onishi et al., 1994). Application of
potassium nitrate in the breaking of alginate capsule has also been reported in a few
plant species (Guerra et al., 2001; Kumar et al., 2005).
According to the literature, somatic embryogenesis for a variety of plants has
been achieved using a variety of media ranging from relatively dilute White’s medium
to the more concentrated formulations of somatic embryogenesis , over 70% of the
successful cases used MS salts or its derivatives. Of the plant growth regulators, auxin
is known to be essential for the induction of somatic embryogenesis in some plant
species, although 2,4-D is the most commonly used auxin. Somatic embryo induction
is usually promoted by auxins (Williams & Maheswaran, 1986). In some plant
species, a combination of 2,4-D or NAA with cytokinin was reported to be essential
for the induction of somatic embryos (Kao & Michayluk, 1981; Gingas & Lineberger,
1989; Schuller et al., 1989). Inorganic components in the medium such as potassium,
and organic components such as proline can modulate the embryogenesis or callus
response, but they cannot replace auxin (Shetty & McKersie, 1993). Embryogenic
callus culture of carrot (Daucus carota L.) was induced from the hypocotyl explants
on MS medium supplemented with casein hydrolysate, 2,4-D and BAP for somatic
embryogenesis in vitro cell suspension cultures for high multiplication rate (Latif et
al., 2007). The highest induction frequencies of somatic embryos were obtained on
MS medium supplemented with NAA 1.0 mg/l and kinetin 2 mg/l and 3% sucrose
(Devendra et al., 2011).
Many investigators have obtained plantlets by encapsulating somatic embryos.
Padmaja et al., (1995) obtained plantlet by encapsulating somatic embryos of
groundnut. Prewein and Wilhelm (2003) obtained plants from encapsulated somatic
embryos of Quercus rubur. Ipekci and Gozukirmizi (2003) obtained direct somatic
embryogenesis and produced synthetic seed in Paulownia elongata. Nieves et al.,
(2003) worked by field performance of artificial seed derived sugarcane plant. Kumar
et al., (2005) enhanced synthetic seed conversion to seedlings in hybrid rice.
109
Malabadi & Staden (2005) have done storability and germination of sodium alginate
encapsulated somatic embryos derived from the vegetative shoot apices of mature
Pinus patula trees. Niranjan and Sudarshana (2005) encapsulated somatic embryos
from leaf derived embryonic callus in Lagerstroemia indica. Germaná et al., (2007)
obtained synthetic seeds by encapsulating somatic embryos from in vitro anther
culture of Citrus reticulata. Daud et al., (2008) reported that 3% sodium alginate is
best in Ananas comosus and Saintpaulia ionantha.
Effect of different type of medium on in vitro morphogenic response of
synseed of Rauvolfia tetraphylla L. (Apocynaceae) an endangered evergreen woody
medicinal shrub was evaluated. The maximum frequency (90.3%) of conversion of
encapsulated beads into plantlets was achieved on woody plant medium (WPM)
containing 7.5 µM BA and 2.5 µM NAA after 6 weeks of culture. Encapsulated nodal
segments stored at 4°C for 1 to 8 weeks also showed successful conversion, followed
by development into complete plantlets when returned to regeneration medium
(Alatar & Faisal, 2012).
Maqsood et al., (2012) have worked on synthetic seed development of
Catharanthus roseus (L.) G. Don of the family Apocyanaceae and is one of the most
widely investigated medicinal plants. They reported that different levels of sodium
alginate and calcium chloride were used in which perfect bead formation was
observed in condition encapsulated with 2.5% sodium alginate and 100 mM calcium
chloride solution. Synthetic seeds were kept at 0, 4 and 25°C as to examine the best
storage temperature; preservation at 4°C was found to be the optimum temperature for
embryo storage and germination purposes. The encapsulated embryos were preserved
up to 10 weeks or more without losing germination abilities.
Research has been done on Clitoria ternatea Linn. an important medicinal
climber. Synthetic seeds were produced by encapsulating embryos in calcium alginate
gel. The highest synthetic seed germination (92 %) was observed on MS medium
supplemented with 2 mg/l BA and 0.5 mg/l NAA. The synthetic seeds were stored at
4°C and lab conditions (25 ± 2 °C) up to 5 months. Synthetic seeds germinated and
were transferred to soil successfully (Krishna Kumar & Thomas, 2012). As synthetic
seed technology is useful for germplasm conservation, present investigation is aimed
to prepare and germinate the synthetic seeds of Trianthema decandra and to record
110
the percentage of germination and subsequently the survivability of the germinated
plant on the soil.
