In Vitro Clonal Propagation of a Fast Growing Legume Tree- Acacia mangium Willd. Employing Cotyledonary Node Explants
Muhammad SHAHINOZZAMAN*, Mustafa Abul Kalam AZAD, Muhammad Nurul AMIN
University of Rajshahi, Faculty of Life and Earth Science, Department of Botany, Rajshahi-6205, Bangladesh; [email protected] (*corresponding author)
Abstract
An efficient protocol for in vitro clonal propagation of A. mangium was developed using seedling derived explants. Out of three different explants tested for shoot proliferation, cotyledonary node showed best performance than leaf node and shoot tip explants. MS (Murashige and Skoog’s) medium was found best for shoot proliferation and cotyledonary nodes were subsequently cultured on MS medium supplemented with BA and Kn alone or in combination with NAA, IBA and GA3 at different concentrations. Maximum number of shoots was formed on MS medium containing 4.0 µM BA. For adventitious rooting, in vitro proliferated shoots were transferred to full strength MS medium fortified with IBA and NAA singly at different concentrations (0-8.0 µM). Best rooting responses were observed in the medium containing 8.0 µM IBA. Plantlets having well developed root system were transferred to soil and successfully acclimatized with 65% survival rate under ex vitro condition.
Keywords: in vitro propagation, multipurpose legume tree, plantation forestry, seedling explants
Introduction
A. mangium Willd., belonging to Mimosaceae family, is one of the most important leguminous tree characterized by its fast growth, nitrogen fixing ability, good growth in adverse soil condition and a tendency to grow well in hu- mid and hot climate (Umezawa et al., 2008). It is native to northern Queensland of Australia, through Papua New Guinea into the Indonesian provinces of Irian Jaya and Maluku. This tree species is now widely used for timber, pulp and fire wood (Galiana et al., 1991). In addition, its bark contains considerable amount of antioxidant phenols (Zhang et al., 2010) offering increasing demand of its bark in different industries as a source of active substance for cosmetic and pharmaceutical composition. Taking into account of its different valuable characteristics, such as fast growth, good growth in adverse soil condition, good quality pulp and fuel-wood production etc., this tree spe- cies was introduced into Bangladesh from Australia dur- ing 1980s (Islam, 2003). Besides, several reports (Amin et al., 1995; Hossain et al., 1997; Khan et al., 2004) recom- mended this species suitable for afforestation and refores- tation in degraded hilly areas, marginal lands and roadside plantations in Bangladesh.
Conventional propagation methods like using seeds, cutting, grafting etc. have limited scope for large scale prop- agation of A. mangium because of poor seed germination and poor rooting ability of cuttings. Compare to conven- tional propagation methods, in vitro clonal propagation is a common technique, which has been extensively applied in large scale multiplication of many important forest tree
species (Ahuja, 1993; Bonga et al., 1992). Due to having recalcitrance nature of adult tissues, however, most of the investigations on in vitro clonal propagation of forest le- gumes are concentrated on juvenile materials especially on seedling derived explants. There are a few successful reports on in vitro clonal propagation of A. mangium us- ing seedling derived explants like seedling nodes (Ahmad, 1991; Galiana et al., 1991; Saito et al., 1993), but no noted reports on its clonal propagation employing cotyledonary node explants have been found yet. This communication, therefore, describes a successful and quick method on large scale in vitro clonal propagation of A. mangium via cotyledonary node explants.
