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A synthetic seed method through encapsulation of in vitro proliferated bulblets of garlic (Allium sativum L.)

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Abstract

A synthetic seed protocol for conservation of garlic as asexual propgated plant was established using in vitro rgenerated bulblets. Shoot cultures were obtained directly from excised shoot apices. Bulblets were formed in vitro on shoot cultures in high frequencies using MS medium containing 2 mg /l benzyladenine (BA) + 1 mg /l gibberellic acid (GA3) or 2 mg/l Jasmonic acid. Among four tested concentrations of sodium alginate used as gel matrix, 3 % gave the highest percentages of survival and conversion of encapsulated bulblets to plantlets. Also 30 min was the best duration of exposure to calcium chloride for hardening of sodium alginate capsules. For plantlets recovery from encapsulated bulblets, different combinations of growth regulators were examined. The highest number of proliferated shoots and their growth parameters (shoot length and fresh mass) were obtained using MS medium containing 2 mg /l BA +2 mg /l naphthaleneacetic acid (NAA). Randomly Amplified Polymorphic DNA (RAPD) analysis has been used to study the genetic stability of convered plantlets of garlic. According to data obtained, plantlets derived from capsulated cultures as well as normally in vitro propagated cultures were generally similar to those the in vivo grown plants.The developed plantlets were successfully adapted to the free living conditions after phase of simple acclimatization.
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
A synthetic seed method through encapsulation of in vitro
proliferated bulblets of garlic (Allium sativum L.)
(Received: 15 .06.2006; Accepted: 30. 06 .2006)
Shawky A. Bekheet
Plant Biotechnology Department, National Research Centre,Egypt
ABSTRACT
A synthetic seed protocol for conservation of garlic as asexual propgated plant was
established using in vitro rgenerated bulblets. Shoot cultures were obtained directly from excised
shoot apices. Bulblets were formed in vitro on shoot cultures in high frequencies using MS medium
containing 2 mg /l benzyladenine (BA) + 1 mg /l gibberellic acid (GA3) or 2 mg/l Jasmonic acid.
Among four tested concentrations of sodium alginate used as gel matrix, 3 % gave the highest
percentages of survival and conversion of encapsulated bulblets to plantlets. Also 30 min was the
best duration of exposure to calcium chloride for hardening of sodium alginate capsules. For
plantlets recovery from encapsulated bulblets, different combinations of growth regulators were
examined. The highest number of proliferated shoots and their growth parameters (shoot length and
fresh mass) were obtained using MS medium containing 2 mg /l BA +2 mg /l naphthaleneacetic acid
(NAA). Randomly Amplified Polymorphic DNA (RAPD) analysis has been used to study the genetic
stability of convered plantlets of garlic. According to data obtained, plantlets derived from
capsulated cultures as well as normally in vitro propagated cultures were generally similar to
those the in vivo grown plants.The developed plantlets were successfully adapted to the free living
conditions after phase of simple acclimatization.
Key words: Garlic, in vitro, bulblet, encapsulation, RAPD analysis.
INTRODUCTION
ome crop species have genotypes which
do not produce seeds and others , have
either sterile genotypes or produce
orthodox seeds which are hihghly heterozygous
and are therefore of limited interest for the
conservation of particular gene combinations.
These species are mainly propagated
vegetatively to maintain clonal genotypes. At
present, the common methods to preserve the
genetic resources of these problem crop species
are as tuber, bulbs and corms. There are,
however, several serious problems with these
methods (Withers and Engels, 1990). The
collections are exposed to natural disasters and
attack by pests and pathogens; moreover,
labour costs and the requirment for technical
personnel are very high. In addition,
distribution and exchange from field genebank
is difficult because of the vegetative nature of
the material and the greater risks of disease
transfer. In vitro techniques are of great interest
for the collecting, multiplication and storage of
plant germplasm (Engelmann, 1991). More-
over, establishment of synthetic seeds have
multiple advantage including ease of handling,
potential long-term storage and low cost of
production and supsequent propagation (Ghosh
and Sen, 1994). In this respect, natural unipolar
S
Shawky A. Bekheet
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
416
propagule like microbulbs, rhizomes,
protocorms (Standardi and Piccioni, 1998 ;
Datta et al., 1999 ; Saiprasad and Polisetty,
2003), nodal cuttings (Danso and Ford, 2003)
and shoot buds (Uozumi and Kobayashi, 1995 ;
Micheli et al., 2002) besides bipolar somatic
embryos (Gary and Purohit, 1991 ; Patel et al.,
2002) have been subjected to encapsulation for
storage, easy handling, transport, delivery and
their establishment under in vitro and/or extra
vitrum conditions. Although a variety of natural
and synthetic polymers are available for
encapsulation, sodium alginate is the most
commonly used gel-matrix because of its easy
gelling properties, non-toxicity and low cost.
Different concentrations of sodium alginate
ranging from 1.5 % to 6 % have been used for
different systems (Bapat et al., 1987; Ahuja et
al., 1989; Sakamoto et al., 1995, Datta et al.,
1999; Vij et al., 2001; Mondal et al., 2002).
Because garlic is sterile, it is vegetatively
propagated by cloves and air bulbils.
Development of efficient in vitro techniques to
ensure its safe conservation is therefore of
paramount importance. Due to its ease of
handling and quick of conversion to plantlets,
in vitro regenerated bulblet seems to be the
sutiable explant for germplasm preservation of
garlic. In vitro bulblets formation of garlic
largely dependent on growth regulators and
sucrose in culture media (Matsubara and Chen,
1989 ; Nagakubo et al., 1993), as well as other
conditions such as cultivar, photoperiod and
temperature. In this respect, BA and Jasmonic
acid are reported more effective factors
promoting in vitro bulblet of garlic shoot,
despite a stimulatory role of the higher
concentration of sucrose on in vitro bulblet
formation (Nagakubo et al., 1993). The
objective of this investigation is to establish a
protocol for mid-term preservation of garlic
germplasm through encapsulation of in vitro
regenerated bulblets
MATERIALS AND METHODS
Establishment of aseptic cultures
The Egyptian cultivar (Balady) of garlic
was used as plant material. The cloves were
taken and the protective leaf sheaths were
removed. Then the naked cloves were washed
by tap water and surface sterilized using 70 %
ethanol for 3 min followed by 50 %
commercial Clorox (contained 5.25 % NaOCl)
for 20 min. Under aseptic conditions of laminar
flow cabinet, shoot tips with basal part of
bullbs were excised and cultured on Murashige
and Skoog, 1962 (MS) medium supplemented
with growth regulators described by Bekheet
(2004) for direct shoot proliferation.
In vitro bulblets formation
The cluster of multiple shoots of garlic
were taken and divided into single shoots and
then cultured on MS medium contained several
concentrations of cytokinins i.e. kinetin (Kin)
or BA in combinations with GA3. Also,
different concentrations of Jasmonic acid i.e.
