Introduction
Citrus is a
major fruit crop that is widely grown in tropical and subtropical regions
around the world. They account for an annual production of over 124.3 million
tones worldwide and around 18% of total global fruit production (Mahato et
al. 2020). Morocco is among the major exporting countries of small-fruited
citrus in the Mediterranean region. In a very competitive fresh fruit market,
mandarins and clementines are today one of the most dynamic sectors of citrus
production and world trade. Mandarins and their diploid hybrids, on the other
hand, produce fruits with a lot of seeds, and their planting near clementine
orchards can result in a lot of seeded clementines. One of the best strategies
to meet the constraints set out above would be to create new triploid varieties
of mandarin (2n=3x=27) characterized by sterility (Aleza et al. 2010b;
Handaji et al. 2020; Lourkisti et al. 2021a). The triploidy has
proved to be a powerful approach breeding programs, particularly in citrus, since
seedlessness is one of the greatest consumer expectations (Lourkisti et al.
2021b). Moreover, Triploidy has the potential to play a significant role in the
coming decades through enhancing biomass and abiotic stress tolerance, both of
which have commercial implications (Costa et al. 2019; Lourkisti et
al. 2020). On the other hand, triploid embryos are mostly found in small
seeds, between one-third and one-sixth larger than normal seeds, which in most
cases, do not germinate in a conventional greenhouse.
To obtain high germination rates, embryo rescue
from these small seeds is required (Navarro et al. 2002; Aleza et al.
2010b). Thus, the rescue of immature embryos in vitro with an evaluation
of ploidy by flow cytometry have been revealed as two decisive methods in the
development of effective programs for the selection of triploid plants
(Ollitrault et al. 1996b; Navarro et al. 2003; Aleza et al.
2010b; Dalel et al. 2020). In addition to
seedlessness, embryo rescue has been applied to breeding programs for early
ripening citrus (Ramming et
al. 1990), triploidy or interspecific crosses among other fruit crops such
as apple (Druart et al. 2000), grape (Angelica et al. 2022),
banana (Uma et al. 2011), mango (Krishna and Singh 2007), persimmon (Hu et
al. 2013), and peach (Yamada and Tao 2007). As a result of continuous efforts to optimize the in vitro
rescue technology, the number of obtained spontaneous triploids is insufficient
to support improvement programs of seedless plants as protocol efficiency is
greatly influenced by several endogenous and exogenous factors: Parental
crossing genotypes (Handaji et
al. 2005; Essalhi et al. 2020 and 2021), culture medium composition
(Ennaciri et al. 2020; Hmimidi et al. 2020), plantlet acclimation
conditions and the addition of growth regulators are two of the most critical
elements impacting rescue efficiency (Mahmoudi et al. 2019 and 2020).
Diploid species hybridization from low-frequency
divalent gamete fusion or intra-and inter-ploidy crossings can result in the
production of new constant triploid hybrids. However, without a highly
effective aseptic approach and ploidy event, considerable breeding work based
on small F1 hybrid seeds created is unfeasible. In this study, natural hybrids
were recovered from nonembryogenic diploid, open-pollinated mandarins using in
vitro embryo culture. The purpose of this study was to see how the size of
the seeds and the genotypes of the female parents affected the efficiency of
the embryo rescue method for the regeneration of triploid seedless hybrids from
mandarin diploid crossings.
Materials
and Methods
Plant
materials
Four varieties of mandarin were
open pollinated in non-block area during the anthesis on Marsh and April: 1.
Nadorcott, 2. Murcott honney, 3. Ortanique and 4.
variant of Murcott Honney (M104). These varieties were planted in
the field of the National Institute of Agronomic Research (INRA Kenitra/El
Menzeh).
Methods
In vitro culture of immature embryos: At maturity stage, the fruits of
the four mandarin’s varieties were harvested. Then, after extraction of the
seeds, two categories were identified: normal (fully developed) and abnormal
seeds (partially developed). The last ones are classified as flat and small
(Fig. 1; Table 1). Seeds were classified by size and developmental stage. Size
was measured and developmental stage was evaluated by morphological parameters.