Materials and methods
Seeds of A. mangium were collected from a mature elite tree grown in Rajshahi University campus, Rajshahi, Bangladesh. Seeds were washed thoroughly under running tap water for 15 minutes and then washed with continu- ous agitation in a few drops Savlon™ containing water for 15 minutes. Washed seeds were pre-treated by immersing them in boiling water for 2-5 minutes followed by soaking in cold water for 20 minutes. The pre-treated seeds were then treated with 0.1% HgCl2 for 5 minutes under lami- nar air flow cabinet to disinfect them. Finally, seeds were washed 3 to 5 times with sterile distilled water and were placed in culture tubes (25 × 150 mm) containing hor- mone free MS (Murashige and Skoog, 1962) medium pre- pared with 3% (w/v) sucrose and 0.8% (w/v) agar (Sigma Chemical Co. USA). The pH of the medium was adjusted Received 11 January 2012; accepted 02 April 2012
A. mangium than leaf node and shoot tip explants. Simi- lar results were noted in a range of legume species, such as A. senegal (Khalafalla and Daffalla, 2008), A. sinuata (Vengadesan et al., 2002b), Clitoria ternatea (Barik et al., 2007), Colutea istria (Hegazi and Gabar, 2010), Dalber- gia sissoo (Pradhan et al., 1998a), D. latifolia (Pradhan et al., 1998b), Pterocarpus marsupium (Anis et al., 2005), P.
santalinus (Rajeswari and Paliwal, 2008) and Peltophorum pterocarpum (Uddin et al., 2005). On the contrary of these findings, Amin et al. (1992) in carambola, Ara et al. (1991) in Sesbania grandiflora, Loh and Rao (1989) in guava and Rahman and Blake (1988) in jackfruit observed increased shoot proliferation from the seedling explants rather than cotyledonary nodes. Debergh and Read (1990) suggested that differential responses of different explants of the same plant are more species specific while Lane (1978) proposed the endogenous hormone level in buds of different regions of the stem as a reason of differential responses of different explants of the same plant. In case of A. mangium, further investigations, based on the present findings, should be carried out to find out the specific reason behind the dif- ferential responses of different explants.
The full strength MS medium affected shoot prolifera- tion from cotyledonary node explants significantly than other three media (WPM, MMS1 and MMS2) tested.
Maximum 98.67 ± 0.37% explants produced highest 3.93
± 0.37 shoots on MS medium. Although explants pro- duced longest shoots (1.63 ± 0.34 cm) in WPM medium, but both percentage of response and multiplication rate were lower in this medium than full strength MS medium.
This study revealed that full strength MS medium was pre- ferred for axillary shoot proliferation from cotyledonary nodes of A. mangium while WPM showed a little effect in terms of shoot proliferation. Full strength MS medium has been proved best for axillary shoot proliferation in many other Acacia species, including A. albida (Ruredzo and Hanson, 1993), A. catechu (Kaur et al., 1998), A. mearnsii (Huang et al., 1994), A. nilotica (Abbas et al., 2010), A.
salicina, A. saligna and A. sclerosperma (Jones et al., 1990).
Similar results were also observed in some other woody to 5.7 before autoclaving at 121°C for 20 minutes at 1.2
kg/cm2 pressure. After successful germination of seeds, three different types of explants viz. cotyledonary node, leaf node and shoot tip (1-1.5 cm in length) were excised from 2-week-old seedlings (Fig. 1A) and cultured on MS basal medium containing 4.0 µM BA (6-Benzyl adenine) alone or in combination with 0.5 µM NAA (α-naphthalene acetic acid) to test the effect of explants on in vitro shoot multiplication.
To test the effect of basal medium on in vitro shoot multiplication, cotyledonary nodes were initially cultured on four different basal medium viz. MS, MMS1, MMS2 and WPM (Woody plant medium) (Lloyd and McCown, 1980) supplemented with 4.0 µM BA. In addition, excised cotyledonary nodes of 2-week-old seedlings were cultured on MS medium containing various concentrations (2.0- 8.0 µM) of BA and Kn (6-furfurylamino purine). Differ- ent concentrations (0.5-2.0 µM) of NAA, IBA (Indole-3- butyric acid) and GA3 (Gibberellic acid) were combined with 4.0 µM BA to test their shoot induction efficiency.
Microshoots of 1-3 cm length were prepared from us- able shoots by snipping off the basal leaves and cultured them individually in 25 × 150 mm culture tubes with 15- 20 ml of full strength MS medium supplemented with NAA or IBA (2.0-8.0 µM).