0.5 ,1.0, 2.0, 3.0, 4.0 and 5.0 mg /l added to
cultute medium were examined for their
potential for in vitro bulblet formation of garlic.
The percentage of bulblet formation, number of
bulblets per culture and bulblet fresh and dry
masses were recorded after six weeks of
culturing.
Table (1): Primers used and their annealing temperatures.
Annealing Tm °C / Sec Sequence 5’- 3’ Primer
36
TGGCGACCTG
GAGGCGTCGC
CCCTACCGAC
K1
K3
K3
A synthetic seed of in vitro bulblets in garlic
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
417
Bulblets encapsulation
In two separate experiments, the effect of
gel matrix concentrations and duration of
exposure to calcium chloride on encapsulation
of in vitro regenerated bulblets of garlic were
tested. In the first experiment, sodium alginate
was examined at 1, 2, 3, and 4 % (w/v).
Bulblets in uniform size were taken and blot
dried using filter paper and then mixed well
with the defferent level of sodium alginate
(from Sigma) prepared in distilled water.
Activated charcoal (0.2%) was added to the
matrix to absorb phenols and other compounds
exudate to the encapsulated bulblets. To avoid
bacterial contamination, antibiotic mixture was
used as described by Ganapathi et al. (1992).
The bulblets were picked up and immersed
into calcium chloride solution (2.5 %) and
stirred continuously up to 20 min on a
magnetic stirrer. Then the encapsulated beads
were washed in autoclaved distilled water and
transferred to autoclaved glass vessel for
storage at 15 or 25˚C. For the second
experiment, bulblets were coated by 3 %
sodium alginate and then immersed in 2.5 %
calcium chloride solution for 10, 20, 30, 40
and 50 min for hardening of alginate capsules.
All other conditions were as described for the
first experiment mentioned above. The
percentage of survival and conversion to
plantlets were recorded after 60 days of storing
Plantlet recovery from encapsulated
bulblets
After the storing at the two temperature
conditions, the encapsulated bulblets of garlic
were washed using steriled water and sodium
alginate was thawed. Then the bulblets were
cultured on MS-medium supplemented with
different combinations of BA and NAA. The
number of proliferated shoots shoot length and
shoot fresh mass were recorded after six weeks
of culturing on recovery media.
Culture conditions and statistical analysis
All culture media were solidfied with 0.7
% agar and adjusted to pH 5.8 before
autoclaving at 121˚C and 1.5 Ib/M² for 25 min.
Cultures were normally incubated at 25˚C and
16 hr photoperiod provided by philips white
fluroescent tubes. Each experiment was set up
as a separate completely randomized design
with 20 replicates per treatment. Data were
statistically analyzed using standard error (SE)
according to the method described by
Snedecor and Cochran (1967).
Randomly Amplified Polymorphic DNA
(RAPD) analysis
DNA isolation was performed using the
Cetyl Trimethyl Ammonium Bromide (CTAB)
method of Doyle and Doyle (1987). Half gram
of fresh samples of shoot buds developed from
encapsulated bulblets, in vitro grown shoot
buds and in vivo growing cultures were ground
to powder in liquid nitrogen with a prechilled
pestle and mortar, suspended in 5 ml preheated
CTAB buffer, and incubated at 65°C for 1
hour with occasional shaking. The suspension
was then mixed with 1/3 volume of
chloroform, mixed gently, centrifuged and the
upper phase was transferred to a new sterilized
tube. Extraction was repeated with an equal
volume of chloroform. The aqueous layer was
transferred to a new tube, 2/3 volume of
isopropanol was added and nucleic acids were
either spooled using a Pasteur pipette or
sedimentated by centrifugation. The pellet was
washed carefully twice with 70% ethanol,
dried at room temperature and resuspended in
0.5 ml TE buffer. The enzyme, RNAse a (20
µg) was added to the resuspended mixture to
digest any contaminating RNA and the tube
was incubated at 37 ˚C for 30 min. To remove
the enzyme and other contaminating proteins,
phenol/chloroform extraction was performed.
Shawky A. Bekheet
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
418
The polymerase chain reaction (PCR)
mixture (25 ul) consisted of 0.8 units of Taq
DNA polymerase, 25 pmol dNTPs, and 25
pmol of random primer, and 50 ng of genomic
DNA. The reaction mixture was placed on a
DNA thermal cycler. The PCR programme
included an initial denaturation step at 94°C
for 2 mins followed by 45 cycles with 94°C for
1 min for DNA denaturation, annealing as
mentioned with each primer, extension at 72°C
for 30 seconds and final extension at 72 °C for
10 minutes were carried out. The amplified
DNA fragments were separated on 2% agarose
gel and stained with ethidium bromide. Three
10-mer primers (Operon technologies Inc.,
Alameda, California) randomly selected were
used in RAPD analysis (Table 1). A 100 bp
DNA ladder (Promga) was used as a Marker
with molecular size of 1000, 900, 800, 700,
600, 500, 400, 300, 200 and 100 bp. The
amplified pattern was visualized on a UV
transilluminator and photographed
Development of plantlets and acclima-
tization
Shoots were elongated and rooted in
vitro using medium contained 1 mg/l indole-3-
acetic acid (IAA). Complete plantlets were
washed with tap water and disinfected by
soaking in benlate solution (1g /l) for 20 min.
Then plantlets were transplanted into plastic
pots containing peatmoss and perlite (1:1). The
pots were covered with clear polyethylene
bags which sprayed with water to maintain a
high relative humidity. Humidity was
gradually reduced and covers were completely
removed within four weeks of transplanting.
RESULTS AND DISCUSSION
In vitro bulblets formation
Influence of growth
The role of different combinations of
growth regulators on in vitro bulblet formation
of garlic from shoot cultures was investigated.
As shown in Fig. (1-A), bulblets were formed
on the basis of shoots after six weeks of
culturing. The frequency of bulblet formation
was varied dependent on type and
concentration of cytokinin added to culture
medium (Table 2). The highest percentage of
bulblet formation and number of bulblets per
culture were found with MS medium
containing 2 mg /l BA + 1 mg /l GA3.
However, the highest values of fresh and dry
masses were observed when MS medium
contained 2 mg /l Kin + 1 mg /l GA3. This may
be due to the large size of bulblets formed on
the last medium. These results are in line with
those reported by Kahane et al. (1992) on
garlic. They mentioned that, bulblet formation
was regularly obtained in garlic after shoot
induction using 2 mg /l BA and it happened
however, at erratic frequencies and rarely with
kinetin (2mg/l). In this connection, cytokinins
were generally reported to improve bulb
formation in garlic plantlets by Matsubara and
Chen (1989). On the other hand, Nagakubo et
al. (1993) observed the in vitro bulblet
formation of garlic on hormone-free medium.
Moreover, Kim et al. (2003) mentioned that,
garlic bulblet formation in vitro was not only
induced by plant growth regulators, but also
largely dependent on the size and quality of
regenerated shoots.