Perfect seeds (PS) were normal in appearance, totally filled out, and without
any malformation. Undeveloped seeds (US) had incomplete development, not
totally filled out, wrinkled, and with split outer integument (Fig. 2). Under a
laminar flow hood, seeds were surface sterilized for 5 min in a solution of 70%
ethanol, followed by immersion for 10 min in 10% sodium hypochlorite. Thereafter, they were cleaned three times with sterile distilled water.
The embryos
should be treated very carefully while decortications of the seeds, in order to
avoiding damage them. Under aseptic conditions, they were cultured on Murashige
and Tucker (1969) culture medium with 1 mg/L of gibberellic acid (GA3), which
contained 50 g/L sucrose, 500 mg/L malt extract and vitamins (100 mg/L
myo-inositol, 1 mg/L pyridoxine hydrochloride, 1 mg/L nicotinic acid, 0.2 mg/L
thiamine hydrochloride, 4 mg/L glycine), and 8 g/L Bacto agar (MT culture media
is adjusted to a pH of 5.7 and sterilized at 120°C for 20 min). Baskets
cultures are placed in an incubator under 16/18 h (light/dark) photoperiod with
light intensity of 1000 lux provided through neon and a temperature of 27°C
during the day and 19°C at night. Embryo germination was conducted for 12 weeks
in a growth chamber under a 16-h photoperiod with a light intensity of 1000 lux
provided by a cool-white-fluorescent lamp, at a temperature of 23±2°C. Embryos
showing newly formed cotyledonal leaves were observed and recorded regularly.
The percentage of embryo germination was calculated as the number of germinated
embryos in the total number of formed embryos. Plant growth allows determining
the rate of regeneration. The triploidy rate is calculated. Also, the formulas
of all parameters were detailed by (Ennaciri et al. 2020).
Ploidy determination by the
flow cytometry: Ploidy levels of seedlings were evaluated by flow
cytometry using a Partec II cytometer. Nuclei suspensions were made from mature
leaf samples and whole seeds. About 0.5 g of leaf tissue was collected from
fully or near fully expanded leaves of seedlings two or three months after germination.
In the presence of a leaf portion of a triploid control, the tissue was placed
in a 60×15 mm polystyrene dish with 0.5 mL of phosphate-buffered saline (PBS)
buffer, dithiothreitol 1 mg/L and 0.1% Triton X 100, chopped to a fine mash
with a single-edge razor blade. The nuclei were then filtered through a 50-μm nylon
filter and stained with 2 mL of 4,6-diamine-2-phenylindole (DAPI) solution
(High Resolution DNA Kit Type P, solution B; Partec). The amount of DNA was
evaluated by the intensity of the fluorescence re-emitted by the nuclei under
UV excitation (365 nm). After a 2-min incubation period, stained samples were
run in a Ploidy Analyzer (Partec, PA) flow cytometer. Histograms were analyzed
using the dpac v 2.18 software (Partec), which determines peak position,
coefficient of variation (CV), and the relative ploidy index of the samples.
The triploid control used in this study is Moroccan cultivar “HANA”.
Table 1: Average length, normal, flat and small seeds of each studied variety
|
Mandarins
varieties |
Length means of seeds en mm² |
|||
|
|
Abnormally
developed |
|||
|
|
Normal |
Small |
Flat
|
|
|
Ortanique |
122.15 |
23.9 |
70.4 |
|
|
Murcott
honney |
116.25 |
26.2 |
67.1 |
|
|
Nadorcott |
122.2 |
25.2 |
55.8 |
|
|
Variant
of Murcott (M104) |
105 |
22.15 |
61.2 |
|
|
Mean
(mm²) |
116.40
(± 8.10) |
24.36
(± 1.75) |
63.63
(± 6.46) |
|
Table 2:
Percentage and interval germination of seeds
from mandarin’s varieties
|
Mandarins
varieties |
Germination
rate (%) |
Germination Interval (days) |
|
Ortanique |
76.66b |
3.00a |
|
Nadorcott |
91.85a |
2.77a |
|
Variant of Murcott M104 |
89.28a |
2.78a |
|
Murcott Honney |
83.73ab |
3.00a |
|
Mean |
83.58 (±
6.73) |
2.88 (±
0.13) |
Duncan's test according to a
level of significance (5%)
%20210-218_files/image002.jpg)
Fig. 1: Mandarin’s varieties with their seeds extracted from ripe fruits and
classified according to theirs shapes and sizes (a: abnormal, b: normal).
