Hormonal Requirements Trigger Different Organogenic Pathways on Tomato Nodal Explants

doi:10.4236/ajps.2013.411263

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American Journal of Plant Sciences, 2013, 4, 2118-2125

Published Online November 2013 (http://www.scirp.org/journal/ajps)

http://dx.doi.org/10.4236/ajps.2013.411263

Open Access AJPS

Hormonal Requirements Trigger Different Organogenic

Pathways on Tomato Nodal Explants

Salem Jehan, A. M. Hassanein

Central Laboratory of Genetic Engineering, Faculty of Science, Sohag University, Sohag, Egypt.

Email: hassanein[email protected]

Received August 21st, 2013; revised September 21st, 2013; accepted October 17th, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

In this work, nodal-disk segments (4 - 6 mm in diameter × 5 - 6 mm in length) were obtained from established shoot

culture, resulted from disinfected tomato seedlings, and they were suitable to induce different organogenic pathway

under the influence of specific hormonal treatment. Application of BAP (1 - 2.5 mg/l) alone or in combination of 0.5

mg/l NAA resulted in induction of shoot formation. Somatic embryogenesis was rarely appeared (6%) when relatively

low concentration of BAP (1.5 mg/l) with low concentration of IAA (0.5 mg/l IAA) was applied. Root induction was

triggered when nodal explants or shoot cuttings were cultured on MS medium with (1 mg/l IAA, IBA or NAA) or

without auxins, but the best result was obtained when 1 mg/l IAA was used. Application of 0.5 mg/l NAA stimulated

callus formation but the best result was obtained when the three different phytohormoes were used (0.5 mg/l 2,4-D + 1

mg/l NAA + 0.5 mg/l BAP). These results indicated that nodal segments, as described in this protocol, can be used as

alternative to other types of explants such as cotyledon, hypocotyl and leaf explants.

Keywords: Growth Regulators; Nodal Segment; Organogenesis; Tissue Culture; Tomato

1. Introduction

Tomato is one of the major vegetable crops for human

being; it belongs to the family Solanaceae. The botanical

name of tomato is Lycopersicon esculentum Mill. It is a

diploid plant with 2n = 24 chromosomes. Tomato is a

natural perennial plant, but it is commercially cultivated

as an annual crop. It is grown in almost every country of

the world—in the field, greenhouses and net houses [1].

Tomato is an important crop worldwide for fresh mar-

ket or processing. It is necessary for the human being

because it contains high amounts of antioxidant vitamins

such as vitamins A and C, fiber, and considerable quanti-

ties of antioxidants such as flavonoids, zeaxanthin,

b-carotenes, lutein, and lycopene [2]. Lycopenes protect

cells and other structures in the human body from harm-

ful oxygen-free radicals, therefore they decrease the risk

of cancers [3,4]. In addition, tomato has also great poten-

tial for transgenic applications, it was used for production

of oral vaccines [5] and creation of a new anthocya-

nin-enriched food for cancer prevention [6], and as

model for functional genomics, proteomics and me-

tabolomics to improve abiotic and biotic stress tolerance

[7-9].

Genes were conventionally transferred by plant breed-

ers from specific varieties to the related species by sexual

hybridization to develop new cultivars with the desirable

traits including high yield, stress and disease resistances.

These methods have been faced a great challenge to de-

velop new cultivars and sustain food production for the

ever-growing human population. The adoption of new

technologies such as plant tissue culture and recombinant

DNA may achieve the goals. Since 1930s, plant tissue

culture has been progressing. Consequently, tissue cul-

ture techniques could be used for mass propagation [10],

and induction of somaclonal variation [11].

In vitro regeneration from cultivated tomato explants

on synthetic medium has been a subject of research be-

cause of the commercial value of the crop and its amena-

bility for further improvement via genetic manipulation.

Consequently, numerous studies on plant regeneration

from a wide range of tissues and organs of wild and cul-

tivated tomato germplasm were conducted for selection

of cell lines for biotic and abiotic stresses [7,12], trans-

formation [13], development of haploids [14], production

of somatic hybrids [15], and mass propagation [16].