The rooted plantlets were transferred on to the small plastic pots containing sterilized soil mix (garden soil and compost in 1:1 ratio). Transferred plantlets were hardened in growth chamber condition for 25 days and then trans- ferred to outdoor condition. The total number of plants transferred to the pots and the number of surviving plants in the outdoor condition were recorded.
All the cultures were maintained at 25 ± 2°C under a 16h light and 8h dark cycle with the light intensity of 2000-3000 lux provided by cool-white fluorescent tubes (36 W). Data were recorded after 8 weeks of culture ex- cept for rooting experiment when the data were recorded after 4 weeks of incubation. In all the experiments, 12-15 explants were used and each experiment was repeated three times. Mean and standard error were calculated for all nu- merical data. The mean data of each treatment were com- pared by using Duncan’s Multiple Range Test (DMRT) at P=0.05%.
Results and discussion
Cotyledonary node showed the best shoot prolifera- tion efficiency irrespective of media type, which followed by leaf node and shoot tip explants (Tab. 1). Cotyledon- ary nodes showed the maximum 99.33 ± 0.67% response and produced 5.10 ± 0.58 shoots on MS + 4.0 µM BA (Fig. 1B) while the highest 80.67 ± 1.33% leaf nodes and 51.67 ± 1.28% shoot tips formed maximum 1.50 ± 0.40 and 0.70 ± 0.33 shoots, respectively on the same media formulation. Results of this experiment indicated the high regenerative capacity of cotyledonary node explants of
Tab. 1. Effects of three different explants, cultured on MS medium containing 4.0 µM BA alone or in combination with 0.5 µM NAA, on axillary shoot proliferation of A. mangium
Type of
explant Plant growth regulators (µM)
Percentage of explant responded
(X ± SE)
Number of shoots (X ± SE) Cotyledonary
node
BA 4.0 99.33 ± 0.67 a 5.10 ± 0.58 a BA 4.0 + NAA 0.5 89.33 ± 2.33 b 2.40 ± 0.40 b Leaf node BA 4.0 80.67 ± 1.33 c 1.50 ± 0.40 bc BA 4.0 + NAA 0.5 80.00 ± 1.61 c 1.20 ± 0.29 cd Shoot tip BA 4.0 51.67 ± 1.28 d 0.70 ± 0.33 cd BA 4.0 + NAA 0.5 42.67 ± 2.33 e 0.30 ± 0.15 d Note: Values represent means ± standard error of 20 explants per treatment in three repeated experiments. Means followed by the same letters are not significantly different by Duncan’s multiple Range Test at 0.05 % probability level
trees, like Colutea istria (Hegazi and Gabr, 2010), Dalber- gia latifolia (Swamy et al., 1992), Lagerstromia parviflora (Tiwari et al., 2002), Pterocarpus marsupium (Hussain et al., 2008), P. santalinus (Rajeswari and Paliwal, 2008), Sterculia urens (Hussain et al., 2007) and Swartzia made- gascariensis (Berger and Schaffner, 1995).
