43 Effect of Cytokinins and Carbenicillin on In vitro Axillary-Shoot Growth of Albizia [Albizia falcataria (L.) Fosberg]

  Effect of Cytokinins and Carbenicillin on In vitro Axillary-Shoot Growth of Albizia [Albizia falcataria (L.) Fosberg]

• )

   Sri Nanan Widiyanto , Mirah Dila Sari, and Rina Ratnasih Irwanto School of Life Sciences and Technology, Bandung Institute of Technology, Bandung e-mail: srinanan@sith.itb.ac.id

  Received 11 March 2008, accepted for publication 22 May 2008

  Abstrak

  6 Penelitian ini dilakukan untuk mengevaluasi pengaruh N -benzilaminopurin (BA), 6-furfurilaminopurin (kinetin) dan karbenisilin terhadap pertumbuhan pucuk aksiler nodus kotiledon albizia [Albizia falcataria (L.) Fosberg] secara in

  vitro. Komposisi medium induksi pucuk terdiri dari medium dasar Murashige dan Skoog (MS) dengan atau tanpa

  

pemberian BA atau kinetin. Sensitivitas terhadap karbenisilin diuji menggunakan medium induksi pucuk dengan

penambahan karbenisilin 10-50 mg/L. Hasil penelitian menunjukkan bahwa pertumbuhan pucuk-aksiler yang optimal terlihat pada medium yang mengandung 2,2 µM BA dan 2,3 µM kinetin, yang menghasilkan panjang pucuk rata-rata

36,0 ±2,2 mm dengan jumlah nodus/pucuk 2,9±1,1 dalam empat minggu. Pemberian karbenisilin menghambat

pertumbuhan tunas aksiler, yang terlihat dari panjang pucuk 6,1-11,5 mm dengan jumlah nodus/pucuk 1,0-1,1.

Sebanyak 60 pucuk hasil regenerasi mengalami pemanjangan dan pembentukan akar secara in vitro pada medium MS tanpa zat pengatur tumbuh. Semua pinak tanaman baik yang dikultur dalam medium dengan atau tanpa penambahan BA atau kinetin tumbuh baik di rumah kaca. Kata Kunci: Mikropropagasi, Albizia falcataria (L.) Fosberg, Sengon Abstract

6 Effect of N -benzylaminopurine (BA), 6-furfurylaminopurine (kinetin) and carbenicillin on axillary-shoot growth of

  albizia [Albizia falcataria (L.) Fosberg] from cotyledonary node-segments were evaluated in vitro. Shoot induction medium was composed of Murashige and Skoog (MS) basal medium with or without the addition of BA or kinetin. The sensitivity to carbenicillin was tested using shoot induction medium with the addition of carbenicillin at 10-50 mg/L. The results showed that the optimal axillary-shoot growth was observed on medium containing 2.2 µM BA and 2.3 µM kinetin, which reached the average of 36.0 ±2,2 mm shoot-length and 2.9±1,1 nodes/shoot in four weeks. The addition of carbenicillin inhibited the axillary-shoot growth, shown by 6.1-11.5 mm of shoot length of and 1.0-11.1 of nodes/shoot. A total of 60 shoots were elongated and rooted in in vitro condition on MS medium without growth regulator. All plantlets were successfully transferred to a soil-sand medium (1:1) and grew well in the green-house.

  Keywords: Micropropagation, Albizia falcataria (L.) Fosberg, Sengon

1. Introduction a possible approach to overcome the limited success of the conventional genetic improvement methods.

  The Latin name of albizia or sengon is

  Agrobacterium -mediated transformation has Paraserianthes falcataria (L.) Nielsen and was

  become one of the favored methods for introduction of formerly known as Albizia falcataria (L.) Fosberg. It is foreign genes into higher plants. Genetic an indigenous plant species to the Eastern provinces of transformation mediated by Agrobacterium requires the Indonesia. Like many other tropical legume trees, P. use of antibiotics in the selection and regeneration

  falcataria is a fast growing species that has natural

  medium to eliminate the bacterial growth. It was nitrogen-fixing capacity, and is also becoming one of indicated that the sensitivity of explant tissues to dedicated energy crops. Because of its potential in re- antibiotics was the main obstacle in regeneration of afforestation, albizia has been extensively planted in selected transformed explants. Currently, carbenicillin poor, dry or marginal lands. However, despite its is one of many antibiotics widely used in increasing potential, improvement program of albizia

