Plant Cell, Tissue and Organ Culture (PCTOC) (2024) 156:39

https://doi.org/10.1007/s11240-023-02660-x

ORIGINAL ARTICLE

Somatic embryogenesis and plant regeneration of Litchi chinensis

Sonn. cv. ‘Zili’ from immature zygotic embryos
Miaoqin Huang1 · Wuyan Guo1 · Xiuyu Wu1 · Yaqi Qin1 · Irfan Ali Sabir1 · Zhike Zhang1 · Yonghua Qin1 · Guibing Hu1 ·
Jietang Zhao1

Received: 18 September 2023 / Accepted: 30 November 2023

© The Author(s), under exclusive licence to Springer Nature B.V. 2023

Abstract
In vitro plant regeneration is a powerful tool for the propagation and conservation of valuable plant species. Among the
diverse range of techniques available, the induction of somatic embryos from immature zygotic embryos has proven to
be an effective approach. In the present study, efficient induction methods and a suitable medium for in vitro plant regen-
eration from callus induced from immature zygotic embryos of Litchi chinensis Sonn. cv. ‘Zili’ have been successfully
established. Callus induction and maintenance were achieved on MS basal medium supplemented with 1 mg/L 2,4-D and
1 mg/L biotin under dark conditions. Somatic embryos were induced on the MS basal medium containing 30 g/L sucrose,
1 mg/L biotin, 1 g/L activated charcoal, and 8 g/L agar. The most effective culture method involved inducing somatic
embryos for 10 d under darkness, followed by maturation and regeneration under a 16-hour photoperiod provided by cool
white fluorescent lights (80 µmol·m-2 s-1). The average plant regeneration rate of ‘Zili’ reached 26.0%, with the fastest
regeneration occurring 83 d after inoculation. Somatic embryos exhibited five distinct morphological types: dicotyledon-
ous embryo, conjoined embryo, flake embryo, irregular swollen embryo, and others. The regeneration rates of different
morphological somatic embryos varied. Among them, conjoined embryos showed the highest regeneration rate, reaching
42.6%. Dicotyledonous embryos followed closely at 30.2%, while the rate of flake embryos was the lowest, at 15.4%.
These findings provide crucial insights for successful in vitro regeneration of litchi and shed light on the morphological
variations that influence regeneration efficiency.

Key Message
Plant regeneration of 'Zili' litchi via indirect somatic embryogenesis from immature zygotic embryos.

Keywords Litchi chinensis Sonn · Somatic embryo · Immature zygotic embryos · Plant regeneration · Morphology

Abbreviations CW coconut water

MS Murashige and Skoog 2,4-D 2,4-dichlorophenoxyacetic acid
AC activated charcoal IBA indole-3-butyric acid
LH lactoalbumin hydrolysate IAA indole-3-acetic acid
KT kinetin
GA3 gibberellic acid
NAA 1-naphthylacetic acid
Communicated by Yi Li. BA 6-benzyladenine
Jietang Zhao BAP 6-benzylaminopurine
jtzhao@scau.edu.cn
1
State Key Laboratory for Conservation and Utilization of
Subtropical Agro-bioresources/Key Laboratory of Biology
and Genetic Improvement of Horticultural Crops (South
China), Ministry of Agriculture and Rural Affairs/Guangdong
Litchi Engineering Research Center, College of Horticulture,
South China Agricultural University, Guangzhou, China