Materials and Methods
Callus induction and establishment of cell suspension
A number of combinations of different media and explants were assessed for
callus induction. Fast growing, light green to creamy white friable callus was
established from the explants and maintained on MS medium supplemented with 1.5
mg/l BAP + 0.5 mg/l NAA and 2.5 mg/l Kn + 0.5mg/l NAA, after 6 weeks of
subculture to induce somatic embryos. Callus was subcultured every 4 weeks on MS
medium. After 4-6 passages on this medium, 2.0 mg fresh weight of fast growing
callus was inoculated into 25ml aliquots of liquid medium in 100 ml Erlenmeyer
flasks. Three types of liquid media were used; MS, B-5 & WM containing BAP (1.5
mg/l) as in the original formulation with the addition of 0.5 mg/l NAA. After
inoculation aseptically they were transferred onto a rotary shaker (100 rpm) under the
same growth conditions. After obtaining fine cell suspension, rpm is adjusted to 60-80
for cell division and embryoid formation. In the mean time viability of the cells was
detected by staining cells using Evan’s blue dye. Observations were made using a
drop of cell suspension for the presence of embryoids stained with Heidenhein’s
haematoxyline stain.
Embryogenic calluses of about 0.2-0.5g were chopped into small pieces and
transferred to 100 ml. Erlenmeyer flasks that contained 25ml of liquid MS medium
supplemented with GA3 alone or GA3 with NAA. The flasks were maintained on a
gyratory shaker at 100 rpm. The suspension cells were subcultured biweekly by
transferring 2ml into 25ml fresh liquid medium. Cultures were incubated at 22oC on a
platform shaker. Suspension cultures were subcultured at two week intervals. After 4
weeks of culture, suspension was observed under stereo microscope for the presence
of embryoids. Once detected, they were harvested and evaluated. After 4 weeks of
growth a finely dispersed homogeneous cell suspension culture was obtained. The
suspension cultures comprised mainly of round, densely cytoplasmic starch
containing cells with distinct nuclei. Some cells are large elongated and highly
vacuolated with little cytoplasm. The suspension cultures were subcultured routinely
on fortnight basis by transferring 5 ml of suspension culture into 25 ml fresh medium
111
using a wide bore pipette. For maintenance, fine suspension is necessary to subculture
them because the cultures tend to form cell clusters of a few cells to aggregates. The
growth curve of suspension cultures indicated that the growth rate of cells were
initially slow during first few days but as the cultures proceed they showed a
remarkable increase from day 10 and significantly accumulated great amount of fresh
weight over a period of 14 days. Maximum increase in fresh weight was reached on
day 28 which was about 4-6 fold over initial fresh weight.
Viability of the cells
One of the requirements for the establishment of cell culture is to count on a
reliable and efficient method to estimate cell viability. The cell viability can be
evaluated by staining the dead or living cells, because the colour is a product of cell
metabolic activity (Widholm, 1972). The most used stain for dead cells is Evans blue
or methylene blue. The Evans blue is reduced by the living cells turning colourless
while the dead cells remain blue. In the present study, Evans blue is used as a staining
method because of economical, reliable and can be observed easily under light
microscope.
Evaluation of suspension cultures
Initial cell suspensions that gave rise to a high frequency of abnormal structures
during the evaluation were discarded while those producing normal bipolar embryos
were retained by subculturing every 4 weeks. Suspensions were evaluated for
embryogenic potential by assessing;
a. Frequency of single elongated vacuolated cells
b. Organized or unorganized clusters of cells with dense cytoplasm.
c. Proembryogenic masses consisting of elongated vacuolated cells and
d. Globular, cordate, spindle, torpedo-shaped embryos.
Encapsulation, preparation of beads and storage of synthetic seeds
Somatic embryos of various shapes such as globular, clavate, spindle, torpedo
& cotyledonary were carefully isolated from embryo cluster and were bio dried on
filter paper. The isolated embryos were dipped in previously autoclaved 1-4% sodium
alginate prepared in distilled water with or without nutritive additives. The embryos
112
were then picked up by forceps inserted into a drop of alginate falling from separating
funnel and dropped into the solution of CaCl2 2H2O. The drops (beads), each
containing single embryo, were kept in this solution for 40 minutes on a gyratory
shaker in light. After the incubation period, the beads were recovered by decanting the
CaCl2 2H2O solution and finally, these beads were washed three times with double
sterile distilled water to remove all the traces of calcium chloride.
One set of encapsulated embryos were then stored in 120 x 20mm petri dishes
sealed with parafilm and left for 0 – 60 days in darkness at low temperature 4oC.
