Development of synthetic seeds involving androgenic and pro-embryos in elite indica rice

Abstract

Synthetic seeds were produced from anther-derived mass-multiplied embryos and pro-embryos of rice (Oryza sativa L.) var. IR 72. A high dose (4-6 mg L -1) of BAP was found to produce a large number of dormant embryos, pro-embryos and embryo-like structures in about 45 d. These were encapsulated in sodium alginate (2.5% w/v) matrix. Germination and plantlet regeneration capacity of the encapsulated seeds were tested by culturing them on MS fortified with different combinations and concentrations of BAP, Kn and NAA. The result indicated that BAP in combination with lower concentrations of NAA increased germination of beaded embryos over control (MS without hormones). High percent of germination (55-87.5%) was observed when MS was supplemented with BAP and lower concentration of NAA; whereas, addition of Kn in MS reduced the germination percentage. The germination of unbeaded pro-embryos was 92.5% on MS basal medium. The reduced rate of germination of artificial seeds may be attributed to the damage incurred while separating the embryos from clusters and/or owing to adverse effects of chemicals used for encapsulation. Moderate germination (40.0%) was seen on sterile sand. Synthetic seeds may be used for in vitro propagation as well as genetic transformation experiments, especially involving biolistics.

Full text

Indian Journal of Biotechnology
Vol 7, October 2008, pp. 515-519
Development of synthetic seeds involving androgenic and pro-embryos
in elite indica rice
Bidhan Roy*1 and Asit B Mandal
Biotechnology Section, Central Agricultural Research Institute, P O Box No. 181, Port Blair 744 105, India
Received 12 February 2007; revised 19 February 2008; accepted 22 April 2008
Synthetic seeds were produced from anther-derived mass-multiplied embryos and pro-embryos of rice (Oryza sativa L.)
var. IR 72. A high dose (4-6 mg L-1) of BAP was found to produce a large number of dormant embryos, pro-embryos and
embryo-like structures in about 45 d. These were encapsulated in sodium alginate (2.5% w/v) matrix. Germination and
plantlet regeneration capacity of the encapsulated seeds were tested by culturing them on MS fortified with different
combinations and concentrations of BAP, Kn and NAA. The result indicated that BAP in combination with lower
concentrations of NAA increased germination of beaded embryos over control (MS without hormones). High percent of
germination (55-87.5%) was observed when MS was supplemented with BAP and lower concentration of NAA; whereas,
addition of Kn in MS reduced the germination percentage. The germination of unbeaded pro-embryos was 92.5% on MS
basal medium. The reduced rate of germination of artificial seeds may be attributed to the damage incurred while separating
the embryos from clusters and/or owing to adverse effects of chemicals used for encapsulation. Moderate germination
(40.0%) was seen on sterile sand. Synthetic seeds may be used for in vitro propagation as well as genetic transformation
experiments, especially involving biolistics.
Keywords: Androgenic embryo, anther culture, artificial seeds, pro-embryo, rice, sodium alginate
Introduction
Production of synthetic seeds, endowed with high
germination rate under in vitro and in vivo conditions,
bears immense potential as an alternative of true
seeds. It is an emerging area with great potential for
large-scale production of propagules at lower cost
with ease in handling and transport of crop plants1,2.
Artificial seeds consisting of culture-derived embryos
encased in a protective matrix were found to be
germinable for mass production of elite varieties3. In
several plants, conversion of artificial seeds into
plantlets was reported4-7. However, information about
production of artificial seeds from androgenic
embryos derived from androgenic callus in rice is
scanty. Initially, the preparation of artificial seeds was
restricted to encapsulation of somatic embryos in a
protective jelly and plantlet development from
artificial seeds was observed in a wide variety of
crops. The same principle has been exploited in the
present study. However, the encapsulation of anther-
derived embryos is extremely limited. Haploid
plant breeding has been well established as a tool
for crop improvement8-12. The induction of pollen
embryogenesis, which genetically differs from
zygotic embryogenesis, may be used for this purpose.
The essential prerequisite for application of the
artificial seed technology en masse involves large-
scale production of high quality micro-propagules,
which at present is a serious limiting factor1.
Embryogenesis in androgenic calli in indica rice has
been comparatively low than japonica and tropical
japonica varieties. Owing to certain inherent
problems in callus induction and plantlet regeneration,
the rate of production of uniform and high quality
embryos was found to be much lower. As a result,
efficient and quality artificial seed production was
found to be not much successful involving anther-
derived callus in rice. To overcome this, appropriate
protocol to produce a large number of embryos, pro-
embryos and embryo-like structures from anther-
derived plantlets has been developed for an elite
indica rice var. IR 72. When androgenic shootlets
were cultured on MS13 medium supplemented with 4
and 6 mg L-1 BAP14, they produced more than 100
_____________
*Author for correspondence:
Tel: 91-3582-270587; Fax: 91-3582-270157
E-mail: bcroy10@yahoo.com
1Present address: Department of Genetics and Plant
Breeding, Uttar Banga Krishi Viswavidyalaya, Pundibari,
Cooch Behar 736 165, India