%20210-218_files/image004.jpg)
Fig. 2: Comparison of seeds Size, with A: Normal; B: Flat and C: Small seeds
Statistical data analysis
Quantitative data were analyzed
using SAS (Statistical Analysis System version 9.1 and version 5.5) and were
subjected to analysis of variance (ANOVA). Means were compared using Duncan's
test at a 5% level of significance. Several analyses of variance were performed
to compare the means of the variables.
Results
Seed germination and
seedling formation
Variance analysis showed a
significant effect between the four mandarin varieties for majority of the
traits (germination rate and interval, stem and root growth rate) (Fig. 3;
Tables 2, 3).
Germination
rate and interval: The germination rate varied from
77 to 92% with an average of 83.58%. Three statistically differentiated groups
were identified. The first group included the varieties Nadorcott and
the variant of Murcott M104: the second group ab contained
the variety Murcott Honney and the group b with the
variety Ortanique. For the germination interval, it appears that there is no significant difference between all the
varieties studied. The germination was taken approximate 3 days.
Stem
growth rate: The aerial growth rate varied
statistically according to the genotypes studied (Table 3). It oscillated from
1.00 mm/day to 3.70 mm/day with an average of 2.03 mm/day and from 0.60 mm/day
to 1.80 mm/day with an average of 1.10 mm/day during the second and third weeks
respectively. Indeed, the growth rate of the stem decreased from the third week
for all the varieties studied. This could be explained by the exhaustion of the
culture medium used. Similarly, the variety Ortanique is relatively
characterized by a better stem growth rate with an average of 3.70 mm/day
followed by the variant M104. A good development of the foliar system is thus
observed for all the regenerated green seedlings.
Root
growth rate: The statistical analyzes
revealed a significant difference between the four varieties of mandarins for
root growth. Thus, three different groups have been highlighted, group b
includes the two varieties Ortanique and Nadorcott, group c
represents the variant of Murcott M104 and group bc
contains the variety Murcott honney. In addition, the average value of
root growth speed varied from 3.03 mm/day (2nd week) to 2.33 mm/day
(3rd week). In addition, a decrease in the evolution of growth took place
during the following weeks for all varieties with the exception of the variant of
Murcott M104.
The
Influence of seed size on the parameters studied
Germination
rate and interval: There was a significant
difference between the four varieties of mandarin in germination rate and seed
shape (Fig. 4). The germination rate was 100% for the normal seeds, but it
varied from 55 to 98% for the small seeds and from 66 to 100% for the one with
the flat shape. For the variety Ortanique, there are two Table 3: Growth rate of stem and root of
four mandarin’s varieties
|
Mandarin varieties |
Growth rate (mm/day) |
|||
|
|
Stem |
Root |
||
|
|
SGR1 |
SGR2 |
RGR1 |
RGR2 |
|
Ortanique |
3.70a |
1.80b |
3.80ab |
2.30ab |
|
Nadorcott |
1.20b |
0.60c |
3.40b |
1.60b |
|
Variant of Murcott M104 |
2.20ab |
1.40bc |
1.90c |
3.30a |
|
Murcott honney |
1.00b |
0.60c |
3.00bc |
2.10b |
|
Mean |
2.03±1.23 |
1.10±0.60 |
3.03±0.82 |
2.33±0.71 |
Duncan's test with
significance (5%)
SGR1: stem growth rate of
seedlings in the second week, SGR2: stem growth rate of seedlings in the
third week, RGR1: root growth rate of seedlings in 2nd week, RGR2: root
growth rate of seedlings in 3rd week
%20210-218_files/image006.jpg)
Fig. 3: Seedlings from immature embryo rescue of three types of seeds; a:
normal; b: small; c: flat; for four varieties of mandarin’s (A: Nadocott;
B: Variant of Murcott M104; C: Ortanique; D: Murcott Honney).
