Introduction
Micropropagation technology or plant propagation through
tissue culture has developed rapidly, especially for synthetic seed production.
Synthetic seeds are considered as one of modern techniques for providing viable
seed through encapsulation of somatic embryo or other meristematic part which can
be regenerated into a plant. Advance propagation through synthetic seeds is
important alternative to scale up seedlings especially for elite genotype of
sugarcane. Synthetic seeds of sugarcane are able to be stored within a certain
period (Ravi and Anand 2012). Damage resistant in synthetic seed is potential
to be utilized for propagation and conservation (Sharma et al. 2013) and expand seedling shipping distribution (Rihan et al. 2017). The application of
synthetic seed technology has promising prospects to support sugarcane
seedlings production, including for conservation and preservation of sugarcane
genotype.
Somatic embryos, zygotic
embryos, buds, apical buds, axillary buds and callus from tissue culture
process could become a source of explant for synthetic seeds production.
Somatic embryo propagation through somatic embryogenesis is widely used for
production of synthetic seeds (Helal 2011). This method forming the
propagules rapidly in relative short time (Raza et al. 2012) which increases the success of its transformation
(Heringer et al. 2015) and production
of virus-free plant especially in sugarcane (Dewanti et al. 2016a). There are several factors influencing the occurrence
of somatic embryo for synthetic seed encapsulation including plant growth regulators
(Dewanti et al. 2016b), explant
types, and plant genotypes (Damayanti et
al. 2018). Application of exogenous auxin (Tahir et al. 2011; Sardar et al. 2016)
and amino acid source also effect the production of somatic embryo. Amino acids
addition stimulates the communication between cells and tissues in
multicellular organs. Nitrogen derived from amino acids is rapidly assimilated
during metabolism, which is used for protein synthesis in cell.
Suitable application of plant
growth regulator and amino acid trigger the formation of somatic embryogenesis
stages in sugarcane. Somatic embryo is initiated from restructured somatic cell
to develop embryogenic cells (Yang and Zhang 2010). These cells experience
several morphological and biochemical pathways through induction,
proliferation, and regeneration stages. Previous studies identified several
genes that play important role in somatic embryogenesis process such as Somatic Embryogenesis Receptor Kinase (SERK) (Ahmadi et al. 2016; Porras-Murillo et
al. 2018), Baby Boom (BBM) (Florez et al. 2015; Horstman et al. 2017),
Leafy Cotyledon (LEC) (Kumar and Van Staden 2017) and Wuschel (WUS) (Bouchabké-Coussa et al.
2013). However, the expression of these genes during embryogenic phase in
sugarcane are still undisclosed.
Induction stage requires an
optimal concentration of auxin to induce competent callus. Proliferation stage
is the crucial stage for producing high quality of somatic embryo. It required
involvement of auxin just as 2, 4-D and combination of amino acids such as
casein hydrolysate and L-Proline. Under optimum media composition, embryogenic
callus develops into pro embryogenic mass (PEM), globular, scutellar, and
coleoptile. Coleoptile was reported to be a suitable stage for synthetic seed
germination (Inpuay and Te-Chato 2012; Ningtiyas et al. 2016).
Encapsulation of sugarcane
coleoptile using proper gelling agent is required to preserve somatic embryo.
Gelling agent using sodium alginate and calcium chloride acts as artificial
endosperm for somatic embryo growth (Rihan et
al. 2017). Proper concentration of sodium alginate is very important to be
optimized to create suitable coating for somatic embryo. This coating
characteristic affects the germination and regeneration level of plantlet. In
Indonesia, production of synthetic seeds particularly in sugarcane is limited.
This study investigated a comprehensive study of the synthetic seeds production
derived from somatic embryo at coleoptile stage in sugarcane including callus
production, molecular analysis of somatic embryogenesis genes, until synthetic
seeds germination and regeneration.
Materials and Methods
Experimental Material
Experiments were performed in the Center for Development
of Advanced Science, University of Jember, Indonesia during February 2018 –
December 2019. A healthy 6 months old sugarcane plants variety NXI 1-3 were
used as experimental material. The outer part of leaves tip were
sterilized using alcohol 70% for 5 min and discarded until 4-5 layers of green
leaves tip. The inner part of leaves tip cleaved to collect spindle leaf and
sliced to 0.5 cm thickness as explants.