INDIAN J BIOTECHNOL, OCTOBER 2008
516
dormant embryos per culture within 45 d of culture.
These dormant embryos, pro-embryos and embryo-
like structures produced shoots and roots en route
embryogenesis on MS with no hormones, and were
used as source materials for preparation of artificial
seeds. The present communication deals with
encapsulation of embryos, pro-embryos and embryos-
like structures of androgenic origin and assessment of
their germination in vitro and in vivo to ascertain the
feasibility of their use as an alternative of true seeds,
and possibility of use in genetic transformation
experiment especially by using biolistic system.
Materials and Methods
Callus Induction and Plantlet Regeneration
'IR 72', an elite indica rice variety obtained from
International Rice Research Institute (IRRI), Manila,
Philippines, was grown in the experimental net-house
of Central Agricultural Research Institute, Port Blair,
India. For anther culture, the primary panicles
enclosed in leaf sheaths were collected from the main
tillers when the microspores were in mid-uninucleate
to early bio-nucleate condition. The panicles were
cold shocked for 8 d at 8°C in a BOD incubator prior
to anther plating. On the day of culture, selected
spikelets were surface sterilized in tissue culture
bottles with 0.1% freshly prepared HgCl2 solution for
10 min. The HgCl2 was then drained off and the
panicles were washed 4 times in sterile distilled water.
Anthers were plated aseptically onto radiation
sterilized Petridishes having callus induction medium
(CIM) containing N6 with 2 mg L-l 2,4-D, 6% sucrose
and 0.8% agar. The cultures were sealed with
parafilm and kept in complete dark at 25±2°C.
Embryogenic calli of at least ~ 2 mm diameter
were transferred to culture tubes containing 10 mL
of regeneration medium (RM) consisting of MS with
1 mg L-l BAP, 1 mg L-l Kn, 0.5 mg L-l NAA, 3%
(w/v) sucrose and 0.8% (w/v) agar. Inoculated
cultures were kept for 4 wk under 16/8 h light
(~ 130 µE m-2 s-1)/dark at 25±2°C. The regenerated
plantlets were used as explants for induction of
microtillering.
Multiplication of Embryos
The regenerated shoots of androgenic origin were
cultured on MS basal supplemented with different
concentrations of BAP, Kn, NAA singly and in
various combination (data not presented) to induce
and optimize microtillering. Two androgenic plantlets
were inoculated onto each culture tube. In each
treatment, 40 androgenic shootlets were cultured.
Culture condition was kept same as used for the
regeneration of plantlets from androgenic calli in
anther culture experiment.
Encapsulation of Embryos
The in vitro produced embryos, pro-embryos and
embryo like structures of androgenic origin were used
for encapsulation. Sodium alginate (Sigma Chemical
Company, Cat.No. A7003) solution (4% w/v) was
prepared by mixing with liquid MS supplemented
with 3% sucrose and autoclaved at 121°C under
104 kPa pressure for 15 min. Individual embryos,
pro-embryos and embryo like structures were isolated
from the clusters under stereomicroscope. Efforts
were made to select embryos only. These embryos
were mixed with sodium alginate solution for a few
seconds, picked up and placed in a sterile aqueous
solution of calcium chloride (CaCl2.2H2O; Sigma
Chemical Company, USA, Cat.No. 1.02382.0500)
with occasional agitation. Calcium alginate beads
were formed within 15-20 min. Beads were taken out
by decanting off the CaCl2 solution, washed with
sterile double distilled water, and surface dried with
sterilized blotting paper. Freshly prepared beads were
directly cultured on MS fortified with different levels
of synthetic hormones (Table 1) and on sterile sand
also. Seedling emergence and plantlet regeneration
were recorded after 10 d of culture. For each
treatment, 40 beads were employed in three
replications. The experiment was conducted
entirely under control environment using laminar
air flow bench.
Results
Callus Induction and Plantlet Regeneration
Cultured anthers of var. 'IR 72' showed profuse
callus induction within 15-30 d on N6 supplemented
with 2 mg L-1 2,4-D. Most of the responding anthers
turned brown and produced 2-4 calli per anther. In a
few cases they produced multiple calli. Response of
anther to callus formation was 60% and regeneration
of green plantlets from calli was only 22.2% (data not
presented).
Multiplication of Embryos
Rapid and high rate of multiplication of embryos
and pro-embryos (Fig. 1a) were observed on MS
containing 2 mg L-l BAP and 4 mg L-l Kn (~ 40.1
embryo and pro-embryo), followed by 4 mg L-l BAP

ROY & MANDAL: ANDROGENIC AND PRO-EMBRYOS OF RICE AS SYNTHETIC SEEDS
517
Fig. 1—Development of synthetic seeds and their germination under in vitro condition: a, stereomicroscopic view of germinating
embryos of androgenic origin before emasculation (10×); b, encapsulated embryos in sodium alginate (4%) beads; c, mass germination of
beaded embryos on MS medium with no hormone; d, seedling elongation from germinating synthetic seeds; and e, growing plantlet under
in vitro culture condition on MS basal medium.