Another set of retrieved encapsulated beads stored at 4oC to 22o C for 30 days, 60
days and 90 days. After storage, the embryos were transferred to maintenance
medium & were incubated under the growth chamber conditions as described earlier.
The cultures were evaluated after 4 and 6 weeks to know the percentage of
germination, elongation and rooting of synthetic seeds. Well-rooted shoots
regenerated from encapsulated embryos were transferred to 10 cm diameter pots
containing a mixture of sand, peat and soil (3:3:4, v/v/v).
Effect of different media and germination conditions
For germination, the synthetic seeds were placed on different types of media
supplemented with various PGR’s. Cultures were incubated at 20ºC ± 2oC under
white fluorescent light intensity of 50 µmol m-² s-¹, at 70-80 % relative humidity with
16/8 hour day/ night photoperiod. 64 seeds were used per treatment.
Effect of percentage of sodium alginate and its composition on growth ability of
encapsulated embryos
This investigation was performed to study the effect of percentage of sodium
alginate (1.0 to 4.0 %) used to encapsulate the somatic embryos. In addition, to study
the effect of alginate bead composition on shoot and root emergence, somatic
embryos were immersed in sterilized mixtures of 2 or 3% (w/v) sodium alginate +
distilled water + sucrose (1.5%) + GA3 (0.5 or 1 mg/l). Percentage of shoot and root
formation was recorded after 6 weeks of culture.
113
Effect of plant growth regulators on non stored synthetic seeds
Encapsulated somatic embryos were cultured on MS medium supplemented
with different combinations and concentrations of growth regulators and additives.
Germination percentage of encapsulated embryo was recorded after 6 weeks of
culture.
Results
Material source
Induction of callus from various explants has been explained in tissue culture
chapter. However, friable and nodular callus has been utilized for the establishment of
suspension cultures.
Establishment of embryogenic suspension culture
Friable calli were subcultured on MS liquid medium supplemented with
different concentrations of growth regulators such as IBA, GA3 and ascorbic acid.
After 7 days, the calluses releases single cells and small clusters of 10-20 cells, and
irregular aggregates into the medium of shaking cultures at 100rpm. Microscopic
observations of crude suspensions showed a great variability in cell size. Upon
transfer to basal suspension medium friable callus released embryogenic cells.
Callogenesis and organogenesis of the cell suspension derived from leaf and internode
calluses were dependent on medium composition and sequences. Suspension cultures
(Fig 3.1) of one week consisted of heterogenous cells, where the embryogenic cells
were spherical, elliptical (Fig 3.2) and elongated in shape with a lot of vascuoles. In
the 3rd week of suspension high percentage of highly vaculated cells, xylem elements
(Fig 3.3) dumble-shaped cells and filamentous cells were observed.
The subculturing procedure and induction of suspension culture was most
successful when the initial callus was in the exponential growth phase. The growth
rate curves of friable and compact biomass of both leaf and stem of Trianthema
decandra L. showed that the rate of friable callus growth was generally higher than
the rate of compact callus growth. This difference was most noticeable during 3rd and
4th weeks. On comparing the biomass growth of leaf and stem suspension cultures of
114
Trianthema decandra significant growth rate was observed in leaf suspension
cultures.
Initiation and proliferation of embryogenic cultures
The effect of various growth regulators on the growth rate of cultures was also
examined. Compact callus placed in the liquid subculture medium containing IBA,
GA3, and ascorbic acid at different concentrations improved the friability of the
callus, allowing for the production of finer cell suspensions. The agitated liquid
medium contained free cells, in the form of cell suspensions. It also possess cell
aggregates , xylem elements and vacuolated cells measuring about 0.5-3.0 mm in
diameter and a few organized ones. Five-week-old clusters became nodular and
somatic embryos at different stages of development with different shapes (Fig 3.4) as
globular, dumbell and cordate with suspensor were visible after 3- weeks. The
embryogenic cells were observed as yellowish white in colour. The yellow
pigmentation of cytoplasm indicated the embryogenic character of the suspension
cells. Cells stained with 0.1% hematoxyline showed more than 95% viability.
Cultures displayed the fastest growth rate and reduced lag phase in the media
supplemented with IBA, GA3, and ascorbic acid. In these media, the cell culture
reached stationary phase after five weeks.
