In Vitro Propagation of Sugar Beet Cultivar Frida, Through...

American Journal of Agricultural Science

2016; 3(3): 27-34

http://www.aascit.org/journal/ajas

ISSN: 2381-1013 (Print); ISSN: 2381-1021 (Online)

Keywords Artificial Seed,

Sugar Beet,

Adventitious Shoots Sodium

Alginate

Received: March 13, 2016

Accepted: March 31, 2016

Published: May 6, 2016

In Vitro Propagation of Sugar Beet Cultivar Frida, Through Encapsulated Different Explants

Roba M. Ismail, Wesam M. Raslan, Gihan M. H. Hussein

Gene Transfer Lab, Plant Genetic Transformation Department, Agricultural Genetic Engineering

Institute (AGERI), Agricultural Research Center (ARC), Giza, Egypt

Email address [email protected] (R. M. Ismail), [email protected] (W. M. Raslan),

[email protected] (G. M. H. Hussein)

Citation Roba M. Ismail, Wesam M. Raslan, Gihan M. H. Hussein. In Vitro Propagation of Sugar Beet

Cultivar Frida, Through Encapsulated Different Explants. American Journal of Agricultural

Science. Vol. 3, No. 3, 2016, pp. 27-34.

Abstract Synthetic or artificial seed protocol is a powerful method for production of seed

analogues. It relies on in vitro encapsulation of any meristematic tissue by its coating

with suitable gelling agents. Here we describe the synthetic seeds production for sugar

beet (Beta vulgaris) cv Frida using encapsulation by sodium alginate. Different factors

affecting the encapsulation efficiency such as explant type, presence of BA and presence

of sucrose were studied. The best combinations for synthetic seed production found to be

using of 4% sodium alginate for encapsulation of shoot tip explants in presence of 1.3

mg/l BA or 4% sucrose. In case of adventitious shoots, the best combinations found to be

using of 4% sodium alginate with 1.3 mg/l BA, 4% sucrose and 1.2% agar.

1. Introduction

Sugar beet (Beta vulgaris) is an important root crop and it considers the main source

for sugar production in the moderate climate regions with five months long growing

season [1]. In Egypt, the total planted sugar beet area in 2014/2015 is about 183,000 ha,

comparing to 174,000 ha in 2013/14 as reported by Sugar Annual, 2015

(http://www.thefarmsite.com/reports/contents/EgyptSugar29April2014.pdf).

According to in vitro culture and genetic transformation, sugar beet considers as an

unruly plant species [2]. Regenerated adventitious shoot from different explants has been

effectively used for the propagation of elite sugar beet genotypes [3], although high

degree of variability in the regeneration frequencies from diverse explants of different

genotypes have been detected [4]. Additionally, the vegetative nature of the material and

the greater risks of disease transfer increase the difficultly of the distribution and replace

from gene bank field.

Artificial seed protocol extends plant biotechnology and agriculture prospect in the

plant propagation [5] as it useful for protecting the best agricultural and endangered plant

species, which are difficult to regenerate through conventional methods and natural

seeds. In addition, artificial seed is very useful way for the large-scale propagation of

superior hybrids of economically important species [6].

Encapsulated somatic embryo firstly reported by Murashige [7]. Afterward,

engineering of somatic embryo into synthetic seed has been continued by the efforts of

Kitto and Janick [8], Gray [9] and Gray and Purohit [10]. Encapsulated micro shoots or

somatic embryos were used to propagate different plant species, such as sandalwood,

mulberry, banana, cardamom, sugar beet, rice and peer [11, 12, 13, 14, 15, and 6].

28 Roba M. Ismail et al.: In Vitro Propagation of Sugar Beet Cultivar Frida, Through Encapsulated Different Explants

Different types of the explants have been previously used to

produce synthetic seeds such as nodeal segment from

Bacopamonnieri L [16] and Menthaarvensis [17]; or somatic

embryos and shoot tip from sugar beet [14, 18],

Begoniaxhiemalis Forch [19] and shoot tip M. arvensis [17]

and Stevia [20]. In general, the synthetic seed was used in

propagating different economically important plant species

such as vegetable crops, forage legumes, industrially

important crops, cereals, spices, plantation crops, fruit crops,

ornamental plants, orchids, medicinal plants and wood

yielding forest trees [21].

In this work, synthetic seeds were produced from inter

node, shoot tip explants and adventitious shoots from sugar

beet cv. Frida and their ability to regenerate in complete

plants was assessed. In addition, adventitious shoots

development from the internode explants was studied.

