World J Microbiol Biotechnol (2012) 28:1459–1466 DOI 10.1007/s11274-011-0947-4

ORIGINAL PAPER

Comparative assessment of the efficacy of bacterial and cyanobacterial phytohormones in plant tissue culture Anwar Hussain • Shahida Hasnain

Received: 26 August 2011 / Accepted: 4 November 2011 / Published online: 13 November 2011 Ó Springer Science+Business Media B.V. 2011

Abstract Efficient callus and explant regeneration medium, using microbial extract (SPE purified) or supernatant has been formulated for Brassica oleracea L. var. capitata. Two cyanobacterial strains (Anabaena sp. Ck1 and Chroococcidiopsis sp. Ck4) and two bacterial strains, (Pseudomonas spp. Am3 and Am4) known to produce a number of cytokinins, tZ, cZ, ZR, DHZR and IAA were selected for the media formulation. Supernatant from strains with high cytokinin to IAA ratio, including Pseudomonas aeruginosa Am3 (2.08) and Chroococcidiopsis sp. Ck4 (0.8) efficiently induced compact calli which were turned green upon exposure to light. The strains producing lower cytokinins to IAA ratio (0.11–0.13) on the other hand induced friable callus which were unable to regenerate on the selected media combinations. Leaf, stem and root explants of Brassica oleracea L. regenerated on MS medium supplemented with phytohormones from microbial origin with efficiency comparable to standard cytokinins and IAA. Supplements from cyanobacterial origin proved to be the best for induction of adventitious roots and shoots on internodal and petiolar segments. Hypocotyl explants however, responded well on MS supplemented with bacterial metabolites. Induction of adventitious shoots on root explants was supported by phytohormones from both origin equally well. Callus induction on the seeds and regeneration of shoots on calli was also

A. Hussain (&) Department of Botany, University College of Science, Abdul Wali Khan University, Shankar Campus, Mardan 23200, Pakistan e-mail: [email protected] S. Hasnain Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan e-mail: [email protected]

observed. Cyanobacteria based media were more efficient to induce calli capable of regeneration upon exposure to light. Internodal explants were highly amenable to regenerate shoot and roots simultaneously. Root explants were the less successful to regenerate shoots. Keywords B. oleracea L.
Cytokinins
IAA
Chroococcidiopsis
Anabaena
P. aeruginosa

Introduction Efficient in vitro regeneration of plants depends upon reliable tissue culture techniques, which can bring about successful application of biotechnology in crop improvement (Ozgen et al. 1998). White Cabbage (Brassica oleracea L. var. capitata) has received great attention as a crop plant forits enormous nutritional value and a potential role in combating cancer (Vermeulen et al. 2006; You et al. 2008). Previous studies have reported that the plant is amenable to tissue culture techniques (Nugent et al. 2006; Ali and Hasnain 2007). Shoot regeneration from hypocotyl and cotyledon explants of Brassica oleracea has been achieved by Qin et al. (2007). Ferreres et al. (2009) reported that in vitro shoot production of B. oleracea is important in obtaining dietary source of compounds with health protective potential. Two classes of phytohormones i.e., cytokinins (CKs) and auxins are very important for in vitro regeneration of plants. CKs constitute a group of mobile phytohormones having a central role in plant growth and development (Kudo et al. 2010). The role of different CKs (zeatin, isopentenyladenine, kinetin, benzyladenine and benzylaminopurine) has been demonstrated in plant tissue culture (Guo et al. 2005; Nikolic et al. 2006). CKs alone as well as

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in combination with auxins induce shoot in cotyledon and hypocotyl explants (Guo et al. 2005). CK production is wide spread in different bacterial genera including Pseudomonas, Azospirillum, Azotobacter, Bacillus and Rhizobium (Garcia de Salamone et al. 2001; Tsavkelova et al. 2006). Bacterial auxin has been demonstrated to induce adventitious shoots in Brassica oleracea internodal explants (Ali and Hasnain 2007). Cyanobacterial extracellular products have also been used for in vitro growth of plants (Rodrı´guez et al. 2006). Extracellular products from cyanobacteria efficiently induced root formation in the endosperm explants of rice, showing auxin-like activity (Manickavelu et al. 2006). Similarly cyanobacterial biomass extract has been reported to promote somatic embryogenesis (Wake et al. 1992).The purpose of the present study was to investigate the impact of phytohormones from bacterial and cyanobacterial origin in Brassica oleracea L. in vitro culture and develop an efficient callus as well as explant regeneration medium based on microbial hormones.