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 39 Page 2 of 11
PVP polyvidone
EDTA ethylenediaminetetraacetic acid
PPFD photosynthetic photon flux
Introduction
Litchi chinensis Sonn. is an important evergreen fruit crop
of China, Vietnam, India and Thailand, which originated in
southern China and was widely grown in the tropical and
sub-tropical areas in the world (Chapman 1984; Hussain et
al. 2021). Litchi belongs to the Napheleae subfamily of Sap-
indaceae (Das et al. 2016). The primary focus in this field
has been the selection and breeding of elite litchi cultivars.
However, the traditional breeding process has proven inef-
ficient, mainly due to the long growth cycle associated with
juvenile period (Das et al. 1999), high heterozygosity (Giri
et al. 2004; Wang et al. 2023), and high polyphenol oxidase
activity of fruit trees (Picinelli et al. 1995; Puchooa 2004;
Galmés et al. 2021; Wang et al. 2023). Fortunately, the in
vitro regeneration method offers a promising solution to this
inefficiency by significantly shortening the breeding period.
This approach proves to be simpler and more efficient, revo-
lutionizing the litchi breeding process and accelerating the
development of elite cultivars (Koroch et al. 2002; Singh
et al. 2005; Kanwar et al. 2010; Shi et al. 2015). Moreover,
the establishment of a robust regeneration protocol not only
expedites traditional breeding but also enhances the appli-
cation of molecular breeding and transgenic techniques.
This advancement opens new avenues for precise genetic
manipulation, enabling the development of litchi cultivars
with desirable traits and improved resistance to environ-
mental challenges. The integration of in vitro regeneration
with modern molecular techniques holds great promise for
the future of litchi breeding and agricultural innovation
(Welander 1988; Sarin et al. 2009; Padilla et al. 2013; Kisku
et al. 2017).
In recent years, there has been breakthroughs on in
vitro regeneration of litchi. Researchers have successfully
induced callus from various explants, including leaf, stem
segments, anther, and immature embryo. Subsequently, they
have achieved plant regeneration through somatic embryo
induction and maturation. Eight litchi cultivars (‘Feizixiao’,
‘Shuidong’, ‘Xiafangzhi’, ‘Gushanjiaohe’, ‘Chenzi’, ‘Tai
So’, ‘Bendana’, and ‘Nuomici’) have been regenerated suc-
cessfully via somatic embryogenesis (Fu and Tang 1983;
Kuang et al. 1997; Yu et al. 2000; Puchooa 2004; Deng
2005; Raharjo and Litz 2007; Das et al. 2016; Wang et al.
2016, 2023). Among these approaches, the use of embryo-
genic callus derived from immature embryos demonstrated
a notably higher regeneration rate. This finding highlights
the importance of selecting appropriate explants for the in
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Plant Cell, Tissue and Organ Culture (PCTOC)
vitro regeneration process, with immature embryos proving
to be a highly effective choice for achieving successful plant
regeneration in litchi.
Despite successful regeneration of plantlets in certain
litchi cultivars, a comprehensive and efficient regeneration
system for litchi has not been reported to date. As with many
other fruit trees, in vitro culture of litchi presents several
challenges, including strong genotype dependence, various
abnormal embryos (Qin et al. 2021), low regeneration rate
(Singh et al. 2005), and long regeneration process (Shi et
al. 2015). These complexities have impeded the develop-
ment of a standardized and reliable regeneration protocol
for litchi. Therefore, it is crucial to persistently explore and
refine in vitro culture techniques to achieve consistent and
widespread success in the regeneration of this valuable fruit
tree species (RH, 1986; Holcroft and Mitcham 1996), The
main objectives of this study were to select the morphol-
ogy of somatic embryos for regeneration, and enhance the
regeneration rate of litchi. Consequently, the study pre-
sented a detailed protocol to achieve a high frequency of
regeneration of L. chinensis Sonn. cv. ‘Zili’ through somatic
embryogenesis. Additionally, we focused on observing the
morphology of somatic embryos induced from callus. This
research contributes valuable insights and practical guide-
lines to advance the successful and efficient regeneration of
litchi through somatic embryogenesis.
Materials and methods
Plant materials and culture medium
Litchi chinensis Sonn. cv. ‘Zili’ was grown at litchi germ-
plasm resource nursery of South China Agricultural Univer-
sity, Guangzhou, China. Fruitlets from 25 d after anthesis
(DAA) were sampled. Thoroughly washed fruitlets were
surface sterilized with 70% ethanol for 1 min, followed by
submersion in 0.1% mercuric chloride for 8 min with occa-
sional agitation. Disinfected fruitlets were rinsed five times
with sterilized distilled water. Disinfected fruitlets were
cut longitudinally, and immature embryos were taken out
before inoculation.
The basal medium was comprised of Murashige and
Skoog (MS) mineral salts and vitamins (Murashige and
Skoog 1962). After pH was adjusted to 5.8 with 1 M KOH
solution, the medium was autoclaved at 121℃ for 20 min.
All cultures were maintained at 25 ± 1℃.
Callus induction and somatic embryogenesis
Immature embryos were cultured on MS basal medium
containing 30 g/L sucrose, 7 g/L agar, in combination with
Plant Cell, Tissue and Organ Culture (PCTOC)
1 mg/L 2,4-D and 1 mg/L biotin to induce callus. Cultures
were maintained in the dark. Callus induction frequencies
were calculated based on the number of explants with callus
versus the total number of explants after 2 months of cul-
ture. Yellow friable callus was picked out and sub-cultured
on the same medium at 45 d intervals. The callus was trans-
ferred to MS basal medium containing 30 g/L sucrose, 8 g/L
agar, supplemented with 1 mg/L biotin and 1 g/L activated