2. Materials & Methods

2.1. Materials

Seeds of sugar beet (Beta vulgaris) cultivar Frida were

kindly obtained from sugar crops institute, Agricultural

Research Center (ARC).

2.2. Methods

Seeds germination

Sugar beet seeds were surface sterilized by soaking in 70%

(v/v) ethanol for 1 min; then rinsed several times with

sterilized distilled water. Seeds were transferred to 40% (v/v)

Clorox solution for 45 min, then washed by rinsing several

times with sterile distilled water for 30 min. Thereafter, seeds

were germinated on MS medium supplemented with 0.5 mg/l

2, 3, 5-Triiodobenzoic Acid (TIBA) medium and incubated

for 45 days.

2.3. Synthetic Seeds of the Shoot Tip and

Internode Cutting Explants

2.3.1. Explants Encapsulation

I. Coating explant with alginate

Shoot tip and internode cutting as explants were coated

with six coating solutions composed 4% sodium alginate

dissolved in water or in MS basal medium with or without

benzyl adenine (BA) and sucrose (Table 1 from T1-T6).

Explants were coated by dropping the explants in sodium

alginate solutions for 20 min; the alginate-coated explants

were transferred to plates containing 1.4% CaCl2 for another

20 min. Then, coated explants were washed with liquid MS

medium for three times. All procedure steps were conducted

under aseptic condition.

II. Synthetic seeds storage

Coated explants were placed on 3.5 cm Petri dishes either

on filter paper or without filter paper and incubated for 1, 2, 4

and 8 weeks at room temperature. To select the best coating

condition, in vitro germination of coating seeds was

performed on half SH9 medium [22]. The following

parameters were recorded: Required time for germination in

days, percentage of germination, number of shoots/explant,

shoots height and root formation.

2.3.2. Shoot Development

For producing microshoots, the inter node segments were

cut from 45-day-old in vitro seedling as explants, then

cultured on different multiplication media composed of

different media, different growth regulators and different

solidifying agent (phytagel and agar, Table 2). Each

multiplication medium had six plates on each five explants

with total number of 30 explants. Obtained micro-shoots

were isolated from their explants and were coated for

producing synthetic seeds.

2.3.3. Encapsulated Microshoots

Obtained shoots were coated with two sodium alginate

solutions (Table1, T7 and T8). Then the coated explants were

then incubated into CaCl2, and washed as mentioned before

with the shoot tip and inter-node explants.

2.3.4. In vitro Germination

To select the best germination medium encapsulated

shoots were transferred to four different media which are: G1

(hormone-free MS medium); G2 (MS with 0.5 mg/l TIBA);

G3 (MS with 40µg NAA) and G4 (half strength SH medium

with 40 µg/l NAA). All germination media were solidified

with agar at concentration of 8 g/l. Germinated sugar beet

seeds were transferred to root formation medium containing

1mg/l IBA. The rooted plantlets were acclimatized on

mixture of beet moss: sand (1:1).

Table 1. Different sodium alginate solution.

Treatments Sodium alginate agar MS BA(mg) sucrose

T1 4% +

T2 4% 1.3

T3 4% + 4%

T4 4% + 1.3

T5 4%

T6 4% 4%

T7 4% 1.2% + 4%

T8 4% 1.2% + 1.3 4%

American Journal of Agricultural Science 2016; 3(3): 27-34 29

Table 2. Different shoot development media composition.

Media medium Growth regulatorsmg/l Solidifying agentg/l

MS SH NAA mg/l TIBA BA mg/l TDZ Phytagel Agar

SD1A half 3 7

SD2A half 3 7

SD3P full 3 3

SD3A full 3 7

SD4P full 3 3

SD4A full 3 7

SD5P full 0.5 3

SD5A full 0.5 7

SD6P full 0.3 3

SD6A full 0.3 7

SD7P half 3

SD7A half 7

SD8P half 3 3

SD8A half 3 7

SD9P half 3 3

SD9A half 3 7

SD10P half 0.3 3

SD10A half 0.3 7

2.3.5. Root Formation

The obtained shoots were transferred to two different

rooting media composed of MS with different concentration

of Indole-3-butyric acid(IBA) (1 or 2 mg /l), individually.

2.3.6. Acclimatization Stage

Plantlets were transferred to greenhouse in pots containing

1:1 mixture of peat moss: sand. The pots were covered with

transparent plastic bags to keep the humidity at 90% for one

week. The plastic bags were removed gradually.