World J Microbiol Biotechnol (2012) 28:1459–1466

Cyanobacteria

Biomass 200 mg homogenized Bieleski buffer/80% aqueous methanol

Bacteria

Supernatant 250 ml concentrate; adjust pH 7 extract 3x nbutanol/Bieleski buffer

Supernatant 250 ml concentrate; adjust pH 2.5 extract 3x ethyl acetate

Reconstitute water; adjust to pH 3

Elute 3 x 2 ml methanol / 5% NH3

MCX/SCX column

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Wash a) 2 ml 0.1 N HCL in water b) 2 ml methanol

Cytokinin/IAA

Fig. 1 Extraction and purification of phytohormones (cytokinins and IAA) from bacterial and cyanobacterial source

Materials and methods

Culture conditions

Microbial strains

Murashige and Skoog (MS) medium containing 3% glucose, 5.0 lM thiamine-HCl and 30 lM nicotinic acid was used as basal medium. Five milliliters of MS medium, supplemented with different combinations of phytohormones, bacterial supernatant (BS), bacterial extract (BE), cyanobacterial supernatant (CS), cyanobacterial extract (CE) and L-broth or BG11 (LB/BG11), was dispensed into each test tube (2.5 9 15 cm). Filter-sterilized BS and CS were supplemented in 100, 150, 200 and 250 ll aliquots. Different medium combinations used with BS and CS are depicted in Table 1. Different concentrations of CKs and IAA (0.001–1 lg ml-1) were tried to finally select the optimum concentration for tissue culture. The optimized concentration of CKs and IAA was adjusted in the microbial extracts (CE and BE) by properly diluting the extracts reconstituted in sterile distilled water. BS, BE, CS, CE and LB/BG11 were added to autoclaved MS medium after filter sterilization.

Microbial strains used in this study were previously isolated and characterized (Hussain et al. 2010; Hussain and Hasnain 2011).Two bacterial strains, namely Pseudomonas aeruginosa Am3 and Am4, were selected for cytokinins/ IAA ratio in their culture. Cyanobacterial strains selected for the study were Anabaena sp. Ck1 and Chroococcidiopsis sp. Ck4. Extraction and quantification of phytohormones Cyanobacterial cultures were grown in 250 ml BG11 medium at 25°C under continuous light (18 lmol photons m-2s-1) for 2 weeks (inoculum density adjusted to 0.1 lg ml-1chl-a). Biomass was separated from culture medium by centrifugation at 4°C and 11,000 rev min-1 for 10 min. The supernatant was collected in a fresh flask and filtered through a cellulose acetate filter of 22 lm pore size to obtain cell/filament-free culture filtrate. Bacterial strains were grown in M9 medium supplemented with 20% glucose, 0.2% casaminoacid and 2 pg l-1 biotin for 72 h at 28°C. 100 ml culture from each strain was centrifuged at 16,000 rev min-1 for 10 min at 4°C to yield supernatant which was then filtered to obtain a cell-free culture filtrate. Extraction and purification of CKs and IAA from cell-free microbial cultures was done according to the scheme given in Fig. 1. Cytokinins and IAA in the extract were determined using UPLC-ESI–MS/MS, as described by Hussain et al. (2010).

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Plant material Seeds of Brassica oleracea L. were surface sterilized with 0.1% HgCl2 for 2 min and rinsed five times with autoclaved distilled water. For calli induction seeds were inoculated in the dark on all the 7 combinations of MS medium (Table 1) as mentioned. Seeds were grown on MS supplemented with sucrose, kinetin and IAA for 2–4 weeks to produce seedlings, which were then used as a source of explants. Two-cm intermodal, hypocotyl, petiolar or root sections of the seedlings were taken aseptically and

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Table 1 Medium combinations used for in vitro growth of Brassica olaraceae var. capitata Media combination

Components

Media combination

Components

C1

MS ? BS/CS (100 ll)

C3

MS ? BE (50 ll) ? IAA

C2

MS ? BS/CS (200 ll)

