in vitro development and germination of immature embryos of … · 2019-03-13 · in vitro...

Journal of Agricultural Science and Technology B 8 (2018) 405-419 doi: 10.17265/2161-6264/2018.07.001

In Vitro Development and Germination of Immature

Embryos of Sweet Oranges Parson Brown and

Pineapple (Citrus sinensis)

Karim Mahmoudi1, 2, Najat Handaji1, Mohammed Ibriz2, Najat Arsalane1, Tarik Aderdour1, 2, Kawtar Label1, 2,

Hanae Ennaciri1, 2 and Hamid Benyahya1

1. Laboratory for the Improvement and Conservation of Plant Genetic Resources, National Institute of Agronomic Research (INRA),

Kenitra, BP 257, Morocco

2. Laboratory of Genetics and Biometry, Faculty of Sciences, University Ibn Tofail, Kenitra, Morocco

Abstract: Oranges are classified as the most traded fruit variety worldwide. The diversification of the varietal profile of this group is among the objectives of the Citrus breeding program. So, the creation of new triploid hybrids via immature embryos rescue, can lead to an increase in gene pool and produce seedless varieties. In addition, flow cytometry was successfully used for analyzing ploidy levels and genetic origin of triploid plants. The present study was aimed at optimizing immature embryos in vitro germination and plantlet development based on medium chemical composition of the two orange varieties, Pineapple and Parson Brown. When the fruit gets matured, they are harvested and the seeds extracted are classified according to their size. Only those which are small are cultured in a sterile Murashige and Tucker (MT) basal medium. The different concentrations of growth regulators are put to the test in order to gain the best medium for seeds development. M1 (MT + 1 mg/L gibberellic acid (GA3)), M2 (MT + 1 mg/L kenitin + 0.5 mg/L 6-benzylaminopurine (BAP) + 0.1 naphthalene acetic acid (NAA)), M3 (MT + 25 mg/L adenine sulfate (ADS)), M4 (MT + 0.5 mg/L kenitin + 0.5 mg/L BAP + 1 mg/L GA3). Percentage of germinated embryos (PGE) is the highest in M3 with 100% and 90% for both Pineapple and Parson Brown varieties, respectively. In addition, the germination interval average (GIA) is shorter in M1 for both varieties, which takes 6 d and 7 d, respectively. As regard to speed seedling growth (mm/week), both varieties undergo significant change in all four mediums. Similarly, the highest rate of acclimatization for the Pineapple and Parson Brown varieties is 80% and 90% in M1. In general, the smaller the embryos are, the more sensitive they are to culture medium composition. It is therefore essential to optimize the medium components in order to stimulate their in vitro development. Therefore, M1 (MT + 1 mg/L GA3) remains the best to promote short term good germination and produce a better rate of acclimatization. Key words: Citrus, orange, triploids, in vitro germination, immature embryos, flow cytometry, genetic.

1. Introduction

In Morocco, the Citrus sector is one of the main

fruit production and plays an important

socio-economic role, with orange varieties dominating

the production, estimated at 962,250 tons in 2016 [1].

Similarly, orange exports increased nearly to 120,500

tons, from which Maroc Late and Navel varieties are

the highest. Citrus varietal diversification based on

Corresponding author: Najat Handaji, Ph.D., research field:

biotechnology.

high quality criteria through extending production

period is considered as the central point around which

this breeding program is rotated. Quality is pursued

based both on the visual and organoleptic criteria

including color, skin texture, caliber, aspermia etc.

and juice content, sugar, acidity, aroma, which is

considered an issue of paramount importance in the

development of this sector. Moreover, the majority of

orange varieties (Sanguine, Navel and Valencia) are at

low seed rate, except Pineapple and Parson Brown

whose rates are high [2]. They are classified among

D DAVID PUBLISHING

In Vitro Development and Germination of Immature Embryos of Sweet Oranges Parson Brown and Pineapple (Citrus sinensis)

406

the most exploited varieties of juice industry [3].

