Plant Tissue Cult. & Biotech.

34(2): 153-164, 2024 (December) ISSN 1817-3721, E-ISSN 1818-8745

DOI: h ps://doi.org/10.3329/ptcb.v34i2.78830
©Bangladesh Assoc. for Plant Tissue Culture & Biotechnology
PTC&B
Optimization of Plant Growth Regulators for Efficient
Embryogenic Callus Induction and Subsequent Plant
Regeneration in Two Indigenous Aromatic Rice Varieties of
Bangladesh
Mimma Afrin1, Sium Ahmed, Tanvir Ahamed, Mir Md. Mahbubur
Rahman, Sayeda Mahfuja Khatun2, A.K.M. Mohiuddin1 and Abdullah
Mohammad Shohael*

Cell Genetics and Plant Biotechnology Laboratory (CGPBL), Department of Biotechnology and
Genetic Engineering, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Key words: Aromatic rice, Callus induction, Kalijira, Somatic embryogenesis, Tissue
culture, Tulshimala

Abstract
An efficient regeneration protocol via somatic embryogenesis for two aromatic Indica rice
varieties, Kalijira and Tulshimala was developed. Using mature, dehusked seeds, the
process involved callus initiation, embryo development and plant regeneration involving
MS medium accompanied with specific concentrations of the auxins 2,4-D and NAA,
along with the cytokinin BAP. Callus induction was optimized at 2 mg/l 2,4-D, and 1
mg/l NAA, yielding initiation rates of 87% for Kalijira and 95% for Tulshimala. The
medium supplemented with 2 mg/l 2,4-D supported robust embryogenic calli
development. Mature embryos were successfully regenerated on media comprising 0.1
mg/l NAA and 2.5 mg/l BAP for Kalijira while 2 mg/l BAP for Tulshimala, with 35% of
Kalijira calli forming multiple shoots and 65% rooting, on the other hand Tulshimala
achieved 70% shoot formation and 100% rooting. Transplanted plantlets adapted well,
showing high survival rates.

Introduction
Rice (Oryza sativa L.) is an annual grass species from the Poaceae family. Aromatic rice
varieties are a unique and limited category of rice as they are regarded as the highest
grade because of their aroma, flavor, and texture (Weber et al. 2000). Although aromatic
rice is highly priced, its market demand is high (Singh et al. 2000). Aromatic rice is valued

*Author for correspondence: <amshohael@juniv.edu>. 1Department of Biotechnology and Genetic

Engineering, Mawlana Bhashani Science and Technology University, Tangail-1902, Bangladesh.
2Holding No-55, East Talaimari, Motihar, Rajshahi, Bangladesh.
154 Afrin et al.

for its culinary qualities and economic importance in global trade, making it a focus of
agricultural research aimed at improving yield, aroma retention, and stress resilience.
Ambemohar, Basmati, Chinigura, Gobindobhog, Jasmine, Kali Mooch, Kalijira, Sona
Masuri, Texmati, Tulaipanji, Tulshimala, Wehani, and wild pecan rice are some of the
popular varieties of aromatic rice (Singh et al. 2000). Some of the well-known local
varieties of Bangladesh are Badshabhog, Chiniatab, Chinigura, Kalijira, Kataribhog,
Khirshapati, Madhumala, Radhunipagal, Shakhorkora, Tulshimala and Zirabhog
(Hossain et al. 2008). Among these, Kalijira, Kataribhog, and Tulshimala rice received
Geographical Indication (GI) product tags in Bangladesh (Bangladesh Trade Portal 2024).
The grain production per hectare of most conventional aromatic rice varieties is
unsatisfactory. Several efforts have been made to produce high-yielding varieties with
superior nutritional quality using classic and mutation breeding, extensive hybridization,
omics approaches, genetic engineering, and gene editing (Zafar and Jianlong 2023).
Genetic engineering methods are employed to create rice plants with increased yield
characteristics and improved stress resistance (Parmar et al. 2017). Usually, tissue culture
methods are employed to generate genetically engineered plants, and establishing an
efficient tissue culture method is considered a requirement in the development process
(Purwantoro et al. 2022). In plant biotechnology, plant tissue culture is an essential
method that enables the in vitro development of plant cells, tissues, or organs on a
nutritional medium. This technique makes it easier to produce genetically uniform
plantlets, faster multiplication of disease-free plants, and conserve rare or endangered
species (Bhojwani and Dantu 2013).
By manipulating hormonal and environmental conditions, the tissue culture
technique can enable somatic embryogenesis, leading to the generation of somatic
embryos, which can further result in complete plants with well-formed roots and shoots
(Desai et al. 2022). Somatic embryogenesis mimics the phases of zygotic embryogenesis
in which somatic cells turn into embryos without fertilization (Desai et al. 2022). This
phenomenon can be induced in vitro by exposing plant tissues to specific growth
regulators under controlled conditions. Somatic embryos can develop into whole plants,
making this technique valuable for clonal propagation, genetic engineering, and
conservation of genetic resources (Bidabadi and Jain 2020).
Rice research focuses on key agronomic traits such as high grain quality, increased
yield, and resistance to diseases, pests, and environmental stress (Rezvi et al. 2023).
However, aromatic rice varieties often exhibit drawbacks, including susceptibility to
pests and diseases, low productivity, taller growth prone to lodging, and susceptibility to
both biotic and abiotic stresses. With the growing global demand for aromatic rice and
the current limitations of existing varieties, there is a pressing need to develop improved
aromatic rice cultivars that combine superior quality with enhanced resilience and yield
potential (Kaewmungkun et al. 2023). Developing indigenous aromatic varieties into
high-yielding varieties with enhanced resilience is imperative to support local economy,
improve food security, and maintain biodiversity. Therefore, integrating biotechnology
Optimization of Plant Growth Regulators for Efficient Embryogenic 155