Embryogenic development was achieved following the transfer of cell clusters
into liquid medium supplemented with IBA, GA3 and ascorbic Acid. Friable callus
derived from MS medium developed into greenish suspension with small cell
aggregates in liquid conditions. Cultures initiated and maintained on the same
medium after 3 weeks sporadically showed well-organized somatic embryos. Transfer
of this from solid medium to the fresh liquid medium of the same composition gave
rise to green suspension clumps of varying size. After stabilization of the cell
suspension, somatic embryos were observed after 6 weeks. During the course of the
culture different types of somatic embryos were observed. The cytoplasmic rich cells
were spherical, elongated or intermediate between these two shapes. From the
meristematic zone of cells a single celled proembryo (Fig 3.5) had taken its origin
with a single stalk cell. Initially the elongated cells divided transversely and produced
two equal or unequal cells. The basal cell by several transverse divisions formed
linear suspensor consisting of 2-4 cells (Fig 3.6). During the second subculture, the
115
pro-embryo like structure consisting of 8-16 cells (Fig 3.7) was developed. In
subsequent subculture further division in the proembryo like structure was at the
periphery rather than near the centre resulting in globular structure. In the next
subculture heart-shaped structure was observed from the globular shape. The heart
shaped structure showed a well developed epidermis and meristematic tissues which
is surrounded by parenchymatous tissues. Typically many of embryos were connected
to each other, and as 2 embryogenic cells divided and became 4 embryogenic cells
(Fig 3.8). The nuclei dividing vacuolated cells are commonly more spherical.
Embryogenic suspension cultures contained yellow calli, embryogenic, non-
embryogenic cell aggregates and free cells after 20 days. The clusters of fused and
individual embryos at various stages of development were observed.
The effects of cytokinins and auxins on the growth of suspension cells were
found to be significant for Trianthema decandra. The initial suspension contained cell
aggregates of which 12% were embryogenic cell aggregates. When callus was
transferred to liquid culture, white, friable callus was broken apart and dispersed into
clumps. Further agitation fragmented these clumps into small cell aggregates. The
cultures grown with 0.5 mg/l GA3, 1.0 mg/l IBA and 60 mg/l ascorbic acid included
mostly calli of non–embryogenic cell aggregates and isolated free cells, as the
concentration of GA3 was increased from 0.5 to 1.0 mg/l with IBA (1.0 mg/l) and
ascorbic acid (60 mg/l), calli were composed of embryogenic and non- embryogenic
cell aggregates and became more numerous besides the production of xylem elements.
Induction of somatic embryogenesis
After 6 weeks of culture all the replicates grown in embryogenic induction
media developed nodular calluses which initially looked like creamy–yellow globular
structures of 1 to 2 mm3 with a smooth and slack appearance. Between 70 to 80% of
the cultures from all treatments showed nodular calluses as against 20 to 30% of
compact ones which showed many round hyaline cells on their periphery. After two
weeks in liquid medium developed embryogenic calluses, creamy white and with
loose granular structure having numerous somatic embryos on their periphery. The
embryogenic cells were usually small, round with dense cytoplasm and a large
nucleus in relation to the cell size. The embryogenic cell aggregates were represented
mainly by cell masses with conspicuous embryogenic cells on their surface. The non –
116
embryogenic cells were larger than the others, had diverse shapes, predominantly
elongated with little cytoplasmic contents, a large vacuole and a small nucleus.
Effect of different media on germination of synthetic seeds
The best nutrient media was found to be MS+1.0 mg/l BAP followed by
B5+1.0 mg/l BAP and MS basal media.
Effect of different concentrations of sodium alginate on germination
For artificial seed production (Fig 3.9), alginate gels seemed to be appropriate
capsule materials for any type of somatic embryos. In preliminary experiments, it was
found that the gel capsule around the somatic embryo was most effective when
formed using a sodium alginate concentration of 2%, gel capsules formed of other
than 2% are found to be unsuitable (Table 3.1 & Graph 3.1). The conversion
frequency was highest (75.0±8%) when 2% sodium alginate was used and decreased
with increase in the percentage of sodium alginate. The encapsulated embryos showed
signs of germination 6-8 days after culture (Fig 3.11). The alignate matrixes ruptured
and shoot tips and roots emerged from the capsule. The plantlets grew vigorously and
were comparable to the plants developed from non-encapsulated embryos grown
under identical conditions. Occasionally, embryogenic calli induction was observed
from the synthetic seed cultures (Fig 3.10).
Effect of storage time and temperature on germination
Encapsulated somatic embryos stored at different temperatures (4 oC, 15 oC
and 22oC) and at different periods (30, 60 and 90 days) showed different survival
rates on MS medium respectively. Conversion frequency of shoots is directly
dependent on storage period and temperature (Table 3.2 &Graph 3.2). It was observed
that 30-day-old somatic embryos resulted in higher emergence of plants (76.7±0.13%)
when compared to the germination response of 60 and 90-day-old somatic embryos
(57.3±0.67% and 48.4±0.57% respectively). The best storage temperature is 22oC
during 30, 60 and 90-day- periods. After 90 days of storage, the percent frequency of
conversion was reduced, along with death and decay of encapsulated somatic
embryos.