Callus Induction
Explants are placed in callus induction media for 6
weeks, at a temperature of 24°C under dark condition to induce embryogenic
callus. Callus induction media consisted of MS (Murashige dan Skoog)
supplemented with 30 g L-1 sucrose + 2.5 g L-1 phytagel +
300 mg L-1 Casein Hydrolisate. The media of callus induction (CI)
comprised of: (CI1) 0 mg L-1 2, 4-D; (CI2) 3 mg L-1 2, 4-D;
(CI3) 4 mg L-1 2, 4-D. Formation and percentage of callus were
observed by macroscopic and microscopic observations using a stereo microscope
(Leica EZ4HD).
RT-PCR Analysis
%20377-383_files/image001.png){kind=small}
Embryogenic and non-embryogenic callus were collected
from induction phase for RNA isolation analysis. RNA isolation was conducted
using iScript cDNA Synthesis Kit (Bio-Rad).
Isolated mRNA from callus converted to cDNA through reverse transcription (RT)
and amplified by PCR (Biorad t 100) using specific primer (SERK, WUS, BBM, and
LEC gene). Electrophoresis using agarose gel 1% conducted for PCR product
visualization.
Proliferation of Somatic
Embryo
Pro Embryo Mass (PEM) from callus induction stage was
transferred to proliferation media to induce somatic embryogenesis formation.
PEM incubated in proliferation media for 6 weeks (4 weeks darkness, 2 weeks
light) under 1600 lux at 24oC. Proliferation media contained MS
media, 30 g L-1 sucrose + 2.5 g L-1 phytagel, and 2 mg L-1
2,4-D + 500 mg L-1 L-Proline + 300 mg L-1 Casein Hydrolisate.
Proliferation stage determined the development of pre-embryo, globular,
scutellar and coleoptile.
Synthetic Seed Production
Preparing Embryo
In coleoptile stage, embryos were collected from
proliferation stage. Coleoptiles from somatic embryo were selected uniformly (±
0.5 cm) from the explants and sown into sodium alginate solution.
Encapsulation
Encapsulation solution comprised of media MS + 1.5 mg L-1 BAP + 0.5 mg L-1 NAA
and three different concentrations (3, 4 and 5%) of sodium alginate.
Coleoptilar embryo was mixed with encapsulation solution, piped by pipette
pasteure and put in hardening solution. Capsule (synthetic seed) placed in CaCl2.2H2O 100 mM,
soaked for 30 min and rinsed with sterile distilled water 3 times, then placed
on a filter paper and air dried.
Germination and Regeneration
Synthetic seeds were grown in media germination and
regeneration. Regeneration media consisted of MS, 30 g L-1 sucrose +
2.5 g L-1 phytagel, 24°C, 1600 lux light intensity, 16 h light and 8
h darkness. Plantlet observed in ±2 cm heights. Germinated synthetic seeds were observed daily.
Shoot growth of plantlet were measured after plant height reached 0.5 cm.
Results
Somatic embryogenesis was initiated from very young leaf
tip of sugarcane, which named commonly as spindle leaf, as meristematic tissue
to induce callus production on induction media containing 2, 4-D under dark
condition (Fig. 1A and 1B). Incubation of spindle leaf on callus induction
media stimulated formation of swollen tissue as callus initiation process. In
consequence of cell division process, swollen tissue developed into embryogenic
callus on outer layer of spindle leaf (Fig. 1C) showed smooth, white and glossy
structure, and this particular spot potentially develop into next stage of
somatic embryogenesis (Fig. 1D).
Addition of exogenous plant
growth regulator particularly auxin to induction media also contributed on callus development. Result showed
that 3 mgL-1 and 4 mgL-1 of 2, 4-D more potential for
accelerating sugarcane callus initiation process than without application of 2,
4-D. Addition of 4 mgL-1 of 2, 4-D (C3) induced callus initiation
after 12.67 days, five days faster than 3 mgL-1 of 2, 4-D (C2). Data
showed that auxin played important role in callus induction. Without
application of 2, 4-D, it required up to 37 days for callus initiation (Fig.
2A).
Application of 3 mgL-1
and 4 mgL-1 of 2,4-D also increased percentage of callus production
during incubation on induction medium 70 and 90% respectively. Otherwise, MS
medium without 2, 4-D delayed percentage of callus production until 6.67% (Fig.
2B). Callus induction without growth
regulator intervention resulted non-embryogenic callus and less of embryogenic
callus. High production of embryogenic callus stimulated higher development of
somatic embryo on proliferation stage.
Identification of embryogenic
and non-embryogenic callus using molecular marker required to convince the
ability of callus to develop into somatic embryo. In this study, the expression
of SERK, BBM and LEC genes on sugarcane embryogenic callus were discovered
(Fig. 3). Interestingly, WUS gene did not identified on both types of sugarcane
callus.