Influence of jasmonic acid
A range of jasmonic acid concentrations
has been experimented for in vitro bulblet
formation in garlic. Results generally indicated
that, incorporation of culture medium by
Jasmonic acid had great effects on bulblet
formation. The frequencies of bulblet formation
increased with increasing of Jasmonic acid till
2 mg/l and then decreased. Also, the highest
number of bulblets per culture as well as
bulblet fresh and dry masses were registered
when using 2 mg/l Jasmonic acid (Table 3).
A synthetic seed of in vitro bulblets in garlic
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
419
However, the in vitro bulblet formation of
garlic was significantly supressed by addition
of high concentration of Jasmonic acid to
culture medium. In similar study, Kim et al.
(2003) mentioned that, single treatment of 2 mg
/l Jasmonic acid formed bulblets at rate of 77%.
They added that fresh weight of bulblets was
markedly increased by addition of 1 mg /l
gibberelic acid to jasmonic acid treated
medium. In this respect, Jasmonic acid was
reported to be an effective factor for promoting
in vitro bulbing of garlic shoots (Nagakubo et
al., 1993.
Table (2): Effect of different combinations of growth regulators on in vitro bulblet formation of
garlic.
Bulblets dry mass
(mg)
Bulblets fresh mass
(mg)
No bulblets /culture
(g)
Bulblets formation
(%)
Growth regulators
(%)
138.80 ± 6.00
189.40 ± 5.50
169.00 ± 6.30
160.50 ±7.00
1.25 ± 0.50
1.80 ± 0.33
1.65 ± 0.40
1.75 ± 0.55
2.50 ± 0.05
3.00 ± 0.09
4.00 ± 0.08
4.00 ± 0.03
10
20
35
55
1 mg/l kin +1 mg/l GA3
2 mg/l kin +1 mg/l GA3
1 mg/l BA+1 mg/l GA3
2 mg/l BA+1mg/l GA3
± SE =Standard Error. Each treatment is the average of 20 replicates.
Table (3): Effect of Jasmonic acid concentrations on in vitro bulblet formation of garlic.
Bulblet dry mass
(mg)
Bulblet fresh mass
(mg)
No
bulblet/culture
Bulblet formation
(%)
Jasmonic acid conc.
(mg/l)
109.00 ± 4.00
166.80 ± 5.30
315.70 ± 7.50
210.50 ± 5.00
142.80 ± 4.50
83.30 ± 4.40
1.20 ± 0.05
1.75 ± 0.09
3.00 ± 0.10
2.00 ± 0.08
1.50 ± 0.05
0.75 ± 0.02
2.00 ± 0.04
3.50 ± 0.08
6.00 ± 0.05
4.00 ± 0.03
3.00 ± 0.06
1.50 ± 0.03
30
40
75
65
35
20
0.5
1.00
2.00
3.00
4.00
5.00
± SE =Standard Error. Each treatment is the average of 20 replicates
Bulblets encapsulation
Effect of gel matrix concentration
In this study, 1, 2, 3 and 4 % of sodium
alginate were investigated as gel matrix for
encapsulation of garlic bulblets formed in
vitro. The results presented in Fig. (1-B) and
Fig. (2) Indicated that increasing of sodium
alginate from 1 up to 3 % increased
percentage of survival and conversion of
encapsulated bulblets since smooth texture is
appropriate for the storage as well as for
survival and conversion was observed with 3
g/l sodium alginate solution. At low
concentration (1%) of sodium alginate,
capsules were weak and bulblets dried and
turned brown. However, at high level (4 %)
of sodium alginate, capsules were so hard and
prevented bulblets proliferation. Results also
revealed that, storage of encapsulated
bulblets at 15 ºC was more effective than
storage at 25 ºC. At low temperature, about
90 percent survival and 80 percent
conversion were observed. In the concern of
sodium alginate level for capsulation, the use
of 3 % for capsulation of shoot tips of banana
was reported by Ganapathi et al. (1992). In
this connection, Castillo et al. (1998)
mentioned that, beads of uniform size and
shape were obtained when 2.5 % sodium
alginate was used for encapsulation of
somatic embryos of papaya. Ghosh and Sen
(1994) achieved a maximum conversion of
encapsultaed somatic embryos of Asparagus
cooperi with 3.5 % sodium alginate. They
added, higher or lower levels of sodium
alginate reduced the conversion frequency.
Shawky A. Bekheet
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
420
However, Rady and Hanafy (2004) in their
study on Gypsophila paniculata reported that,
high percent of vitrification was observed in
shoots derived from shoot tips coated by 2 %
sodium alginate.
Effect of duration of exposure to calcium
chloride
The encapsulated bulblets (3 % sodium
alginate) were tested for different periods of
exposure to 2.5 % calcium chloride
solution.The duration of exposure to calcium
chloride during the hardening process
strongly affected the frequency of
germination from encapsulated bulblets of
garlic (Fig.3). The highest percentage of
survival and conversion to plantlets were
obtained when coated beads were submerged
in calcium chloride (2.5 %) for 30 min for
hardening. There was no dehydration and
desiccation of beads during the storage of
encapsulated bulblets hardened with this
period. Following more than 30 min of
exposure to calcium chloride a very hard
bead was formed and a lower percentage of
regeneration into plantlets was achieved.
Capsules were made for most of the synthetic
seeds production researches either by mixing
the explants in sodium alginate followed by
dropping into calcium salt solution or by
inserting an explant into a drop of sodium
alginate just as it was falling into the calcium
solution. Calcium alginate beads form as a
result of the ion exchange between Na+ and
Ca++ ions (Redenbaugh et al., 1987). The
present results are in line with those obtained
by Ganapathi et al. (1992). They followed 30
min duration of exposure to calcium chloride
for hardening of encapsulated shoot tips of
banana. However, Dave et al.(2004)
mentioned that four millimeter long shoot
buds of Chloroohytum borivilianum
encapsulated in 3% sodium alginate matrix
polymerised for 40 min by CaCl2 2H2O
yielded the best results of regrowth. On the
other hand, Castillo et al. (1998) obtained the
highest germination frequency of
encapsulated somatic embryos of papaya
when coats were hardened for only 10 min
Plant recovery
Shoot regeneration from encapsulated
bulblets
After thawing the capsules, the garlic
bulblets were cultured on medium containing
different combinatios of BA and NAA for
plantlets recovery. Normal growth was
achieved in encapsulated bulblets and the
shoots proliferated normally without any
morphological aberrations (Fig.1-C). Results
of Table (4) show that, growth paramaters
presented as number of shoots, shoot length
and fresh mass varied depending on the
balance of BA to NAA.The highest value of
shoot number (6.30), shoot length (6.00 cm)
as well as shoot fresh mass (3.80 g) were
obtained when using medium containing 2
mg /l BA + 2 mg /l NAA (Fig. 1-D). These
results are accordance with that obtained by
Changeup et al.(1995). They mentioned that,
supplementation of MS culture medium with
1-2 mg /l BA and 0.1 mg /l NAA increased
the frequency of in vitro regenerated shoot
buds of garlic. In this respect, Masuda et al.