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Effect of low temperature storage of encapsulated embryo
The encapsulated embryos showed high frequency germination when stored at
4ºC for a few days and thereafter showed a decline in the percentage of germination
(Table 3.3 & Graph 3.3).
Effect of additives on the germination of synthetic seeds
In order to establish a suitable medium composition of artificial seed,
different concentrations and combinations of phytohormones are added to MS
medium to germinate synthetic seed embryos. For this purpose, BAP and Kn alone or
in combination with NAA, ascorbic acid and GA3 were used in the medium. There
was a significant difference in percent frequency and nature of response due to
different growth regulators. Data were taken on germination percentage, number of
shoots per explant and shoot length. Best result was obtained when 2.0 mg/l BAP with
1.0 mg/l GA3 and 0.5 mg/l NAA were used in the medium. In this combination
85±8% of synthetic seeds germinated within eight to ten days of culture on MS
medium. Second highest germination rate was observed in the medium supplemented
with 2.0 mg/l Kn with 0.5 mg/l NAA and 1.0 mg/l GA3, but when MS basal was used
artificial seeds failed to germinate. Highest elongation on shoots was observed on MS
medium supplemented with Kn (2.0 mg/l) + NAA (0.5 mg/l) and GA3 (1.0 mg/l) with
3±0.7 cm of elongation (Table 3.4).
Effect of alginate matrix composition
We found that the growth of encapsulated embryos to shoots and the rooting
of emerged shoots depend on the bead composition. Shoots emerged (Fig 3.12) from
nonstored encapsulated embryos, breaking the capsule wall at the end of the first
week and did so most intensively in the second week of incubation on agar medium.
After 4–6 weeks roots are formed at the base of shoots on the same medium. Among
the various types of tested gel matrices 2% or 3% sodium alginate solution with or
without the addition of sucrose and GA3 alone or in combination, the results for shoot
and root emergence were best with embryo encapsulated in 2% sodium alginate with
sucrose (1.5%) and GA3 (1.0 mg/l) (Table 3.5). Almost 76±8% of these beads
regenerated into shoots with normal morphology and 36±5% of the obtained shoots
had roots. The plantlets with elongation shoots and roots (Fig 3.13) were transferred
118
to plastic pots containing sterile soilrite and covered with transparent polythene bags.
After 1 month these were planted in earthen pots containing normal garden soil and
maintained in greenhouse (Fig 3.14).
Discussion
To obtain a fine suspension culture, it is of prime importance to initiate
suspension cultures from a friable callus source. Hence, friable callus obtained from
our callus culture experiments were taken as a source material. As the friability of the
callus increases, it becomes much easier to achieve a full separation of the cells. In the
present research work, calli were induced on MS medium supplemented with different
combinations and concentrations of auxins and cytokinins. For example MS medium
supplemented with lower concentrations of NAA upto 1.0 mg/l and BAP (1.0 to 2.5
mg/l) is suitable for the induction of callus in T. decandra. So a suitable concentration
of growth regulator is fruitful in tissue culture for further propagation. Similar results
has been reported by Qureshi et al., (2012) in leptadenia pyrotechnica.
Cell suspension cultures growing in the MS liquid medium supplemented with
GA3, IBA and ascorbic acid at all concentrations showed a finer and more
homogenous suspension, a better specific growth rate and higher cellular viability
(70-80%) than the other media. Subculturing the callus from BAP and NAA
containing medium to regular maintenance medium produced more friable callus.
Embryogenesis is affected by several factors such as concentration and type of
phytohormones, media used and light. In the present investigation, higher rates of cell
growth were observed when the cells were cultured in a liquid medium containing
GA3 (1.0 mg/l), IBA (1.0 mg/l) and ascorbic acid (60 mg/l) when compared to much
lower rates of cell division when the culture medium contained GA3 alone. In another
published work, it is reported that, liquid MS medium containing 2 mg/l NAA
induced high frequency of somatic embryos formation (47.67 ± 4.53 per ml)
(Armiyanti et al., 2010). In the present investigation, the frequency of germination of
synthetic seed was higher on MS basal medium when compared to Nitsch medium
and the difference was found to be significant. This is due to the fact that regeneration
varies with the culture media and also with the species. In contrast, Chetia et al.,
(1998) have reported that there was not much difference between the germination
percentage between MS and Nitsch’s media in orchids. MS medium with or without
119
BAP gave 60-64 % results whereas, in the other media viz., B5 and Nitsch media
supplemented with 0.5 to 1mg/l BAP gave better results than basal B5 and Nitsch.