In tomato, adventious shoot formation was obtained

Hormonal Requirements Trigger Different Organogenic Pathways on Tomato Nodal Explants 2119

directly [17], or indirectly through the callus formation

[18], and both the shoot and root could be obtained to-

gether [19]. Response of shoot formation on tomato ex-

plants higher in the order of leaves, cotyledons and hy-

pocotyls for the cell cultivars were proved [20]. Any part

of plant species (preferably young explants) was used to

induce specific type of organogenesis under the influence

of phytohormones [21]. In general, nodal explants were

rarely used as plant material for induction of different

organogenic pathway in tomato. Therefore, this study

was used to describe a simple protocol for induction of

different type of organogenesis on nodal tomato explants

which can be used to improve tomato via gene biotech-

nology.

2. Materials and Methods

2.1. Preparation of Plant Materials

Tomato seeds (Cassel rock) were surface-sterilized in 5%

commercial bleach solution for 10 min followed by 5

min treatment in 75% (v/v) ethanol. After three succes-

sive rinses in sterile distilled water for 5 min each, seeds

were placed on MS medium [22] supplemented with 3%

sucrose, without growth regulators, for seed germination.

The medium was solidified with 8 g/l agar at pH 5.8.

Vitamins (mgl-1) were: myo-inositol (100), vitamin

B1-hydrochloride (4), nicotinic acid (4), pyridoxal hy-

drochloride (0.7), biotin (0.04) and folic acid (0.5). Seeds

were germinated at 25˚C ± 2˚C with 16-h photoperoid. A

two-three cm section of seedling containing shoot apical

meristem was cut and transferred for further growth to

establish shoot culture. Subculture of these shoot cultures

was fulfilled on basal MS medium in 11 cm Petri dishes.

These shoot cultures were used as a source of tomato

nodal segments. The nodal-disk segments (4 - 5 mm ra-

dius × 4 - 6 mm long) were cut and cultured vertically or

horizontally on MS medium where the half of the explant

was immersed in the medium containing different hor-

monal treatments. In each treatment, fifteen explants

were placed horizontally on prepared medium in 11 cm

width Petri dishes. Three replicates of each treatment

were incubated at tissue culture rooms at 25˚C ± 2˚C.

After 6 weeks, the type of organogenesis and other pa-

rameters were determined.

2.2. Induction of Root Formation

To obtain roots, shoot cuttings or nodal explants (4 - 5

mm radius × 5 - 10 mm long) obtained from established

shoot culture were vertically cultured on MS medium

containing 1.5% sucrose and 1 mg/l of NAA, IAA or

IBA. Three replicates of each treatment were incubated,

each one containing 15 segments, at tissue culture rooms

at 25˚C ± 2˚C. After 6 weeks, root frequency, root num-

ber and the length of root system/plantlet were deter-

mined.

2.3. Induction of Compact Callus Formation

To obtain callus, nodal explants (4 - 5 mm radius × 5 - 10

mm long) obtained from established shoot culture were

placed vertically or horizontally on MS medium con-

taining 3% sucrose and different concentration of hor-

mones as indicated in Table 1. Three replicates of each

treatment were incubated at tissue culture rooms at 25˚C

± 2˚C. After 6 weeks, frequency and the weight of callus

were determined.

3. Results and Discussion

Tomato is a subject for research in many research insti-

tutes in Egypt aiming to improve its productivity to fulfill

the market needs and export. There are several species of

the genus Lycopersicon, and they represent an important

source of genes, conferring resistance to various diseases

and pests of cultivated L. esculentum [23]. They require

the delivery of appropriate genes into the plant cells and

are followed by regeneration of the transformed cells

[15,24]. Consequently, establishment of tissue culture

protocol, as has been described in this work, is essential

prerequisite for improvement of tomato plants.

The suitable size of the explants was easily obtained as

a nodal-disk segment from in vitro grown shoots. The

cultured shoots were grown on basal MS medium for 6

weeks; therefore they reached about 10 cm long with 4 -

6 mm in diameter. The obtained nodal segments were

like nodal disk and they were suitable to induce different

organogenic pathway under the influence of specific

hormonal treatment. The obtained explant size of the

nodal segment was suitable for shoot regeneration where

the very small structures such as individual cell, cell

clumps, and shoot tip meristems were in general consid-

ered much more difficult for the induction of organo-

genesis [25], but more shoots were formed from small

explants than from large ones [26,27].

Nodal explants were obtained easily and in large quan-

tity higher than other types of explants such as shoot tip,

cotyledons and hypocotyls. The influence of explant on

organogenesis depends on several factors, including the

Table 1. Effect of explant position on shoot regeneration.

Nodal segments were cultured on MS medium supple-

mented with 2 mg/l BAP and 0.5 mg/l NAA. Means ± stan-

dard deviation of three independent experiments, 30 ex-

plants were used for each one.