(1994) in their study on micro propagation of
garlic reported that, basal segments of the
bulblets regenerated multiple shoots on MS
medium containing BA and NAA
RAPD analysis
RAPD analysis has been used
successfully for measuring diversity in plants,
and the patterns of variation observed have
been shown to be closely resemble to those
obtained using more classical characters
(Howell et al., 1994). In the present
investigation, RAPD-DNA analysis was used
to compare the shoot buds developed from
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Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
421
encapsulated bulblets of garlic to those grown
in vitro and their source which was growing
in vivo. Three randomly selected primers
were used in this investigation. The three
primers gave sufficient and reproducible
amplification products (Fig. 4). Six
polymorphic bands (900, 780, 650, 550, 400,
and 100 bp) were detected with primer k1.
Most of bands were similar in the three types
of cultures except one band (100 bp) which
was absent in the encapsulated bulblet-
derived plantlets. However, the polymorphic
bands of primer k3 were identical in the three
types of cultures. The results of banding
reveal that the three types of cultures were
similar and there is no effective variation and
for this reason we did not investigate more
primers. It is particularly important to
confirm that encapsulated bulblet cultures of
garlic produce plantlets genetically similar to
both nontreated and plant grown in free-
living conditions. In this respect, DNA-based
analysis was applied to study the genetic
stability of plant tissue cultures (Williams et
al., 1990). Other genetic marker analysis has
been used to study the degree of genetic
change in plants regenerated in vitro such as
pea (Cecchini et al., 1992), sugarbeet (Sabir
et al., 1992) and wheat (Brown et al., 1993).
Table (4): Effect of different combinations of BA and NAA on plantlets recovery from
encapsulated bulblets of garlic.
Shoots fresh
mass (g)
Shoot length
(cm)
No. proliferated
Shoots
Combinations of
BA and NAA
2.50 ± 0.10
2.30 ± 0.15
2.60 ± 0.14
2.75 ± 0.20
3.50 ± 0.50
3.80 ± 0.24
1.30 ± 0.12
2.10 ± 0.20
3.75 ± 0.25
4.00 ± 0.17
4.80 ± 0.30
6.00 ± 0.30
3.90 ± 0.12
3.75 ± 0.20
3.20 ± 0.25
4.80 ± 0.17
5.60 ± 0.30
6.30 ± 0.32
1mg/l BA + 0.5 mg/l NAA
1mg/l BA + 1 mg/l NAA
1mg/l BA + 2 mg/l NAA
2 mg/l BA + 0.5 mg/l NAA
2 mg/l BA + 1 mg/l NAA
2 mg/l BA + 2 mg/l NAA
± SE =Standard Error. Each treatment is the average of 20 replicates.
Plantlets development and acclimatization
Shoots of garlic derived from
encapsulated bulblets were cultured on MS
medium containing 1 mg /l IAA for
elongation and rooting. On this medium
suffecient root system was initiated without
bulb formation. Within short period of
acclimatization, plantlets were successfully
adapted to free-living conditions when strong
rooted shoots were transplanted into pots
containing perlite and peatmoss (1:1) (Fig. 1-
E).
Finally, we can conclude that
encapsulation of in vitro grown bulblets of
garlic is a suitable system for mid-term
storage of garlic tissue cultures since encap-
sulation saves space, time and resources and
it demonstrates advantages over conventional
method is of shoot multiplication repeated by
subculturing. Also this method considered a
very good tool to exchange the garlic
germplasm between countries and intern-
ational plant genebanks.
Shawky A. Bekheet
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
422
0
10
20
30
40
50
60
70
80
90
Survival
Conversion
Survival
Conversion
Incubation at 25 ºCIncubation at 15 ºC
%
1%
2%
3%
4%
Sodium
alginate
Conc.
Fig. (2): Effect of gel matrix concentration on survival and conversion of encapsulated bulblets
of garlic.
Fig. (1) : A- Bulblets formation on the basis of
shoots of garlic cultured on MS medium
containing 2 mg/l Kin + 1 mg/l GA3.
B- Encapsulated bulblets of garlic using 3 %
sodium alginate .
C- Culturing of garlic bulblets after thawing
of sodium alginate capsules.
D- Emerged shoots from recovery
encapsulated bulblets of garlic using
MS medium containing 2 mg /l BA +
2 mg /l NAA.
E- Adapted plantlet of garlic
transplanted onto pots containing
perlite and peatmoss (1:1).
A synthetic seed of in vitro bulblets in garlic
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
423
0
10
20
30
40
50
60
70
80
90
100
Survival
Conversion
Survival
Conversion
Incubation at 25 ºCIncubation at 15 ºC
%
10
20
30
40
50
Exposure
duration
(min)
Fig. (3): Effect of duration of exposure to calcium chloride on survival and conversion of
encapsulated bulblets of garlic.
Fig (4): RAPD profile of in vivo grown plant (lane 1), normally in vitro propagated plantlets
(lane2), encapsulated bulblets-derived plantlets (lane 3) and the DNA marker (M) from
left to right using random primers i.e.K1, K2, and K3.
Shawky A. Bekheet
Arab J. Biotech. Vol. 9, No. (3) Oct. (2006): 415-426.
424
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426
אא
ﺎﻴﻠﻤﻌﻤ ﺓﺩﻟﻭﺘﺴﻤﻟﺍ ﻡﻭﺜﻟﺍ ﺕﺎﺒﻨ ﺕﻼﻴﺼﺒ ﻑﻴﻠﻐﺘ لﻼﺨ ﻥﻤ ﺔﻴﻋﺎﻨﺼﻟﺍ ﺭﻭﺫﺒﻠﻟ ﺔﻘﻴﺭﻁ
ﺕﻴﺨﺒ ﺩﻴﻤﺤﻟﺍ ﺩﺒﻋ ﻰﻗﻭﺸ ﺔﻴﺘﺎﺒﻨﻟﺍ ﺔﻴﻭﻴﺤﻟﺍ ﺎﻴﺠﻭﻟﻭﻨﻜﺘﻟﺍ ﻡﺴﻗ-ﺙﻭﺤﺒﻠﻟ ﻰﻤﻭﻘﻟﺍ ﺯﻜﺭﻤﻟﺍ -ﺓﺭﻫﺎﻘﻟﺍ -ﺭﺼﻤ .
