Agroforest Syst DOI 10.1007/s10457-012-9583-8
In vitro regeneration and multiplication for mass propagation of Acacia ehrenbergiana Hayne: a potential reclaiment of denude arid lands S. B. Javed • M. Anis • P. R. Khan • I. M. Aref
Received: 30 March 2012 / Accepted: 14 November 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract Acacia ehrenbergiana Hayne, an indigenous legume of the Middle East is an abiotic stress resistant nitrogen fixing legume which possesses valuable medicinal and economic properties. It has great potential to be used as reclaiment of denuded barren lands if only it could be propagated at a large scale. However, low seed germination and high seedling mortality apprehend its uses. Use of tissue culture technology could overcome these limitations but unfortunately there is no report describing micropropagation protocol for this species. The present study thus describes the first successful report on micropropagation of A. ehrenbergiana and also discusses the possible physiological and molecular reasons responsible to make particular culture condition, type and concentration of PGR most effective. It uses cotyledonary node explants cultured on PGR augmented Murashige and Skoog (MS) medium. A maximum of 90.3 ± 2.4 % of culture showed regeneration on benzyl adenine (BA) (10 lM) supplemented medium, maximum number of shoots (7.3 ± 0.15) were obtained on BA and NAA (0.1 lM) supplemented S. B. Javed M. Anis (&) Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202002, UP, India e-mail:
[email protected] M. Anis P. R. Khan I. M. Aref Department of Plant Production, College of Food and Agriculture Sciences, King Saud University, PO Box 2460, Riyadh 11451, Kingdom of Saudi Arabia
medium. Culture conditions were optimized by manipulation of pH of the medium, 5.8 being the best. Subculturing up to three passages for multiplication and thereafter induction of in vitro rooting was found to be most economical both in terms of time and money. Rizogenesis was observed best in MS medium supplemented with IBA (5.0 lM). 80 % of the rooted plants were successfully transferred to the natural condition under the sun. Keywords Micropropagation Acacia ehrenbergiana Shoot multiplication speed Woody tree species Plant growth regulators (PGRs)
Introduction Fast decreasing fertile land is certainly a major hurdle in attempts to provide food security to the world, which has increasing requirements of arable lands. Clearing of forests for cultivation also needs to be compensated to encounter the ill effects of global warming. There is a dire need to recover the lost fertility of the barren lands. The situation in arid environment is even graver where a short favorable season is followed by prolonged dry season. Slow growth and indiscriminate logging of trees for various human requirements have also contributed to a dearth of harvestable trees.
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Acacia, a genus of the tropical and subtropical regions of Australia, South America, Asia and Africa, is a conglomeration of about 1,350 species (Seigler 2003) that cater to the human requirements for fuel wood, timber, fodder, tannin, pulpwood, shelterbelts, medicine, and soil improvement (Jones et al. 1990; Simmons 1987). These plants, predominantly trees, are well adept to endure the extreme temperature and moisture stresses and therefore, can be grown both in arid and moist regions of the world on a wide range of tropical soils. The presence of antecedent properties concurrently make them highly valuable for reclamation of denuded lands by providing a permanent green cover in harsh environment and simultaneously helping the soil to convalesce. A large scale cultivation of acacia trees on waste lands would be an efficient way to maintain soil fertility. However, traditional methods of cultivation are not sufficient to cope with the increasing demand because of poor seed germination rate and high mortality of seedlings in the natural conditions (Nanda and Rout 2003). Acacia ehrenbergiana Hayne, an indigenous legume of the Middle Eastern part of the world, provides quality honey with antioxidant properties (Al-Mamary et al. 2002), charcoal, fire wood and Qatran (a fluid used in the treatment of animal skin diseases and also in painting furniture to protect them from insects) (Al-Jeffri and Haile 2009). The plant extract shows antibacterial and anti-fungal activities (Al-Jeffri and Haile 2009) and its bioactive constituents include gallic acid, rutin 200 -O-a-L-rhamnopyranosyl, myricetin 3-O-b-D-rutinoside, rutin, myricetin 3-O-b-D-glucoside,quercetin 3-O-b-D-glucoside (isoquercitrin), myricetin, quercetin and catechin (Gaara et al. 2008). The ground cover of this multipurpose tree is decreasing rapidly due to overgrazing, establishment of new agricultural farms, charcoal production and indiscriminate harvesting for timber (Al-Jeffri and Haile 2009). A large scale cultivation of this droughtresistant tree on arid lands would help in conserving its gene pool and reclaiming the wastelands. The benefits mentioned hereof could be best achieved with the application of tissue culture techniques. Tissue culture technology has been used successfully to overcome the limitations of traditional plant breeding methods and can produce large number of elite plant apace (Pence 1999). Plant regeneration by tissue culture has been attempted for few species of Acacia (Vengadesan
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et al. 2002) which unfortunately do not include A. ehrenbergiana. Keeping in view of the prevalent situation, in the present study we describe a micropropagation protocol for A. ehrenbergiana using the cotyledonary node explant, with a hope that its application would help to annihilate contingency of its availability for the future while simultaneously increasing fertile land.