%20210-218_files/image008.jpg)
Fig. 4: Comparison of immature embryos germination rate according to the seeds
size (normal, small and flat) of the mandarin’s varieties.
statistically different groups,
the first group a includes the normal and flat seeds which are characterized
by a very high germination rate followed by the second group b
which includes only the small form. For Nadorcott, the normal seeds
forming group a gave a higher germination rate than the small and
flat seeds ab. In the majority of citrus varieties, the
germination rate of normal seeds is higher than that of abnormal seeds (flat
and small). For germination rate of Murcott Honney varieties and its
variant M104, normal and small seeds showed the highest germination rate
compared to the flat form.
For the interval of germination, there is not a significant difference
between the three forms of seeds of the two varieties Ortanique and Murcott
Honney, their seeds gave all an average interval of three days. Unlike the
other two genotypes, there is a statistically significant difference. The
variety Nadorcott presented two groups a which includes
the normal seeds and ab the small and flat seeds, while for the
variant of Murcott M104 three statistical groups were distinguished a
which includes the normal seeds, group ab for the small seeds and
b for the flat seeds (Fig. 5).
Stem and
root growth rate: For the growth speed of the
stem during the first two weeks, the small seeds of the variety Ortanique
are characterized by a better aerial growth, followed by the normal and flat
seeds. For the Nadorcott variety, there is no significant difference
between the three seed forms, they all gave the same growth rate. For the
variant of Murcott M104, the small seeds were characterized by a better
stem growth. Also, the small seeds gave a fast growth rate compared to the flat
and normal seeds. At the fourth week, the stem growth rate decreased for all
four tangerine varieties, this can be explained by the depletion of growing
medium for the tangerine varieties during the first two weeks.
The seed forms that are characterized by better root growth rate in the
first two weeks are small seeds for Ortanique variety, normal seeds for
varieties Nadorcott and Murcott honney. The flat seeds of the
variant of Murcott M104 showed a better growth rate, at the third week,
the root growth rate decreased for the three seed forms of the four varieties
studied and this can be explained by the depletion of the culture medium (Fig.
6 and 7).
Effect
of size seeds on different parameters studied
According to a statistical
comparison of the values of the studied parameters (Table 4), for
the germination rate of the embryos there is a significant difference for the
three forms of seeds, which detects two large groups, the group a
including the flat form giving a maximum germination rate (87.69%) and group b
including the two small and normal forms with an average germination rate of
84.68%. While there is no significant difference for the other variables
studied (germination interval, stem growth rate (SGR1 and SGR2), root growth
rate (RGR1 and RGR2) in relation to the tested seed form (small, flat and
normal). The average values vary respectively as follows: 2.88 mm/d; 1.9 mm/d;
1.7 mm/d; 3.5 mm/d and 2.3 mm/d.
The results
obtained showed a statistical variation of the variables according to the
genotype and all depending on the seed shape (Table 5), the interaction between
the seed shape and the variety studied varied for each character, the
interaction is highly significant for the germination percentage (P < 0.
001), it is significant for germination interval and root growth rate between
the fourth and third week (RGR2), however the interaction is statistically
insignificant for aerial growth rate during the four weeks (SGR1 and SGR2) as well
as root growth rate between the second and first week (RGR1) with (P > 0.05).