Several methods are employed on these two varieties

in order to increase genetic variability, such as the

irradiation of seeds and axillary buds that produce

seedless clones for Pineapple [4, 5]. In vitro culture of

Pineapple buds (Citrus sinensis) results in the highest

bud induction according to Murashige and Skoog (MS)

medium which contains 3.0 mg/L

6-benzylaminopurine (BAP) [6-9]. Similarly, the

hybridization method which is employed on

Clementine and tetraploid variety ‘Pineapple’ results

in the formation of 17 triploid hybrids which are

obtained from undeveloped seeds [10, 11]. In addition,

the increase of genetic variability due to triploids has

become an interesting method for seedless cultivars

development [12, 13]. Soost and Cameron [14] stated

that triploid hybrids are vigorous, productive and

seedless. Thus, the gametic sterility which

characterizes triploid hybrids is due the fact that there

is an imbalance between chromosomes when the

embryonic development occurs in the ovum, as it was

claimed by Fatta et al. [15]. This meiotic disorder

removes seeds from fruits. In general, the triploidy

which is obtained through crossbreeding and

immature embryos rescue is considered the most

commonly used process [16]. In vitro germination of

immature mandarin embryos are obtained based on

Murashige and Tucker (MT) medium and

supplemented with gibberellic acid (GA3) (0.5 mg/L

GA3) [17] while orange varieties are obtained based

on MS medium which contains adenine sulfate (ADS)

(0.25 g /L) [18]. In general, seed germination is the

most frequently used procedure for propagation but

embryos rescue is way to produce seedless triploids

hybrids in Citrus [19, 20]. The present study was

aimed at optimizing in vitro germination of immature

embryos of Pineapple and Parson Brown varieties

based on different culture medium.

2. Materials and Methods

2.1 Plant Material Preparation

Two oranges varieties (Pineapple and Parson

Brown) are used to study in vitro germination

mediums for their immature embryos. The samples are

collected from the Citrus collection of the National

Institute of Agronomic Research “El Menzeh” during

the period from November to February.

2.2 Preparation and Sterilization of Seeds

At maturity, all seeds are extracted from the ripe

fruits and classified in two categories on the basis of

their morphology; either they are normal with fully

developed seeds, or abnormal with partially developed

seeds having flat shape or small one (Fig. 1).

Under the laminar flow cabinet, the flat and small

seeds were placed in tea bags and disinfected for 10

min in a solution of Lauryl mercyl followed by 70%

alcohol for 5 min, then surface sterilized with a

sodium hypochlorite solution 4% for 2 min. After that,

they are washed. The seeds are decorticated very

gently to avoid causing damage for the embryos. The

germination medium is composed of macro and micro

elements of MT medium (1969) [21]. Then they are

supplemented with 1 mg/L of GA3, 0.5 mg/L of

kinetin, 0.5 mg/L of BAP, and 0.1 mg/L of

naphthalene acetic acid (NAA) and 25 mg/L of ADS

(Table 1).

Table 1 Composition of the medium examined for immature orange embryos rescue

Code Basic medium Constituents Dose (mg/L)

M1 MT GA3 1

M2 MT Ken + BAP + NAA 0.5 + 0.5 + 0.1

M3 MT ADS 25

M4 MT Ken + BAP + NAA + GA3 0.5 + 0.5 + 0.1 + 2

MT: Murashige and Tucker; NAA: naphthalene acetic acid; BAP: 6-benzylaminopurine; ADS: adenine sulfate; Ken: kenitin; GA3: gibberellic acid.


407

Fig. 1 Seeds extracted from oranges varieties and classified according to their size. a: normal; b: flat; c: small.

2.3 Culture Conditions

Based on the all experiments carried out, the

medium which constitutes the basis is that of

Murashige and Tucker, 1969. Four compositions

come under examination.

All medium in this study are sterilized by an

autoclave of 120 °C under a pressure of 1 bar for 30

min. Then they are distributed into the testing tubes.

These tubes are incubated under 2,000 of lux light, a

16 h of photoperiod and a temperature of 27 °C during

the day and 19 °C at the night.

2.4 Studied Variables

Percentage of germinated embryos (PGE):

PGE (%) = NGS

TNS (1)

NGS: number of germinated seeds; TNS: total number

of seeds cultured.

Germination interval average (GIA):

GIA (d) = GD – CD (2)

CD: in vitro culture date; GD: germination date.

Speed seedling growth (SSG): The period of

development is generally identified from an initial

date that corresponds to the date of sowing and the

date of plantlets emergence. The development period

is the one which separates the two successive events.

The velocity is as follows:

SSG (mm/week) = D D

T (3)

D2: stem length (mm) of the second week; D1: stem

length (mm) of the first week; T: time (week).

Average number of plantlets leaves is counted after

seven weeks.