  Agrobacterium -mediated gene transfer, although it may

  tree through conventional breeding is still a slow interfere with the growth of plant tissues. It was process (Lemmens and Soerianegara, 1994; Bassam, reported that carbenicillin inhibited the regeneration 1998). Therefore, it is important to develop alternative ability of Picea omorika embryogenic cells tools to improve the genetic quality of the planting

  (Milhaljevi ć et al., 2001) but promoted callus growth

  6

• benzylaminopurine (Sigma) at 2.2 and 4.4 µM, alone or in combination with 6-furfurylaminopurine (Sigma) at 2.3 and 4.6 µM. For the carbenicillin sensitivity test, the optimal shoot induction medium was used with the addition of carbenicillin (PhytoTechnology) at 10, 20, 30, 40 and 50 mg/L. Carbenicillin was filter-sterilized and added to the medium after autoclaving and cooling to 40-50

  6

  44 JURNAL MATEMATIKA DAN SAINS, JUNI 2008, VOL. 13 NO. 2 and shoot regeneration in loblolly pine transgenic tissues (Tang et al., 2004). Effects of various antibiotics on the growth of plant tissues were also studied in carnation (Estopa et al., 2001), papaya (Yu et al., 2001), and apricot (Petri et al., 2005).

  The in vitro regeneration procedure on albizia was successfully developed through the single node- cutting technique. The use of cytokinins, i.e. N

• benzylaminopurine (BA) and 6-furfurylaminopurine (kinetin) seemed to be the critical factor in stimulating

  in vitro shoot development of albizia (Bon et al., 1998;

  C under a pressure of 1.2 kg/cm

  It was indicated that the seed viability of albizia was very high.

  3.1 Seed germination Seeds started to germinate after 3-5 days and were growing fast since then. After six days, roots were elongated, cotyledons were expanded and compound leaves were obviously visible in 4-6 cm albizia seedlings. The results showed a high percentage of germination ability of seeds (90-92 %, data not shown).

  3. Results and discussion

  randomized-block design. Each treatment consisted of 4-5 replicate culture vessels with 3-4 explants per vessel. Explants were randomly allocated in each vessel. Experiments were repeated twice. Data were analyzed using ANOVA and Duncan’s New Multiple Range Test (DNMRT).

  al ., 2005), and grown naturally in the green house. In vitro experiments were arranged as a complete-

  2.3 Experimental procedures Elongated seedlings (4-6 cm) were used as the source of explants in shoot induction experiments and carbenicillin sensitivity tests. Shoot-tips and roots from seedlings were removed to obtain about 8-10 mm long of cotyledonary node-cuttings (Figure 1), which were cultured on various shoot induction medium with or without addition of carbenicillin. After four weeks, the shoot length (mm) and the numbers of nodes per elongated axillary shoot were measured and used as the growth parameters. All in vitro cultures were maintained in a culture room at 25±2 ºC under continuous light emitted by 40W Philips cool white fluorescent tubes. A large part of regenerated shoots was maintained in in vitro culture condition for being used in further experiments. A total of 60 shoots (6-8 cm shoot-length and 3-5 cm root-length) were collected from medium containing 2.2 µM BA and 2.3 µM kinetin. Those plantlets were transferred to soil:sand mixture (1:1) for acclimatization, following the procedure previously developed (Sasmitamihardja et

  2 for 15 min.

  o

  Sasmitamihardja et al., 2005). Comparative studies of cytokinins were also reported in axillary shoot development of node micro-cuttings of other leguminous tree species, such as Pterocarpus

  C. The medium pH was adjusted to 5.7-5.8 with addition of 1-2 drops of 0.1 N NaOH or 0.1 N HCl. All culture media were autoclaved at 121

  o

• benzylaminopurine (BA) and 6-furfurylaminopurine (kinetin) on the axillary shoot-growth of albizia from cotyledonary node-cuttings.

