Cotton is an economically important crop that is grown throughout the world.
It is grown as a source of fibre, food and feed Lint, the most economically
important product from the cotton plant, provides a source of high quality fibre
of the textile industry. Cotton seed is an important source of oil and cotton
seed meal is a high protein product used as livestock feed (Keshamma
et al., 2008). Although, great progress has been made in the field
of improvement of cotton with conventional breeding methodology, it is time-consuming
and commercialization of new cotton varieties often takes 6 to 10 years. Compatibility
limitations narrow the gene pool available for this process.
A number of these shortcomings may be overcome by plant biotechnology. For
example, control can be exerted over selection of the gene(s) and its expression.
The gene pool can be expanded to all living organisms (plants, animals, bacteria
and fungi). Researcher notes that as technology is refined, custom-made synthetic
genes will become another source for desired traits (Zhang
et al., 2000). Thus, cotton biotechnology can be significantly applied
for the improvement of cotton. Although, plant biotechnology is an attractive
means for improving cotton, its use requires an effective regeneration system
from somatic tissues of cotton plants (Zhang et al.,
One of the major problems for many tissue culture system is browning and subsequent
death of the cultured explants that usually depended on the phenolic compounds
and the quantity of total phenols Ozyigit (2008).
Phenolic compounds occur as secondary metabolites in all plant species and
they are generally characterized by a benzene ring and one hydroxyl group (Antolovich
et al., 2000; Kefeli et al., 2003).
They are also extremely diverse compounds, for example carnosol and rosmanol,
which are diterpenes were identified in herbs and spices while the main phenolics
are isoflavone glycosides and several phenolic acids like ferulic, caffeic and
chlorogenic acids which are present in soybean (Robards et
al., 1999). Plant phenols are classified into major groupings distinguished
by the number of constitutive carbon atoms in conjunction with the structure
of basic phenolic skeleton (Robards et al., 1999;
Antolovich et al., 2000). Shikimate is the starting
product for the biosynthesis of most phenolic compounds. They are also acidic
substances, due to the dissociability of their -OH groups. Many phenols are
rather reactive compounds and as long as no steric inhibition due to additional
side chains occurs, they form hydrogen bonds. The composition and synthesis
of phenolics in plant tissues may determine by genetic and environmental conditions
like oxidative reactions during culturing, processing and storage (Lux-Endrich
et al., 2000). It seems that there is a relation between chemical
compound of media and phenolic exudation, media discoloration, rooting deficiencies
and explant browning and death. For example plant phenolics are modulators of
Indole Acetic Acid (IAA) catabolism. Some monophenols like synaptic acid and
ferulic acid, at low concentrations, inhibit enzymatic oxidation of IAA and
this results in cell elongation and cell division and subsequent plant growth
and development (Volpert et al., 1995; Arnaldos
et al., 2001). It was noticed that plant phenolics increase the
rigidity of plant cell walls acting as molecular bridges between cell wall components
In this study effect of plant growth regulators for organ formation and phenolic compound in cotton tissue culture media were examined.
MATERIALS AND METHODS
This study conducted in Iranian Research Institute of Plant Protection from Aug. 2008 to Sep. 2009
Seed Germination and Culture of Aseptic Seedlings
Mature seeds of cotton (Gossypium hirsutum L.) were surface sterilized
by commercial bleach, ethanol 70%, flame of an alcohol burner for a moment,
sterile distilled water, tap water, H2O2 and sterile filter
papers. The surface sterilized seeds were germinated on MS Murashige
and Skoog (1962) medium. Medium supplemented with 1% agar and 3% sucrose
for germination at 25±2°C under 24 h photoperiod conditions with
the light intensity of approximately 2000 Lux.
Induction and Proliferation of Callus and Organogenesis
Hypocotyl sections (6-9 mm), cotyledon pieces (10-16 mm 2) area,
root segments (4-6 mm) and meristematic shoot tips (1-2 mm) of 7 days old sterile
seedlings were placed on MS medium supplemented with various concentrations
of Plant Growth Regulators (PGRs) (BA, IBA, 2,4-D and KIN) for the induction
of callus and organogenesis. Cultures were maintained in 24 h photoperiod conditions
with the light intensity of approximately 2000 Lux at 25±2°C. After
establishment, cultures were subcultured at 5-6 weeks intervals on fresh media
and 2 months later the means number of browned and died cultured explants in
different media were counted.
Experimental Design, Data Collection and Analysis
Experiments were set up in Completely Randomized Design and repeated four
times. Each treatment has 16 replications. Observation on the browned or died
explants and the Number of explants with callus or organ were recorded. Data
were subjected to SD and ANOVA test.
Different seeds and concentrations of disinfecting material were used in this experiments for establishment of sterile seeds cotton. Percentage of sterilization and germination of seeds varied in different manners. These variation are shown in Table 1. In Ex5 that commercial sodium hypochlorite 30% were used for 30 min cotton seeds were establishment in the best way.
