Continuous Culture of Acacia mangium Willd.

 

Afaque Quraishi*

School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, India

 

ABSTRACT:

Acacia mangium, is a fast growing leguminous tree. It forms hybrids with A. auriculiformis, by air pollination; thus, micropropagation may provide genetically pure quality planting material. Therefore in vitro cultures were initiated from nodal explants of 1-year-old Mangium plant, on Driver and Kuniyuki medium with 6-benzyl adenine and polyvinyl pyrrolidone. Shoot cultures were further proliferated using on very low level of 6-benzyl adenine. Different adjuvant were tested for shoot multiplication. Shoot cultures were maintained up to 1 ˝ years with retaining the shoot proliferation capacity, by normal subculture cycles. These 1 ˝ years old shoot cultures were successfully rooted in vitro on half strength medium in presence of auxin, to regenerate complete plants.    

 

KEYWORDS: Micropropagation, DKW medium, propagule proliferation, adjuvant, subculture, rooting

 

INTRODUCTION:

Acacia mangium Willd. is a leguminous tree, native to northern Queensland in Australia, Papua New Guinea, and Indonesia (Gunn and Midgley, 1991). The tree has been planted for pulpwood throughout the tropics. A. mangium is a fast-growing, medium-sized, evergreen tree with phyllodes. Its capacity to grow well on barren soils makes it a favorite for re-afforestation because of its nitrogen-fixing capacity. In vitro propagating provides a good option for fast multiplication of selected Mangium for large scale plantations. Moreover, in nature Mangium forms hybrids with A. auriculiformis. Thus, micropropagation can provide genetically pure quality planting material of this important tree.  Micropropagation of A. mangium has been reported by several workers - Galiana et al. (1991), Bon et al. (1998), Monteuuis and Bon (2000), Monteuuis (2004 a and b). Present study is about the long term in vitro shoot culture of Mangium via routine subculture cycles; using five to ten times lower level of cytokinin in comparison to the previous reports, with the help of adjuvant.

 

MATERIAL AND METHOD:

Shoot tips with 5 – 6 nodes were obtained from 1-year-old plant of Mangium in the campus of Pt. Ravishankar Shukla University, Raipur; in growing season (April). After removing the leaves, these shoots were thoroughly washed in running tap water and surface disinfested. Each nodal segment with single axillary bud was cut to approx 1.5 cm long and inoculated aseptically on Driver and Kuniyuki (DKW) medium (1984) with or without 6-benzyladenine (BA) plus 25 μM polyvinyl pyrrolidone (PVP). Sucrose (3%) was used as carbon source and media were solidified with 0.8% agar. The media were adjusted to pH 5.7 with 1 N NaOH and sterilized by autoclaving for 20 min at 1.05 kg/cm2 pressure at 121oC. The cultures were kept under 16/8 (day/night) photoperiod with 40 μmol m-2s-1 light intensity provided by cool white fluorescent tubes. The bud break percentage, number and length of shoots per explant; and number of nodes per culture were recorded after 45 days.


After 45 days, the shoots which had elongated from the nodal explants of Mangium, were excised aseptically and divided into nodes and transferred onto multiplication medium for further shoot proliferation. This procedure was repeated at 60 – day intervals to study the shoot production potential of the regenerated nodes. At this shoot proliferation stage different adjuvant – adenine sulfate (AdS), casein hydrolysate (CH) and PVP were tested in different combinations to optimize shoot production. For rooting of regenerated shoots, half-strength DKW medium with different concentrations of indole-3-butyric acid (IBA) was used. Elongated microshoots were excised and placed vertically on the rooting medium.

 

In all experiments, 10 replicates were taken in each treatment, and each experiment was repeated three times. The shoot length, shoot number and node number were analyzed by analysis of variance (ANOVA).

 

RESULTS AND DISCUSSION:

Nodal explants of Mangium released excessive phenolics into the culture medium from their cut ends and eventually died. This problem was overcome by suspending explants in a sterile solution of PVP (25 μM) for 10 min before inoculation and by adding 25 μM PVP also in the explant establishment medium. Nodal explants exhibited 90% bud break response with 0.44 μM BA on DKW medium (Table-1). Here normal and healthy shoots were obtained from explants. DKW medium is used in the present study, it is more concentrated in inorganic nutrients than Murashige and Skoog (MS) medium (1962) and it also contains L-glutamine (250mg l−1).

 

After 45 days, the shoots which were elongated from the original explants were excised and divided into nodes for further axillary shoot proliferation. These nodes were placed on shoot proliferation medium (DKW with 0.44 μM BA). After 60 days at 1st subculture, all the shoots produced by each node were harvested and again cut into nodes, used for shoot production in further subcultures. Best proliferation response was recorded with the combination of all the 3 adjuvant tested- adenine sulfate (200 mg/l), casein hydrolysate (50 mg/l) and PVP (1 g/l) on DKW medium with 0.44 μM BA (data of 4th subculture cycle) (Figure 1). On this medium, shoot production capacity of regenerated nodes has been continue and increasing at further subcultures (Table 2 and Figure 2). These Mangium shoot cultures were continuously produced shoots up to 1 ˝ years and further up to 2 years (data not shown), without losing the multiplication rate. It is estimated that with this rate of multiplication, from a single nodal explant of Mangium more than 10 thousand shoots may be produced in vitro, within 1 ˝ years.