  2.2 Culture media All in vitro experiments used MS medium composition based on Murashige and Skoog (1962) which was added with 3% (v/v) sucrose and 0.8% (w/v) agar (Sigma) to solidify. For seed germination, the medium was prepared without the addition of growth regulator. For the shoot induction medium optimalization, the media were supplemented with N

  2.1 Plant material Albizia seeds were obtained from a tree grown in our campus area (Bandung Institute of Technology) and collected during the dry season in June-July 2006. Seeds were washed thoroughly with a little amount of detergent under running tap water for 30 min and disinfected by immersing them in 70% (v/v) alcohol for 1 min and another 10 min in 50% household bleach solution (active ingredient: 5.25% sodium hypochlorite) with drops of Tween-20. Sterile seeds were rinsed in sterile distilled water three times and imbibed with sterile distilled water over night before placing them on germination medium. After 5-6 days, the percentage of seed germination was calculated to evaluate the viability of albizia seeds. Six day-old seedlings were used as the sources of cotyledonary node-cuttings.

  6

  a transformation procedure using Agrobacterium as the mediator, the effects of carbenicillin on in vitro regeneration of albizia need to be evaluated. The objective of our research was to evaluate the influence of carbenicillin and cytokinins, i.e. N

  marsupium (Chand and Singh, 2004), Albizia odoratissima (Rajeswari and Paliwal, 2006), Albizia chinensis (Sinha et al., 2000). In an attempt to develop

2. Materials and methods

  Widiyanto et al., Effects of Cytokinin and Carbenicillin on In vitro Axillary-Shoot

  45 demonstrated in Pterocarpus marsupium cotyledonary nodes (Chand and Singh, 2004).

  Table 1. Responses on axillary shoot-buds of albizia

  cotyledonary node-cuttings by various concentrations of BA and kinetin, after four weeks in shoot induction experiments

  Cytokinins Growth responses of*) BA Kinetin Shoot Length No.of Nodes

  (µM) (µM) (mm) per Shoot

  b xy

  29.7 ± 4.2 2.3 ± 0.9

  b y

  2.3 29.7 ± 7.3 2.4 ± 0.7

  ab xy

  4.6 27.2 ± 7.7 2.2 ± 1.3

  ab x

  2.2 23.5 ± 7.7 1.2 ± 0.4

  c y

  2.2 2.3 36.0 ± 2.2 2.9 ± 1.1

  a y

  2.2 4.6 22.4 ± 2.8 2.6 ± 0.7

  Figure 1. Cotyledonary node-cuttings of albizia (the a xy

  4.4 23.2 ± 3.7 2.0 ± 0.5 arrow indicates the cotyledon). Bar = 5 mm.

  b y

  4.4 2.3 29.4 ± 1.5 2.7 ± 0.5

  ab xy

  4.4 4.6 25.9 ± 6.3 2.3 ± 0.9

• ) Each value represents the mean ± SE of 4-5 replicates, each treatment with 10-12 explants. Value followed by the same letters, within each column, are not significantly different at the 0.05 level as determined by Duncan’s Multiple Range Test (DMRT).

  The different effects of BA and kinetin on axillary shoot growth might be due to different mode of action of BA and kinetin in shoot development. Like other cytokinins, both BA and kinetin are known to promote cell division and cell expansion in plant development. However, suitable cytokinin types and concentrations to stimulate shoot formation and growth vary depending on plant species. Many studies show that kinetin is effective in stimulating axillary shoot

  

Figure. 2. Elongated axillary shoots of albizia with 2-3 elongation but ineffective for shoot multiplication. In

  nodes. Bar = 5 mm. contrary, BA is known to be very effective in stimulating axillary shoot multiplication rather than