ﺘﻟﺍ ﺔﻴﺴﻨﺠ ﺭﻴﻐﻟﺍ ﺕﺎﺘﺎﺒﻨﻟﺍ ﺩﺤﺄﻜ ﻡﻭﺜﻠﻟ ﺔﻴﺜﺍﺭﻭﻟﺍ لﻭﺼﻷﺍ ﻅﻔﺤﻟ لﻭﻜﻭﺘﻭﺭﺒ ﺦﻴﺴﺭﺘ ﻡﺘ ﺓﺩﻟﻭﺘﺴـﻤﻟﺍ ﺕﻼﻴﺼـﺒﻟﺍ ﻡﺍﺩﺨﺘﺴﺎﺒ ﺭﺜﺎﻜ ﺎﻴﻠﻤﻌﻤ . ﺔﻴﻤﻴﺘﺴﺭﻤﻟﺍ ﻡﻤﻘﻟﺍ ﺔﻋﺍﺭﺯ ﺩﻌﺒ ﺏﻴﺒﺎﻨﻷﺍ ﻰﻓ ﻥﺎﻘﻴﺴﻟﺍ ﻉﺭﺍﺯﻤ ﻰﻠﻋ لﻭﺼﺤﻟﺍ ﻡﺘ . ﻉﺭﺍﺯـﻤ ﻰﻠﻋ ﺔﻴﻟﺎﻋ ﺔﻓﺎﺜﻜﺒ ﺕﻼﻴﺼﺒﻟﺍ ﺕﻨﻭﻜﺘ ﻰﻠﻋ ﺕﻭﺘﺤﺍ ﺔﺌﻴﺒ ﻡﺍﺩﺨﺘﺴﺎﺒ ﻥﺎﻘﻴﺴﻟﺍ٢ ﻡﺠﻠﻤ / ﻥﻴﻨﻴﺩﺃ لﻴﺯﻨﺒ ﺭﺘﻟ +١ ﻡﻠﺠﻤ / ﺕﻭـﺘﺤﺍ ﺔـﺌﻴﺒ ﻰـﻠﻋ ﻙﻟﺫـﻜﻭ ﻥﻴﻠﻠﻴﺭﺒﺠﻟﺍ ﺽﻤﺤ ﺭﺘﻟ٢ ﻠﻤﻡﺠ/ﺘﻟ ﻙﻴﻨﻭﻤﺴﺎﺠﻟﺍ ﺽﻤﺤ . ﺕﻼﻴﺼـﺒﻠﻟ ﺔﻔﻠﻐﻤ ﺓﺩﺎﻤﻜ ﻡﻭﻴﺩﻭﺼﻟﺍ ﺕﺎﻨﻴﺠﻟﺃ ﺓﺩﺎﻤ ﻥﻤ ﺎﻬﻤﺍﺩﺨﺘﺴﺍ ﻡﺘ ﺕﺍﺯﻴﻜﺭﺘ ﺔﻌﺒﺭﺃ ﻥﻴﺒ ﻥﻤ ، ﻥﺎـﻜ ﺯﻴﻜﺭﺘﻟﺍ٣ % ﺏﻴﺒﺎﻨﻷﺍ ﻰﻗ ﺕﺎﺘﺎﺒﻨ ﻰﻟﺍ ﻙﻟﺫ ﺩﻌﺒ ﺎﻬﻟﻭﺤﺘﻟ ﺔﺒﺴﻨ ﻰﻠﻋﺃ ﻙﻟﺫﻜﻭ ﺔﻴﺤ ﺕﻼﻴﺼﺒﻟﺍ ﺀﺎﻘﺒﻟ ﺔﺒﺴﻨ ﻰﻠﻋﺃ ﻰﻁﻋﺃ ﺩﻗ . ﻥﻭﺜﻼﺜ ﺎﻀﻴﺃ ﺕﻼﻴﺼﺒﻟﺍ ﺽﻴﺭﻌﺘﻟ ﺓﺭﺘﻓ لﻀﻓﺃ ﺕﻨﺎﻜ ﺔﻘﻴﻗﺩ ﻡﻭﻴﺩﻭﺼـﻟﺍ ﺕﺎﻨﻴﺠﻟﺍ ﺕﻻﻭﺴﺒﻜ ﺏﻠﺼﺘﻟ ﻡﺯﻼﻟﺍ ﻡﻭﻴﺴﻟﺎﻜﻟﺍ ﺩﻴﺭﻭﻠﻜ ﺓﺩﺎﻤ ﻰﻟﺍ ﺎﻬﻔﻴﻠﻐﺘ ﺩﻨﻋ . ﺔﻔﻠﻐﻤﻟﺍ ﺕﻼﻴﺼﺒﻟﺍ ﻥﻤ ﺕﺍﻭﻤﻨﻟﺍ ﺭﻭﻁﺘ ﻰﻠﻋ ﺔﻋﺍﺭﺯﻟﺍ ﺔﺌﻴﺒ ﻰﻟﺍﻭﻤﻨﻟﺍ ﺕﺎﻤﻅﻨﻤ ﻥﻤ ﺔﻔﻠﺘﺨﻤ ﻕﻴﻓﺍﻭﺘ ﺔﻓﺎﻀﺍﺭﻴﺜﺄﺘﺭﺎﺒﺘﺨﺍ ﻡﺘ . لﺩـﻌﻤ ﻰﻠﻋﺃ ﺎﻫﻭﻤﻨ ﺱﻴﻴﺎﻘﻤ ﻙﻟﺫﻜﻭ ﺕﺍﻭﻤﻨﻟﺍ ﺦﻴﺭﻔﺘﻟ ) ﻑﺎﺠﻟﺍﻭ ﺝﺯﺎﻁﻟﺍ ﻥﺯﻭﻟﺍ ( ﻭﺼﺤﻟﺍ ﻡﺘ ﻰﻠﻋ ﺕﻭﺘﺤﺍ ﺔﺌﻴﺒ ﻡﺍﺩﺨﺘﺴﺎﺒ ﺎﻬﻴﻠﻋ ل٢ ﻡﺠﻠﻤ / لﻴﺯﻨﺒ ﺭﺘﻟ ﻥﻴﻨﻴﺩﺃ +٢ ﻡﻠﺠﻤ / ﻙﻴﻠﺨﻟﺍ ﺽﻤﺤ ﻥﻴﻟﺎﺜﻔﻨ ﺭﺘﻟ . ﺔـﻨﻴﺎﺒﺘﻤﻟﺍ ﻯﻭﻭـﻨﻟﺍ ﺽﻤﺤﻟﺍ ﻊﻁﺎﻘﻤﻟ ﻰﺌﺍﻭﺸﻌﻟﺍ ﺭﻴﺒﻜﺘﻟﺍ لﻴﻠﺤﺘ ﻡﺍﺩﺨﺘﺴﺍ ﻡﺘ)RAPD ( ﺎﻴﻠﻤﻌﻤ ﺔﻔﻠﻐﻤﻟﺍ ﺕﻼﻴﺼﺒﻟﺍ ﻥﻤ ﺓﺭﻭﻁﺘﻤﻟﺍ ﺕﺎﺘﺎﺒﻨﻠﻟ ﻰﺜﺍﺭﻭﻟﺍ ﺕﺎﺒﺜﻟﺍ ﺔﺴﺍﺭﺩﻟ . ﺘﻤﻟﺍ ﺕﺎـﻨﺎﻴﺒﻟﺍ ﻰﻟﺍ ﺩﺎﻨﺘﺴﻻﺎﺒ ﺎـﻬﻴﻠﻋ لﺼـﺤ ، ﺕﺎـﺘﺎﺒﻨﻟﺍ
ﻰـﻓ ﺔـﻴﻤﺎﻨﻟﺍ ﺕﺎـﻋﺍﺭﺯﻟﺍ ﻙـﻠﺘﻟ ﺔﻬﺒﺎﺸﻤﻭ ﺔﻬﺒﺎﺸﺘﻤ ﺕﻨﺎﻜ ﺏﻴﺒﺎﻨﻻﺍ ﻰﻓ ﺓﺭﺸﺎﺒﻤ ﺓﺩﻟﻭﺘﺴﻤﻟﺍ ﻙﻟﺫﻜ ﺔﻔﻠﻐﻤﻟﺍ ﺕﻼﻴﺼﺒﻟﺍ ﻥﻤ ﺔﻟﻭﺤﺘﻤﻟﺍ
ﺓﺭﺤﻟﺍ ﺔﻴﺌﻴﺒﻟﺍ ﻑﻭﺭﻅﻟﺍ .ﺔﻤﻠﻗﻻﺍ ﺕﺍﺀﺍﺭﺠﺍ ﻥﻤ ﺔﻁﻴﺴﺒ ﺔﻠﺤﺭﻤﺒ ﺭﻭﺭﻤﻟﺍ ﺩﻌﺒ ﺏﻴﺒﺎﻨﻷﺍ ﺝﺭﺎﺨ ﻭﻤﻨﻠﻟ ﺡﺎﺠﻨﺒ ﺎﻬﺘﻤﻠﻗﺃ ﻡﺘ ﺔﻟﻭﺤﺘﻤﻟﺍ ﺕﺎﺘﺎﺒﻨﻟﺍ .