Composition media of
proliferation also played important role to accelerate embryogenic callus
production. Application of amino acid such as proline and casein hydrolysate
influenced total callus weight and the number of formed coleoptiles. Presence
of single casein hydrolysate produced lower total callus weight per 100 mg than
combination of casein hydrolysate with proline (Fig. 4A). Based on the data,
300 mgL-1 of casein hydrolysate was the optimum concentration for
somatic embryo development at proliferation. After combining with proline,
total callus weight
significantly increased approximately 45%. However, application of proline at
concentration 550 mgL-1 tended to decline total callus weight per
100 mg about 43%
(Fig. 4B).
%20377-383_files/image003.png)
Fig. 1: Callus induction of sugarcane
for synthetic seeds production: (A)
Young leaf tip from the field for explant; (B) Spindle leaf section cultured on callus induction media; (C) Embryogenic callus on outer layer of
spindle leaf; (D) Microscopic
observation of embrogenic callus on 12.5 x
magnification
%20377-383_files/image005.png)
Fig. 2: Time of callus formation: (A) First callus formation and its
percentage; (B) Percentage of callus
formation during 6 weeks embryogenic callus induction period (C1 media: 0 mg L-1
2, 4-D, C2 media: MS
+ 3 mg L-1 2, 4-D, and C3 media: MS + 4 mg L-1
2, 4-D
%20377-383_files/image007.jpg)
Fig. 3: Expression of SERK,
WUS, BBM, and LEC genes on non-embryogenic (NE) and embryogenic callus (E)
in sugarcane
%20377-383_files/image009.png)
Fig. 4: Callus growth and development
during proliferation (A) Total
callus weight per 100 mg and (B)
Number of coleoptiles under different combination of proline and casein
hydrolysate concentration. (P0CH1: 0 mg L-1 proline and 250 mg L-1
casein hydrolysate; P0CH2: 0 mg L-1 proline and 300 mg L-1
casein hydrolysate; P1CH1: 500 mg L-1 proline and 250 mg L-1
casein hydrolysate; P1CH2: 500 mg L-1 proline and 300 mg L-1
casein hydrolysate; P2CH1: 550 mg L-1 proline and 250 mg L-1
casein hydrolysate; P2CH2: 550 mg L-1 proline and 300 mg L-1
casein hydrolysate). NA: not available
%20377-383_files/image011.png)
Fig. 5: Development of somatic embryogensis stages during proliferation
%20377-383_files/image013.png)
Fig. 6: Synthetic seeds germination and regeneration of
plantlet: (A) Synthetic seeds encapsulation;
(B) seed germination after 7 days; (C) Regeneration of plantlet after 14
days; and (D) after 48 days
Proper composition and
concentration of media determined optimum development of somatic embryogenesis
on both induction and proliferation stage. Each stage had various time
initiations dependent on specific nutrient applied on media (Fig. 5). In this research, development of SE initiated from
callus to Pro Embryo Mass (PEM) structure. Production of PEM structure from
callus induced globular formation after 14 days incubation under optimum
condition. Totally,
induction phase from callus to globular structure required 42 days under dark
condition.
Globular stage characterized by
yellowish white to glossy of smooth clumps. After incubating under dark
condition, embryo somatic transferred into light condition to promote scutellar
stage. Scutellar initiation required shorter time than globular initiation.
Round clumps of globular stage turned into heart shape. Some parts displayed
green spots and further developed into elongation shape. Coleoptile structure
formed after 26 days, characterized by the emergence of bipolar structure green
leaves. Coleoptile from embryo somatic was collected for encapsulation of
synthetic seeds (Fig. 6A).
Synthetic endosperm for
encapsulation used sodium alginate as nutrition storage for somatic embryos
growth. Nutrient absorption of synthetic seeds was influenced by concentration
of sodium alginate. Proper sodium alginate concentration enables synthetic
seeds germinated after 7 days (Fig. 6B).
Data showed that optimum concentration of sodium alginate for synthetic
seeds encapsulation was 4%. Lower germination percentage of synthetic seeds in
5% sodium alginate concentration expected inhibit embryos growth due to hard structure
of seed to be penetrated. Concentration
of sodium alginate also influenced number of shoot growth after 14 days (Fig.
6C). Concentration of 3 and 4% of sodium alginate supported high number of
shoot production. However, high of number of shoot production did not decide
the quantity and quality of plantlet formed. After 48 days incubation in
regeneration media, plantlet in 3% of sodium alginate showed better growth
particularly in number of plantlet formation and its height.