charcoal (AC), for 10 d under dark, and then maintained
under the PPFD of 80 µmol·m− 2 s− 1·with a 16 h photope-
riod for somatic embryogenesis.
Plant regeneration
Green globular embryos were selected and transferred to
MS basal medium containing 30 g/L sucrose, 8 g/L agar,
supplemented with 1 mg/L biotin and 1 g/L AC for embryo
maturation and plant regeneration. The plant regeneration
rate, calculated as the percentage of the number of normal
plantlets obtained from selected somatic embryos, was
recorded after 4 months of culture. To investigate the effects
of somatic embryo morphology on plant regeneration, dis-
tinct morphology of somatic embryos, including conjoined
embryo, dicotyledonous embryo, flake embryo, irregular
swollen embryo, were placed on the same medium for plant
regeneration.
Histological observation
As described by Lee and Mu (1966), callus and various
stages of somatic embryos (globular embryo, heart-shaped
embryo, torpedo-shaped embryo and cotyledonous embryo)
were collected and fixed in FAA (formalin: glacial ace-
tic acid: 70% alcohol at 5: 5: 90 by volume) for 96 h, and
then were rinsed three times with 70% alcohol. FeSO4 and
hematoxylin were used for consequent mordant dyeing and
staining. Tissues were dehydrated by increased ethanol
series. After transparent treatment by T0 transparent agent
(Guangxi Cenxi Rosin factory, China), the samples were
embedded in paraffin. After dewaxing, 8 μm thick sections
were further dried to make a permanent seal, which were
observed under a microscope.
Fig. 1 Callus induction of ‘Zili’
from immature embryo. (A).
Fruitlets of ‘Zili’ from 25 d after
anthesis and its section (arrow
indicated immature embryo); (B).
Callus obtained from imma-
ture embryo cultured on callus
induction medium for 30 d. (C).
Embryogenic callus obtained
after 4 subcultures. Bar = 1 mm
Page 3 of 11 39
Acclimatization and transplantation of regenerated
plantlets
When the plantlets with two or more leaves and well-
developed roots (more than 2 roots or a length longer than 2
centimeters), the culture bottles were transferred to an incu-
bation chamber under the PPFD of 80 µmol·m− 2 s− 1·with
a 16 h photoperiod at 25 ± 1℃ to acclimatize for 8 d. The
plantlets were carefully removed from the culture bottles
and rinsed in water to remove medium. Then, plantlets were
transplanted into pots filled with a nutrient-rich soil and
vermiculite mix in a ratio of 3:1. The pots were covered
with a transparency film and maintained in the incubation
chamber with the same conditions described above. A daily
spraying routine which included removing the film, spray-
ing and replacing the film was implemented to provide ade-
quate moisture for the plantlets. After 5 d, the transparency
film was removed and maintained with the same conditions
to facilitate gradual acclimatization of the plantlets to the
external environment.
Statistical analysis
Data collected was analyzed using SPSS software. One-Way
ANOVA was utilized to analyze variance among means, fol-
lowed by Duncan’s multiple comparison test for compari-
sons. The significant difference was set at P ≤ 0.05 level.
Results
Plant regeneration through indirect somatic
embryogenesis
Immature zygotic embryos (Fig. 1A) of ‘Zili’ were used as
explants for callus induction. Callus started to form from the
surface of the explants (Fig. 1B). The frequency of callus
formation was 78.5% after 2 months of dark culture. Yellow
friable callus (Fig. 1C) was obtained after 4 subcultures. The
embryogenic callus was used for somatic embryo induction.
The callus exhibited rapid proliferation within 14 d
on callus subculture medium under darkness (Fig. 2A).
White globular somatic embryos were observed 10 d after
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 39 Page 4 of 11
Fig. 2 Somatic embryogenesis of ‘Zili’ from immature embryo. (A)
Friable and light-yellow callus obtained after subculturing for two
weeks; (B) Somatic embryos obtained after plating callus on somatic
transferring the callus on somatic embryo induction medium
under dark conditions. The somatic embryos gradually
changed to light green and eventually dark green (Fig. 2B).
Dicotyledonous embryos underwent various developmental
stages, including globular, heart-shape, torpedo, and coty-
ledonous stages (Fig. 2C). The first germination of somatic
embryos occurred on the 83 d after inoculation. After 4
months of plating the callus on the somatic embryo induc-
tion medium, the regeneration rate reached approximately
26.0%.
Effect of distinct morphology of somatic embryo on
plant regeneration
As described by Kuang et al. (1996), the somatic embryos
were classified into distinct categories, including dicotyle-
donous embryos, conjoined embryos, flake embryos, irreg-
ular swollen embryos, and other embryos. Notably, each
13
Plant Cell, Tissue and Organ Culture (PCTOC)
embryo induction medium for 6 weeks; (C) Developmental progress
from globular embryo to cotyledonous embryo. Bar = 1 mm
specific embryo morphology yielded corresponding regen-
erated plantlets (Fig. 3). The regeneration frequencies of
various somatic embryo morphologies exhibited significant
differences (Table 1). Conjoined embryos demonstrated
the highest regeneration rate, reaching 42.6% (Fig. 3A, B),
closely followed by dicotyledonous embryos with a rate
of 30.2% (Fig. 3C, D). The regeneration frequencies of
flake embryos (Fig. 3E, F) and irregular swollen embryos
(Fig. 3G, H) were significantly lower, 15.4% and 16.2%,
respectively.
Histological observation
Histological observations of the callus revealed a tightly
arranged structure, exhibiting significant embryogenic poten-
tial characterized by regularity and a high nuclear to cyto-
plasmic ratio (Fig. 4A). Globular somatic embryos appeared
when the callus was transferred to the somatic embryo
Plant Cell, Tissue and Organ Culture (PCTOC) Page 5 of 11 39