In this study various explants and media for induction and growth of calli, organogenesis, direct regeneration response and effect of PGRs compound and amounts of phenolic compound were investigated. Present studies showed that either various explants or different media are very effective (Table 2). All of root explants on all of media browned and died due to excretion and oxidation of phenolic compound.
|| Sterilization and germination of cotton cv varamin seeds
with various disinfecting materials and manners
||Callus induction and organogenesis of cotton Gossypium
hirsutum L. variety varamin from different explants and different concentrations
of PGRs (μM L-1)
|Data represent means of 16 replicates. There is significant
difference between treatment and control at p<0.01
Cotyledon explants on media with 0.492 μM IBA+0.929 μM KIN, 0.984 μM IBA+0.464 μM KIN, 4.92 μM IBA+9.29 μM KIN, 9.84 μM IBA+4.64 μM KIN, did not synthesize phenolic compound. In this media cotyledon explants at first growth and increased their diameters then in medium with 0.492 μM IBA + 0.928 μM KIN induced callus. Growth of calli in this conditions was very slow (Fig. 1).
Response of hypocotyl in different media was very various. This variation related to PGRs content of media. In media with KIN and IBA callus was induced and growth. These calli were two kinds. Organoge and nonorganogen. In organogene calli when diameter of calli increased to 5-6 mm induced roots. These roots was white, either length or thickness of them growth very well and browned in media (Fig. 2). Some of calli in these media only growth and browned severity (Fig. 3). Also in these media regenerated roots and regenerated leaves were induced on one hypocotyl explants (Fig. 4).
|| Callusing on cotyledon explants in 0.492μM IBA and 0.929
|| Callusing and rooting on hypocotyl explants in 0.492 μM
IBA and 0.929 μM KIN
|| Callusing on hypocotyl explants in 9.84 μM IBA and 4.64
|| Regenerations of leaves on hypocotyl explants in 0.984 μM
IBA and 0.464 μM KIN
||Callusing on hypocotyl explants in 27.12 μM 2, 4-D and
8.87 μM BA
||Regeneration of four week old plantlets on meristematic shoot
tip in 0.492 μM IBA and 0.929 μM KIN
In medium with 27.12 μM 2, 4-D and 2 8.87 μM BA severity of phenolic compound excretion was very slow and nearly was zero (Fig. 5). On hypocotyl explants in this medium calli induced and growth very well (Fig. 5).
As could been seen in Fig. 3 and 5 these calli that induced on hypocotyl explants had two important differences due to explant polarity for inducing of calli (polar and bipolar).
Meristematic shoot tips in media induced regenerated plants without excretion of phenolic compound abundantly. In Fig. 6 four week old regenerated plantlets on MS medium supplemented with 0.492 μM IBA and 0.929 μM KIN were seen.
There are many methods for surface sterilization of cotton seed (Zhang
et al., 2001), Ozyigit (2008), Ikram-ul-Haq
(2005), (Zhang et al., 2003). All of these
methods were tested in this experiments but use of commercial bleach (30%) for
30 min were suitable for Iranian cotton seed, it is clear that natural conditions
and normal floral of Iranian field are effective.
In this study different explants and media were used and were confirmed that
amount of phenolic compound and their inhibitor effectives could be varied.
Volpert et al. (1995) and Arnaldos
et al. (2001) declared that plant phenolics are modulators of Indole
Acetic Acid (IAA) catabolism. Some monophenols like synaptic acid and ferulic
acid, at low concentrations, inhibit enzymatic oxidation of IAA and this results
in cell elongation and cell division and subsequent plant growth and development.
As it is known, phenolics are synthesized in leaves and then carried to other
tissues and organs. Therefore, amounts of total phenolic compounds in leaves
are more than the other tissues and organs of the plants (Ozyigit,
2008). But in this study phenolic compounds are synthesized in roots was
more than to other explants, also phenolic compounds were synthesized in leaves
were less to other explants.
As was noticed above composition of media influenced on total phenolic compounds and excretion of them into media also. Hypocotyl explants in medium with 27.12 μM 2,4-D and 8.8 μM BA had the least production of phenolic compound. These explants in medium 0.492 μM IBA and 0.929 μM KIN browned severely (Fig. 3). Therefore, we can decrease inhibitor effectives of phenolic compound in cotton tissue culture with election of suitable explants and suitable media.
Meristematic shoot tips in MS medium plus 0.492 IBA μM and 0.929 μM
KIN regenerated whole plantlets. On the other hand, these explants had very
little phenolic compounds. This resulted in research of Ozyigit
(2008) with 4.64 μM KIN.
In medium supplemented by 27.12 μM 2,4-D and 8.87 μM BA hypocotyl
explants induced and growth of calli at the highest level, whereas the calli
induced and growth very well in 0.57 μM IAA, 0.452 μM 2,4-D and 0.464
μM KIN (Zhang et al., 2003).
Zhang et al. (2001) stated that for callus inducing
in cotton the best explants are hypocotyls. Also, in this study hypocotyls explants
induced calli with powerful growth.
As were explained earlier meristematic shoot tips in MS medium plus 0.492 μM IBA and 0.929 μM KIN regenerated whole plantlets. Also expressed that meristematic shoot tips had low level of phenolic compound in media.
Based on our results the most important factors for reduction of phenolic compound
in tissue culture media are composition of media and type of explants, whereas
there are researchers that believed another factors such as age of explants
and genotype are the most effective factors for total amount of phenolic compound
in media (Ozyigit et al., 2007; Gupta
et al., 2000; Kumria et al., 2003;
Lorenzo and Angeles, 2001).
In this research we report that could been overcome phenolic compound excretion
in tissue culture media simply by variation of explants and PGRs for callusing
and organogenesis without to need addition of external material such as activated
charcoal Wang and Huang (2009).