MS ? BE (100 ll) ? IAA

MS ? BS/CS (300 ll)

MS ? BE (150 ll) ? IAA

MS ? BE/CE (50 ll)

C4

MS ? Kinetin

MS ? BE/CE (100 ll)

C5

MS ? kinetin ? IAA

MS ? BE/CE (150 ll)

C6

MS

C7

MS ? LB

BS Bacterial supernatant, CS cyanobacterial supernatant, BE bacterial extract, CE cyanobacterial extract (both bacterial and cyanobacterial extracts contain cytokinins, purified by SPE and reconstituted in sterile distilled water); LB, Extract from blank LB

incubated on our seven media formulation under photoperiod of 8 h light and 16 h darkness. Cultures were observed daily for organogenesis. All the experiments were performed in Versatile Environmental Test Chamber (Sanyo MRL-350H) at 25 ± 1°C and exposed to a 16-h photoperiod provided by cool white fluorescent lamps (50–55 lmol m-2s-1) and humidity was maintained at 80% level.

Results Phytohormones in microbial cultures Four different CKs species (cZ, tZ, ZR and DHZR) were determined in microbial cultures. Major CK was ZR in 96-h-old culture of Am3 and Am4 (Table 2). In contrast to bacterial cultures, DHZR was the most abundant CK in Anabaena CK1 and Chroococcidiopsis Ck4 incubated for 2 weeks. Bacterial cultures produced greater amount of phytohormones than cyanobacterial cultures (Table 2).

IAA was also secreted in microbial cultures (Table 2). In contrast to the high CKs/IAA ratio in cultures of Chroococcidiopsis Ck4 and P. aeruginosa Am3, low CKs/IAA ratio was observed in cultures of Anabaena CK1 and P. aeruginosa Am4. Callus induction and maintenance Four strains including two strains of cyanobacteria (Anabaena sp. Ck1 and Chroococcidiopsis sp. Ck4) and bacteria (P. aeruginosa Am3 and P. aeruginosa Am4) each were selected for in vitro study on the basis of the phytohormone (CKs/IAA) ratio in their culture media. The cytokinin/IAA ratios in cultures of P. aeruginosa Am3, P. aeruginosa Am4, Anabaena sp. Sk1 and Chroococcidiopsis sp. Ck4 were 2.08, 0.13, 0.11 and 0.8, respectively. Seeds inoculated on MS medium alone (control) or supplemented with different combinations of phytohormones, microbial extract and supernatant, germinated within 3–4 days in the dark. The optimum concentrations of kinetin and IAA for callus induction in the MS medium was 0.1 lg ml-1 and 0.5 lg ml-1, respectively (low CKs to IAA ratio). Callus induction were observed in seeds incubated on MS supplemented with 100, 150, 200 and 250 ll supernatant from all four strains. However, the optimum concentration was 200 ll. Only the optimum concentration of supernatant for callus induction is discussed. Off-white calli were induced in seeds inoculated on C1 (MS ? BS), C3 (MS ? BE ? IAA), and C5 (MS ? kinetin ? IAA) within 2 weeks after germination while C2 (MS ? BE), C4 (MS ? kinetin), C6 (MS) and C7 (MS ? LB) did not support callus induction. Calli formed on MS supplemented with supernatant from P. aeruginosa Am3 and Ck4 were compact while supplements from P. aeruginosa Am4 induced friable calli (Fig. 2). However, in the case of Anabaena sp. Ck1, medium combination C1 failed to induce callus in the seeds of cabbage (Table 3). Maximum callus induction by C1 was 70% in case of Am4 followed by Am3 and Ck4 where 48 and 45% seeds formed calli, respectively. C3 supported callus induction from all the selected strains. Callus induction was 74% on C3-Am3 (C3 formulation containing extract from Am3) as well as C3-Ck4 (C3 formulation

Table 2 Phytohormones (CKs and IAA) determined in microbial cultures incubated for 96 h (bacterial) and 2 weeks (cyanobacterial) by UPLCESI–MS/MS Strains

cZ (ng ml-1)

tZ (ng ml-1)

ZR (ng ml-1)

Anabaena Ck1 Chroococcidiopsis Ck4 P. aeruginosa Am3 P. aeruginosa Am4

DHZR (ng ml-1)

IAA (ng ml-1)