Materials and methods Seed viability Tetrazolium test (TZ) was used to determine the seed viability. It is a reliable test which work on the principal that all living tissues which respire are capable of reducing a colorless chemical 2,3,5-triphenyltetrazolium chloride into red colored formazan by H? transfer reaction. Formazan being non diffusible, stain the living tissue, thus the living part of the seed is stained red. To test the viability, seeds were cut in half and incubated in dark with standard tetrazolium salt solution for 30 min. Thereafter, the seeds were observed for pink color development in viable seeds. Plant material and establishment of aseptic seedlings Seeds were obtained from mature pods collected in Medina Munawwara (KSA) and were authenticated from King Saud University, Riyadh. The seeds were scared with a sharp blade on the opposite side of embryo before being washed in running tap water followed by soaking in 5 % (v/v) solution of a detergent, Labolene (Qualigens, Mumbai, India) for 5 min. The seeds were sterilized through 0.1 % HgCl2 treatment for 3 min. Following repeated washes with sterile distilled water, the seeds were inoculated on semi solid Murashige and Skoog (1962) (MS) medium solidified with 0.6 % agar for germination. Cotyledonary node (CN) explants excised from 10 days old aseptic seedlings were used as explants. Culture media and culture conditions The culture media tested was MS with 3 % sucrose (w/v) and 0.6 % (w/v) agar. Plant growth regulators
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were added to the medium as specified below. The pH of the MS medium was adjusted to 5.8 by using 1 N NaOH or 1 N HCl prior to autoclaving at 121 °C for 20 min except for the experiment in which the effect of pH of the media was tested where the pH was adjusted to various levels (5.0, 5.4, 5.8, 6.2 and 6.4). All the cultures were maintained in a culture room at 24 ± 2 °C under a 16 h photoperiod with a photosynthetic photon flux density (PPFD) of 50 lmol m-2 s-1 provided by cool white fluorescent lamps (Phillips, India). Shoot multiplication For multiple shoot induction, CN excised from 10 days aseptic grown seedlings were placed on MS medium devoid of or supplemented with different cytokinins viz. benzyl adenine, kinetin and 2-isopantenyl adenine (BA, KIN, 2iP) at varying concentrations (2.5, 5.0, 10.0, 12.5 lM) either singly or in combination with different concentrations (0.01,0.1, 1.0 lM) of a-naphthalene acetic acid (NAA). Cultures were transferred to the same fresh medium after every 4 weeks. Data were recorded for frequency of explants producing shoots, number of shoots per explant and shoot lengths after every week of culture. Rooting of shoots in vitro Elongated healthy shoots (3–4 cm) were excised and cultured on root induction media comprising MS medium supplemented with indole-3-butyric acid (IBA) or NAA at different concentrations (0.5, 2.5, 5.0, 7.5 lM). Data were recorded on percentage of rooting and number of roots after 4 weeks of culture. Rooted plantlets were removed from root induction medium and were washed in sterile distilled water to remove all the adherent traces of agar and basal callus. The plantlets were then transferred to plastic pots (6 cm diameter) containing sterile soilrite (Keltech Pvt Ltd., Bangalore). The plastic pots were covered with polythene bags to maintain relative humidity. These pots were maintained at 24 ± 2 °C and with a 16 h light photoperiod and watered every 3 days with half strength MS salt solution for 2 weeks. Polythene bags were opened after 2 weeks in order to acclimatize plants to field conditions. After 4 weeks, acclimatized plants were transferred to pots containing normal garden soil and maintained in a greenhouse under natural light.
