Introduction: plant regeneration protocol via adventitious shoot regeneration

Introduction:

Dianthus caryophyllus L.
Is the most important cut flower plant. This plant is one of the valuable
commercial cut flowers around the world. Due to its excellent storage quality, wide range of forms, ability
to withstand longdistance transportation, and remarkable ability to rehydrate
after continuous shipping, it is preferred by growers to roses and
chrysanthemums in several fl ower-exporting countries. Invitro micropropagation
technique is widely used in ornamental crops. The global cut flower market by
the introduction of new improved cultivars is maintaining. Standard carnation generally
propagating through stem cuttings. However, this method is slow and rely on collecting
and cutting mother plants. To strengthen the conservation, development,
and utilization of this plant resource, it is important to establish an
efficient in vitro micropropagation and plant regeneration protocol. The
genetic variability within carnation is relatively poor; therefore, the
breeding potential for new flower colors and patterns as well as resistance to
biotic and abiotic stresses is also very limited. Carnation is a vegetatively
propagated plant, which further reduces its genetic pool availability. The
modern cell and molecular techniques could be regarded as an alternative and an
additional complementation tool to the classical improvement methods of
carnation. A few articles for tissue culture and plant
regeneration protocol via adventitious shoot regeneration exists for carnation,
but because of variation amoung the cultivars of this plant, set and optimize
for all of varaieties is nessessory. In this study, we established efficient protocols
for high-frequency regeneration via two pathways direct and indirect shoot
organogenesis from some leaf explants carnation cultivars. Although callus-mediated shoot formation is not only a useful method
for plant propagation but also a powerful tool for plant genetic improvement
(such as in vitro mutagenesis and gene transformation), germplasm conservation, and production of useful compounds.
We also investigated the effects of different types and concentrations of plant
growth regulators and media tightness for Overcoming to Hyperhydricity shoot produced.

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 Material and methods:

Location
and plant materials:

Young leaf explants were
taken from six species of carnation cultivars. We cutting the leaves into 0.5 to
0.7 cm2 explants and inoculated adaxial-side down on MS basal medium (Murashige
and Skoog 1962). The pH of MS basal medium, which contained 30 g L?1 sucrose,
was adjusted to 5.8 before being solidified with 0.7% (w/v) agar .We used from sterile
Motherhood plant that they established at the MS media  Containing PGR (Kin 1 mg/l and Naa .1 mg/l). Leaf
explant planted in 80 mm petri and after callus induction transfer to Culture
jars (10 cm high and 6 cm in diameter) were placed in an air-conditioned
culture room at 25 ± 2°C with a 16-h photoperiod under 100-?M m?2 s?1 fluorescent light. These culture conditions
were identical for all experiments.

Callus
induction and indirect plant regeneration 

Leaf segments adaxial-side
down on MS medium supplemented with different concentration of 2.4,D (0, 0.2,
0.5, 1, 2, 3 mg/L) , NAA (0, 0.2, 0.5, 1, 2 mg/L)  and BA (0, 0.5, 1 mg) in factorial combination
were inoculated. After 3 weeks, callus driven from leaf explant transferred to
MS media supplemented with different concentration of BA (1, 2, 3 mg/L), NAA
(0, 0.2, 0.5, 1 mg/L) and adenine sulfate (0, 20, 40 and 60 mg/l). Among the
PGRs tested, GA3 was filter-sterilized through a 0.24-?m filter and added after autoclaved
medium had cooled while 2,4-D, BA, indole-3-butyric acid (IBA), and NAA were added
prior to autoclaving at 121°C for 15 min.

Direct adventitious
shoots regeneration from leaf explants:

For direct regeneration,
we used different combination of Kin, BA, NAA, adenine sulfate, casein
hydrolysate and AgNo3.

Hyperhydricity
overcoming and Shoot proliferation:

 

Root
formation and acclimatization:

Shoots with 2–3 leaves
that had reached 2–3 cm in height on MS medium supplemented with 2.0 ?MBA and
0.5 ?MNAAwere isolated from multiple shoot clusters, cut off at the base, and
ransferred to half-strength MS medium supplemented with 0.5 ?M IBA, 0.5 ?M NAA, 0.5 ?M IBA, and 0.5 ?M NAA or
no PGRs. After culture for a total of 30 days on these media, root
formation was investigated (Table 4). A total of 270 rooted plantlets with 4–5
leaves and 3–4 cm tall were removed from jars representing the above four
treatments. The agar was gently washed off roots in tap water. Plantlets were
transplanted to square plastic trays (50 cm×40 cm× 10 cm) containing three
substrates: sand, loessal clay, and sand: loessal clay: vermiculite (1:1:1,
v/v). A total of 90 plantlets were planted per

Data
collection and Statistical analyses:

Each treatment contained
seven explants per culture jar and tree jars per treatment. The data were reported
as mean±SD (standard deviation). Percentage values were arcsine transformed
prior to analysis. Means were statistically analyzed by one-way analysis of
variance (ANOVA), and treatment means were considered to be significantly
different from controls by Duncan’s multiple range test at P? 0.05 using SPSS
v. 19.0 (IBM, New York, NY).