with traditional breeding can accelerate the development of aromatic rice varieties that
maintain their characteristic fragrance while exhibiting be er performance under diverse
agricultural conditions (Prodhan and Qingyao 2020). Establishing a reliable tissue culture
method is crucial for supporting the application of modern biotechnology and new
breeding techniques in aromatic rice.
In light of these conditions, this study aimed to evaluate in vitro callus induction,
embryogenesis, multiple shoot formation, root formation, and subsequent regeneration
in nutrient media with various amalgamations of growth regulators for two indigenous
aromatic rice varieties, Kalijira and Tulshimala. The primary objective was determining
the optimal concentrations and combinations of growth regulators needed for effective
callus initiation, somatic embryogenesis, and regeneration in these varieties. Ultimately,
the goal was to develop an effective regeneration protocol to support future genetic
improvement efforts for Kalijira and Tulshimala.

Materials and Methods

The experiment was conducted at the Cell Genetics and Plant Biotechnology Laboratory,
Department of Biotechnology and Genetic Engineering, Jahangirnagar University,
Dhaka-1342, Bangladesh (23°53’14” N 90°15’56” E). Kalijira and Tulshimala rice seeds
were collected from the local farmers of Tangail district, Bangladesh, and used as
explants in this study.
The study utilized two auxins, 2,4-Dichlorophenoxyacetic Acid (2,4-D) and α-
Naphthalene Acetic Acid (NAA), along with one cytokinin, 6-Benzylaminopurine (BAP).
Different concentrations and combinations of these phytohormones were incorporated
into the MS medium. For scutellum-derived callus induction, three hormonal treatment
groups were employed. The first group contained 2,4-D at concentrations of 1.0, 2.0, 3.0
and 4.0 mg/l. The second group contained NAA at concentrations of 0.5, 1.0, 2.0 and 3.0
mg/l. The third group contained a constant 2.0 mg/l concentration of 2,4-D combined
with four different concentrations of NAA (0.5, 1.0, 1.5 and 2.0 mg/l). For embryogenic
callus production, varying 2,4-D concentrations of 1.0, 2.0, 3.0 and 4.0 mg/l were
employed. For high-frequency plant regeneration, different concentrations of BAP (1.5, 2,
2.5 and 3 mg/l) were paired with a fixed concentration of NAA (0.1 mg/l). A quantity of
30 mg/l (3%) sucrose was used as the carbon source, and 8 mg/l agar (0.8%) was used as a
solidifying agent. The pH of the media was maintained at 5.6-5.8 before the addition of
solidifying agent. Following this, the medium was sterilized by autoclaving.
For surface sterilization of the explants, the seeds were dehusked and rinsed with
distilled water. Then, the seeds were dipped in 70% ethanol for 30 sec followed by
thorough rinsing with sterile distilled water three times. A 10% sodium hypochlorite
solution was prepared using the commercial bleach Clorox (5.25% sodium hypochlorite)
with a few drops of Tween-20. The seeds were treated with this Clorox solution for 25-30
156 Afrin et al.