Discussion
Somatic embryogenesis has several specific stages
starting with the formation of pro-embryonic mas (PEM) followed by somatic
embryo formation maturation, and regeneration (Fehér 2015). Somatic
embryogenesis was induced from young leaf tip in sugarcane to provide optimal
source for callus induction due to its totipotent character (Yasmin et al. 2011). Callus from meristematic
tissue appeared from sliced region of spindle leaf explant and developed into
embryogenic callus (Fig. 1).
Embryogenic callus were discovered and characterized by dry and yellowish-white
which indicated that callus had a dense cytoplasm and high ratio of nucleus or
cytoplasm (Alcantara et al. 2014).
These characteristics showed that cell have meristematic zones for somatic
embryo development. Non embryogenic callus were also identified
during this study. Non embryogenic callus mostly compact, wet, transparent
structure and some showed browing region (Widuri et al. 2016).
There are several different features
of embryogenic and non-embryogenic callus based on histology studies. Embryogenic
callus produces somatic embryos with globular structure while non embryogenic
callus only show meristematic part without somatic
embryos formation (Silveira et al. 2013).
The absence of somatic embryo formation affected by lower cell differentiation
related to protein degradation process and resulted in lower metabolic activity
which inhibit somatic embryo development (Heringer et al. 2015). Somatic embryogenesis development required proper
growth regulators to produce embryogenic callus. Plant growth regulators play a
key role in both zygotic and somatic embryogenesis. Among all of them, auxin is
the most effective for induction of somatic embryogenesis (Gulzar et al. 2020). Auxin reported to play
important roles for callus induction, particularly 2, 4-D (Fig. 2) (Tahir et al. 2011). Addition of 2, 4-D was widely
used for inducing rapid cell division and differentiation during callus
induction in Gramminae family (Lee et al.
2012). High auxin concentration applied during induction stage; promote somatic
cell to produce important substance for globular phase development in
proliferation stage.
Previous studies stated that
application of single 2, 4-D induced high performance of callus induction
(Jahangir et al. 2010; Altaf et al. 2013). Induction media without 2,
4-D treatment mostly experienced slower callus growth even cell death (Alfian et al. 2019). Appearance of browning
region supposed to be a limitation factor of lower callus production. Phenolic
compound from explant tissue inhibit nutrition absorption
of explant, disturb enzyme activity and other metabolic process, and caused
death of explants (Ahmad et al. 2013).
Application of plant growth
regulator contributes to gene regulation during somatic embryogenesis initiation
(Fig. 3). Genes involved during callus formation in sugarcane were SERK, BBM,
and LEC. Somatic Embryogenesis Receptor
Kinase (SERK) gene remarks embryogenic cell development and plays important
role in early somatic embryogenesis pathway (Steiner et al. 2012). SERK gene was highly expressed in embryogenic callus
of sugarcane. The expression patterns of SERK gene in sugarcane was similar to
those described for the Momordica
charantia (Talapatra et al. 2014). Baby boom (BBM) gene was responsible
to promote the alteration of somatic cell. Over expression of BBM gene resulted
in increasing SE development. Maulidiya et
al. (2020) mentioned that BBM gene expressed at a high level in the
globular stage and lower on the next proliferation stage. The expression of BBM
gene was used as biomarker for SE initiation in Theobroma cacao (Florez et
al. 2015). LEC also play important role in somatic embryo development and
differentiation. Interestingly, our data revealed that WUS gene did not
identified in embryogenic and non-embryogenic callus of sugarcane. It supported
by that WUS gene regulation first localized not in callus tissue but in shoot
meristem part of heart stage embryo. These finding justified the involvement of
at least three genes during somatic embryogenesis induction in sugarcane.
Expression of genes in embryogenic callus tended to support high production of
somatic embryo through somatic embryogenesis pathway.
Six weeks callus induction
considered as optimal age for transferring callus into proliferation media to
produce somatic embryo. Proliferation is important stage for somatic
embryogenesis development. Cell elongation occurs to develop somatic embryo
during proliferation stage. This process involve role of 2, 4-D in lower
concentration than induction to trigger continued cell division and produce
somatic cell. Embryo somatic developments also require application of amino
acid.
This research revealed the
synergy of casein hydrolysate and proline combination for somatic embryo
development. Both amino acids were very crucial for proliferation stage,
particularly for increasing callus weight and number of coleoptile (Fig. 4).