Fig. 3 Different morphologies

of somatic embryos of ‘Zili’ and
the corresponding regeneration
plantlets.(A) Conjoined embryo; (B)
Plant regeneration from conjoined
embryo; (C) Dicotyledonous
embryo; (D) Plant regeneration from
dicotyledonous embryo; (E) Flake
embryo; (F) Plant regeneration from
flake embryo; (G) Irregular swollen
embryo; (H) Plant regeneration
from irregular swollen embryo. Bar
= 1 mm

induction medium. The cells of globular somatic embryo
had similar size and shape, showing a closely arranged
structure (Fig. 4B). The globular somatic embryo devel-
oped rapidly and two cotyledon primordia raised, forming
white-transparent heart-shaped structures. The round cells
of heart-shaped displayed the obvious nuclear and vascular
initials (Fig. 4C). The cells located in the depression of the
heart-shaped embryo exhibited accelerated division, giving
rise to white-opaque torpedo-shaped embryos (Fig. 4D).
Finally, the torpedo-shaped embryos differentiated into
two cotyledons, ultimately maturing into dicotyledonous
somatic embryos. The root primordium which consisted
of tiny cells emerged from the middle of the dicotyledon-
ous somatic embryos and the provascular strand grew out
(Fig. 4E). The cells of the developing shoot meristem region

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 39 Page 6 of 11 Plant Cell, Tissue and Organ Culture (PCTOC)

Table 1 Effects of distinct mor-
phology of somatic embryos on
regeneration rate of ‘Zili’ after 4
months of culture
Regeneration rates with the same
letter do not differ significantly
(P = 0.05) according to Duncan’s
multiple comparison test
Morphology of somatic Replicate Number of somatic Number of Regen-
embryos embryos regenerative eration
somatic embryos rate (%)
Dicotyledonous embryo 1 9 3 30.2 ± 3.0
2 11 3 ab
3 10 3
Conjoined embryo 1 9 4 42.6 ± 8.5
2 9 3 a
3 8 4
Flake embryo 1 8 2 15.4 ± 8.4
2 9 1 c
3 10 1
Irregular swollen 1 8 1 16.2 ± 7.7
embryo 2 8 2 c
3 9 1
Others 1 10 2 23.3 ± 5.8
2 10 3 bc
3 10 2