min, after which they were thoroughly rinsed with sterile distilled water 3-5 times.
Finally, the seeds were gently kept in sterile tissue paper to remove excess moisture.
All inoculation procedures were conducted in a sterile, aseptic environment using a
laminar airflow cabinet, following previously described protocols (Saha et al. 2015 2017).
For callus initiation, dehusked sterilized seeds were inoculated. The inoculated explants
were incubated in the growth chamber for 10-15 days at 25 ± 2°C in dark conditions. Data
on callus initiation frequency and days to callus initiation were recorded. Callus initiation
frequency was calculated as follows:

No. of calli
Callus initiation frequency (%) = × 100
Number of inoculated seeds

After 15 days, the scutellum-derived calli were transferred to the same medium,
producing nodular and compact calli. Selected calli were sub-cultured two times in
a new, freshly prepared medium. During subculturing, two types of calli were observed-
friable, yellowish, more prominent embryogenic calli and compact, translucent, slimy,
non-embryogenic calli. Only the embryogenic sections were sub-cultured after being
isolated from the non-embryogenic parts to facilitate embryogenic callus production.
Subculturing was conducted twice, with 15-day intervals between each transfer. Data on
average callus weight, properties of callus and degree of necrosis were recorded. For
high-frequency plant regeneration, fragile and fast-growing embryogenic calli (nodular,
white to pale yellow) were moved to the medium. Regeneration frequencies were
observed after 20 days. Data on days of shoot initiation, number of shoots per callus,
percentage of callus forming multiple shoots, and percentage of callus forming roots
were recorded. During the callus induction and regeneration, the culture conditions in
the growth chamber were kept at 23-25°C, 50% humidity, and a regulated photoperiod of
16 hrs of light and 8 hrs of darkness.
Regenerated shoots with well-developed root systems were removed from the jar
and gently washed with sterile distilled water to eliminate any remaining medium.
Afterwards, they were moved to pots filled with sterile soil and kept in a growth room at
27°C and 70% relative humidity for 7-10 days. The pots were covered with clear
polythene bags to keep the plantlets from drying out, and frequently, water was sprayed
on them to maintain adequate humidity levels. The plantlets were relocated to a
polyhouse to acclimate to the natural environment. After they were completely
established, the plantlets were moved to pots with a 1:1 mixture of autoclaved sand and
soil for hardening and further growth.
The experiment was conducted in a completely randomized design (CRD). All data
were displayed as the mean ± standard deviation (SD) of three independent biological
replications. Significant differences among mean values were compared by Tukey's
honestly significant difference (HSD) test at a level of significance of p ≤0.05. Statistical
analyses were performed using IBM SPSS Statistics 27.0.
Optimization of Plant Growth Regulators for Efficient Embryogenic 157

Results and Discussion

Following two weeks of seed inoculation, creamy white swelling (pre-embryogenic
masses) close to the root-shoot junction (scutellum area) indicated the onset of callus
induction. The optimal 2,4-D and NAA levels for the highest callus initiation were
assessed. Tables 1-2 present the findings. The table 1 shows that the callus formation was
lowest in the MS medium supplemented with 4 mg/l 2,4-D for Tulshimala, 1 mg/l 2,4-D
for Kalijira and also MS medium without any growth regulators (considered as control
and data not shown in the table). In contrast, the callusing response of explants was more
robust in the MS medium supplemented with 2 mg/l 2,4-D. The percentages of callus
formation across different treatment groups for the two varieties were statistically
significant.

Table 1. Effects of 2,4-D concentrations on callus initiation from scutellum of mature seeds of
Kalijira and Tulshimala.

Rice varieties Kalijira Tulshimala

Concentrations of 2,4-D (mg/l) 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0
Days to callus initiation 10 ± 1 a 8±1a 9±0a 10 ± 1 a 9 ± 0 ab 7±2b 10 ± 1 a 11 ± 0 a
Callus formation (%) 52.22 ± 0.2 d 86.66 ± 0.1 a 75 ± 3 b 66 ± 1.5 c 76 ± 1 c 89 ± 2 a 70 ± 1 b 62 ± 2 d

Results are shown as Mean ± SD. Means with same le er within each row are not significantly different
according to Tukey’s HSD tests (P <0.05).