INDIAN J BIOTECHNOL, OCTOBER 2008
518
and 1 mg L-l K n (~ 26.4). BAP singly at a concentra-
tion of 4, 6 and 8 mg L-l showed average of 22.6, 26.7
and 14.8 embryos, respectively (data not presented).
Germination of Encapsulated Embryos
Embryos and pro-embryos developed into plantlets
on MS basal medium without any phytohormones.
Hence, MS alone was used for artificial
encapsulation, followed by germination and plantlet
establishment (Figs 1b-e). Beads were cultured on MS
supplemented with different concentrations and
combination of growth regulators and on sterile sand
too (Table 1). The germination varied between 20.0 to
87.5%. Maximum germination (87.5%) was recoded
on MS supplemented with 1 mg L-1 BAP, 1 mg L-1 Kn
and 0.5 mg L-1 NAA, followed by ½ MS fortified
with 2 mg L-1 BAP and 0.5 mg L-1 NAA (62.0%) and
full MS with 2 mg L-1 BAP, 0.5 mg L-1 NAA (55.0%).
Minimum germination was observed when the
medium was supplemented with 4 mg L-1 Kn (20.0%).
Discussion
High rate of multiplication of embryos, pro-
embryos and embryo like structures were recorded on
MS supplemented with medium dose (4-6 mg L-1) of
BAP. Whereas MS fortified with Kn produced
multiple shootlets. This suggests that BAP at high
concentration exerts inhibitory effect on shootlet
development under in vitro.
The present results indicate that BAP in
combination with lower concentrations of NAA
increased germination of beaded embryos over
control (MS without hormones); whereas, addition of
Kn in MS reduced the germination percentage. The
germination of unbeaded pro-embryos was 92.5% on
MS basal medium. The reduced rate of germination of
artificial seeds may be attributed to the damage
incurred while separating the embryos from clusters
and/or owing to adverse effects of chemicals used for
encapsulation. By trail and error, the germination rate
may be improvised. The low germination rate of
encapsulated embryos of Santalum album15 and
Solanum melongena16 was also reported. In contrast,
however, superiority of encapsulated embryos was
reported in horseradish6, microspore-derived
embryos of barley17 and in rice7. The germinated
artificial seeds produced roots and established
into complete plantlets on transfer to MS basal
medium. Moderate germination (40.0%) was
observed when the artificial seeds were evaluated
for germination on sterile sand in vivo.
Recent advances in the production of artificial
seeds revealed that beside somatic embryos,
encapsulation of cells and tissues developed in vitro is
becoming popular. It offers a simple way of handling
cell and tissues, protecting them against strong
external gradients and as an efficient delivery
system17. Brar et al18 emphasized the need for
intensive research on artificial seeds and outlined its
impact on mass propagation of true breeding hybrids.
Storage of encapsulated embryos for a considerable
period of time allows the preservation of valuable
elite germplasm. The judicious and intelligent
coupling of artificial seed technology with that of
microcomputer would be important in achieving the
automated encapsulation and regeneration of
plantlets. This would tremendously increase the
efficiency of encapsulation and production of
homogenous and high quality artificial seeds, and
deems to be revolutionizing the current concept of
commercial micro-propagation method19. It is
important to mention, though, that information on the
development of synthetic seeds from anther-derived
embryos is scanty. Therefore, the present findings
would generate a new vista for rapid multiplication of
androgenic embryos and production of uniform
synthetic seeds. Encapsulation of embryos makes
easier shipment from one laboratory to another and
storage of elite materials for research purposes.
Furthermore, synthetic seeds are expected to offer an
Table 1—Germination of synthetic seeds of androgenic origin
on different media under in vitro and in vivo conditions
Medium and treatment No. of synthetic
seeds germinated*
Germination
(%)
½ MS + 2 mg L-1 Kn +
0.5 mg L-1 NAA
21d 52.5
MS + 2 mg L-1 Kn +
0.5 mg L-1 NAA
15f 37.5
½ MS + 2 mg L-1 BAP +
0.5 mg L-1 NAA
26c 62.0
MS + 2 mg L-1 BAP +
0.5 mg L-1 NAA
22d 55.0
½ MS only 19e 47.5
MS + 1 mg L-1 BAP +
1 mg L-1 Kn + 0.5 mg L-1
NAA
35b 87.5
MS + 4 mg L-1 Kn 08g 20.0
Sterile sand 16ef 40.0
Unbeaded embryos on MS
basal
37a 92.5
*Values bearing same letter in the column are not significantly
different at P=0.05 of DMRT

ROY & MANDAL: ANDROGENIC AND PRO-EMBRYOS OF RICE AS SYNTHETIC SEEDS
519
appropriate recipient system for alien gene transfer in
micro-projectile based gene delivery system which of
course has to be explored in course of time.
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