Fig. 4 Histological sections of embryogenic callus and somatic embryo development of ‘Zili’. (A) Embryogenic callus; (B) Globular embryo; (C)
Heart-shape embryo; (D) Torpedo-shape embryo; (E) Cotyledonous embryo, arrow indicated root primordium (rp). Bar = 100 μm

that situated in the depression of the dicotyledonous somatic
embryos divided rapidly (Fig. 4E).
Transplantation
The thickness of the roots played a pivotal role in determin-
ing the transplant survival rate. Plantlets with thicker roots
had higher rates of successful transplantation. Plantlets
regenerated from somatic embryo (Fig. 5A, C) were trans-
planted to greenhouse under conditions identical to those in
the incubation chamber. After 30 d of greenhouse growth,
new leaves emerged from the lateral buds (Fig. 5B, D), indi-
cating successful ex intro transplanting.
Discussion
Somatic embryogenesis has been become the most common
method for litchi in vitro regeneration (Mir et al. 2018).
Eight litchi cultivars have been successfully regenerated via
somatic embryogenesis (Table 2). Among them, three culti-
vars (‘Bendana’, ‘Nuomici’, and ‘Xiafanzhi’) used imma-
ture zygotic embryos as explants, four cultivars (‘Chenzi’,
‘Gushanjiaohe’, ‘Feizixiao’, and ‘Shuidong’) used anther as
explants, two cultivars (‘Brewster’ and ‘Taiso’) used leaves
as explants. Callus induced from various explants differed
in growth rate, morphology, color and texture. Comparing
with other explants, embryogenic callus from immature
zygotic embryos showed vigorous growth and light-yellow
color (Kuang et al. 1997). In this study, a simple and effi-
cient method of ‘Zili’ regeneration via somatic embryogen-
esis from immature embryo was established.Initially, the
embryogenic callus was stimulated to develop into the pro-
embryo stage under darkness. Subsequently, the culture was
shifted to light condition with a 16-hour photoperiod from
the proembryo stage until the regeneration stage. In the sec-
ond stage, somatic embryos developed gradually through
various stages, including globular embryo, heart-shaped
embryo, torpedo-shaped embryo and cotyledonous embryo.
The two-step culture method employed in the study proved
to be instrumental in achieving successful regeneration. This
finding is consistent with the protocol reported by Kuang et
al. (1997), further confirming the significance of darkness
treatment in promoting successful somatic embryogenesis
and subsequent plant regeneration in litchi tissue culture.
The primary rationale behind using darkness culture is that
it enables the callus to gradually acclimate to light condi-
tions and prevents somatic embryos to turn brown (Gow et

13
Plant Cell, Tissue and Organ Culture (PCTOC)
al. 2009). This process ultimately enhances the regeneration
rate. Additionally, during the maturation stage, light pro-
motes the maturation and regeneration of somatic embryos.
These complementary effects of darkness and light culture
stages contribute significantly to the overall success and
efficiency of the regeneration process in litchi tissue culture
(Michler and Lineberger 1987; Kintzios et al. 1998).
The optimal culture medium corresponding to different
cultivars often varies (Raharjo and Litz 2007). MS basal
medium supplemented with 2,4-D has been successfully
used to induce callus of ‘Xiafangzhi’ (Yu et al. 2000), ‘Ben-
dana’ (Das et al. 2016) and ‘Nuomici’ (Kuang et al. 1997)
from immature embryos. In the present study, yellow and
friable callus of ‘Zili’ was induced from immature embryos
on the MS basal medium supplemented with 1 mg/L 2,4-D
(Fig. 1). These results were consistent with the reports
that 2,4-D is the common hormone in callus induction in
other fruit trees (Azim 2011). Previously, three types of
media were utilized to induce plant regeneration from cal-
lus via somatic embryogenesis, including somatic embryo
induction medium, somatic embryo maturation medium,
and germination medium. Phytohormones or plant growth
Fig. 5 Plantlets obtained from
regenerated somatic embryo of
‘Zili’ and transplanted in the pot
for 3 months. (A and C). Plant-
lets regenerated from somatic
embryo; (B). Recovered plant
from plant A (arrow showed new
leaves); (D). Recovered plant
from plant C (arrows showed
new leaves)
Page 7 of 11 39
regulators have been widely used in the media for somatic
embryogenesis. Low concentration of NAA was proved to
accelerate the formation of somatic embryos (Fu and Tang
1983; Kuang et al. 1997; Wang et al. 2016). GA3 was added
to promote the root elongation and shoot development in
the germination medium (Yu et al. 2000; Das et al. 2016;
Wang et al. 2016;). During the maturation and germina-
tion stage, extra nutrients were needed for different culti-
vars. Glutamine played important roles in the maturation of
somatic embryos (Das et al. 2016), and loyal jelly improved
the differentiation of shoots (Fu and Tang 1983). Two spe-
cific media compositions, CW and LH, were considered to
be instrumental in promoting the successful development
of somatic embryos and facilitating efficient plant regen-
eration in litchi (Raharjo and Litz 2007; Nath et al. 2017).
10–20% (v/v) CW were critical for achieving opaque-white
somatic embryos of ‘Feizixiao’ which had higher regenera-
tion rate (Raharjo and Litz 2007; Wang et al. 2016). How-
ever, the media compositions CW and LH were not effective
for inducing somatic embryos and promoting regeneration
in ‘Zili’ (data not shown). Here, MS basal medium supple-
mented with 1 mg/L biotin and 1 g/L AC was proved to be
13
 39