For the encapsulation, sodium alginate at 1–4 % (w/v) was used as the gelling
agent and 100 mM calcium chloride was used as the complexing agent. In the present
study, the presence of 2% sodium alginate and 100 mM calcium chloride was found
to be the best composition for gel complexation, which produces firm, clear and
isodiametric beads. Lower concentrations of sodium alginate (1%) not only prolonged
the polymerization duration, but also resulted in fragile beads, which were difficult to
handle, whereas at higher concentrations of sodium alginate (4%), beads were too
hard causing considerable delay in shoot emergence. In another research work, it was
observed that 2% sodium alginate dipped in 100 mM CaCl2 solution and incubated for
30 min in orbital shaker was found to be the best matrix and complexing agent
respectively to produce firm, transparent and uniform synthetic seeds (Nagananda et
al., 2011). It also has reported that different levels of sodium alginate and calcium
chloride were used in which perfect bead formation was observed in condition
encapsulated with 2.5% sodium alginate and 100 mM calcium chloride solution
(Maqsood et al., 2012). It has been shown that the percentage of sodium alginate
employed for maximum conversion ability depends on the species of the plant under
investigation (Redenbaugh et al., 1987).
The result of the study on the effect of storage time and temperature on
germination showed that the viability percentage decreased with increase in storage
time on all tested temperatures. At higher temperature (22�) there is reduction in the
survival percentage of embryos. There is decline in the germination frequency among
the encapsulated seeds stored at low temperature and it may be due to inhibited
respiration of plant tissues by alginate (Redenbaugh et al., 1987). In the present study,
longer duration of cold storage (4�) of encapsulated embryos resulted in a significant
reduction in percentage of germination. Encapsulated embryos of T. decandra could
be stored at 4� for 60 days; however, conversion percentage decreased about 2.4 fold
in comparison to control (Table 3.3). The experiments carried out and observations
made show that synthetic seed could be stored for 60 days that is sufficient for
germplasm exchange. The most desirable feature of the encapsulated embryos is their
capability to retain viability after storage for a reasonable period required for the
120
exchange of germplasm between laboratories (Micheli et al., 2007; Rai et al., 2008).
Taha et al., (2008) have worked on the production of synthetic seeds from
microshoots and somatic embryos of Gerbera jamesonii and they reported that the
viability of the seeds after storage period at 4°C was also determined. High
germination rate (75-95%) was achieved after one to three months storage, whereas
low germination rate (8-50%) was obtained after four to six months storage.
To investigate the effect of additives on the germination of artificial seeds,
different concentrations and combinations of phytohormones are added to MS
medium to germinate synthetic seeds. Best result was obtained when 2.0 mg/l BAP
with 1.0 mg/l GA3 and 0.5 mg/l NAA were used in synthetic seed medium. In this
combination 85±8% of synthetic seeds germinated within eight to ten days of culture
on MS medium. Our results are in agreement with earlier reports that the presence of
cytokinins in medium with full strength of MS salts i.e. kinetin for zingiber (Sharma
et al., 1994) and BA for Cassava (Danso & Ford-Lloyd, 2003) and zingiber
(Sundararaj et al., 2010) supported better proliferation of shoots developed from
synseeds. Perhaps, the major limiting factor for conversion is the non-availability of
nutrients for synseeds to develop balanced shoot and root systems (Fujii et al., 1987;
Ganapathi et al., 1992). In other studies also, synseeds sown on autoclaved soil or soil
rite moistened with distilled water or tap water failed to respond in encapsulated shoot
tips of pineapple (Soneji et al., 2000) and pomegranate (Naik & Chand, 2006).
Similarly, in encapsulated shoot buds of banana, 10% of synseeds sown on sterilized
soil showed the emergence of shoots, but failed to form complete plantlets in the
absence of roots (Ganapathi et al., 1992). To evaluate the effect of composition of
alginate matrix on conversion, somatic embryo encapsulated in sodium alginate (2%
and 3%) and 100 mM calcium chloride using a sterile pipette of appropriate diameter.