... Better clonal plants; preservation of rare plant species extending biodiversity and more consistent and synchronized harvesting of important agricultural crops would become a reality are the few advantages among many other possibilities of synthetic seed technology (Khor and Loh, 2005). Besides this; ease of handling, potential long-term storage and low cost of production and subsequent propagation are other benefits (Bekheet, 2006). There would also be a channel for new transgenic plants produced through biotechnological techniques to be transferred directly to the greenhouse or field. ...
... But from last two decades vegetative propagules e.g. axillary buds in Camellia sinensis (Mondal et al., 2002), calli in Allium sativum (Kim and Park, 2002), bulblets in A. sativum (Bekheet, 2006), hairy roots in Horseradish (Phunchindawan et al., 1997), and shoot tips in Cucumis sativus (Adhikari et al., 2014) were also used (Table 1). In addition to the other in vitro derived meristematic tissues like microtubers, rhizomes and corms can also be used (Bapat and Mhatre, 2005). ...
... Since garlic is sterile, it is vegetatively propagated by cloves and air bulbils. Development of efficient in vitro techniques to ensure its safe conservation is therefore of paramount im-KHATOON ET AL S133 portance (Bekheet, 2006). Kim et al., (2002) encapsulated calli obtained from shoot tip of garlic in a calcium alginate gel. ...
Article
Synthetic seed technology has great potential in conservation and large-scale multiplication of plant propagules. In recent years lot of research has been carried out for the production of good plant propagules, better conversion rate, good germination rate and its preservation. This technology could be more useful where there is a requirement of the huge amount of propagating material or costly hybrid seed. So applicability of this technology is more in vegetable industry and research so that farmers could get quality planting material at a cheaper rate and could produce more vegetable at lower seed rates. Synthetic seeds production by encapsulating somatic embryos, shoot buds or any other meristematic tissue has the advantage of minimizing the cost of micro-propagated plantlets for commercialization and final delivery. This review attempts to focus on available information regarding the applicability of synthetic seed technology in vegetable crops.
... Synthetic seeds are employed in biotechnology to cultivate a variety of important plant species, and they are considered as a promising method for propagating plants that don not produce viable seeds, such as orchids and some medicinal plants [22]. They have various advantages, including inexpensive manufacturing costs, convenience of handling and the possibility of long-term storage [23]. They also possess the ability to produce superior and clonal plants in the same way that real seeds do, preserving rare plant species and thus increasing biodiversity, producing uniform plantlets, continuous mass propagation in less time and season independent production of identical plantlets, and more consistent and synchronized harvesting critical crops [17,24]. ...
... The encapsulated garlic calli had a significantly greater shoot length than the naked garlic calli [62]. It was noted that among four sodium alginate gel matrix concentrations studied, 3% offered the best percentages of encapsulated bulblet survival and conversion to garlic plantlets [23]. When MS media with 2 mg/l BA + 2 mg/l NAA was used, the most proliferating shoots and their growth parameters (shoot length and fresh mass) were obtained. ...
... Synthetic seeds are employed in biotechnology to cultivate a variety of important plant species, and they are considered as a promising method for propagating plants that don not produce viable seeds, such as orchids and some medicinal plants [22]. They have various advantages, including inexpensive manufacturing costs, convenience of handling and the possibility of long-term storage [23]. They also possess the ability to produce superior and clonal plants in the same way that real seeds do, preserving rare plant species and thus increasing biodiversity, producing uniform plantlets, continuous mass propagation in less time and season independent production of identical plantlets, and more consistent and synchronized harvesting critical crops [17,24]. ...
... The encapsulated garlic calli had a significantly greater shoot length than the naked garlic calli [62]. It was noted that among four sodium alginate gel matrix concentrations studied, 3% offered the best percentages of encapsulated bulblet survival and conversion to garlic plantlets [23]. When MS media with 2 mg/l BA + 2 mg/l NAA was used, the most proliferating shoots and their growth parameters (shoot length and fresh mass) were obtained. ...
Article
Full-text available
Over the last few decades, biotechnology research has advanced to the point where it is now possible to improve essential agricultural crops. Synthetic seed manufacturing has opened up new possibilities of in vitro plant propagation technique since it has various practical benefits on a commercial scale for the cultivation of a wide range of agricultural crops. It is one of the most important tools for plant tissue culture breeders and scientists, since it provides significant benefits for massive production of distinct (elite) plant species. Synthetic seeds offer a promising strategy for mass plant production in a way of encapsulation of non�embryonic or embryonic (somatic embryos) tissues in a gel-like matrix, such as shoot buds, shoot tips, microshoots, protocoms and nodal segments. When the strategies minimize the cost of the production of elite plant genotypes, this strategy can be adapted for commercial production. The technology of the synthetic seed necessitates the use of in vitro growth systems in order to produce viable materials that can be converted into plants on a wide scale. The discovery of an artificial seed technology opens up new possibilities of a promising strategy for improving a wide range of commercially significant plant species, including cereals, fruit crops, medicinal plants, and vegetable crops, which we focus on in this review article.