Treatment Shoot formation

frequency (%) Number os

shoots/explant

Vertical position 76 7 ± 1.5

Horizontal position 86 3.33 ± 0.57

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genotype, the age of explant, the size of explants, the

method of inoculation [28], the time of the year and the

environment where the source plant was grown [29].

Tomato shoot culture was used as donor plant materials

(nodal segment) where they existed under elite environ-

ment and could be obtained at any time through the year.

In general, the response of tomato explants from seed-

lings grown in vitro culture differed from those raised in

a greenhouse [30]. The established shoot culture, as was

used in this work, was preferable as plant materials for

induction of different forms of organogenesis as was

reported in other works [7,11,21,29].

The in vitro organogenic responses of cultured ex-

plants of tomato were affected by the type and the con-

centration of growth hormones (Table 2) as was de-

scribed in this work and others [1,28]. Response of shoot

regeneration from the explants higher in the order of

leaves, cotyledons and hypocotyls for the cell cultivars

were proved [31]. In this work, shoot organogenesis was

observed when nodal explant segments were cultured on

MS medium supplemented with various concentrations

of BAP (1.0 - 2.5 mg/l), but maximum shoot organo-

genesis was obtained when BAP was used in combina-

tion with NAA (2 mg/l BAP and 0.5 mg/l NAA). These

results indicated that nodal explant segments, as de-

scribed in this protocol, can be used as alternative to

other types of explants (cotyledon, hypocotyl and leaf

explants) in tomato without negative effect on the num-

ber of shoots when appropriate concentration of phyto-

hormones was used (Table 3). Combination between

BAP (2 mg/l) and NAA (1.5 mg/l) was previously re-

ported to induct shoot formation [32,33], and it was af-

fected by the type of explants [34].

Using tomato nodal segments, organogenesis pathway

was controlled using specific hormonal treatments. Ap-

plication of BAP (1.0 - 2.5 mg/l) alone or in combination

of 0.5 mg/l NAA resulted in induction of shoot formation

(Table 2, Figures 1-3). Somatic embryogenesis (Figures

3 and 4) rarely appeared (6%) when relatively low con-

centration of BAP (1.5 mg/l) and IAA (0.5 mg/l IAA)

was applied. Root induction was triggered when 1 mg/l

NAA was used but application of 0.5 mg/l NAA stimu-

lated callus formation (Table 2). Shoot formation can be

initiated with or without the appearance of callus growth.

Table 2. Effect of different growth regulators on organogenic pathways of tomato explants under the influence of growth

regulators. Means ± standard deviation of three independent experiments, 30 explants were used for each one.

BAP (mg/l) NAA (mg/l) Morphogenetic

frequency (%) Organogenesis typeNo. of shoots/explantNo. of roots/explant Fresh weight/explant (gm)

2.5 - 55 Buds 3.33 ± 0.57 - 2.07 ± 0.37

2.0 - 63 Buds 3.67 ± 0.57 - 2.30 ± 0.17

1.5 - 43 Buds 2.33 ± 0.57 - 1.57 ± 0.15

1.0 - 40 buds 1.67 ± 0.57 - 1.33 ± 0.15

- 1.0 82 Roots - 3.67 ± 0.57 1.53 ± 0.30

- 0.5 43 Callus - - 0.93 ± 0.15

2.5 0.5 80 Buds 5.33 ± 0.57 - 2.03 ± 0.40

2.0 0.5 90 Buds 8.00 ± 1.00 - 2.40 ± 0.10

-Buds

1.5 0.5 67

-Embryos

4.00 ± 1.00 - 1.47 ± 0.05

1.0 0.5 55 Buds 2.33 ± 0.57 - 1.27 ± 0.20

Table 3. Effect of explant type on shoot organogenesis on tomato nodal segments. Different segment types were cultured on

MS medium supplemented with 2 mg/l BAP and 0.5 mg/l NAA. Means ± standard deviation of three independent experi-

ments, 30 explants were used for each one.

Explant type Shoot formation frequency (%) Number of shoots/explant

Nodal segment 82 7 ± 1.0

Cotyledons 85 6 ± 1.5

Hypocotyl 56 5 ± 1.5

Leaf 64 3 ± 1.5

Hormonal Requirements Trigger Different Organogenic Pathways on Tomato Nodal Explants 2121

Figure 1. Shoot organogenesis on nodal explant segment cultured for 6 weeks on MS medium supplanted with 2 mg/l BAP.