For NAA, the results clearly showed that callus initiation of explants did not occur at
lower NAA values. Callusing and embryogenic responses were found when NAA
concentrations were higher. Among these hormonal concentrations, the highest
percentage of callus formation was found at 3.0 mg/l NAA for Tulshimala (55%) and
Kalijira (45%) (Table 2). Days to callus initiation were varied among three varieties. The
percentages of callus formation across different treatment groups for the two varieties
were statistically significant.
Table 3 shows the results obtained from the experiments where the callusing
responses were observed with four distinct NAA concentrations and a fixed 2,4-D
concentration (2 mg/l). From the table, it is apparent that the response was much be er
when two auxins were used in combination rather than separately. Among these
hormonal concentrations, callus induction was maximum for both varieties when the
medium was supplemented with 2 mg/l 2,4-D and 1 mg/l NAA. A variation in callus
initiation between varieties under identical experimental conditions suggests that callus
initiation is genotype-dependent. Previous studies reported the same findings (Alam
et al. 2003, Kaur et al. 1999, Summart et al. 2008). There were no significant differences in
days to callus initiation in regard to concentrations of growth hormone applications,
while significant differences were observed for callus formation.
158 Afrin et al.

Table 2. Effects of NAA concentrations on callus initiation from scutellum of mature seeds of Kalijira and
Tulshimala.

Rice varieties Kalijira Tulshimala

Concentrations of NAA (mg/l) 0.5 1.0 2.0 3.0 0.5 1.0 2.0 3.0
Days to callus initiation NCI NCI 13 ± 1 a 11 ± 0 b NCI NCI 10 ± 0 a 11 ± 1 a
Callus formation (%) N/A N/A 40 ± 2 b 45 ± 1 a N/A N/A 45 ± 3 b 55 ± 2 a

Results are shown as Mean ± SD. Means with same le er within each row are not significantly different
according to Tukey’s HSD tests (P <0.05). NCI- No callus initiation.

Table 3. Influence of NAA and 2,4-D combination on the percentage of callus formation and the days of
callus initiation.

Concentrations of 2,4-D Days to callus Callus formation

Rice varieties
and NAA initiation (%)

2 mg/l 2,4-D + 0.5 mg/l NAA 12 ± 1 a 70 ± 2 cd

2 mg/l 2,4-D + 1 mg/l NAA 10 ± 2 a 87 ± 3 a
Kalijira
2 mg/l 2,4-D + 1.5 mg/l NAA 11 ± 3 a 75 ± 2 bc
2 mg/l 2,4-D + 2 mg/l NAA 14 ± 3 a 68 ± 3 d
2 mg/l 2,4-D + 0.5 mg/l NAA 12 ± 1 a 70 ± 2 cd
2 mg/l 2,4-D + 1 mg/l NAA 9±2a 95 ± 2 a
Tulshimala
2 mg/l 2,4-D + 1.5 mg/l NAA 10 ± 2 a 80 ± 3 b
2 mg/l 2,4-D + 2 mg/l NAA 14 ± 3 a 75 ± 3 bc

Results are shown as Mean ± SD. Means with same le er within each column are not significantly different
according to Tukey’s HSD tests (P < 0.05).

The generated calli were sub-cultured, leading to multiplication and the

development and maturation of somatic embryos in the medium. The development of
pre-embryogenic masses following several cultures in auxin-supplemented media
provided evidence of an indirect somatic embryogenesis pathway. The results obtained
from this experiment are shown in Table 4. The resulting calli were compared
structurally based on their external features (Fig. 1) under a microscope. It was noticed
that MS media supplemented with 2.0 mg/l 2,4-D produced fragile, yellowish, nodular
embryogenic callus for Kalijira and Tulshimala. The texture of the callus was primarily
seen as compact at higher hormone doses and friable at lower concentrations. Of the four
treatments, MS medium supplemented with 2 mg/l 2,4-D was the most efficient
hormonal treatment for maximum proliferation in terms of the average weight of somatic
embryogenic callus of both rice varieties. However, there was a lack of statistical
significance. Proliferated calli were mostly friable, loose textured, and creamy-yellow,
which was an indication of somatic embryos.
Optimization of Plant Growth Regulators for Efficient Embryogenic 159

Table 4. Impact of varying 2,4-D concentrations on the production of embryogenic calli and determining the
physical characteristics of the resulting embryogenic calli.