... It was reported (2010) that the main producer of garlic is China at any phase of plant development (Abdel-halim, 2014). Therefore, molecular characterization of garlic germplasms has been performed around the world with different molecular markers such as RAPD marker (Bekheet, 2006;Paredes et al., 2008), AFLP marker (Volk et al., 2004;Phillips et al., 2008), SSR marker (Zhao et al., 2011;Jo et al., 2012) and ISSR marker (Jabbes et al., 2011;Chen et al., 2014;El-Nagar and El-Zohiri, 2015). ...
... It was shown that increased JAs level occurs also in bulb forming plants suggesting that this phytohormone is involved in the formation of storage organs other than tubers. JAs plays role in the formation and enlargement of bulblets on N. triandus (Santos and Salema 2000), A. sativum (Bekheet 2006), Allium victorialis (Park et al. 2004) shoot explants placed on MS medium, and A. sativum basal plates transferred onto B5 medium (Ravnikar et al. 1993) (Table 2; Fig. 3). For A. sativum and Narcissus papyraceus it was also noted that JA and MeJA, respectively, stimulated bulblet formation and increased number and weight of bulblets in combination with NAA (Kim et al. 2003;Hosseini et al. 2013), although in N. papyraceus MeJA showed an inhibitory effect on a size of bulblets (Hosseini et al. 2013). ...
Article
Full-text available
Jasmonates (JAs), such as jasmonic acid and its methyl ester, are lipid-derived compounds with signal functions in plant growth and development, as well as in responses to stress. JAs are widely distributed in plants as natural plant growth regulators. JAs do not work independently but work as a part of a complex signaling network with other phytohormones. They are deployed to induce response during wounding and are often used for elicitation and stimulation of secondary metabolites production in different in vitro culture systems. Application of JAs seems to be promising during different steps of the micro-propagation system for different species. JAs stimulate proliferation rate of shoots, roots, callus and induce microtubers and bulblets formation. However, negative effects of JAs on the condition of plant tissues are also reported, e.g. leaf senescence, reduced growth and inhibited somatic embryogenesis. This review summarizes the current knowledge of the application and properties of jasmonates under in vitro conditions in terms of cell division, explant growth, proliferation ability, storage organ formation and stress response.
... In such situation, the induction of mass-scale embryo formation is essential and encapsulation of SE by forming synthetic seed has huge applications. This methodology is noted to be highly successful in many plants like Adhatoda vesica (Anand and Bansal 2002), Allium sativa (Bekheet 2006), and Daucus carota (Latif et al. 2007). In C. roseus, an encapsulation process was developed for making synthetic seeds by using sodium alginate (2.5%) + calcium chloride (100 mM) added solution. ...
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Artificial seeds are produced by encapsulating somatic embryos, shoot tips, or any other micropropagule which have the ability to convert into a plant in vitro or ex vitro. The need of artificial seed production was felt due to failed seed propagation in some crop species due to very small seed size, seed heterozygosity, reduced endosperm, no germination in the absence of seed–mycorrhizal association as in case of orchids and also time-consuming vegetative means of propagation in some seedless varieties of crops such as Citrullus lanatus and vitis vinifera, etc. Effective seed coating of micropropagules is done using different gelling agents such as alginate, agar, carrageenan, gellan gum, sodium pectate and carboxy methyl cellulose. However, sodium alginate has been documented as most frequently used gelling agent. The absence of seed coat and endosperm in somatic embryos necessitates the encapsulation matrix to be supplemented with nutrients and growth regulators such as 0.5 mg/L indoleacetic acid (IAA), 0.5 mg/L naphthalene acetic acid (NAA), 2 mg/L 6-benzyl aminopurine (BA), 2 mg/L Fe-EDTA and 30 g/L sucrose. In many plant species such as Allium sativum, Ananas comosus, Dioscorea bulbifera, Cineraria maritima, Cucumis sativus, etc. genetic stability of the plants derived from artificial seeds has also been examined with the help of biochemical and molecular markers and found them genetically consistent.KeywordsArtificial seedEncapsulationSodium alginateSomatic embryos
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Date palm (Phoenix dactylifera L.), a monocotyledonous species of the Arecaceae family, is widely cultivated in the arid regions of the Middle East and North Africa. Considering the prolonged generation cycle, the dioecious nature of date palm trees, and high heterozygosity, the traditional breeding approaches in date palm are lengthy and laborious, and numerous crosses and back-crosses all have led to intangible advancement in date palm breeding. In recent years, the powerful potential of biotechnology has been considered for resolving fundamental difficulties associated with date palm breeding. Plant tissue culture, an important application of biotechnology, is an essential tool for vegetative propagation and a prerequisite for genetic modification. Genomic studies and molecular tools are integrated with modern plant breeding programs for precise determination of genetic diversity, identification of desired traits, germplasm conservation, and genetic drift control. This technology clarifies how the genome works in a specific evolutionary or environmental condition, determines relationships between genes, identifies the role of coding and non-coding parts of the genome, and identifies key points in regulating evolutionary processes and responding to plant internal and external factors. A comprehensive and clear picture of functional genomics pave the way for plant genetic engineering to improve the desired traits. This review surveys the recent approaches and applications of biotechnology in date palm breeding.
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The asexual, non-zygotic (somatic) embryogenesis was reported over 60 years ago. It is a unique process in which somatic cell transforms into embryo in cultured condition affirming cellular totipotency. Since its first report, somatic embryogenesis has been noted in different plant genera of a dicot, monocot, and lower fern groups. In the present chapter, an attempt has been made in describing somatic embryogenesis and its current status in periwinkle, Catharanthus roseus—an immensely important medicinal plant. The plant has anticancerous properties, the yield of alkaloids specific to this disease is however low and needs enrichment. The early embryogenic cells could serve as an important tissue for generating new cell lines with improved yield and related traits. Here, the influence of explants, the involvement of plant growth regulators, and the role of other regulating factors in establishing Catharanthus embryogenic tissues are discussed. The somatic embryo development, structure, progress, i.e., maturation, germination of embryo are also discussed with the help of Scanning Electron Microscopic method. The secondary somatic embryogenesis cycle, the embryo irregularities, and the role of protoplast culture in somatic hybridization is briefly deliberated taking Catharanthus as an example. Liquid overlaying at varying levels are described of which 0.50 ml overlaying on solid medium improved embryogenic growth and embryo number significantly. Successful artificial seed production, storage at low temperature, and plant regeneration are discussed by optimizing sodium alginate (2.5%) and calcium chloride (100 mM) solution. The proteomic approach has recently been exploited to investigate non-embryogenic, embryogenic tissues, embryo development, and other events at molecular level. In Catharanthus, over 1000 new proteins were identified from embryogenic callus, which was absent in non-embryogenic tissues. Some important proteins identified at various stages of embryogenesis are catalase, glutathione S transferase, superoxide dismutase, heat shock protein 70 (HSP70), HSP 90, and cytochrome C. Conservation of embryogenic tissues is important as it produces embryos and plantlets continuously. The cryopreservation protocol of Catharanthus is discussed by mentioning the role of osmotica, various cryoprotective agents, and the exposure of dehydration. Somatic embryo regenerated plants are true to type when developed directly on explant, but various types of variations are noted in plants developed from callus, suspension, and similar other differentiated tissues. In Catharanthus, the genome size or the 2C DNA of somatic embryo regenerated plant was noted to be 1.516 pg, which is similar to field grown plant DNA. No alteration in genome size was noted although the plants were produced through callusing. All this accumulated information on Catharanthus will be very valuable in practical and basic applications as it broadens the knowledge of cellular differentiation and dedifferentiation processes; at the same time, the methodology will augur fast multiplication of raw materials for the synthesis and enrichment of alkaloids.