Cotyledonary node explants cultured on MS medium devoid of growth regulator produced 0.92 ± 0.19 shoots with 0.64 ± 0.14 cm lengths, which may be due to the pres- ence of endogenous cytokinin in cotyledonary nodes, sug- gested by Rajeswari and Paliwal (2008) in Pterocarpus san- talinus. However, the addition of exogenous cytokinin to MS medium induced shoot multiplication rate remarkably,
indicating the requirement of exogenous cytokinin supply in the medium for better axillary shoot proliferation. Out of two different cytokinin-BA and Kn, best shoot prolif- eration was observed on medium containing BA. Highest number of shoots (4.08 ± 0.60) and longest shoots (1.20 ± 0.15 cm) were formed on 4.0 µM BA containing medium (Fig. 1. C) while 3.25 ± 0.43 shoots with maximum 1.16 ± 0.18 cm length were found in medium containing 6.0 µM Kn. The results indicated that BA was superior to Kn for axillary shoot proliferation from cotyledonary nodes of A. mangium. The superiority of BA over Kn has also been reported in in vitro propagation of other species of Acacia (Badji et al., 1993; Beck et al., 1998; Dewan et al., 1992;
Fig. 1. In vitro propagation of A. mangium Willd. from cotyledonary node explants. A. Two-week old aseptically germinated seed- lings; B. Multiple shoot formation from cotyledonary node explant after 4 weeks of culture on MS + 4.0 µM BA; C. In vitro proliferated shoots from cotyledonary node explant after 8 weeks of culture on MS + 4.0 µM BA; D. Rooted plantlets ready for transplantation; E. Acclimatized plantlets after 7 days of transplantation onto soil mix
Galiana et al., 1991; Junior et al., 2004; Khalafalla and Daffalla, 2008; Mittal et al., 1989; Nandwani, 1995; Rout et al., 2008; Singh et al., 1993; Vengadesan et al., 2002b).
In addition, Jeyakumar and Jayabalan (2002) in Psoralea corylifolia, Husain et al. (2008) in Pterocarpus marsupium, Pradhan et al. (1998a) in Dalbergia sissoo, Rajeswari and Paliwal (2008) in Pterocarpus santalinus, Shyamkumar et al. (2003) in Terminalia chebula, Widiyanto et al. (2008) in Albizia falcataria also observed the superiority of BA over Kn on axillary shoot proliferation from seedling ex- plants. On the contrary, Nandwani and Ramawat (1993) in Prosopsis cinerarea and Kumar (1992) in Bauhinia pur- purea found Kn as superior cytokinin to BA in in vitro shoot multiplication. The differential effects of BA and Kn on in vitro axillay shoot proliferation might be due to the different mode of action of BA and Kn during shoot development (Widiyanto et al., 2008).
Inclusion of NAA or IBA along with BA had no sig- nificant effect on both proliferation and elongation of ax- illary shoots. Average number of shoots and average length of shoots both were markedly reduced in all auxin-cytoki- nin combinations. Cotyledonary nodes produced highest 3.75 ± 0.70 shoots on medium containing 4.0 µM BA + 0.5 µM NAA and the length of shoots was 1.00 ± 0.10 cm in that medium. The results revealed that exogenous auxin was not essential to initiate shoot bud formation, which also indicated the antagonistic effect of NAA or IBA with BA on in vitro shoot proliferation of A. man- gium. Vengadesan et al. (2002b) also observed that auxins (NAA, IBA and IAA) along with BA were not effective for shoot proliferation from cotyledonary nodes of Aca- cia sinuata. In Acacia senegal similar results were reported by Khalafalla and Daffalla (2008). This finding is also in agreement with Mallikarjuna and Rajendrudu (2009) in Holarrhena antidysenterica, Hussain et al. (2007) in Ster- culia urens. Garland and Stoltz (1981) demonstrated that