Rice Concentrations Average callus Degree of

Properties of callus
varieties of 2,4-D (mg/l) weight (g) necrotic callus
Friable, brown embryogenic callus
1.0 0.109 ± 0.03 a +
initiation
Fragile, yellowish, nodular embryogenic
2.0 0.143 ± 0.04 a -
callus initiation
Kalijira
Yellowish-white nodular callus
3.0 0.088 ± 0.03 a ++
initiation
Translucent, slimy, and yellowish
4.0 0.079 ± 0.03 a ++
compact callus initiation
Soft, friable, brown color non-
1.0 0.128 ± 0.03 ab +
embryogenic callus initiation
Fragile, yellowish, nodular large
2.0 0.185 ± 0.04 a -
embryogenic callus initiation
Tulshimala
Yellowish-white, translucent, and slimy
3.0 0.093 ± 0.02 b +
compact callus initiation
Yellowish-brown, compact callus
4.0 0.092 ± 0.02 b ++
initiation

+++ = High; ++ = Moderate; + = Low; - = Negative. Results are shown as Mean ± SD. Means with same le er
within the column are not significantly different according to Tukey’s HSD tests (P < 0.05).

Fig. 1. Initiation and maturation of embryogenic calli of Kalijira and Tulshimala at different concentrations of
2,4-D. (A) Somatic embryogenesis at 1 mg/l 2,4-D for Kalijira rice seeds; (B) Somatic embryogenesis at 2
mg/l 2,4-D for Kalijira rice seeds; (C) Somatic embryogenesis at 3 mg/l 2,4-D for Kalijira rice seeds; (D)
Somatic embryogenesis at 4 mg/l 2,4-D for Kalijira rice seeds; (E) Somatic embryogenesis at 1 mg/l 2,4-D
for Tulshimala rice seeds; (F) Somatic embryogenesis at 2 mg/l 2,4-D for Tulshimala rice seeds; (G) Somatic
embryogenesis at 3 mg/l 2,4-D for Tulshimala rice seeds; (H) Somatic embryogenesis at 4 mg/l 2,4-D for
Tulshimala rice seeds.
160 Afrin et al.

Embryogenic calli showed high responsiveness to regeneration (Seraj et al. 1997). In

addition, scutellum explants have the benefit of rapid and straightforward callus
production, and no seedling germination is required to obtain explants (He and Lazzeri
2001, Joyia and Khan 2013). Although all somatic plant cells are theoretically totipotent,
the success of callus induction and regeneration in rice is strongly influenced by factors
such as explant origin, media composition, and genetic background (Hartke and Lörz
1989, Long et al. 2022, Pasternak and Steinmacher 2024).
Regeneration was performed with four different hormonal combinations of NAA
and BAP supplemented in MS medium to find the appropriate concentrations for
maximum regeneration of shoots. Data was documented across 6 weeks of inoculation
and given in Table 5. The table shows that the regeneration responses significantly varied
with the varying combinations of auxin and cytokinin. It was also evident from the
results that the two varieties differed slightly in regeneration performance; Tulshimala
had the highest regeneration efficiency, followed by Kalijira. While the embryogenic
callus was put into MS media supplemented with 0.1 mg/l NAA + 2 mg/l BAP, 70% of the
embryogenic callus produced multiple shoots in Tulshimala.

Table 5. Impact of regeneration media supplemented with varying quantities of BAP and NAA on multiple
shoots and root development parameters.

Days to No. of Callus forming Callus

Rice Concentrations of NAA
shoot shoots per multiple shoots forming roots
varieties and BAP
initiation callus (%) (%)
0.1 mg/l NAA + 1.5 mg/l BAP 23 ± 1.73 a 1 ± 0.00 d 15 ± 1 c 50 ± 2 c
0.1 mg/l NAA + 2 mg/l BAP 21 ± 1 a 3 ± 0.58 b 20 ± 2 b 55 ± 2 bc
Kalijira
0.1 mg/l NAA + 2.5 mg/l BAP 21 ± 1 a 4 ± 0.18 a 35 ± 1 a 65 ± 3 a
0.1 mg/l NAA + 3 mg/l BAP 25 ± 3 a 2 ± 0.25 c 15 ± 2 c 60 ± 2 ab
0.1 mg/l NAA + 1.5 mg/l BAP 22.3 ± 2.52 a 5 ± 0.00 bc 45 ± 5 bc 100 ± 0 a
0.1 mg/l NAA + 2 mg/l BAP 20 ± 2 a 7 ± 0.58 a 70 ± 1 a 100 ± 0 a
Tulshimala
0.1 mg/l NAA + 2.5 mg/l BAP 21 ± 1 a 5 ± 0.18 b 50 ± 3 b 100 ± 0 a
0.1 mg/l NAA + 3 mg/l BAP 24.6 ± 3.1 a 3 ± 0.25 c 40 ± 4 c 100 ± 0 a

Results are shown as Mean ± SD. Means with same le er within each column are not significantly different
according to Tukey’s HSD tests (P <0.05).