2.4. Synthetic Seeds of the Adventitious

Shoots

Synthetic seeds were also produced from adventitious

shoots that were developed from the inter-node explants.

3. Results

3.1. Synthetic Seeds of the Shoot Tip and

Internode Cutting Explants

Explants (shoot tip & internode cutting) encapsulation:

In order to select the best solution of encapsulation, the

encapsulated explants (synthetic seeds) were germinated

after different storage periods. All coated explants from

different coating treatment were germinated on half SH9

medium. Results showed that all explants incubated on a

filter paper were dried after one weak. All encapsulated

explants with T1, T2, T5 and T6 coating solutions did not

germinate. While, the encapsulated shoot tip explants with

T3 and T4 germinated easily. The internode coated explants

with T3 or T4 that stored for one week, two weeks and a

month germinated but those stored for two months did not

germinate (Table 3). It was also showed that there were

approximately no differences of the germination rate

between the coating solutions (T3 and T4). All coated

explants that were stored for one week started to germinate

after three days, whereas, the encapsulated explants that

incubated for two weeks started to germinate after four days.

In addition, the germination time was varied from 6-12 days

for the coated explants that incubated for one month.

Furthermore, the internode coated explants incubated for

two months did not germinate while the shoot tip coated

explants incubated for two months were germinated after 7

days. The germination percentage of synthetic seeds was

varied among the treatments from 0-100 (Table 3). Shoot

heights were ranged 8.5-13 cm among the treatments. The

highest germination percentage was in the synthetic seeds

incubated for one week, while the lowest germination

percentage was in the encapsulated explants incubated for

two month. Thus shoot tip as an explant and T3 or T4

coating solutions reveled the best condition for producing

synthetic seeds.

Not all treatments formed root on the half SH9 medium

therefore the shoots were rooted on medium containing IBA

(Fig. 1). It was observed that medium containing IBA at

concentration of 1 mg/l was better than that containing 2mg/l

(Data not shown).


Table 3. Germination of both encapsulated explants (shoot tip-inter node) with T3 & T4 solution.

explant Coating

treatment

Storage

time

Total No.

coating seeds

Germinating

seeds

% Germinating

seeds

Time of

germination

Obtaining

shoots

Shoot heights

par cm

Shoot tip

T3

week 15 15 100 3days 7 13

Two

weeks 15 11 76 4 days 7 10

month 15 5 30 6 days 2 10

Two

moths 15 3 20 7 days 1 9

T4

week 15 15 100 3 days 6 12

Two

weeks 15 12 80 4 days 7 10

month 15 5 33.3 7 days 2 9

Two

moths 15 3 20 7 days 1 8.5

Inter node

T3

week 15 15 100 3 days 10 11

Two

weeks 15 12 80 4 days 10 12

month 15 3 20 10 days 1 8

Two

moths 15 0 0 0 0 0

T4

week 15 15 100 3 days 12 13

Two

weeks 15 15 100 4 days 10 13

month 15 3 20 10 days 3 11.5

Two

moths 15 0 0 0 0 0

Fig. 1. Germination of encapsulated shoot tip (A) & interned segments (B).

A, Encapsulated shoot tips on germination medium after 5 days (1)and after 10 days (2); germinated shoots on rooting medium after 3 weeks (3) and sugar

beet plant after 45 days under acclimatize condition.

B, Encapsulated inter-nod segments on germination medium after 5 days (1) and after 2 weeks; germinated shoots after 3 weeks incubation on rooting medium

and sugar beet plant after 45 days under acclimatize condition

3.2. Synthetic Seeds of the Adventitious

Shoots

3.2.1. Adventitious-Shoot Differentiation

Results showed that the average of developed shoots per

each explants ranged from 1-1.5 shoots. Among all media,

SD2A and SD1A revealed highest response of shoot

development. Medium SD2A containing 3mg/l TDZ revealed

higher percentage rather than SD1A that contained 3mg/l

BA. Whereas, the medium SD8P revealed highest percentage

of shoot development but it resulted in the highest

vitrification percentage as well. Media composed of half SH

media (SD7P-SD10A) had low frequency of shoot

production except SD8P medium which contained TDZ at


concentration of 3 mg/l. It was observed that the percentage

of vitrification was ranged from 7-38% in shoots that

produced on media solidified with phytagel (Table 4).

Shoots developed on medium containing MS medium and

BA was earliest than all other media as shoots started to

germinate within 4-7 days of culturing, while adventitious

shoots on other media started to differentiate after 10 days. It

was observed that shoots developed on MS medium were

greener, stronger and taller than those that developed on SH

media (Fig. 2). Thus, medium SD1A composed of half MS,

3mg/l BA and 8g/l agar was selected for developing

adventitious shoots from sugar beet internodes.