Figure 2. Shoot formed on MS medium supplemented with 2 mg/l BAP and 0.5 mg/l NAA after 8 wee ks culture .

Figure 3. Somatic embryo (arrow ) and shoots initiated on nodal explant cultured for 8 weeks on MS medium supplemented

with 1.5 mg/l BAP and 0.5 mg/l NAA.

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Figure 4. Pro-embryo structure on nodal explant cultured for 6 weeks on MS medium supplemented with 1.5 mg/l BAP and

0.5 mg/l NAA.

Figure 5. Root organogenesis on nodal explant segment cultured for 8 weeks on MS medium supplemented with 1 mg/l IAA.

Application of BAP alone stimulated direct shoot or-

ganogenesis (Figure 1) but application of BAP (2 mg/l)

with NAA (0.5 mg/l) resulted in induction of shoot for-

mation mediated with the appearance of callus (Figures

2 and 3). In tomato, adventitious shoot regeneration can

be achieved either directly [17] or indirectly through an

intermediate callus phase [18]. The application of phy-

tohormones to trigger specific molecular processes on the

explant tissues resulting specific organogenesis pathway

was previously reported [21]. As was observed in this

work (Table 2), variations in quantity and type of PGRs

influence both the percentage of explants responding,

and the number of shoots/explant. These differences may

be governed by both cytoplasmic and nuclear genes, as

illustrated in the reciprocal hybrids developed by [35].

Nodal explants can be used for application of tissue cul-

ture in the fields of tomato improvements and molecular

studies [12,36-39].

Nodal segments of the used tomato cultivar were in-

oculated on the culture media in polar (straight up, with

the physiological base in the medium). The polar orienta-

tion resulted in shoot formation higher than horizontall

orientation (Table 1), where the chemical and physical

conditions of the polar culture were more easily than

non-polar orientation. In this work, progressive enlarge-

ment of the explants was proceeded with the shoot for-

mation (Figure 1), but root formation was obtained with-

out enlargement (Figure 5). In another work, more shoots

Hormonal Requirements Trigger Different Organogenic Pathways on Tomato Nodal Explants 2123

Table 4. Effect of hormone type on callus induction from node segments of tomato shoots. Means ± standard deviation of

three independent experiments, 30 explants were used for each one.

2,4-D (0.5 mg/l) NAA (1 mg/l) BAP (0.5 mg/l) Frequency (%) Weight/explants (gm)

+ − − 55 2.49 ± 0.14

+ + − 63 2.11 ± 0.17

+ + + 100 2.69 ± 0.11

Table 5. Effect of MS medium supplemented with 1 mg/l auxin (IAA, IBA, or NAA) on root induction from 2.5 cm length

shoot segments of tomato plantlets after four weeks culture. Means ± standard deviation of three independent experiments,

30 explants were used for each one.

Type of auxin Frequency (%) No. of roots/plantlets Root length/plantlets

Without auxin 76 4.33 ± 1.15 5.07 ± 0.90

IAA 90 11.00 ± 1.00 6.97 ± 0.87

IBA 86 6.00 ± 1.00 3.17 ± 0.28

NAA 80 4.00 ± 1.00 2.17 ± 0.76

were produced from leaf and cotyledon explants placed

horizontally than from the ones placed vertically, and

hypocotyls explants placed horizontally produce more

shoots than those placed vertically straight or upside

down [20].

Nodal segments cultured on MS medium supple-

mented with 1 mg/l of 2,4-D alone or in combination of

NAA with or without BAP resulted in callus formation

on the nodal explants irrespective the position of the ex-

plants on the medium surface, the best results were ob-

tained when the three different phytohormoes were used

(Table 4).

Root formation on nodal explants or shoot cuttings

were obtained on MS medium with or without auxins,

but the number and the length of root systems were in-

fluenced by the type of auxin, the best result was ob-

tained when 1 mg/l IAA was used (Table 5 and Figure

5). These rooting plantlets were transferred to soil after

adaptation under plastic bags for three weeks under green

house condition. Root formation was detected when

nodal explants were vertically placed on MS medium

without phytohormones, which gave an indication about

the importance of indigenous auxins in the induction of

root formation. Many researchers have speculated that

tomato has a high level of endogenous auxin, based on

the observations of shoot cultures producing roots with-

out the addition of auxins in the medium [1].

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