Table 4: Multiple comparisons of the average values of the different parameters
studied according to the shape of the seeds (small, flat and normal)
|
Variables |
Seed shape |
|||
|
|
Small |
Flat |
Normal |
Mean |
|
Germination rate (%) |
78.67b |
87.69a |
87.68a |
84.68±5.20 |
|
Germination interval (days) |
3.00a |
2.79a |
2.87a |
2.88±0.11 |
|
Stem Growth Rate
(mm/days) |
|
|
|
|
|
SGR1 |
2.51a |
1.72a |
1.60a |
1.93±0.49 |
|
SGR2 |
1.85a |
1.65a |
1.94a |
1.76±0.15 |
|
Root Growth Rate
(mm/days) |
|
|
|
|
|
RGR1 |
3.62a |
3.14a |
3.89a |
3.50±0.38 |
|
RGR2 |
2.64a |
2.37a |
2.23a |
2.36±0.21 |
SGR1: stem growth rate
of seedlings in the second week, SGR2: stem growth rate of seedlings in
the third week, RGR1: root growth rate of seedlings in 2nd week, RGR2:
root growth rate of seedlings in 3rd week.
Table 5:
Multiple comparisons of genotype*seeds size
interaction
|
Variables |
CV (%) |
F Value |
Pr > F |
|
Germination rate (%) |
20.48 |
74.22 |
<0.0001*** |
|
Germination interval (days) |
21.08 |
2.33 |
0.0226* |
|
Stem Growth Rate
(mm/days) |
|
|
|
|
SGR1 |
178.37 |
0.40 |
0.9190 |
|
SGR2 |
89.17 |
1.07 |
0.3870 |
|
Root Growth Rate
(mm/days) |
|
|
|
|
RGR1 |
60.61 |
1.45 |
0.1817 |
|
RGR2 |
76.81 |
2.25 |
0.0281* |
SGR1: stem growth rate of
seedlings in the second week, SGR2: stem growth rate of seedlings in the
third week, RGR1: root growth rate of seedlings in 2nd week, RGR2: root
growth rate of seedlings in 3rd week.
%20210-218_files/image010.jpg)
Fig. 5: Comparison of immature embryos germination interval according to the
seeds size (normal, small and flat) of the mandarin’s varieties.
%20210-218_files/image012.png)
Fig. 6: Comparison of the Stem growth rate for the four mandarin varieties.
SGR1: Stem growth rate during two weeks; SGR2: Stem growth rate during three
weeks
Leaf
analysis by flow cytometry
A substantial number of triploid
plants have been recovered from immature embryos of citrus varieties in M1
medium containing GA3, through leaf analysis by flow cytometry technique. The
triploidy rate varied according to the size of the seed and the variety
studied.
Study of
the genotype effect on triploidy: The variety Ortanique
showed a higher triploidy rate than the other varieties with a percentage of
13.51%, followed by the variant of Murcott M104 and Nadorcott
with an average percentage of 8.11 and 5.40% respectively. Then the variety Murcott
honney which is in last place producing a low percentage of triploid
vitroplants at about 2.70%. According to
these results, mandarin varieties are able to give a very high triploidy rate
but depending on the genotype studied (Table 6).
Study of
the influence of seed size on triploidy: In the
case for the variety Ortanique, 8.11% of the triploid plants were
derived from small seeds compared to diploid hybrids, in contrast to the flat
seeds which gave a percentage of 5.41% of triploid plants. The variant M104
also showed similar results due to the fact that only small seeds gave a high
percentage of triploid plants in order of 8,11% and no triploid hybrids were
regenerated from flat seeds. For the variety Nadorcott, there is an
equality of percentage between the small and flat seeds, both had the same
chance to give triploids. Lastly and the case of the variety Murcott Honney
that was found in this study only one triploid hybrid, this last one was
regenerated from the germination of a flat seed while the small seeds of this
variety studied gave a percentage in the order of 2.70% and 0% for the small
seeds.
Results showed the significant effect of genotype and seed shape on the
percentage of triploidy of all the mandarins varieties studied, the variety Ortanique
showed a very good ability to regenerate triploid hybrids in comparison with
the other varieties and for the small seeds are the best to produce these
hybrids sought in the case of the three varieties Ortanique, variant of Murcott
M104 and the Nadorcott compared to the flat seeds giving low percentages
of triploidy. Only the variety Murcott honney from which a single
triploid hybrid was obtained from a flat seed (Table 7). The variety Ortanique
appeared to be the best genotype to produce triploid in vitro plants,
which are mainly derived from small seed germination.