Fig. 2. Effects of auxins (IBA and NAA) on adventitious rooting of in vitro derived microshoots of A. mangium
Tab. 3. Effects of plant growth regulators on axillary shoot proliferation from cotyledonary node explants of A. mangium
Plant growth regulators (µM)
Number of shoots (X ± SE)
Length of shoots (cm) (X ± SE)
BA Kn
0 0 0.92 ± 0.19 hi 0.64 ± 0.14 bcd
2.0 - 1.42 ± 0.19 ghi 0.86 ± 0.10 bcd
4.0 - 4.08 ± 0.60 a 1.20 ± 0.15 b
6.0 - 1.67 ± 0.43 fghi 0.63 ± 0.19 bcd
8.0 - 2.42 ± 0.29 bcdefg 1.05 ± 0.14 bcd
- 2.0 1.17 ± 0.21 ghi 0.80 ± 0.16 bcd
- 4.0 1.58 ± 0.23 fghi 0.85 ± 0.16 bcd
- 6.0 3.25 ± 0.43 abcd 1.16 ± 0.18 bc
- 8.0 2.08 ± 0.34 defgh 0.83 ± 0.12 bcd
BA NAA IBA GA3
2.0 0.5 - - 1.25 ± 0.25 ghi 0.85 ± 0.19 bcd 2.0 1.0 - - 1.08 ± 0.26 ghi 0.83 ± 0.18 bcd
2.0 2.0 - - 0.67 ± 0.19 i 0.59 ± 0.16 cd
4.0 0.5 - - 3.75 ± 0.70 a 1.00 ± 0.10 bcd
4.0 1.0 - - 2.25 ± 0.45 cdefgh 0.94 ± 0.09 bcd 4.0 2.0 - - 1.92 ± 0.43 efghi 0.66 ± 0.13 bcd 2.0 - 0.5 - 1.08 ± 0.26 ghi 0.66 ± 0.15 bcd
2.0 - 1.0 - 0.92 ± 0.23 hi 0.49 ± 0.18 d
2.0 - 2.0 - 0.67 ± 0.19 i 0.48 ± 0.13 d
4.0 - 0.5 - 3.50 ± 0.67 abc 0.86 ± 0.14 bcd 4.0 - 1.0 - 2.25 ± 0.46 cdefgh 0.79 ± 0.14 bcd 4.0 - 2.0 - 1.67 ± 0.41 fghi 0.71 ± 0.15 bcd
2.0 - - 0.5 1.38 ± 0.32 ghi 1.72 ± 0.32 a
2.0 - - 1.0 1.13 ± 0.23 ghi 0.73 ± 0.18 bcd
2.0 - - 2.0 0.88 ± 0.23 hi 0.65 ± 0.18 bcd
4.0 - - 0.5 2.88 ± 0.61 abcdef 1.82 ± 0.17 a
4.0 - - 1.0 3.63 ± 0.65 ab 2.09 ± 0.30 a
4.0 - - 2.0 3.13 ± 0.55 abcde 0.96 ± 0.21 bcd Note: Values represent means ± standard error of 20 explants per treatment.
Means followed by the same letters are not significantly different by Duncan’s multiple Range Test at 0.05% probability level
Tab. 2. Effects of different basal media containing 4 µM BA on in vitro shoot multiplication from cotyledonary node explants of A. mangium
Basal mediumq
Percentage of explant responded
(X± SE)
Number of shoots (X ± SE)
Length of shoots (cm) (X ± SE) MS 98.67 ± 0.37 a 3.93 ± 0.37 a 1.43 ± 0.18 ab MMS1 98.33 ± 1.67 a 2.87 ± 0.36 ab 0.90 ± 0.13 bc MMS2 63.33 ± 1.69 c 2.13 ± 0.55 b 0.61 ± 0.18 c WPM 78.33 ± 2.41 b 2.87 ± 0.52 ab 1.63 ± 0.34 a Note: Values represent means ± standard error of 20 explants per treatment in three repeated experiments. Means followed by the same letters are not significantly different by Duncan’s multiple Range Test at 0.05% probability level.
qMS = Full strength MS medium; MMS1 = MS with ½ strength of major salts only; MMS2 = MS with ½ strength of both major and minor salts
humid ex vitro condition in the growth room (Fig. 1F).
The in vitro derived plantlets acclimated better under ex vitro condition when they were maintained in growth room for 25 days before transferring them to outdoor con- dition. Finally, 65% transplanted plantlets were survived and acclimated well under ex vitro condition after 25 days of transplantation.
Conclusions
The described strategy demonstrates an efficient sys- tem of in vitro clonal propagation of A. mangium via coty- ledonary node explants, which could play a significant role in large scale plantlet production all around the year, as well as in wide plantation and in conservation of this plant’s genetic resources.
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