Statistical analysis All the experiments were set up in a completely randomized design and repeated three times with ten replicates for each treatment, five cultures were randomly selected from each treatment for conducting the statistical analysis. The data was subjected to analysis of variance (ANOVA) to detect significant difference between means. Means differing significantly were compared using Duncan’s multiple range test at P = 0.05. All the statistical analysis was done by using SPSS Ver 17 (SPSS Inc., Chicago, USA) Statistical software package. Shoot multiplication speed (SMS) and Shoot multiplication acceleration (SMA) were calculated using the following formulas. SMS ¼ Number of shoots in one passage =duration of passage ðweeksÞ SMA ¼ Change in SMS=duration ðweeksÞ Results Effect of cytokinin Morphogenesis was successfully induced in all the cultures except those inoc ulated on cytokinin-free media. Irrespective of the type of cytokinin, increase in concentration increased the parameters concerned up to an optimum concentration, to be followed by a slight decrease on any further increase in the concentration of the cytokinins. All the cytokinins tested were most effective at 10.0 lM concentration. Among the different cytokinins tested BA was found to be most efficient in inducing the morphogenic response by inducing bud formation in the explant (Fig. 1a), giving a 90.3 ± 2.4 % regeneration frequency at the optimum concentration, while KIN and 2iP at the same concentration could evoke morphogenesis in only 87.3 ± 1.2 % and 84.6 ± 0.8 % of cultures respectively. A similar pattern was also observed in multiple shoot induction, with BA inducing the maximum number of shoots (6.60 ± 0.24) per explant followed by KIN (4.0 ± 0.12) and 2iP (2.7 ± 0.12) at the optimum concentration (Table 1). However, KIN was most effective in stimulating the elongation of regenerated shoots, resulting in the greatest shoot length (4.0 ± 0.12) per shoot at 10.0 lM. A steep increase in
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Fig. 1 a Induction of shoots after 4 weeks of culture on BA (10 lM). b Multiplication after 4 weeks on BA (10 lM) ? NAA (0.1 lM) supplemented MS medium. c Culture 12 weeks after inoculation, with elongated shoots. d Rooted
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plantlet. e Acclimatized plantlet after 2 weeks acclimatization. f Hardened plantlet 6 weeks after transfer to the natural conditions
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height could also be noticed in BA treated cultures on moving from suboptimal to optimal concentration however, KIN was superior at all concentrations tested, while 2iP lagged behind both the cytokinins (Table 1). Effect of auxin Addition of auxin acted synergistically for multiple shoot formation as well as shoot length. Among the different concentrations tested 0.1 lM concentration of NAA gave the best result in terms of number of shoots giving 7.3 ± 0.15 (Fig. 1b, c) shoots per explant, any increase beyond this optimum concentration, a detrimental effect was observed on shoot multiplication however, shoot length continued an increasing trend (Fig. 2). Effect of culture period Time is a crucial factor when it comes to commercial application of tissue culture protocols. Therefore, we calculated multiplication speed and acceleration to find out best possible time to move from one stage of the protocol to the next, which would help us to make fullest utilization of the resources with minimum possible wastage of time and money. It was observed that soon after second sub-culturing passage SMS decreased gradually and became negligible at the fifth sub-culturing (Fig. 3). While SMA remained positive till the second sub-culturing (Fig. 4). These results helped us to decide the time for extraction