Fig. 2. Adventitious shoots differentiation on different media: obtained adventitious-shoots on half SH based medium (A), MS based medium (B). Difference on

normal and vitrified shoots on medium solidified by phytagel (C).

Table 4. Effect of different media on shoot differentiation.

Media Total n of

explants

No of explants

producing shoots

% explants

producing shoots

Total No of

shoots

Means of

shoot/explants

Number of Vitrified

plants(v) %(v)

SD1A 30 30 100 39 1.3 0 0

SD2A 30 30 100 42 1.4 0 0

SD3P 30 30 100 37 1.2 13 35%

SD3A 30 30 100 30 1.0 0 0

SD4P 30 30 100 42 1.4 15 36%

SD4A 30 25 83 25 1 0 0

SD5P 30 30 100 30 1 9 30%

SD5A 30 30 100 31 1 0 0

SD6P 30 30 100 30 1 6 20%

SD6A 30 30 100 30 1 0 0

SD7P 30 28 93 28 1 10 36

SD7A 30 10 33 10 1.0 0 0

SD8P 30 30 100 45 1.5 17 38

SD8A 30 30 100 32 1.06 0 0

SD9P 30 30 100 30 1 2 7

SD9A 30 30 100 30 1 0 0

SD10P 30 20 66.6 20 1 3 15

SD10A 30 30 100 35 1.16 0 0

3.2.2. Explants Encapsulation

Obtained adventitious shoots were coated with two coating

solutions composed of 4% sodium alginate, 1.2% agar and

4% sucrose (T7) or 4% sodium alienate, 1.2% agar, 4%

sucrose and 1.3 mg/l BA (T8) dissolved into liquid MS

medium. After one-week incubation at 25°C, the

encapsulated explants were germinated on four different

media. Seeds started to germinate after 3-4 days on all media;

shoots reach 2-11 cm height during 3-4 weeks. From the data

in Table (5), the best conditions for germination were at

hormone free MS medium and T8 coating solution, because

it counted the height seed germination, shoots and root

formation (Fig. 2). The germinated shoots that did not form

roots were transferred to root formation medium containing

1mg/l IBA (Fig. 3).


Table 5. Encapsulation adventitious shoot explant germination on different media.

Germination

media

Coating

treatment

Total No.

coating seeds

Germinating

seeds

% Germinating

seeds

Obtaining

shoots

Shoot heights

par cm Root formation

G1 T7 15 8 53.3 8 11 0

T8 15 15 100 30 4 1

G2 T7 15 8 53.3 8 2 0

T8 15 6 66.7 10 2.5 0

G3 T7 15 6 40 9 6.3 1

T8 15 10 66.7 20 3.2 0

G4 T7 15 8 53.3 8 3.5 1

T8 15 8 53.3 18 3.1 0

Fig. 3. Adventitious shoot explant before coating: Adventitious shoot used for coating (A); coated adventitious shoots with alginate/agar (B); coating shoots

after one week incubation (C); germinated of synthetic seeds after 2 weeks on MS medium (D); Shoots after 3 weeks on rooting medium (E); acclimatized

plants after 45 days (F) and after 100 days (G) and produced sugar beet tap-root after 100 days (H).

4. Discussion

The artificial seed technology has opened a new scope for

handling, transplantations and maintenance of rare and

attractive genotypes [23]. In this investigation the production

of the synthetic seed from in vitro materials of sugar beet cv

Freda was studied. Three sugar beet explants were used, i.e.,

shoot tip, internode cutting and adventitious shoots that were

developed from the internode cutting explants. Several

explants were used previously to produce synthetic seeds

such as: the nodal segment in Bacopamonnieri L [16];

Cannabis sativa L. [24] and M. arvensis[17]; shoot tip of M.

arvensis[17]; Stevia [20].Synthetic seeds of sugar beet were

produced previously from somatic embryos [14] and shoot

tip [17].

Kitto and Janick [25] used many encapsulating agents such

as agar, agarose, alginate, carrageenan, gelrite and

polyacrylamide. Alginate is one of the most commonly used

polymers for immobilization of plant cells and production of

synthetic seeds because it is available in large quantities,

inert, non-toxic, cheap and can be easily handled [26, 27].