 

Table 1: Effect of BA concentration in DKW medium, on shoot bud establishment response from nodal explants of A. mangium, after 45 days.

BA                                   Bud Break                             Shoot number                    Shoot length (cm)    Nodes / culture

 (µM)                               (%)                                        Mean ±SE                          Mean ±SE                                Mean ±SE

0                                      50                                          1.0 ± 0               0.7 ± 0.08                                1.2 ± 0.18

0.44                                 90                                          1.0 ± 0               1.0 ± 0.08                                2.1 ± 0.15

Each treatment consisted of 10 replicates, and each experiment was repeated three times.

ANOVA (Effect of BA conc.):                            

                                        Shoot length:                df =1; F=8.7;                                      p=0.0064

                                        Nodes/culture:             df =1; F=21.51;                                  p=0.0001

 

Figure 1: Effect of adjuvant on shoot production of single node of A. mangium on DKW medium with 0.44 µM BA, after 60 days of culture in 4th subculture cycle.

 

 

1= Control; 2 = Adenine sulfate 200 mg/l; 3 = Adenine sulfate 200 mg/l + Casein hydrolysate 50 mg/l; 4 = Adenine sulfate 200 mg/l + Casein hydrolysate 50 mg/l + PVP40 1 g/l

Each treatment consisted of ten replicates, and each experiment was repeated three times.

ANOVA:

Effect of Adjuvant:                Shoot no.:                      df=3;    F=4.49;            p=0.0051;                          LSD=0.5

                                              Nodes/culture:               df=3;    F=6.54;            p=0.0004;                          LSD=1.6

Table 2:In vitro shoot production potential of a single node of A. mangium in DKW with 0.44 µM BA, 200 mg/l Adenine sulfate, 50 mg/l Casein hydrolysate and 1 g/l polyvinyl pyrrolidone, on different subculture.

Subculture                             Shoot number                            Shoot length (in cm)                            Nodes / Culture

                                              Mean ±SE                                  Mean ±SE                                            Mean ±SE

Initiation (45days)                 1.0 ± 0                                       1.0 ± 0.15                                            2.0 ± 0.13

1st (105 days)                        1.1 ± 0.06                                  0.9 ± 0.08                                            1.6 ± 0.12

2nd (165 days)                       1.5 ± 0.11                                  1.5 ± 0.20                                            2.5 ± 0.13

3rd (225 days)                        1.8 ± 0.13                                  1.3 ± 0.20                                            2.4 ± 0.10

4th (285 days)                        2.4 ± 0.11                                  1.9 ± 0.20                                            5.9 ± 0.30

5th (345 days)                        1.9 ± 0.07                                  2.0 ± 0.20                                            5.4 ± 0.25

6th (405 days)                        1.8 ± 0.09                                  1.9 ± 0.10                                            5.5 ± 0.31   

7th (465 days)                        2.0 ± 0.12                                  2.1 ± 0.10                                            5.7 ± 0.31

8th (525 days)                        2.1 ± 0.12                                  1.8 ± 0.10                                            5.8 ± 0.29

Each treatment consisted of ten replicates, and each experiment was repeated three times.

 

ANOVA:

Effect of subculture:              Shoot no.:                     df =8;   F=22.6828;      p=0.0001;        LSD=0.28

                                              Nodes/culture:              df =8;   F=66.1795;      p=0.0001;        LSD=0.65

 

Figure 2: In vitro shoot multiplication of A. mangium in DKW with 0.44 µM BA, 200 mg/l Adenine sulfate, 50 mg/l Casein hydrolysate and 1 g/l polyvinyl pyrrolidone.

 

 

Table 3: In vitro rooting in Mangium shoots of 8th subculture, in half-strength DKW medium; observation recorded after 45 days.

IBA                Rooting                          Root Number               Root Length (in cm)                   Intervening Callus

(µM)              %                                   Mean ±SE                          Mean ±SE 

0                    0                                    -                                   -                                                 -

2.4                 40                                  1.3 ± 0.1                      2.4 ± 0.4                                    -

4.9                 80                                  2.1 ± 0.2                      2.2 ± 0.3                                    -

9.8                 30                                  2.4 ± 0.4                      1.6 ± 0.2                                    +

Each treatment consisted of ten replicates, and each experiment was repeated three times.

 

 

 

For rooting, regenerated shoots derived from 8th subculture cycle (1˝ years) were cut and placed vertically on half strength DKW medium. Shoots exhibited 80% rooting response with 4.9 μM IBA (Table 3). Higher concentrations of IBA induced callusing. 