Table 2 The regeneration systems of different litchi cultivars

Cultivar Explant Callus induction medium >Somatic embryo induction Somatic embryo matura- Germination medium Highest Refer-
medium tion medium regeneration ence

13
rate
Bendana Immature MS salts + B5 vitamins + 2 mg·L-1 NN + 0.1 mg·L-1 IBA + 30 g·L-1 1/4 B5 macrosalts + 1/2 B5 macrosalts + MS mic- 16% Das et
zygotic 2,4-D + 2 g·L-1 ascorbic acid + 3 sucrose + 8 g·L-1 agar MS microsalts + 100 rosalts + iron-EDTA + 2.74 al., 2016
Page 8 of 11

embryos g·L-1 citric acid + 50 g·L-1 sucrose + mM Cacl2 + 2% sodium mM glutamine + 30 g·L-1
8 g·L-1 agar alginate sucrose (in liquid medium)
and 1/4 B5 macrosalts +
MS microsalts + iron-EDTA
+ 2.9 μM GA3 + 30 g·L-1
sucrose + 7 g·L-1 agar
Brewster Leaf B5 + 4.52 mM 2,4-D + 9.30 mM KT 1/2 MS + 4.52 mM 2,4-D + 0.91 MS + 5–20% (v/v) CW + MS + 100 mg·L-1 AC + 30 Not Raharjo
(Chenzi) + 400 m g·L-1 glutamine + 200 m mM zeatin + 100 mg·L-1 insitol + 45 g·L-1 sucrose+3 g·L-1 g·L-1 sucrose + 4 g·L-1 agar mentioned and Litz,
g·L-1 casein hydrolysate + 30 g·L-1 30 g·L-1 sucrose + 4 g·L-1 agar gellan gum 2007
sucrose + 3 g·L-1 gellan gum
Chenzi; Anther MS + 2 mg·L-1 2,4-D + 2 mg·L-1 MS/B5 + 0.5 mg·L-1 KT+ 0.1 MS/B5 + 400 mg·L-1 1/2 B5 + 0.1 mg·L-1 KT 26% Fu and
Gushanjiaohe KT+ 0.5 mg·L-1 NAA + 500 mg·L-1 mg·L-1 NAA + 400 mg·L-1 loyal loyal jelly + 500 mg·L-1 + 0.01 mg·L-1 IAA + 500 Tang,
LH + 30 g·L-1 sucrose + 7 g·L-1 agar jelly + 500 mg·L-1 LH +30 g·L-1 LH + 500 mg·L-1 gluta- mg·L-1 LH + 1600 mg·L-1 1983
sucrose + 7 g·L-1 agar mine + 30 g·L-1 sucrose glutamine + 10 g·L-1 sucrose
+ 7 g·L-1 agar + 7 g·L-1 agar
Feizixiao Anther MS + 13.57 μM 2,4-D + 2.22 μM BA MS + 0.54 μM NAA + 23.23 μM KT + 0.4 g·L-1 LH + 0.56 1/2MS + 1.44 μM GA3 + 30 7% Wang et
+ 2.69 μM NAA + 30 g·L-1 sucrose + μM inositol + 10% CW + 60 g·L-1 sucrose + 7 g·L-1 agar g·L-1 sucrose + 7 g·L-1 agar al., 2016
7 g·L-1 agar
Feizixiao Anther MS + 2.0 mg·L-1 2,4-D + 0.5 mg·L-1 MS + 0.5 mg·L-1 KT + 0.1 MS + 1 mg·L-1 BA + 1 1/2 MS + 20 g·L-1 sucrose + 17.77% Deng,
NAA + 500 mg·L-1 PVP + 50 g·L-1 mg·L-1 NAA + 30 g·L-1 sucrose mg·L-1 KT + 0.5 mg·L-1 6.8 g·L-1 agar 2005
sucrose + 7 g/L agar + 0.5% AC + 6.8 g·L-1 agar NAA + 30 g·L-1 sucrose
+ 7 g·L-1 agar
Shuidong Anther -1 16.57% Deng,
MS + 2.0 mg·L-1 2,4-D + 0.5 mg·L-1 MS + 0.5 mg·L BA + 0.5 MS + 1 mg·L-1 BA + 1 1/2 MS + 20 g·L-1 sucrose +
-1 -1 2005
NAA+ 500 mg·L-1 PVP + 50 g·L-1 mg·L KT + 500 mg·L LH + mg·L-1 KT + 0.5 mg·L-1 6.8 g·L-1 agar
sucrose + 7 g/L agar 400 mg·L-1 royal jelly + 60 g·L-1 NAA + 30 g·L-1 sucrose
sucrose + 10 g·L-1 agar + 7 g·L-1 agar
Nuomici Immature MS + 2.0 mg·L-1 2,4-D + 0.5 mg·L-1 -1 22% Kuang et
MS + 5-8 mg·L 2,4-D + 30 MS + 0.2 mg·L-1 NAA 1/2 MS + 0.2 mg·L-1 NAA
zygotic NAA + 30 g·L-1 sucrose g·L-1 sucrose + 0.1 mg·L-1 IBA + 30 + 1 mg·L-1 IBA + AC + 20 al., 1997
embryos g·L-1 sucrose g·L-1 sucrose
Taiso Leaf -1 -1 Not Puchooa,
MS + 1.5 mg·L-1 2,4-D + 1.0 mg·L-1 MS + 3 mg·L IAA + 2.0 mg·L BAP + 30 g·L-1 sucrose + MS + 2.0 mg·L-1 IBA + 30
BAP + 225 mg·L-1 ascorbic acid + 2.5 g·L-1 agar g·L-1 sucrose + 2.5 g·L-1 agar mentioned 2004
225 mg·L-1 citric acid + 30 g·L-1