Sodium alginate is recommended as most suitable

encapsulating agent due to its solubility at room temperature

and its ability to form completely permeable gel with calcium

chloride forming a hydrogel in the presence of calcium ions

[28, 29]. In this study, sodium alginate has been used as an

encapsulating agent for coating explants in different matrix

solutions. It was found that the presence of MS, sucrose and

BA in encapsulating solutions is important in the artificial

seed germination. Results showed that all explants coated

with sodium alginate solution did not germinate. Moreover,

the synthetic seeds that coated with solution of sodium

alginate and sucrose dissolved in water did not germinate.

While, the synthetic seeds that coated with sodium alginate

with sucrose dissolved in MS germinated. Explants coated

with encapsulation solution containing 4% sodium alginate

dissolved in MS medium with either 1.3 mg/l BA or 40 g/l

sucrose revealed the same germination frequency. This

indicates the importance of sucrose, BA or MS for synthetic

seed germination. Furthermore, the adventitious shoot

explants were coated with matrix solution that containing BA

and sucrose revealed higher germination rate than those

coated with solution contained only sucrose. This result

indicates that the combination of BA and sucrose in coating

solution affected positively on seed germination as well as

the shoot development frequencies from the synthetic seeds.

We concluded that the best coating (encapsulating) matrix

solution was 4% sodium alginate, 1.2% agar as a solidified

agent, 1.3 mg/l BA and 3% sucrose. The enhancement effect


of sucrose in producing synthetic seeds was previously

reported by Rizkalla et al., [18] while conducting the

synthetic seeds production of sugar beet cvs Francesca and

Toro by encapsulating shoot tip with matrix solution

composed of 4% sodium alginate and 30 g/l sucrose. They

found that the addition of sucrose at 30 g/l to encapsulation

matrix solution gave high germination rate of synthetic seeds

than addition of 40, 50 or 60 g/l sucrose. Tsvetkov et al. [30]

and Maqsood et al. [31] reported that supplying 3% sucrose

to the alginate solution was very important in the starting

stage of the re-growth and synthetic seeds germination of

hybrid aspen and Catharanthusroseus L, respectively.

Murthy et al. [32] found that the shoot developing

percentage, a high number of shoots obtained of each

encapsulated explant and maximum shoot length were

achieved with the encapsulation matrix prepared with MS

supplemented with 3 mg/l BA, 3% sucrose and 3% sodium

alginate in Ceropegiaspiralis. Our results showed also that

the coating solution containing both sucrose and BA has also

influenced positively on producing synthetic seeds from

adventitious shoot explants as it reveled higher shoot

development than the explants coated by solution without

BA. Tsvetkov et al. [30] reported the positive effect of

combining BA and sucrose in the coating solution. They

observed that the highest fresh mass was shown when using

MS media with either BA and sucrose or MS with only

sucrose. They also demonstrated that BA increases the mass

weight twenty fold. Contrarily, Nower [20] encapsulated

stevia explants in MS liquid supplemented with only 4%

sodium alginate. It was indicated that the encapsulation

solution optimization is species-dependent and there is no

general encapsulation solution for every plant.

Nature of explants was found to be an important factor in

producing synthetic seed. The synthetic seeds from shoot tip

explants were earlier in germination than those produced

from internode. In addition, shoot tip synthetic seeds could

be stored viable for two months while, the synthetic seed

from inter node lost its germination ability after two months.

This was agreed with Islam and Bari [17] as they reported

that the artificial seed of shoot tip of M. arvensis exhibited

highest percentage of multiple shoot formation than nodal

segment explants.

The storage time of the synthetic seeds was also found to

be affecting the required germination time. The required

germination time was prolonged when the storage time was

increased. It was three or four days in case of seeds stored for

one or two weeks, respectively, and varied from 6-10 days in

seeds that stored for month or two months. In our result only

4 days required to germinate the one week stored synthetic

seeds on hormone free MS. Whereas, Sionget al. [6] reported

that the artificial seeds produced from adventitious shoots of

cauliflower, Brassica oleracea var. botrytis required 12 days

(after 7 days storage) and 14 days (after 30 days storage) to

germinate on MS basal medium.

Artificial seeds of sugar beet in current study were stored

at empty Petri dishes, while Rizkalla et al. [18] stored their

seeds on MS medium with sorbitol or mannitol.

5. Conclusion

In this study, synthetic seeds of sugar beet (Beta vulgaris)

cv Frida from different explants were produced. Our

established protocol showed efficient germination rate and

storage time. We also concluded the importance of sucrose in

the encapsulation by alginate.

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In Vitro Propagation of Sugar Beet Cultivar Frida, Through...

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