–

3.94 ± 0.1

4.48 ± 0.3

4.73 ± 0.2

6.52 ± 0.4

12.11 ± 0.2

199.95 ± 2.6

4.03 ± 0.5

9.20 ± 0.4

67.2 ± 1.9 36.1 ± 0.8

115.2 ± 2.5 101.1 ± 2.1

25.8 ± 0.6

162.5 ± 3.1 146.6 ± 2.6

73.3 ± 1.1 21.2 ± 0.7

200 ± 4.3 2280 ± 23.5

Values are means of three replicates ± SE of means

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Fig. 2 Different stages of callus induction and de novo shoot induction in B. olaraceae L. seeds cultured on C3-Am3and C3-Ck4; compact offwhite after 1 week of incubation (stage I), green patches (visible as

black patches) induction upon exposure to light (stage II), De novo shoot bud induction and shoot emergence (stage III) and shoot elongation in 1 month old cultures (stage IV). (Color figure online)

Table 3 Callus induction in seeds of B. oleraceae var. capitata on MS media supplemented with bacterial/cyanobacterial metabolites and standard phytohormones Strain

Medium

Designation

Callus induction

Callus differentiation Calli differentiated

Am3

Am4

Ck1

Ck4

Cont

Shoots per callus

MS ? BS

C1

13.6 ± 0.05

15.4 ± 0.32

3 ± 0.01

MS ? BE

C2

–

–

–

MS ? BE ? IAA MS ? BS

C3 C1

22.3 ± 1 20.9 ± 0.6

16.8 ± 1.2 –

7 ± 0.06 –

MS ? BE

C2

–

–

– 6.5 ± 0.02

MS ? BE ? IAA

C3

15.4 ± 1

15.1 ± 1.1

MS ? CS

C1

–

–

–

MS ? CE

C2

–

–

– 6.5 ± 0.06

MS ? CE ? IAA

C3

20.4 ± 1.4

13.5 ± 0.35

MS ? CS

C1

14.4 ± 1.3

16.5 ± 1

2.3 ± 0.03

MS ? CE

C2

–

–

–

MS ? CE ? IAA

C3

21.9 ± 2

19.32 ± 1.5

8 ± 0.06

MS ? Kinetin

C4

–

–

–

MS ? Kinetin ? IAA

C5

25.8 ± 1.3

18.6 ± 1.13

8.1 ± 0.32

MS

C6

–

–

–

MS ? LB

C7

–

–

–

Each treatment was replicated 10 times Mean of 30 replicates with standard error from three experiments

containing extract from Ck4). In case of positive control C5 the callus induction was recorded in 86% of the seeds. Callus differentiation Calli were shifted to fresh media and incubated under a photoperiod of 8 h light and 16 h dark. Greening occurred in calli incubated on MS supplemented with BS (C1) from

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P. aeruginosa Am3 and Chroococcidiopsis sp. Ck4 (Fig. 2). However, C1 from P. aeruginosa Am4 and Anabaena sp. Ck1 failed to facilitate greening in the calli. BE from all the selected bacterial strains in combination with a low amount of IAA supplemented to MS affected greening in the calli. Optimum concentration of standard CK (kinetin) and IAA was 0.1 lg ml-1 and 0.05 lg ml-1, respectively for callus differentiation. After an incubation period of 2 weeks the green patches in calli regenerated

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adventitious shoots (Fig. 2). Maximum caulogenesis was 64%, observed in case of C3-Ck4. 56% Calli incubated on C3-Am3, developed adventitious shoots. C1-Am3 also supported callus differentiation in more than 50% of the calli (Table 3). Adventitious shoot induction per callus was in the range of 3–9. On C5, 8.1 shoots were induced per callus. C3 was more efficient than C1 regarding shoot emergence per callus. 8, 7, 6.5 and 6.5 shoots were formed per callus when cultured on C3-Am3, C3-Ck4, C3-Am4 and C3-Ck1, respectively. Organogenesis in explants Seven different media were tried for organogenesis in B. oleracea var. capitata explants (Table 1). For morphogenesis the optimum concentration of IAA was 0.05 lg ml-1 along with 0.1 lg kinetin ml-1. In all cases 200 ll BS and BE (equivalent to 0.1 lg l-1) showed best results regarding organogenesis in explants and hence only these quantities are discussed. Organogenesis in the selected explants cultured on C3-Am3 and C3-Ck4 is shown in Fig. 3. MS ? BS (C1) and MS ? BE ? IAA (C3) proved efficient media for plantlet regeneration from the selected explants. Generally shoot bud formation on the explants