sucrose + 2.5 g·L-1 agar
Xiafanzhi Immature MS salts + B5 vitamins + 2 mg·L-1 MS salts + B5 vitamins + 1 MS salts + B5 vitamins + MS salts + B5 vitamins + 1 33 (33.1%) Yu et al.,
zygotic 2,4-D + 50 g·L-1 sucrose + 8 g·L-1 mg·L-1 KT + 0.1 mg·L-1 NAA + 500 mg·L-1 glutamine + mg·L-1 KT+ 5 mg·L-1 GA3 2000
embryos agar 500 mg·L-1 glutamine + 80 g·L-1 50 ml·L-1 CW + 50 g·L-1 + 50 ml·L-1 CW + 30 g·L-1
sucrose + 15 g·L-1 agar sucrose + 9 g·L-1 agar sucrose + 7 g·L-1 agar
Zili Immature MS + 1.0 mg·L-1 2,4-D + 1 mg·L-1 -1 -1 -1 42.6% This
MS + 1 mg·L biotin + 1 g·L AC + 30 g·L sucrose + 8 g/L agar
zygotic biotin + 30 g·L-1 sucrose + 7 g/L agar study
embryos
Plant Cell, Tissue and Organ Culture (PCTOC)
Plant Cell, Tissue and Organ Culture (PCTOC)
effective for the entire process of somatic embryo induc-
tion, maturation, and regeneration (Fig. 2). The inclusion of
AC in the medium was particularly important, which could
reduce light intensity (Wang et al. 1994), effectively absorb
harmful substances such as the phenols produced during
plant growth (Mattson et al. 1969; Dąbrowski et al. 2005;
Alam et al. 2009), and induce roots of regenerated plants
(Lau et al. 2008; Yaseen et al. 2013). AC was also added
in the germination medium of ‘Brewster’ and ‘Nuomici’
(Kuang et al. 1997; Raharjo and Litz 2007). Biotin, known
as vitamin B7, is a water-soluble vitamin, which is important
in carboxylation reactions (Alban et al. 2000; Alban 2011)
and somatic embryo development (Wurtele and Nikolau
1992). The role of biotin in somatic embryo induction and
plant regeneration has not been defined. In this study, callus
induction, somatic embryo growth and plant regeneration
of ‘Zili’ were significantly influenced by biotin. During the
period of callus induction, biotin played a role of antioxi-
dant to avoid callus browning. Other antioxidants such as
PVP, ascorbic acid and citric acid were also used in callus
induction medium (Deng 2005; Das et al. 2016). Al-Khayri
(2001) demonstrated that 1 mg/L biotin could significantly
stimulated callus proliferation and somatic embryogenesis
of date palm. The effect of biotin on somatic embryo growth
and plant regeneration was also observed in Eucalyptus tree
(Hajari et al. 2006) and banana (Jalil et al. 2003; Khalil and
Elbanna 2004).
Abnormal somatic embryos which could not regener-
ate is still one of the main problems in somatic embryo-
genesis (Garcia et al. 2019; Bidabadi and Jain 2020). Here,
we investigated the correlation between somatic embryo
morphology and regeneration rate in ‘Zili’ (Fig. 3). Nota-
bly, significant differences in both morphology and size
were observed during the developmental stages of somatic
embryos. The regeneration rates of dicotyledonous and con-
joined embryos reached 30.2% and 42.6%, respectively,
which were higher than those of flake embryos and irregular
swollen embryos (Table 1). Wang et al. (2016) displayed
that conjoined embryos and dicotyledonous embryos were
better than other abnormal embryos, as they had good
development of plumule, hypocotyl, radicle and cotyledon.
Kuang et al. (1997) showed that the regeneration rate of