The ploidy level profiles of the varieties: Nadorcott, Murcott
honney, Ortanique and variant of Murcott M104, resulting from the
immature embryo rescue are presented as histograms (Fig. 8). The comparison of
the plants tested, which are derived from in vitro immature embryo
rescue, with a triploid control profile (Moroccan Mandarin Hana) and a diploid
control profile (Fig. 9), allows us to conclude the amount of DNA in the in
vitro plantlet tested and thus its ploidy level.
The Seedlings confirmed as
triploid by flow cytometry are transplanted into pots in a greenhouse. After
two to three weeks, they are transplanted into black plastic bags. Thus, the
triploid seedlings will be grafted and transplanted into Table 6: Percentage of diploid and triploid seedlings from in
vitro germination of abnormal seeds (flat and small) analyzed by flow
cytometry
|
Mandarins
varieties |
Percentage
of plantlets |
|
|
|
Diploids |
Triploids |
|
Ortanique |
86.49 |
13.51 |
|
Variant
of Murcott M104 |
91.89 |
8.11 |
|
Nadorcott |
94.59 |
5.40 |
|
Murcott
Honney |
97.30 |
2.70 |
|
Total
Mean |
92.57±4.61 |
7.43±4.62 |
Table 7: Percentage
of polyploid plants analyzed by flow cytometry of the four varieties studied
according to seed shape.
|
Mandarins
varieties |
Percentage of polyploids |
||
|
|
Seeds shape |
Diploids |
Triploids |
|
Ortanique |
Small |
40.54 |
8.11 |
|
Flat |
45.95 |
5.41 |
|
|
Variant of Murcott M104 |
Small |
48.65 |
8.11 |
|
Flat |
45.95 |
0.00 |
|
|
Nadorcott |
Small |
56.76 |
2.70 |
|
Flat |
35.14 |
2.70 |
|
|
Murcott Honney |
Small |
54.05 |
0.00 |
|
Flat |
43.24 |
2.70 |
|
|
Total Mean |
46.28±6.99 |
3.72±3.21 |
|
%20210-218_files/image014.png)
Fig. 7: Comparison of the root growth rate for the four mandarin varieties.
RGR1: Root growth rate during the first two weeks; RGR2: Root growth
rate during three weeks.
large pots (Fig. 10). They were placed in a greenhouse under conditions
of temperature 25±2°C and humidity 80%.
Discussion
The average germination percentage was
statistically different among the four mandarin varieties, suggesting an
important distinction in the mechanisms controlling germination in these citrus
cultivars. This finding is similar to those found by (Fucik et al.
1974,1978); Orbović et al. (2013) who found statistical differences
for the germination rate of the cultivated seeds in citrus. Similarly, the
statistical distinction of germination rates among the studied genotypes as
well as stem growth; they may be related to the existence of seed coat during
the cultivation. This was proved by Cimen et al. (2020) and Azim et
al. (2013) who indicated that the shoot formation of Citrus sinensis
is immensely related to the presence of seed coat during in vitro
culture, which leads to a reduction in the germination percentage. In this
study and according to mandarin’s varieties studied, the germination rate
varied from 55 to 98% for the small seeds and from 66 to 100% for flat-shaped
seeds, these results showed similarity some previous studies (Aleza et al. 2010a; Ennaciri et al. 2020; Hmimidi et
al. 2020; Mahmoudi et al. 2020),
but it was higher than found by Ollitrault et al. (1996b)
who reported the same for small clementine seeds.
In
citrus, many studies have demonstrated the significant importance of selecting
the optimal in vitro culture medium to ensure successful embryo rescue (Hmimidi et
al. 2020; Ennaciri et al. 2020;
Mahmoudi et al. 2020). In studying the germination of
immature and mature seeds, we found a significantly difference in their
development as a function of time, between seed types (small, flat and normal).