2.5 Acclimatization

The rooted in vitro plants go through a hardening

phase so that they become ready for the acclimation

stage; this phase involves the test tubes to be opened,

and the roots to be cleaned as the culture medium is

solidified by agar. Then the seeds are transplanted into

a

b c


408

pots containing 50% of sand and 50% of peat in a

greenhouse; these plants are covered by plastics

during a period ranges from three weeks to a month so

that a high hygrometry is maintained around young

plantlets. Also, they are transplanted into bags to

promote their development.

2.6 Flow Cytometry Analyses

Ploidy levels of seedlings from the rescue of

immature embryos were evaluated by flow cytometry

using a Partec II cytometer. The leaf tissues of each

sample were finely cut in the presence of the triploid

control (Moroccan mandarin Hana) with a razor blade,

in a Petri dish containing 0.5 mL of

phosphate-buffered saline (PBS) buffer, dithiothreitol

1 mg/L and 0.1% Triton 100. The core suspension

was filtered using a 40 μm nylon filter. Half a

milliliter of a propidium iodide solution at 1 mg/mL

was added and the mixture was incubated at room

temperature for 5 min. This fluorochrome specifically

binds to the DNA and under ultraviolet (UV)

excitation at 365 nm; the fluorescence intensity

re-emitted by the nuclei is proportional to the amount

of visible DNA on the abscissa axis. Measurements of

several thousand nuclei are retranscribed as a

histogram.

2.7 Statistical Analysis of Data

Using statistical analysis system, several variance

analyses are carried out in order to draw a comparison

between the averages of the following variables:

germination rate (%), GIA (d), SSG (mm/week) and

the leaves average number in terms of culture medium

effects. For acclimation, the Chi-square test is used to

determine its rate.

3. Results

3.1 Study of the Effect of Culture Medium on the

Studied Parameters

3.1.1 Percentage of Germination and GIA

The reason behind the germination of immature

embryos from the cultivars of the two oranges

varieties is that growth hormone exists in the four

culture mediums. PGE ranges from 65% to 100%

across the four culture mediums of both Pineapple and

Parson Brown varieties (Tables 2 and 3). The MT

medium which contains only ADS with

concentrations of 25 mg/L is the best performing

(100%).

For the GIA, there is no significant difference

between the two varieties “Pineapple and Parson

Brown” in the four cultures medium. The shortest and

most efficient one is the MT medium contains GA3 (1

mg/L), which takes 7 d.

3.1.2 Speed Seedling Growth (SSG)

SSG varies statistically based on the variety and the

medium (Table 3). Regarding M1, the growth rate is

the same for the two orange varieties during the first

week; however, statistical treatment through the Duncan’s

Table 2 Effect of different medium on the germination variables.

Variables Medium Varieties

Mean Pineapple Parson Brown

Percentage of germination (%)

M1 89.00a 88.00b 88.50

M2 91.00a 87.00b 89.00

M3 100.00a 90.00b 95.00

M4 65.00b 85.00a 75.00

Germination interval average (GIA)

M1 6.48a 7.02a 6.75

M2 7.27a 9.18a 8.22

M3 24.09a 22.40a 23.24

M4 16.42a 13.15a 14.78

Comparison between the averages of the two varieties for the same medium (Duncan’s test); in the same column followed by the same letters do not differ significantly at the level 5% (Duncan’s test); M: culture medium.


409

Table 3 Variation of growth rate of the two orange varieties across the four culture mediums.

SSG (mm/week)

Varieties Medium SSG1 SSG2 SSG3 SSG4 SSG5 SSG6 Mean

Pineapple

M1 0.87a 0.70b 1.17a 0.29b 0.25a 0.58a 0.64

M2 0.27a 0.66b 0.42a 0.26a 0.31a 0.55a 0.41

M3 0.00a 0.00b 0.88a 0.09a 1.30a 0.54a 0.46

M4 0.00b 0.00b 0.26a 0.73a 0.60a 0.57a 0.36

Parson Brown

M1 0.71a 11.46a 0.63b 0.52a 0.00b 0.00b 0.62

M2 0.00b 2.31a 0.14a 0.19a 0.64a 0.36a 0.60

M3 0.00a 2.05a 0.36b 0.27a 0.75b 0.49a 0.65

M4 0.40a 3.87a 0.34a 0.07b 0.76a 0.69a 1.02

Comparison between the two varieties’ growth rate within the same medium (Duncan’s test); figures in the same column followed by the same letters do not differ significantly at level 5% (Duncan’s test); M: culture medium, SSG: speed seedling growth (mm/week).

test reveals a significant difference during the next

five weeks. As for the M2, the first two weeks marks a

significant variance, but in the following week

Pineapple and Parson Brown stops to grow.