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was recorded after 1 week of incubation followed by shoot elongation while rooting occurred on the same medium after 7 weeks of incubation. However, there were cases where only rooting occurred but the proportion of such explants was very low. The proportion of explants which did not root after caulogenesis was also low in general (Fig. 4). Internodal explants were the most amenable to organogenesis, whereas root segments showed the highest degree of recalcitrance. Explants cultured on variable media combinations behaved differently (Fig. 4). Combination of MS, kinetin and IAA (positive control; C5) was an excellent medium to support organogenesis in 81% internodal explants (Fig. 4a). This was a significantly high proportion of plantlet regeneration in internodal explants relative to other media combinations. Among microbial phytohormone-based medium the best combination was MS supplemented with extract from P. aeruginosa Am3 and IAA (C3-Am3) where shoot induction followed by rooting was observed in 76% of the internodal explants. However, difference in the rate of plantlets regeneration on C3-Am3 and C3-Ck4 was not significant (P = 0.05). Plantlet regeneration frequency in internodal explants was 63% when incubated on MS supplemented with BS alone (C1-Am3). C1 of other strains supported significantly less

Fig. 3 Organogenesis in internodal, petiolar and hypocotyl explants inoculated on C3 media combinations

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% organogenesis

a

Plantlet

C1

d

d fg

% Organogenesis

d

c b

C2

C3

c

bb

b ab b

C1

C2

C3

C1

C2

Am4

b

C3

C1

C2

Ck1

C3

C4

C5

C6

C7

Ck4

Root

Shoot f

e

e d

d de

c b

C2

C3

b

C1

c

c

c

bc ab

C2

C3

C1

C2

Am4

f

cd

c c

C3

d

d

c

C1

C2

Ck1

Plantlet

f g

80 fg 70 60 50 40 30 bb 20 10 0

d e

c

c bc

Am3

C1

b

b a

Plantlet 90 80 70 60 50 40 cde 30 bc 20 10 0

c

c

b ab

g

efg

def

c

b

C1

Shoot

d

Am3

% organogenesis

Root

e

100 90 80 70 de 60 50 40 30 b 20 10 0

C3

C4

C5

bc

C6

C7

Ck4

Root

Shoot

g e f

d

d

d

e

de

d

d

cd c

c

b ab

C2 Am3

C3

c bc b

b

b

ab

a

C1

C2 Am4

C3

C1

C2 Ck1

C3

C1

C2

C3

C4

C5

C6

C7

Ck4

Fig. 4 Organogenesis in internodal (a), petiolar (b) and hypocotyl (c) explants incubated on different media combinations. Similar bars labeled with different letters indicate significant difference among means determined by using Duncan’s multiple-range test (P = 0.05)

number of explants to rejuvenate into plantlets. C3 proved to give better plantlet regeneration in cabbage explants than C1 (Fig. 4). A mixture of MS with extract alone from all strains (C2) and mixture of MS with kinetin (C4) were efficient shooting medium for the types of explants studied, however this combination did not support rhizogenesis, even after incubation for 8 weeks (Fig. 4). Nevertheless shoot formed on this medium formed adventitious roots upon transfer to C1, C3 or C5. No organogenesis with MS alone or MS supplemented with LB was recorded in any of the explants. The majority of internodal, hypocotyl and petiolar segments regenerated plantlets in C1 and C3 based on microbial metabolites (Fig. 4). The exception was C1-

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Am4 and C1-Ck, where only adventitious roots were induced in the majority of the explants. On MS supplemented with supernatant (C1), caulogenesis in intermodal explants was always accompanied by rooting, whereas some explants developed roots alone with no shooting. In hypocotyl explants on the other hand caulogenesis without rhizogenesis was also recorded although in small proportion when incubated on C1 (Fig. 4c). The majority of hypocotyl explants regenerated plantlets on C1 from bacterial strains (33–66%). On some of these explants, either adventitious shoots (10–50%) or roots (10–33%) were induced alone on the same medium from these strains. In case of cyanobacteria on the other hand, shooting (C1-Ck4)