Then synseeds prepared in sodium alginate with MS (1.5 % sucrose + 0.5 – 1.0 mg/l
GA3) were cultured in test tubes containing ½ MS (3% sucrose) + 1.0 mg/l IBA + 0.8
% agar medium. We found that the growth of encapsulated embryos to shoots and the
rooting of emerged shoots depended on the bead composition. Among the various
types of tested gel matrices (2% or 3% sodium alginate solution with or without
addition of sucrose and GA3 alone or in combination) , the results for shoot and root
emergence were best with somatic embryos encapsulated in 2% sodium alginate
with sucrose (1.5%) and GA3 (1.0 mg/l). Pattnaik et al., (1995) reported that
121
supplementing the alginate matrix with 0.3 mg/l GA3 increased shoot formation from
encapsulated axillary buds of different Morus species, and attributed the effect of GA3
to improvement of shoot internode elongation and/or stimulation of vegetative bud
germination.
Considerable progress has been made in the recent past of in vitro propagation
via synthetic seeds in several plant species. Encapsulation technology offers
tremendous scope for the conservation and germplasm exchange of several plants.
Despite these advantages, direct sowing of synthetic seeds in the field for commercial
use remains a limitation of synthetic seed application due to low soil survival (Jung et
al., 2004). In many plant species either embryogenic system does not exist or produce
low quality somatic embryos. Therefore, refinements in protocols are necessary to get
high quality somatic embryos to improve the propagation system through synthetic
seeds. However, in most cases, in vitro raised plantlets were used as the source of
explant for encapsulation because explants from mature trees exhibit recalcitrance
under aseptic conditions. In some plant species poor conversion of encapsulated
propagules into plants is another major problem and still remains one of the factors
limiting commercial application of this technology. Manipulation of medium and
addition of correct formulation of medium, growth regulators, carbohydrate sources,
antibiotics and fungicides in the synthetic endosperm can help to enhance the
conversion frequency of encapsulated propagules and requires detailed studies.
Encapsulation technology is a promising technique for conservation and transport of
transgenic plants, non-seed producing plants, elite traits and plant lines with problems
in seed propagation (Saiprasad, 2001). However, further detailed research is needed
mainly for improvement in conversion of synthetic seeds and subsequent plant growth
in soil and the limitations mentioned above should be taken into considerations while
working on synthetic seeds particularly in rare, endemic and medicinal plants.
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Table 3.1 Effect of different concentrations of alginate on germination of
synthetic seed of T. decandra L.
Alginate (%) No. of cultured
seeds
No. of germinated
seeds Germination (%)
1 64 36±3b 56.2±6
2 64 48±6d 75.0±8
3 64 44±5c 68.7±7
4 64 28±3 a 43.7±5
Note: Date scored at the end of 4 weeks on MS medium
Results represent mean± SD of three replicated experiments.
The values with different letters are significantly different (P<0.05) using the Tukey, s test
Graph 3.1 Effect of different concentrations of alginate on germination of
synthetic seed of T. decandra L.
36
4844
28
56
7568
43
0
10
20
30
40
50
60
70
80
1 2 3 4
Alginate (%)
No. of Germinated Seeds Germination (%)
123
Table 3.2 Effect of storage period and temperature on germination of
encapsulated embryos from T. decandra
Storage period
(days)
Temperature
(oC)
Conversion frequency
(%)
30
30
30
4
15
22
52.3±4.3f
60.4 ±7.1h
76.7±7.8i
60
60
60
4
15
22
36.2±2.9b
45.4±5.1d
57.3±4.7g
90
90
90
4
15
22
30.1±2.8a
40.7±4.6c
48.4±5.4e
Note: Date scored at the end of 4 weeks on MS medium
Results represent mean± SD of three replicated experiments. The values with different letters are significantly different (P<0.05) using the Tukey, s test