According to M3, in the first week, the plantlets grow

alike, while in the following weeks, Duncan’s test

reveals a significant difference and this will continue

until the last week with a marked difference for both

varieties.

For M4, the statistical analysis discloses a

significant variation for the two orange varieties

during the first two weeks, whereas it shows the same

development during the following weeks.

3.1.3 Variation of Average of the Leaves Number

after Seven Weeks in the Four Culture Mediums

Concerning the average number of leaves, the

analysis show a significant difference between

Pineapple and Parson Brown in M2, M3 and M4,

whereas M1 (Table 4), no significant difference

occurs. Thus, the leaves’ average number is relatively

high in M2 with 3.1 and M3 with 3.72, respectively,

for Pineapple variety (Fig. 2).

3.2 Study of Acclimatization

3.2.1 Acclimatization Rate (AR)

The different medium studied (M1, M2 and M3)

showed a significant difference of acclimation rate

between Pineapple and Parson Brown varieties based

on the Chi-square test (Table 5). During the

experiment, in vivo acclimation rate (Fig. 3) range

from 30% to 80% for Pineapple variety (Fig. 4) and

from 35% to 90% for Parson Brown variety (Fig. 5).

Furthermore, M4 medium showed no result of

acclimation because its root system is weak when in

vitro culture takes place (Fig. 3).

3.2.2 Test of Chi-square

For Chi-square test, the p-value is less than alpha =

0.05 (Table 6), which explains why there is a

difference between rows (survival and mortality) and

columns (culture medium). This proves that there is a

close relationship between medium, survival of these

varieties and their mortality. This difference is

explained by Chi-square observed value (135.95)

which is higher than the expected one estimated at

11.07. Explicitly, Fig. 6 revealed that Pineapple and

Parson Brown have higher chance to survive with

80% and 90%, respectively. But in M2, there is a great

difference between Pineapple’s survival rate with 55%

and Parson Brown’s with 30%. For the last medium,

the survival rates are almost the same with 30% for

Pineapple and 35% for Parson Brown (Figs. 4 and 5).

3.3 Study of Immature and Mature Seeds Germination

Fig. 6 showed the comparison between variables of

three types of seeds within the same observation period.

At the end of the first week, the stem length average

was observed the same for small, flat and normal

seeds (Fig. 7). The same thing occurs in the 2nd, 3rd

and 4th weeks (Fig. 6). In the 4th week, the same stem

length average was found for the three seeds, as well


410

as the same root length average (Fig. 6), which

explains that there is a growth harmony in stem and

root length.

The stem length average of the Parson Brown

variety shows a significant difference in the second

week and this continues in the sixth one. After that,

there is similarity between these seeds (Fig. 6).

Moreover, the root length average of Parson Brown

variety shows from the first week to the fifth one a

significant difference between small, flat and normal

seeds, whereas it reveals a similarity in the sixth week

(Fig. 6).

3.4 Result of Flow Cytometry Analysis

A significant number of polyploids recovered from

underdeveloped seeds of Citrus cultivars. Only the

Table 4 Effect of different basal medium on the leaves’ average number variation after seven weeks according to medium culture.

Varieties Average of leaves’ number

M1 M2 M3 M4

Pineapple 2.32bc 3.10ab 3.72a 2.66bc

Parson Brown 2.20bc 1.81c 2.80abc 1.80c

Comparison between averages of the two varieties of the same medium (Duncan’s test); the numbers in the same rows followed by the same letters do not differ significantly at level 5% (Duncan’s test); M: culture medium.

Fig. 2 In vitro plantlets development through embryo rescue after seven weeks of incubation on different basal medium. M: culture medium.

M2

M4M 3

M 1 M1

M3


411

Table 5 Effect of culture medium on the acclimation rate for two orange varieties.

Varieties Acclimatization rate (%)

M1 M2 M3 M4

Pineapple 80 55 30 0

Parson Brown 90 50 35 0

Table 6 Chi-square test of independence between rows and columns.