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or rooting (C1-Ck1) was recorded in the highest proportion of the segments. Hypocotyl explants behaved more or less similarly on C3. Plantlet regeneration frequency was greatest (75%) when hypocotyl explants were incubated on C5 (MS ? kinetin ? IAA) or C3-Am3. Petiolar explants were relatively difficult to regenerate on the selected media (Fig. 4b). The majority of the explants developed shoots which were easily transformed to plantlets on C1-Am3 (27%) and C1-Ck4 (25%). However, when MS was supplemented with supernatant from Ck1 and Am4, the greater proportion formed adventitious roots. Extract-based media (C2) though showed good potential for shoot induction (50–80%) but rooting was not supported in this combination. C2-Ck4 was the most efficient shooting medium. While a high proportion of petiolar explants regenerated plantlets on combination of extract and IAA (C3), the number of explants where either shoots or roots were formed alone, was also reasonable. C3-Ck1 was the most efficient plantlet medium where the proportion of explant regeneration (31%) was very close to C5 (43%). Adventitious shoot induction was recorded in root explants incubated on media containing both CKs and IAA (Fig. 5). In roots explants, shooting was observed only on C3-Ck4 (47%) and C3-Am3 (45%).

Discussion High CKs to IAA ratios in microbial culture were found to induce formation of a compact callus while lowering the ratio decreased the compactness. Compact callus amenable to regenerate shoots was formed on extract equivalent to 0.1 lg ml-1 in combination with 0.01 lg ml-1 IAA. It was previously reported that the CKs to auxins ratio is very important in shoot or plantlet regeneration. Lowering the CKs to auxin ratio may adversely affect the in vitro regeneration of shoots in explants (Ning et al. 2007). It has already been established that a high CKs to auxin ratio favors shoot regeneration in B. oleracea (Qin et al. 2007). Only compact calli were capable of turning green upon

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exposure to light followed by shoot bud induction and regeneration. Among four different explants (internodal, hypocotyl, petiolar and root) the most efficient to regenerate plantlet were internodal explants. According to a previous report, internodal and hypocotyl segments are the most regenerative of the explants (Cheng et al. 2001). Previous studies have reported that different explants from the same plant show variable response in tissue culture (Radhika et al. 2006; Dagustu et al. 2008). There was no difference in the overall efficacy of bacterial and cyanobacterial phytohormones to support the in vitro growth of B. oleracea. Nevertheless, the efficacy of extract/supernatant from Ck4 and Am3 was greater than the efficiency of Ck1 and Am4. Extract and supernatant from the former two strains constituted the most valuable source of supplements for the in vitro growth of the selected plant. The only significant difference in the efficiency of Ck4 and Am3 was recorded in case of shooting on the selected explants. Cultures from both the strain had high CKs to IAA ratio evidencing the importance of this ratio in tissue culture of cabbage. In the case of high IAA producing strains Am4 and Ck1 root explants failed to regenerate on all media combinations. Extract from different strains with same amount of CKs initiated different responses in the explants evidencing the involvement of certain other metabolites which might be present in the extract. Shoot induction was not observed on root explants incubated on media with CKs alone. In one study it was demonstrated that CK along with IAA and picloram offered the best combination for shoot induction in root segments (Lazzeri and Dunwell 1984). It was previously established that a short pre-culture of the root segment on 2,4-D is essential for shoot induction and immediate incubation of the explants on shooting medium leads to shoot formation at the proximal end of the root only (Valvekens et al. 1988). In contrast, we found shoot induction in B. oleracea var. capitata root segment incubated on shooting medium bypassing the pre-culture on 2,4-D. It may be concluded that instead of some differences in the efficacy of bacterial and cyanobacterial

Fig. 5 Adventitious shoot induction on root explants cultured on C3-Am3 (a); C3-Ck4 (b); C5 (kinetin control, c) and C7 (LB control, d)

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phytohormones supplements to support in vitro growth of B. oleracea, their overall performance is essentially similar. Hence phytohormones from both sources can be used as replacement of standard CKs and IAA. Cabbage may be regenerated efficiently and cheaply from internodal explants on simple formulation based on bacterial and cyanobacterial metabolites.

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