Sharma et al.

eluted amplified DNA products of two selected

on germination medium i.e. half strength MS basal

Pseudomonas sp. (An-1-kul, An-13-kul) was first

medium. Aseptically grown 10-12 days old seedlings

purified followed by sequencing in Bioserve Private

of cabbage inoculated on shoot regeneration medium

limited (Hyderabad, India). Similarity searches of

and in vitro regenerated shoots were used as a source

the GenBank database were performed with BLAST.

of explants for root regeneration.

Tissue culture bioassay for detection of plant

Result: All isolates were found to be Gram negative

growth regulators produced by Pseudomonas sp.

rods, non-spore forming, and fluorescent with

on plant tissue culture

transparent to translucent and gave greenish,

brownish, grayish and yellowish pigmentation on

Effect of partially purified plant growth regulators

King’s B medium plates. These strains were also

viz., auxins and cytokinins was studied in vitro by

found to be positive for both catalase and oxidase

plant tissue culture technique (Rashid, R. and Bal,

test. They all were found to be positive for gelatin

S.S. 2010). In this case MS medium supplement with

liquification, oxidative metabolism, denitrification

100mg/L meso-ionositol, 3% sucrose and 0.8% agar

tests and showed growth at 25° C, 37° C, 41° C by

was used as basal medium (Murasshige and Skoog,

plate method but none of the isolates showed growth

1962). Different concentrations of partially purified

at 4° C (Table 1).

cytokinins (1.0mg –1 , 1.5mg –1 and 2.0mg –1 ) and auxins

(0.1mg –1 , 0.15mg –1 and 0.2mg –1 ) from An-1-kul and

Production of plant growth regulators viz ., auxins,

An-13-kul were used in MS basal medium.

gibberellins and cytokinins

Shoot regeneration from cotyledon explants of

The production of auxins like substances by all the

broccoli ( Brassica oleracea var. capitata )

thirty strains of Pseudomonas sp. ranges from 1.83

to 21μg/ml. The maximum production of auxins

Cotyledon explants were excised from 10-12 old days

was shown by Pseudomonas isolates in the range of

aseptically grown seedlings. The edges of cotyledon

(14.72-21.00μg/ml). In supernatants production of

explants were cut and their surface was gently

gibberellins like substances showed the production

tapped with sterile scalpel blade to injure them.

in the range of 116.1-485.8μg/ml. The maximum

These explants were cultured in flasks containing

gibberellins production was shown by Pseudomonas

MS full strength basal medium supplemented with

isolates An-1-kul (485.8μg/ml) followed by Pn-1-kul,

different concentrations (i.e. 1mg –1 , 1.5ml –1 , 2mg –1

Pn-3-kul (419.2μg/ml). All isolates of Pseudomonas

of partially purified cytokinins from An-1-kul and

species were found to produce cytokinins in the

An-13-kul at 25 ± 2° C incubation period and 35

range of 45.4-295.4μg/ml. The maximum production

μmolmeter -2 sec -1 photosynthetic photon of lux (PPF)

of cytokinins shown by Pseudomonas isolates Pn-1-

white fluorescence light emitted by Philips bulb.

kul (295.4μg/ml) followed by Ar-3-kul (227.2 μg/ml)

Observations were taken at the interval of every 7

and Pn-3-kul (249.9 μg/ml) (Table 2).

days. Explants of broccoli were evaluated for callus

formation; percent shoot regeneration and average

Molecular characterization

number of shoots per explants.

Using the universal primer set, DNA fragment of

Cabbage ( Brassica oleracea var. capitata ) root

the 16S rDNA gene was amplified by PCR. The PCR

regeneration in in vitro developed shoots

amplified 16S rDNA region was sequenced and

sequence data were analyzed by BLAST and the

Seedsofcabbageweresoakedinwaterforhalfanhour

nearest match from the GenBank data was reported.

and then surface sterilized for one minute in 0.1%

DNA sequencing and phylogenetic analysis

HgCl ₂ solution. After that seeds were rinsed three

revealed that strain An-1-kul and An-13-kul was

to four time in sterile distilled water and cultured

shown to have a 98% similarity with Pseudomonas

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