Chi-square test (observed value) 139.9554

Chi-square test (critical value) 11.0705

Degrees of freedom 5

p-value < 0.0001

Alpha 0.05

Fig. 3 Steps of acclimatization. a: in vitro plantlets; b: plants covered with plastics; c: plantlets transplanted in a pot; d: recovered triploid grafting on troyer citrange.

a b

c d

412

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413

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414

medium M1 which is the best medium containing

GA3 in terms of the embryo rescue and recovering of

plant triploids, Citrus cultivars were examined for

seed size underdeveloped. Maximum triploids plants

66.67% were found in small seed of variety Parson

Brown, followed by flat seed 40.00%. Furthermore,

25.00% of triploids plants were observed in flat seed

of variety Pineapple whereas the minimum value

(0.0%) was observed in small seed (Table 7).

The ploidy level profiles of two Parson Brown and

Pineapple varieties from the immature embryo rescue

with that of the control are presented in the form of

histograms (Fig. 8), the fluorescence intensity emitted

appearing on the abscissa axis and the number of

nuclei on the ordinate.

Triploids seedling of two orange varieties Pineapple

and Parson Brown selected by cytometry analysis

were budded on Troyer citrange and planted in

National Institute of Agronomic Research (INRA)

domain (Fig. 9).

Fig. 7 Seedling regeneration after six weeks of culture. a: small; b: flat; c: normal.

Table 7 Number of plants analyzed and ploidy level of plants recovered in oranges ‘Parson Brown’ and ‘Pineapple’ in medium M1.

Cultivars Seed size Percentage of polyploids

Haploids (N) Diploids (2N) Triploid (3N) Tetraploids (4N)

Parson Brown Small - 33.33 66.67 -

Flat - 60.00 40.00 -

Pineapple Small - 100.00 0.00 -

Flat - 75.00 25.00 -

a b c


415

Fig. 8 Histogram of flow cytometry for DNA amount counting.

416

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e

n

m


417

with supplements of various hormonal concentrations

including BAP, kinetin and ADS are useful in shoot

proliferation [47]. The use of additives such as ADS

as seen in the study is a key factor in the production of

shoots from Jatropha curcas, a concept that supports

reports by Sarika and Meenakshi [48]. Munde and

Gajbhiye [49] studied the effect of regulators GA3 on

seedling growth of mango stones and producing more

number of leaves.

Furthermore, the combination of three hormones

namely BAP, kinetin, NAA leads to a better leaf

production for of mandarin variety [50]. Also, the

medium which contains GA3 is the one which

stimulate root growth [41], besides, Singh and Kaur

[51] showed that the use of auxins is so important for

root system for citrus species such as limonia, sinensis

and jambhiri citrus.

The study of immature and mature seeds

germination revealed a significant variation between

seed type (small, flat and normal), so their

development was different according to the time. In

fact, embryo rescue depends on the stages of

embryonic development and the composition of the

nutritional medium [52]. A study carried out by

Caraimi et al. [53] showed that the embryos sized (<

0.3 mm) had 76% of germination based on MT

medium which contains 150 mm of sucrose and 14.4

μM of GA3. While embryos whose size is 0.3-0.8 mm

had 95% of germination based on the same medium

supplemented with 150 mm of sucrose and 2.9 μM of

GA3.

MT basal medium containing 1 mg/L GA3 recovered

85 triploid plants Citrus [54]. Triploids were obtained

from underdeveloped seeds ranging 1/3 to 1/6 [55, 56].

Similarly spontaneous triploids were obtained among

progenies of Lisbon and Eureka lemon, Ruby sweet

orange and Imperial grapefruit [57].

5. Conclusions

Triploid plants are rare in nature because of their

inviable seeds and resulting lack of progeny, so it is

challenging to detect naturally occurring triploid

plants. However, due to their faster growth and

seedlessness, they will be useful for improving

biomass, fruit and flower traits, and other qualities of

economically important food, The use of in vitro

embryo rescue and ploidy level assessment by flow

cytometry gave highly efficient recovery of Citrus

triploid hybrids from small and flat seeds. This study

of in vitro germination of immature embryos for two

orange varieties Pineapple and Parson Brown showed

that the favored MT medium containing GA3 is

efficient in terms of the GIA great advantage for in

vitro germination of citrus varieties. For MT medium

which contains ADS, a large average number of

leaves are produced for both varieties, while the M4

could not produce any adequate roots in Pineapple and

Parson Brown acclimatization with a value of 0%.

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