  3.2 Axillary shoot elongation inducing shoot elongation (Bon et al., 1998; Sinha et In the period of four weeks, axillary shoots al ., 2000; Rajeswari and Paliwal, 2006). The were growing from cotyledonary node-buds of albizia effectiveness of BA in inducing shoot proliferation of in all the shoot multiplication media, although no nodal cuttings was reported in many plant species, such multiple shoot formation was observed (Figure 2). as in various species of Albizia (Bon et al., 1998; Sinha Different effects of BA and kinetin were indicated on et al ., 2000; Rajeswari and Paliwal, 2006), in Acacia the stimulation of axillary shoot growth (Table 1). mangium (Bon et al., 1998), in Bacopa monniera Generally, the shoot length is in the range of 22.4-36.0 (Tiwari et al., 2001) and in pear (Kadota and Niimi, mm. In comparison to the shoot length of the control 2003). Our previous research (Sasmitamihardja et al., (29.7 mm), the addition of BA alone at 2.2 and 4.4 µM 2005) showed that axillary shoots of albizia were also caused a significant negative response in shoot length able to proliferate from nodal explants on media (23.2 and 23.5 mm). On the other hand, the addition of containing 0.1-1.0 µM of BA in combination with kinetin at 2.3 and 4.6 µM gave rise to the averages of 0.01-1.0 µM indole-acetic acid (IAA). However, the 29.7 and 27.2 mm shoot length. It indicated that kinetin influence of BA-IAA combination in shoot was more likely to influence shoot elongation rather proliferation apparently depends on the concentration. than BA. The different effect of BA and kinetin in The highest number of shoot proliferation was obtained

  46 JURNAL MATEMATIKA DAN SAINS, JUNI 2008, VOL. 13 NO. 2 0.1 µM IAA and 0.5 µM of BA. On the other hand, the addition of IAA in combination with BA strongly inhibited shoot elongation and induced callus formation at the cut ends of cotyledonary nodal cuttings.

  To provide with an efficient procedure of micro-propagation using the node-microcutting technique, beside the length of shoots, the number of nodes per shoot was also a crucial parameter. The reason is that more nodes per shoot would produce more node-cuttings per shoot. The efficiency of shoot- multiplication from node-cuttings can be calculated with the optimal size of one node micro-cutting is 8-10 mm length (Borthakur et al., 2000).

  Our experimental results showed that the addition of BA and kinetin also affected the formation of nodes per shoot. On medium with BA only at 2.2- 4.4 µM, the average numbers of nodes per shoot (1.2- 2.0) was significantly lower compared to that of the control (2.3 nodes per shoot). The addition of kinetin alone induced a higher average numbers of nodes per shoot (2.4) at 2.3 µM and decreased the average numbers of nodes per shoot at 4.6 µM (2.2). However, the combination of BA and kinetin seemed to influence both the axillary shoot elongation and node formation in albizia. The highest average of shoot-length (36.0 mm) with highest numbers of nodes per shoot (2.9) was observed on medium added with 2.2 µM BA in combination with 2.3 µM kinetin (Table 1). Similarly, Bon et al. (1998) reported that the use of BA in combination with kinetin in appropriate concentration would yield the optimal shoot multiplication of albizia.

  Based on the results, in this research, the shoot induction medium containing 2.2 µM BA and 2.3 µM kinetin was determined as the optimal shoot induction medium and used as the control medium in carbenicillin sensitivity-test experiments. A total of 60 representative shoots, which were collected from medium containing 2.2 µM BA and 2.3 µM kinetin, were sub-cultured on MS medium without growth regulator in the second subculture by following the procedure previously developed (Sasmitamihardja et

  al ., 2005). The result showed that all shoots were able

  to elongate and form roots (Figure 3), and grow into plantlets with 6-8 cm shoot-length and 3-5 cm root- length in four weeks. Complete plantlets were successfully transferred to a soil-sand medium (1:1) and acclimatized. All sixty plantlets survived and grew well in the green house (Figure 4).

  Figure. 3. Rooted shoots of albizia, ready to be transferred to soil-medium. Bar = 10 mm.

  Figure 4. In vitro regenerated plants of albizia survived and grew well in the green-house. Bar = 10 mm.

  3.3 Carbenicillin sensitivity The effects of carbenicillin were evaluated after 4 weeks of nodal explants cultured on shoot induction medium containing 2.2 µM of BA, 2.3 µM of kinetin and 10-50 mg/L of carbenicillin. The results indicated that cotyledonary shoot-buds of albizia were very sensitive to carbenicillin. In 2 - 3 weeks, toxicity of carbenicillin was observed on medium with 50 mg/L, which was indicated when the stems and shoot- buds became yellowish, and cotyledons were necrotized (Figure 5). In general, the average growth of albizia shoot-buds reduced with the addition of carbenicillin (Figure 6). In comparison to the optimal growth on shoot induction medium (averagely 36.0 mm shoot length with 2.9 numbers of nodes per shoot), the addition of carbenicillin decreased the range of average shoot length and numbers of nodes that gave rise to 6.1-11.5 mm shoot length and 1.0-1.1 nodes per shoot (Figure 7).