Amino acid such as casein hydrolysate and proline act as precursor for nucleic
acids and other cellular process during somatic embryogenesis development. Casein
hydrolysate applied as organic nitrogen sources for triggering callus growth
and differentiation. Application of single casein hydrolysate could increase
total callus weight per 100, even combination with proline showed higher total
callus weight (Fig. 4A). Interestingly, presence of single casein hydrolysate
only induced callus formation but did not promote callus differentiation until
coleoptilar stage (Fig. 4B). This finding demonstrated that amino acid
combination simultaneously supports somatic embryogenesis development. None
coleoptile formed during late proliferation stage, expected as the result of
failure callus growth due to the absence of L-Proline. L-proline promotes
callus growth, enhance callus size and elongation (Kishor et al. 2015). However, higher than 500 mg L-1 proline
concentration tended to decline total callus weight and number of coleoptile.
Development of somatic
embryogenesis of sugarcane was recorded in this study. Best performance of
somatic embryogenesis stages in each treatment during induction and
proliferation (Fig. 5). Pro embryogenic mass (PEM) initiation occurred during
14 days after callus formation, followed by globular stage in next 14 weeks.
Globular stage formed structure like embryo (embryoid) with two meristemoid regions
(bipolar) (Pandey et al. 2018).
Oliveira et al. (2017) claimed the
existence of protodermal-dividing cell as competent region during morphogenetic
process in somatic embryogenesis. This state contains high accumulated protein
and starch contents for callus differentiation. It supported the finding of
nodular structure after globular stage started which performed green spot,
indicated meristematic tissue during scutellar stage. Green spot displayed
faster in 7 days and appeared like heart structure. Cell elongation occurrence
in late scutellar indicated shoot initiation and followed by coleoptile with
bipolar structure after 26 days.
Coleoptile was the optimum stage
used for embryo in synthetic seed encapsulation. Previous studies reported
application of suitable coating material (capsule)
such as sodium alginate and CaCl2 required to produce and preserve
synthetic seeds (Ali et al. 2012;
Maqsood et al. 2012). The material of
this capsule acts as an endosperm containing carbon sources, nutrients and growth
regulators that affect the life of embryo, particulary on the viability of
embryo in the seed.
Synthetic seeds germinate when
the encapsulated embryos break the gel. This germination is influenced by the
concentration of sodium alginate. The use of sodium alginate in different
concentrations determines seed production and germination. Good synthetic seed
characterized by compact, transparent, and firmly to wrap somatic embryo from
mechanical damage (Gantait et al. 2015).
Seed germination of 3–4% sodium
alginate proved to be better than 5% sodium alginate. Optimum concentration of
sodium alginate promoted better shoot growth in regeneration stage.
Concentration of 3 and 4% of sodium alginate together induced high number of
shoot production. However, both concentrations resulted in different quantity
and quality of plantlet. Plantlet in 3% of sodium alginate expected to provide
optimum structure of capsule which is supported sugarcane embryo growth during germination
stage. Higher concentration than 4% sodium alginate caused difficulty of the
embryo to break the seed coat. It resulted in stunted growth, lower seed
germination, and the seeds turned brown (browning) at regeneration stage.
Conclusion
Higher auxin concentration 4 mg L-1 was
proper concentration to induce embryogenic callus production. Embryogenic and non-embryogenic
callus formed during induction stage were regulated by somatic embryogenesis
genes including SERK, BBM, and LEC. During proliferation stage, sugarcane callus
required lower auxin concentration combined with proline and casein hydrolysate
to increase cell growth and the number of coleoptile
formed. Coleoptiles were selected as optimum stage for synthetic seeds embryo.
Sugarcane coleoptiles were encapsulated in sodium alginate with 3% concentration
for optimum embryo growth and development during germination stage.
Acknowledgements
This work was supported by grants from
PTUPT Project 2019 number of contract 1767/UN25.3.1/LT/2019 for Dr. Ir. Parawita
Dewanti, M.P. in the cooperation of Ministry of Research Technology and Higher Education
and the University of Jember, and Prof. Dr. Ir. Bambang Sugiharto, M.Agr.Sc the
Chief Director of Center fo Development of Advanced Science and Technology
(CDAST) of University of Jember.
Author Contributions
PD, BS, and PO planned the experiments, AUKM and FNA
interpreted the results, LIW and FNA made the write up and statistically
analyzed the data and made illustrations.
Conflict of Interest
All authors declare no conflict of
interest
Data Availability
Data presented in this study will be
available on a fair request to the corresponding author
Ethics Approval
Not applicable in this paper
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