Graph 3.2 Effect of storage period and temperature on germination of
encapsulated embryos from T. decandra
0
10
20
30
40
50
60
70
80
30 (4) 30 (15) 30 (22) 60 (4) 60 (15) 60 (22) 90 (4) 90 (15) 90 (22)
Co
nv
ersi
on
Fre
qu
ency
(%
)
Storage Period days (Temprature oC)
124
Table 3.3 Effect of low temperature (4oC) storage on germination of
encapsulated embryos of T. decandra
Days of storage No. of cultured synthetic seeds
No. of germinated synthetic seeds
Germination (%)
0 64 56.3±6 a 88.2±5
5 64 53.7±7 b 84.4±2
10 64 50.5±7 c 79.2±6
15 64 46.7±5 d 73.3±9
20 64 42.2±7 e 66.1±8
25 64 37.7±3 f 59.3±7
30 64 33.2±8 g 52.4±9
45 64 28.1±7 h 44.6±3
60 64 23.0±6i 36.7±4
Note: Data scored at the end of 4 weeks on MS medium Results represent mean± SD of three replicated experiments. The values with different letters are significantly different (P<0.05) using the Tukey, s test
Graph 3.3 Effect of low temperature (4oC) storage on germination of
encapsulated embryos of T. decandra
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25 30 45 60
Storage Period (day)
Germination (%) No. of Germinated Capsules
125
Table 3.4 Effect of additives on the development of non stored artificial seeds of
T. decandra
Plant growth regulators and additives
(mg/l) Germination
(%)
Number of shoots per
explant
Shoot length (cm) BAP Kn NAA GA3 A. acid
1.0 _ _ _ _ 68±7ab 1.1±0.4cd 1.4±0.3de
2.0 _ _ _ 70±6bc 1.7±0.5fg 1.8±0.4f
1.0 _ 0.5 _ _ 75±5ef 1.4±0.4def 0.9±0.3bc
2.0 _ 0.5 _ _ 78±7fg 1.6±0.5efg 1.9±0.5f
1.0 _ 0.5 0.5 _ 83±6hi 2.8±0.9j 2.6±0.8 g
2.0 _ 0.5 1.0 _ 85±8i 3.1±1.0j 3.2±0.9 h
1.0 _ 0.5 _ 60 78±6fg 2.3±0.7i 1.2±0.2cd
2.0 _ 0.5 _ 60 80±4gh 2.8±0.7j 2.3±0.6g
_ 1.0 _ _ _ 66±5a 0.3±0.1a 0.5±0.2 a
_ 2.0 _ _ _ 68±5ab 0.8±0.3bc 1.4±0.4de
_ 1.0 0.5 _ _ 73±4cd 0.5±0.2ab 0.7±0.2ab
_ 2.0 0.5 _ _ 75±7ef 1.3±0.5de 1.7±0.3ef
_ 1.0 0.5 0.5 _ 79±9g 1.9±0.6gh 1.9±0.4f
_ 2.0 0.5 1.0 _ 83±5hi 2.2±0.8hi 2.4±0.7g
_ 1.0 0.5 _ 60 77±4fg 1.4±0.5def 0.8±0.3ab
_ 2.0 0.5 _ 60 78±7fg 1.9±0.7gh 1.2±0.4cd
Note: Data recorded after 6 weeks, MS media used, Results represent mean± SD of three replicated experiments. The values with different letters are significantly different (P<0.05) using the Tukey, s test
126
Table 3.5 Effect of gel matrix composition on shoot and root emergence
from non-stored synthetic seeds of T. decandra. Data were recorded
after 6 weeks of culture on regrowth medium (1/2 MS medium with
1.0 mg/l IBA)
Alginate matrix composition Formation
Alginate concentration
(%) Sucrose (%) GA3 (mg/l)
Shoots*
(%)
Shoots with roots**
(%)
2 - - 48±6c 12±2a
2 1.5 - 62±9e 27±4e
2 - 0.5 69±8g 12±3a
2 1.5 0.5 69±9g 27±5e
2 - 1.0 66±5f 22±2d
2 1.5 1.0 76±8h 36±5g
3 - - 44±3b 18±2bc
3 1.5 - 32±2a 16±5b
3 - 0.5 54±5d 22±3d
3 1.5 0.5 64±6ef 17±2b
3 - 1.0 63±8e 20±7cd
3 1.5 1.0 70±9g 30±5f
Note: * Percentage of encapsulated embryo that formed shoots with normal morphology. ** Percentage of obtained shoots that formed roots. Results represent mean± SD of three replicated experiments. The values with different letters are significantly different (P<0.05) using the Tukey, s test
127
Fig.3.1. Stable suspension line of T. decandra
Fig 3.2 Elliptical cells in suspension culture
128
Fig 3.3.Tracheary elements
Fig 3.4. Embryos in suspension culture
129
Fig 3.5. Single celled proembryo with a stalk had its origin
from the embryogenic mass of cells
Fig 3.6. A filamentous four-celled structure
130
Fig 3.7. Homogenous cell suspension culture showing rounded
cells dividing intensely
Fig 3.8. Four celled proembryo with suspensor
131
Production and development of synthetic seeds
A (3.9): Synthetic seeds B (3.10): Induction of embryogenic calli from synthetic seed
C (3.11): Germination of synthetic seeds on MS+ 1.0 mg/l BAP
D (3.12): Induction of shoots from synthetic seeds on MS + 2.0 mg/l BAP+ 0.5 mg/l NAA+ 1.0 mg/l GA3
E (3.13): Elongation of shoots and roots on the same medium composition
F (3.14): Hardened potted plants
A B
C D
E F