  Widiyanto et al., Effects of Cytokinin and Carbenicillin on In vitro Axillary-Shoot

  47 Seedling tissues and organs, such as meristem shoot-tips, hypocotyl, cotyledons, cotyledonary-node segments, are often used as the target tissues in plant genetic transformation due to the high regeneration ability of those tissues. However, the application of antibiotics strongly affects growth and regeneration of meristematic and seedling tissues in a number of plant species. Therefore, effects of antibiotics, either inhibiting or promoting, on growth of various plant tissues and embryogenic cells have been widely studied (Yu et al., 2001; Tang et al., 2004; Petri et al., 2005).

  The sensitivity of plant tissues to antibiotics seems to be species-specific and depends on a large extent on the plant growth conditions. In papaya, carbenicillin at 125 mg/L stimulated the formation of

  Figure 5. Toxicity of carbenicillin caused the embryogenic callus from leaf tissues. Further obser-

  yellowish color of stem and shoot-bud, and necrosis of vation indicated that the addition of carbenicillin at the cotyledons (as indicated by arrow). Bar =10 mm. 125-250 mg/L seemed to promote the development of abnormal somatic embryos from papaya embryogenic callus (Yu et al. , 2001). In apricot, antibiotic sensitivity seemed to be dependent on the concentration of antibiotics used. At the inhibitory concentration (8.4- 16.8 mg/L), the development of adventitious shoots from leaf tissues of apricot were inhibited. However, at the lower concentration (4.2-8.4 mg/L), antibiotics (streptomycin, paromomycin, and geneticin) allowed leaf tissues to grow and regenerate into shoots. Among the antibiotics used, geneticin was the most toxic and highly lethal to the apricot shoot tissues (Petri at al., 2005). Inhibitory effect of carbenicillin (at 200-500 mg/L) on regeneration ability was also reported in embryogenic tissues of Picea omorika (Milhaljevi

  ć et

  al ., 2001). Interestingly, in transgenic tissues of Agrobacterium -mediated transformed loblolly pine, the Figure 6. Elongated axillary shoot of albizia on

  addition of antibiotics such as carbenicillin, claforan medium containing carbenicillin 40 mg/L. Bar = 10 and timentin at high concentration (350-500 mg/L) mm. ₄₀ increased the differenttiation ability of transgenic calli ₃₅ to form adventitious shoots compared to that of the _{rs 30} control (Tang et al., 2004). In carnation, the use of _{m e te 25} different antibiotic combinations (kanamycin, _{P a ra 20} carbenicillin, vancomycin, and cefotaxime) at various _{s of e 15} concentrations (125-250 mg/L) was very effective not _{V a lu 10} only to eliminate Agrobacterium growth after cocultivation, but also influenced the growth and

  Value of Parameter ₅ differentiation of transgenic calli (Estopa et al., 2001). _{10 20 30 40 50} Carbenicillin is a ß-lactam antibiotic, which _{Carbenicillin Concentration (mg/l)} belongs to the penicillin group, and has a chemical Carbenicillin Concentration (mg/L) _{Shoot Length (mm) No.of Nodes/shoot} structure similar to that of penicillin-G, ampicillin,

  Shoot Length (mm) No. of Nodes/shoot

  amoxicillin, cefotaxime, timentin, and claforan. Like other ß-lactam antibiotics, carbenicillin is found to

  Figure 7. The combined data of the shoot length (mm)

  possess auxin-like structural feature. When it is broken and the numbers of nodes per shoot on media with down, carbenicillin displays effects similar to those of various concentrations of carbenicillin. Data were the weak auxin phenylacetic acid in the culture adopted from the carbenicillin sensitivity test