This is reported by several studies (Silvertown 1981; Gross 1984; Stanton 1984)
indicating that the shape of the seeds has an effect on the interval and the rate
of germination and that the development differed over time according to the
seeds size (Singh et al. 2003; Mahmoudi et al. 2020). Indeed,
embryo rescue depends on the stages of embryonic development as well as the
composition of the nutritional environment (Esen et al. 1973). Similarly, the choice of the best male (Handaji
et al. 2005) and female (Essalhi et al. 2020) parents has been
the subject of several studies to optimize the triploidy rate. Triploid embryos are found in small seeds that do not
germinate conventionally under greenhouse conditions (Cuenca et al.
2010). In this study, triploid embryos were contained in seeds between 1/4 and
1/5 smaller than normal seeds (Table 1). The
Embryo rescue from these small seeds is necessary to achieve higher germination
rates (Navarro et al. 2002; Cuenca et
al. 2010) indicating that triploids were obtained from underdeveloped seeds
and possessing a size between 1/3 and 1/6 smaller than normal seeds (Esen et
al. 1973; Thorpe et al. 1994; Aleza et al. 2010a). Likewise,
spontaneous triploids have been detected in the progeny of Eureka lemon and
lime, ruby sweet orange, and imperial grapefruit (Husband et al. 2004).
The results of the latter studies confirmed that seed size is highly correlated
with ploidy level. Indeed, the size of the seeds, carrying triploid embryos, of
the order of l/3 to l/6 is smaller than those carrying diploids (Esen et al.
1973; Handaji et al. 2005).
The probability of obtaining triploid citrus hybrids
spontaneously is higher than 5% (Handaji et al. 2005). The frequency of
unreduced megagametophyte production is a function of maternal genotype, and
this information should be known before beginning extensive breeding programs
(Aleza et al. 2010a). Furthermore, seed size is highly related to ploidy
(Dalel et al. 2020). The study
revealed that triploid hybrids were all obtained from partially developed
seeds, mainly small seeds and this last character allows early selection of
triploid plants.
%20210-218_files/image016.png)
Fig. 8: Histograms
detected by the flow cytometry technique for counting the amount of DNA; a:
profile of a triploid in vitro plantlet; b: profile of a diploid in
vitro plantlet
%20210-218_files/image018.png)
Fig. 9: Histograms generated by flow cytometry for
counting DNA amounts; c: diploid control profile; d: triploid control profile
%20210-218_files/image020.jpg)
Fig. 10: Regeneration
of triploid green seedlings. A: triploid seedlings from small seeds; B:
seedlings transplanted in pots in the greenhouse; C: triploid seedlings from
grafting of triploid mother plants; D: acclimatization of grafted plants in the
greenhouse in large pots
Conclusion
The statistical
variation of triploidy rate depends on two factors: genotypes and seeds shape.
The embryo rescue linked to flow cytometry allowed to obtain and better detect
triploid hybrids. The study of the genotype effect showed that only the variety
Ortanique represented a good ability to produce triploids with a very
high triploidy rate compared to the other genotypes and contrary to the Murcott
honney which presented the lowest triploidy percentage. The seed shape
effect revealed that triploid vitroplants were mainly derived from abnormal seeds
(flat and small) especially the small seeds which have a high ability to
produce and regenerate the desired triploid hybrids.
Acknowledgement
This work is
supported by the National Center of Scientific and Technical Research (CNRST)
through the scholarship program of excellence (2017 edition). A special
gratitude and warm thanks go to Dr. Handaji Najat and Pr. Brhadda Najiba for
their support and their particular help in the development of this work.
Author
Contributions
All authors have
read and agreed to the published version of this manuscript.
Conflicts
of Interest
The Authors declare that there is no conflict of
interests that could possibly arise.
Data
Availability
Data is
available with the corresponding author
Ethics
Approval
No
applicable to this study
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