of shoots for rooting (at third passage) and thus saved time and money by avoiding further sub-culturing. Effect of pH of the medium pH of the media was found to affect the shoot multiplication significantly. pH 5.8 was found to be ideal for regeneration any shift on either side of this pH adversely affected the shoot multiplication. However, shift towards more acidic pH was more damaging then a shift towards neutral pH (Fig. 5). In vitro rooting A threshold concentration (2.5 lM) of auxins was found necessary for inducing rizogenesis in the regenerated shoots of A ehrenbergiana. The MS medium
devoid of or supplemented with auxin concentration lower than the threshold failed to develop roots even after 4 weeks of incubation. The number of roots per shoot produced by the two auxins (IBA and NAA) did not differ significantly. However, the percentage response differed significantly (Fig. 6). The best medium to induce rooting was found to be MS medium supplemented with IBA (5.0 lM) which amounted for 3.6 ± 0.23 roots per shoot in 80 % of the regenerated shoots transferred to rooting medium (Fig. 1d). While at the same concentration NAA was able to induce 3.0 ± 0.25 roots per shoot in only about 40 % shoots transferred (Fig. 6). Proving IBA is far superior for in vitro rooting in this particular species. Hardening and acclimatization Plantlets with 3–4 internodes and well developed root system were removed and transferred to pots containing sterile soilrite. The shoots were kept for 2 weeks under low illumination covered with poly bag and for another 2 weeks without poly bags (Fig. 1e), before being established eventually to the natural soil. About 80 % of the plantlets survived during acclimatization. The regenerated plants did not show detectable variation in morphological or growth characteristics compared with the normal seedling (Fig. 1f).
Discussion Low seed germination has been frequently reported in woody trees especially in legumes (Aliero 2004; Ayisire et al. 2009; Holmes et al. 1987). In the species under study only 13.3 % of seed germination has been reported under in vitro conditions (Al-Khalifah 2004). This poses a major hurdle in its propagation both in vivo and in vitro as it not only significantly increases the cost but also increases the time, labor and space required many fold. Therefore, it becomes important to investigate into the reasons for low seed germination and do necessary manipulations to overcome this problem. Since, our experiment revealed high seed viability, physical barrier to the water and nutrient was found responsible for low germination rate. We have successfully alleviated this problem through mechanical scarification, about 98 % of the seeds germinated within 3 days of inoculation instead of 13.3 % seeds in about 8 days (Table 2). Seed-coat scarification has
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Agroforest Syst Table 1 Effect of different Cytokinins on Multiple shoot regeneration in Acacia ehrenbergiana after 12 weeks of culture BA
KIN
2-iP
2.5
Percentage regeneration
Shoot no./explant
Shoot length/shoot
73.0 ± 1.5e
2.2 ± 0.18de
1.3 ± 0.08h
bc
5.0
83.3 ± 1.7
10.0 12.5 2.5
3.8 ± 0.14
b
2.0 ± 0.30g
a
a
90.3 ± 2.4 84.0 ± 0.5d
6.6 ± 0.20 4.0 ± 0.25b
3.8 ± 0.17a 3.3 ± 0.05bc
78.0 ± 1.1d
1.2 ± 0.18f
2.3 ± 0.12efg
cd
2.6 ± 0.11
cd
3.0 ± 0.14cd
3.9 ± 0.08
b
4.0 ± 0.12a
80.0 ± 0.5
3.7 ± 0.15
b
3.6 ± 0.13ab
2.5
67.0 ± 2.5f
0.9 ± 0.17f
5.0
e
1.2 ± 0.20
f
2.2 ± 0.12fg
2.8 ± 0.12
c
2.7 ± 0.12de
2.0 ± 0.26
e
2.5 ± 0.17ef
5.0
81.0 ± 0.8
ab
10.0
87.3 ± 1.2
cd
12.5
71.6 ± 1.2
bc
10.0
84.6 ± 0.8
d
12.5
77.3 ± 1.1
1.9 ± 0.08g
Values represent mean ± SE of three randomly selected readings of 10 replicates per treatment in three repeated experiments. Means sharing the same letter are not significantly different (P = 0.05) using Duncan’s multiple range test