There were seven shoots on average per regenerated callus. All calli were found to
have roots. In Kalijira, 35% of the embryogenic callus formed multiple shoots when the
embryogenic calli were transferred to MS medium supplemented with 0.1 mg/l NAA and
2.5 mg/l BAP. There were four shoots on average per regenerated callus. On the other
hand, roots were noticed in 65% of calli, which was much lower than Tulshimala. These
results indicated that Tulshimala had the maximum regeneration capacity, followed by
Optimization of Plant Growth Regulators for Efficient Embryogenic 161

Kalijira. Fig. 2 shows the progression of callus initiation, callus proliferation, somatic
embryogenesis induction, somatic embryo regeneration, and acclimatization of
regenerated plantlets of Kalijira and Tulshimala. Comparative Regeneration responses
for two different aromatic rice varieties are presented in the graph shown in Fig. 3.

Fig. 2. (A-H) Callus initiation, callus proliferation, induction of somatic embryogenesis, regeneration of somatic
embryos, and acclimatization of regenerated plantlets of Kalijira; (I-P) Callus initiation, callus proliferation,
induction of somatic embryogenesis, regeneration of somatic embryos, and acclimatization of regenerated
plantlets of Tulshimala.
162 Afrin et al.

Fig. 3. Percentage of callus-forming roots and multiple shoots. Results are shown as Mean ± SD.

Indirect plant regeneration involves the sequential developmental steps of callus

induction and proliferation, embryogenic callus development, and plant regeneration
(Gandonou et al. 2005). To regenerate complete plants, the totipotent cells in the calli
followed distinct developmental routes. Plant development and morphogenesis are
regulated mainly by varying dosages of growth regulators. In general, shoot
morphogenesis is induced in a medium with a high concentration of cytokinin and a low
quantity of auxin. The induction of root primordia requires auxin either by itself or in
combination with low levels of cytokinin (Evans et al. 1981).
In the present study, calli grew in the regeneration medium with low concentrations
of NAA in combination with varying concentrations of BAP to develop the shoots. It has
previously been documented that the stimulatory action of BAP and NAA together
promotes regeneration in Indica rice callus cultures (Mandal et al. 2003, Ramesh et al.
2009). Jubair et al. (2008) employed the same hormone combinations in MS media to
regenerate the rice variety Topa, where they observed 20% regeneration frequency with
an average of 2 shoots per explant at 0.5 mg/l NAA + 3 mg/l BAP (Jubair et al. 2008).
Goswami et al. (2022) observed the regeneration responses of the aromatic rice variety
Doairgura, where 100% regeneration response and 15.3 shoots per callus were found at 1
mg/l NAA + 2.0 mg/l BAP combination (Goswami et al. 2022).
In vitro regenerated plants survived after being transferred to pots, indicating good
adaptability of somaclones in ambient environmental conditions. Additionally, all
plantlets regenerated from mature embryo-derived calli developed good root systems,
which was a significant aspect of the current study.
In conclusion, this study establishes an efficient and reliable somatic embryogenesis
and regeneration protocol for the Bangladeshi indigenous aromatic rice varieties Kalijira
and Tulshimala. This protocol holds promise for enhancing crop improvement efforts
through biotechnological applications. However, improving aromatic rice remains
Optimization of Plant Growth Regulators for Efficient Embryogenic 163

challenging due to its unique genetic complexity and sensitivity to environmental

conditions. In this context, advanced genetic transformation or genome editing
techniques offer exciting opportunities for precise trait enhancement, potentially
overcoming these limitations while preserving the distinctive qualities of aromatic rice.
Notably, Tulshimala displayed a superior tissue culture response compared to Kalijira,
suggesting it may be a particularly promising candidate. Nonetheless, both varieties
demonstrated potential for use in genetic modification studies, underscoring the value of
further research on these aromatic rice genotypes. By advancing a streamlined tissue
culture and regeneration system, this work paves the way for more effective breeding
and transformation efforts to unlock the genetic potential of aromatic rice varieties.

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(Manuscript received on 08 November, 2024; revised on 30 November, 2024)