dicotyledonous embryos of ‘Nuomici’ was 22%, and abnor-
mal somatic embryos could germinate with lower regenera-
tion rates. Abnormal embryos were difficult to grow roots
and prone to browning due to their weak photosynthetic effi-
ciency and poor stomatal performance (Garcia et al. 2019;
Bidabadi and Jain 2020). In the present study, abnormal
embryos were not prone to browning after being sub-cul-
tured, especially the conjoined embryos. It is suspected that
conjoined embryos had higher regeneration rate because
they were consisted of more than one embryo to provide the
Page 9 of 11 39
nutrients needed for regeneration. During the development
of somatic embryo, white opaque embryos were considered
to be easier matured and germinated in several cultivars, in
which ‘Xiafanzhi’ achieved the highest regeneration rate,
reaching 33.1% (Yu et al. 2000). We found that the majority
of white opaque embryos could turn dark-green and eventu-
ally germinate. The historical observation of the procam-
bium and shoot meristem in the dicotyledonous embryos
also confirmed the successful regeneration of ‘Zili’ (Fig. 4),
as root and bud originated from the procambium and shoot
meristem region, respectively (Bowman and Eshed 2000;
Belehu et al. 2004). These findings offer crucial insights into
the impact of somatic embryo morphology on the regenera-
tion process and emphasize the importance of selecting the
most appropriate somatic embryo type to optimize the suc-
cess of plant regeneration in litchi.
Conclusion
In conclusion, this study successfully established a simple
and efficient regeneration system for ‘Zili’ litchi through
indirect somatic embryogenesis from immature embryo.
This achievement holds significant implications for future
genetic transformation and breeding endeavors in litchi
research. Furthermore, the correlation between somatic
embryo morphology and regeneration rate provides valu-
able guidance and reference for other litchi cultivars.
Acknowledgements Authors thank Mr. He Wang for performing
explant disinfection and callus induction. This work was supported
by the National Natural Science Foundation of China (No. 32172528,
32272663), the Science and Technology Planning Project of Guang-
zhou (No. 202103000057, 2023B01J2002), and China Litchi and Lon-
gan Industry Technology Research System (No. CARS-32-05).
Author Contributions Jietang Zhao conceived this study. Miaoqin
Huang, Wuyan Guo, Xiuyu Wu, and Yaqi Qin conducted the experi-
ments. Miaoqin Huang wrote the manuscript. Irfan Ali Sabir, Zhike
Zhang, Yonghua Qin, Guibing Hu, and Jietang Zhao reviewed the
manuscript.
Data Availability The data and materials generated and/or analyzed
during the current study are available from the corresponding author
on reasonable request.
Code Availability Not applicable.
Declarations
Ethical approval Not applicable.
Consent to participate Not applicable.
Consent for publication Not applicable.
13
 39 Page 10 of 11
Conflict of interest All authors have no relevant financial or nonfinan-
cial interests to disclose.
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