8
7
b
SL vs NAA SN vs NAA
ab
6
c
5
ab
a
bc
4
c
1.2
Shoot multiplication's speed
Shoot number and Shoot length
a
1.0
0.8
0.6
0.4
0.2
3 0.00
0.01
0.10
1.00
Concentration of NAA(µM)
Fig. 2 Line graph shows the effect of various concentration of NAA on shoot number and shoot length per shoot. Values represent mean ± SE. Means sharing the same letter are not significantly different (P = 0.05) using Duncan’s multiple range test
been reported to be beneficial in similar cases by various workers (Doran et al. 1983; Kaul and Manohar 1966; Sambe et al. 2010; Abari et al. 2012). Presence of exogenous cytokinin in the media in our study was found essential for regeneration as was found in Vitex negundo (Ahmad and Anis 2007), Melia azaderach (Husain and Anis 2009), Blanites aegyptica (Siddique and Anis 2009). It was observed that the death of the explants in cytokinin free media was preceded by the release of phenolic compounds
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0.0 0
1
2
3
4
5
6
Subculturing passages
Fig. 3 Graph shows the effect of type of time on Shoot multiplication’s speed
which shows the inability of the endogenous hormones to remove the ill-effects of stress endured by the explant due to mechanical injury. Phenolic compounds such as nicotine, caffeine, morphine, colchicines, ergoline, strychnine etc. which are released by Acacia species under stress readily oxidises to form quinones resulting in blacking of the tissue and the medium. This inactivates the growth of the tissue in the culture. Increase in concentration of cytokinin in the media favored growth and multiplication and showed positive correlation up to an optimum concentration beyond which a slight decrease in the parameters was recorded.
Agroforest Syst 5 IBA NAA
0.2
0.1
0.0 0
1
2
3
4
5
6
Number of roots per shoot
Shoot multiplication's acceleration
0.3
80 a
4
40 b
60 b
3 50 c
40 bc
15 d
2
1
-0.1
e
e
e
0
0
0
0.5
1.0
0 0.0 -0.2
2.5
7.5
5.0
Concentration of auxin (µM) Subculturing passages
Fig. 4 Graph shows the effect of the type of time on Shoot multiplication’s acceleration
Fig. 6 Bar graph shows the effect of different concentrations of auxins on number of root induced per shoot. Values represent mean ± SE. Means sharing the same letter are not significantly different (P = 0.05) using Duncan’s multiple range test
8
Number of shoots per explant
a ab
b
Table 2 The effect of different scarification treatments given to the seeds of Acacia ehrenbergiana before inoculation on to MS medium without PGRs
6 e
4
d
2
0 5.0
5.4
5.8
6.2
6.4
Treatment
Days to germination
Percentage germination
Control
4–12 days
11
Hot water treatment
3–8 days
32
Mechanical scarification
2–4 days
98
pH of the media
Fig. 5 Bar graph shows the effect pH of media on shoot number per explant. Values represent mean ± SE. Means sharing the same letter are not significantly different (P = 0.05) using Duncan’s multiple range test
The optimum concentrations of different cytokinin were recorded to be similar, this might be because of the existence of a threshold level of cytokinin that can be stored in the plant, then exceeding this concentration would evoke a protective defense response resulting in formation of N-conjugate product or N6 side chain cleavage product of cytokinins through oxidative cleavage of the side chain making it inactive (van Staden and Crouch 1996) also the energy required in doing so becomes unavailable for growth and development of shoots thus causing reduction in the growth parameters. BA evoking a better response than other cytokinins can be because of the specific nature of the receptors or the metabolic enzymes. Synergistic effect of auxin in combination with the optimum level of cytokinin has been well documented in various species (Faisal et al. 2006; Siddique et al.