 

In the present study, for shoot production of Mangium, low BA concentration (0.44 μM) had been used which is much lower level of BA used previously in A. mangium micropropagation : 4.4 μM BA- Galiana et al. (1991); 4.4 μM BA - Bon et al. (1998); 4.4 μM BA - Monteuuis and Bon (2000); and 2.2 μM BA - Monteuuis (2004 a and b). Although high concentrations of cytokinin induced higher shoot production in Mangium (data not shown) and also reported by Bon et al. (1988); but after subsequent subcultures, they lost vigor and organogenicity. Monteuuis (2004a) also reported that these high cytokinin regenerated shoots further proliferated in high numbers but resulted in quickly and often irreversible organogenic culture decline. As above mentioned, synthetic BA, reported most effective for micropropagation of A. mangium, but may become phytotoxic after a few subcultures (Monteuuis 2004a). Therefore in the present strategy very low BA level used which induced slight less shoot proliferation rate but healthy propagules were produced continually for further shoot proliferation cycles for long time culture under the normal incubation conditions, with good rooting efficiency and without any phytotoxic effect. May be the low BA level worked for healthy shoot production in presence of  AdS, CH, PVP and the DKW medium, which is more concentrated in inorganic nutrients than MS and contains L-glutamine too.

 

The effectiveness of CH could be attributed to different amino acids present which affect nitrogen assimilation, alleviation of phosphate deficiency, replacement of toxic ammonium ions and chelation of divalent metals; hence the overall response (George 1993).  CH was also incorporated in MS medium for Mangium shoot production by Monteuuis and Bon (2000), Monteuuis (2004a and b); but they used much higher levels of BA than this report.

 

Similarly, in many woody plant species - Melia azedarach (Husain and Anis, 2009), Pterocarpus marsupium (Husain et al., 2008), Acacia sinuate (Vengadesan et al., 2003), etc., the shoot proliferation was improved by the addition of AdS in the medium. Adenine in the form of AdS can stimulate cell growth and greatly enhance shoot formation (Murashige, 1974). Also in Syzygium cuminii, adding of CH, amino acids and PVP was reported beneficial for shoot development (Jain and Babbar, 2003).  DKW medium was reported significantly better than MS with respect to the number of shoots of Neem cultures (Quraishi et al., 2004).

 

CONCLUSION:

Thus the present observation is for long term culture cycles under normal incubation regime using very low BA level in the culture medium and supplemented with the supportive additives, for Mangium.

 

ACKNOWLEDGEMENTS:

Author is thankful to Head, School of Life Sciences, Pt. Ravishankar Shukla University, Raipur (C.G.), India, for providing the laboratory facilities; and Council of Scientific and Industrial Research (C.S.I.R.), New Delhi, India, for financial support.

 

REFERENCES:

Bon, M.C., Bonal, D., Goh, D.K.S. and Monteuuis, O. (1998). Influence of different macronutrient solutions and growth regulators on micropropagation of juvenile Acacia mangium and Paraserianthes falcataria explants. Plant Cell Tiss. Organ Cult. 53:171–177.

Driver, J.A. and Kuniyuki, A.H. (1984). In vitro propagation of paradox walnut rootstock. Hort Sci. 19: 507–509.

Galiana, A., Tibok, A. and Duhoux, E. (1991). In vitro propagation of the nitrogen-fixing tree-legume Acacia mangium Willd. Plant Soil 135:151–159.

Gunn, B.V. and S.J. Midgley (1991). Genetic resources and tree improvement: exploring and assessing the genetic resources of four selected tropical acacias. No. 35 (pp 57–63), Edited by J.W. Turnbull, CACIAR Proceedings, Canberra.

Husain, M.K. and Anis, M. (2009). Rapid in vitro multiplication of Melia azedarach L. (a multipurpose woody tree). Acta Physiol Plant 31:765–772.

Husain, M.K., Anis, M. and Shahzad, A. (2008). In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants. Acta Physiol Plant 30:353–359.

Jain, N. and Babbar, S.B. (2003). Regeneration of ’juvenile’ plants of black plum, Syzygium cuminii Skeels, from nodal exp of mature trees. Plant Cell, Tissue and Organ Culture 73: 257–263.

Monteuuis, O. (2004a). In vitro micropropagation and rooting of Acacia mangium microshoots from juvenile and mature origins. In Vitro Cell. Dev. Biol.—Plant 40:102–107.

Monteuuis, O. (2004b). In vitro rooting of juvenile and mature Acacia mangium microcuttings with reference to leaf morphology as a phase change marker. Trees 18:77–82.

Monteuuis, O. and Bon, M.C. (2000). Influence of auxins and darkness on in vitro rooting of micropropagated shoots from mature and juvenile Acacia Mangium. Plant Cell, Tissue and Organ Culture 63: 173–177.

Quraishi, A., Koche, V., Sharma, P. and Mishra, S.K. (2004). In vitro clonal propagation of neem (Azadirachta indica). Plant Cell, Tissue and Organ Culture 78: 281–284.

Vengadesan G., Ganpathi A., Prem R. and Selvaraj N. (2003). In vitro propagation of Acacia sinuata (Lour.) Merr. from nodal segments of a 10-year-old tree, In Vitro Cell. Dev. Biol.—Plant, 39: 409–414.

 

Received on 11.07.2012

Modified on 14.08.2012

Accepted on 30.08.2012              

© A&V Publication all right reserved

Research J. Science and Tech.  4(4): July-August. 2012: 168-171