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Diabetes mellitus is becoming a prime area of public health concern around the world that is shifting its focus toward plants having anti-diabetic properties, which can be used for the treatment of diabetes. To meet the demand based on the pharmaceutical importance, approaches on rapid propagation, conservation, and germplasm exchange of anti-diabetic plant materials are need of the hour. In this context, artificial seed production technique can be employed for large-scale propagation, conservation of germplasms, and exchange of anti-diabetic plants materials among laboratories to facilitate the production of secondary metabolites for drug development. Artificial seed technology is mainly attempted for easier exchange and short- to mid-term storage of germplasm. In addition, long-term storage can be achieved through encapsulation-dehydration or encapsulation-vitrification followed by liquid nitrogen exposure, i.e., cryopreservation (storing at ultra-low temperature). The alginate encapsulation technology is being used in various anti-diabetic plants to produce artificial seeds using different kinds of explants depending on the plant species, which can be stored and regenerated after definite storage duration. To attain uniform, isodiametric, firm beads, a range of sodium alginate with calcium chloride was found to be effective across the concerned anti-diabetic plant species. Relatively low progress has been made regarding the long-term storage of artificial seeds retaining its viability. This chapter primarily addresses the factors affecting the artificial seed production and their storage of some anti-diabetic plants across various families.
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The importance of tissue culture for clonal propagation in agriculture continues to increase each year. In general, commercial use of tissue culture propagation has been limited to crops that have a high per-unit value, such as ornamentals and fruit and nut trees. A lowcost, high-volume propagation system is not available, but could be of significant value to medium per-unit value crops such as lettuce, celery, and many others (Table 1). For these crops, highvolume propagation potential of somatic embryogenesis combined with formation of synthetic seeds for low-cost delivery would open a new field for clonal propagation. Candidate crops for synthetic seed production can be classified into two categories: 1) those that have a strong technological basis, such that high quality somatic embryos can currently be produced, and 2) those with a strong commercial basis. The latter category of crops are those in which seed costs are high because of fertility problems, gamete instability, labor-intensive hybrid seed formation, or a number of other reasons. Currently, there are few crops (Table 1) that meet both requirements and are suitable for synthetic seed technology.
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Somatic embryos of Asparagus cooperi were encapsulated as single embryos approximately 4–6 mm in diameter to produce individual synthetic seeds. The frequency of conversion of artificial seeds to plants was 34%. This frequency was affected by the concentration of calcium chloride, the commercial sources of sodium alginate, and the nutrient medium. The conversion frequency of artificial seeds to seedling plants was 8.3% after storage for 90 days at 2°C.
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In vitro propagation of rare Indian medicinal herb 'Safed Musli' (Chlorophytum borivilianum) was achieved using encapsulated shoot buds. Four millimetre long shoot buds encapsulated in 3.0 % sodium alginate matrix polymerised by 100 mM solution of CaCl2·2H2O yielded best results. Storage conditions, gel matrix media and period in storage influenced the in vitro regrowth potential of shoot buds when transferred on standard multiplication (SM) medium containing MS salts and 5.0 mg l -1 BAP. Encapsulated shoot buds stored on wet agar-gel and kept under culture room conditions of light and temperature (45 μmol m-2 s-1 and 28±2°C, respectively) showed more than 80 % sprouting within 3 weeks. Dark stored (4°C) encapsulated shoot buds on agar-gelled wet medium showed more than 90 % sprouting after 7 d of storage. The per cent response in terms of regrowth potential declined to 60 % when buds were stored for more than 30 d attaining a low of 20 % in another 30 days. Supplementing alginate matrix with sucrose and MS salts (ALMSS) produced better results as compared to that with MS salts alone (AL MS) or without any of them (ALW). All the sprouted shoot buds, irrespective of their storage conditions, produced normal shoots on SM and multiplied at a rate of 2.5-fold per subculture of 21 d each. More than 70 % micro shoots could be rooted on the medium containing 3/4 MS salts and 2.0 mg l-1 IBA. Rooted plantlets were hardened successfully and grown under greenhouse conditions where they produced normal tuberous roots. This method of micropropagation opens possibilities for storage of shoot buds during off-season of production and facilitate transport of germplasm with ease.
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The delivery system, using artificial seed, is of great value in germplasm storage, propagation, and in the production of chemicals. Artificial seed for plant propagation has been investigated in somatic embryogenesis (Kitto and Janick 1985; Redenbaugh et al. 1991). We have extended this concept to the hairy root because of the successful regeneration of the whole plant from Ri-transformed cells. The micropropagation system using these hairy roots promises practical application in the fields of cellular biology, agriculture, and bioengineering.
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The research communication reports the production of artificial seeds through encapsulation of protocorm-like bodies (PLBs) of Geodorum densiflorum (Lam) Schltr. - an endangered orchid taxon of Terai Hills, North-eastern Himalaya. 30-day-old PLBs were encapsulated in sodium alginate. Germination and regeneration capacity of the encapsulated seeds were tested by germinating such seeds in modified Knudson C (KnC) medium supplemented with coconut milk 15% (v/v), peptone (2 g1-1), 6-benzyl-aminopurine (2 mg 1-1), and α-napthaleneacetic acid (1 mg 1-1). 88% Germination was recorded. Artificial seeds stored at 4°C for 120 days showed no reduction in viability. Non-encapsulated PLBs showed no viability after 30 days at 4°C. Artificial seeds showed 28% viability when directly transferred to nonsterile soil condition after incorporating food preservative and fungicide in its encapsulating gel.
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Axillary buds of mulberry (Morus indica L) were encapsulated in alginate and agar to produce individual beads. The beads could be stored at 4°C for 45 days without loss of viability. Amongst the encapsulating agents tested, sodium alginate was found to be a better matrix. Encapsulated buds regenerated complete plantlets on an appropriate medium. This technique would provide an easy and novel propagation system for the elite as well as difficult-to-root species of mulberry.