2006) as was recorded in our study. However, the concentration of auxin required was found to be very low which indicate towards a high endogenous level of auxin in the explant, this is also reinforced by high requirement of exogenous cytokinin as the auxin has been shown to inhibit the transcription of cytokinin biosynthesis gene ISOPENTENYLTRANSFERASE 1 and ISOPENTENYLTRANSFERASE 2 (PsIPT 1 and PsIPT 2), which in turn causes reduced cytokinin biosynthesis in the stem (Tanaka et al. 2006; Ferguson and Beveridge 2009). Exogenous auxin also has a similar effect on the gene expression (Muller and Leyser 2011). A comparatively lower rate of shoot multiplication was observed in the initial stages of culture (Fig. 3) owing to the oxidative stress faced by the explants because of the mechanical injury endured during inoculation. Some of the cytokinin is used up in overcoming this stress as is evident from the fact that cultures devoid of cytokinin die after release of
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phenolic compounds which are indicators of stress. Explants also spend some of its energy to adapt to the culture condition which would otherwise be used in the growth and development of explants, this is evident from the fact that explants taken from in vitro source perform better than similar explants taken from ex vitro source (data not shown). As the harmful compounds are removed and explants adapts to the culture condition a better response is observed, at this stage optimum utilization of all the resources occur. This phase continue for 4–6 weeks thereafter a slight decrease in multiplication is observed (Fig. 3), which is accompanied with a greater increase in the height of the regenerated shoots, this trade-off between shoot number and shoot length can be attributed to various endogenous biochemical changes. As the number of shoots increases so does the site of auxin biosynthesis resulting in increased endogenous auxin concentration which on one hand down regulates cytokinin biosynthesis and induces strigolactone biosynthesis which has been proved to inhibit branching and induce elongation (Hayward et al. 2009). pH of the medium was found to have a significant effect on shoot multiplication and growth of the regenerants. It not only affects the uptake of medium’s ingredients but also have a influences on the chemical reactions especially those catalysed by enzymes (Thorpe et al. 2008). We found that adjustment of medium pH to 5.8 before autoclaving to be most conducive for the growth and development of regenerants, similar results have also been reported in Azadirachta indica (Gautam et al. 1993), Calophyllum apetalum (Nair and Seeni 2003), Aralia elata (Karim et al. 2007), etc. any shift on either side of this pH resulted in noticeable reduction in growth parameters which might be because of the loss in energy available for growth and development as it is used in adapting to the substrate pH (Thorpe et al. 2008). Rooting of isolated shootlets was achieved by transferring them onto rooting media containing auxins (IBA, NAA). Of the two auxins tried IBA proved most effective at 5.0 lM concentration. The effectiveness of IBA for inducing in vitro rooting in woody plants has been well documented (Ahmad et al. 2010; Siddique et al. 2010). Initiation of rooting takes relatively a longer time which might be a side effect of incubation under high cytokinin levels, which might cause a modified metabolic enzymes activity that depends on PGR concentration, a modification of the
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receptor site might also occur which makes the auxin less effective a detailed evaluation of endogenous biochemical environment is however necessary to reach a definite conclusion as to the cause of this problem.
Future prospects The study described here evaluates the regenerative capacity of the somatic tissues of A. ehrenbergiana. This can be used to develop commercial scale protocol with slight modification according to the scale and place of production. This would provide a reservoir of transplantable hardened plantlets which could be planted in barren arid land where the conditions are unfavorable for seed germination and seedling survival, where it would provide permanent green cover, fodder for cattle, source of food for bees and thus honey with high antioxidant properties and good quality harvestable timber while it would also replenish the soil fertility through increasing the nitrogen content and organic matter. This would also annihilate the concern about the extinction of the species generated due to its fast decreasing numbers in the wild. However further research in the physiological aspect of this tree is required to increase the growth rate after field transfer. Acknowledgments Corresponding author greatfully acknowledge King Saud University for giving him Visiting Professorship of KSU. We are also thankful to UGC for financial support under DRS-I SAP (2009–2014) programme.
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