  48 JURNAL MATEMATIKA DAN SAINS, JUNI 2008, VOL. 13 NO. 2 of cultured explants (Tang et al., 2004). The poor response of axillary shoot-buds of albizia cotyledonary- node explants to carbenicillin in the present work might be a result of the possible release of auxin-like compounds that affects the balance of the exogenous and endogenous growth regulators of explants. As we mentioned above, our previous research showed that the addition of auxin (indole acetic acid) at 0.01-1.0 µM, in combination with 0.1-1.0 µM of BA, inhibited elongation of axillary shoot-buds of albizia nodal cuttings (Sasmitamihardja et al., 2005). Similar results was also reported by Bon et al. (1998) that BA (2.2-4.4 µM) or kinetin (2.3-4.6 µM) combined to auxins, i.e. indole butyric acid (1.5-2.5 µM ) or naphthalene acetic acid (1.6-2.7 µM), resulted in a strong inhibition of the growth capacity of albizia axillary shoots from nodal cuttings. Another possible reason of the antibiotic inhibition is suggested that like other antibiotics, carbenicillin may cause DNA hypermethylation, and so it affects expression of plant genes that control the production of hormones or growth regulators in young organ tissues. Therefore, meristematic and embryogenic cells are the most sensitive targets for antibiotics (Milhaljevi ć et al., 2001).

  Tissue Culture, Physiol. Plant, 15, 473-497. Petri, C., N. Albuquerque and L. Burgos, 2005, The

  Cult. , 72:3, 261-265.

  Lemmens, R. H. M. J. and I. Soerianegara, 1994, Timber trees: Major Commercial Timbers.

  Plant Resources of South-East Asia, PROSEA, Bogor. Milhaljevi

  ć, S., M. Perić, and S. Jelaska, 2001, The Sensitivity of Embryogenic Tissue of Picea

  omorika (Panc.) Purl. to Antibiotics, Plant Cell Tiss. Org. Cult

  ., 67:3, 287-293. Murashige, T. and F. Skoog, 1962, A Revised Medium for Rapid Growth and Bioassay with Tobacco

  Effect of Aminoglycoside Antibiotics on the Adventitious Regeneration from Apricot Leaves and Selection of nptII-transformed Leaf Tissue, Plant Cell Tiss. Org. Cult., 80:3, 271-276.

  Dev. Biol-Plant , 40:2, 167-170.

  Rajeswari, V. and K. Paliwal, 2006, In vitro Propagation of Albizia odoratissima L.F.

  (Benth.) from Cotiledonary Node and Leaf nodal explants, In Vitro Cell. Dev. Biol-Plant,

  42:5, 399-404.

  Sasmitamihardja, D., J. S. Hadisutanto, and S. N.

  Widiyanto, 2005, In vitro Regeneration of

  Paraserianthes falcataria (L.), Acta

  Kadota, M. and Y. Niimi, 2003, Effect of Cytokinin Types and Their Concentrations on Shoot Proliferation and Hyperhydricity in In vitro Pear Cultivar Shoots, Plant Cell Tiss. Org.

  Regeneration from Cotyledonary Node Explants of a Multipurpose Leguminous Tree, Pterocarpus marsupium Roxb, In Vitro Cell.

  This study provides important indications regarding the influence of BA, kinetin and carbenicillin in axillary shoot development from cotyledonary node- cuttings of albizia. Kinetin and BA stimulated the shoot elongation of albizia from node-cuttings differentially when added alone. In this research the medium containing 2.2 µM BA in combination with 2.3 µM kinetin was determined as the optimal medium for axillary shoot elongation of albizia, which induced the averages of 36.0 ± 2.2 mm of shoot length and 2.9 ± 1.1 numbers of nodes per shoot. Experimental results indicated that albizia shoot-buds were sensitive to carbenicillin. The addition of carbenicillin in shoot induction medium reduced the growth of axillary shoot-buds. For the future experiments, the use of other kinds of antibiotics rather than carbenicillin should become a consideration.

  James & James Sci. Publ. Ltd., London.

  Acknowledgement

  This study is a part of the research topics on In

  vitro Regeneration of Dedicated Energy Crops, which

  was financially supported by the ITB Research Fund 2006-2007 (SK No. 0018/K01.03.2/PL2.1.5/ I/2006).

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4. Conclusions

  Widiyanto et al., Effects of Cytokinin and Carbenicillin on In vitro Axillary-Shoot

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