Acta Physiologiae Plantarum

, Volume 34, Issue 3, pp 1207–1215

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume

Authors

    • Department of BiotechnologySchool of Life Sciences, Mizoram University
  • Lingaraj Sahoo
    • Department of BiotechnologyIndian Institute of Technology Guwahati
Original Paper

DOI: 10.1007/s11738-011-0917-3

Cite this article as:
Thangjam, R. & Sahoo, L. Acta Physiol Plant (2012) 34: 1207. doi:10.1007/s11738-011-0917-3
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Abstract

In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%) formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage of GUS+ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR+ plants.

Keywords

Parkia timorianaCotyledonary nodeIn vitro regenerationAgrobacterium-mediated genetic transformation

Abbreviations

MS

Murashige and Skoog (1962)

B-5

Gamborg et al. (1968)

NAA

α-Naphthalene acetic acid

BA

6-Benzylaminopurine

IBA

Indole-3-butyric acid

GUS

β-Glucuronidase

CaMV

35S 35S promoter of the cauliflower mosaic virus

nptII

Neomycin phosphotransferase

PCR

Polymerase chain reaction

OD600

Optical density at 600 nm

Introduction

Several conventional and non-conventional legumes are traditionally used by the locals of northeast India for food, medicine, fodder, dye and fish poison. At least 28 legume taxa growing wild in the region supplement the diet of the inhabitants and through ingenious ways of selection has gradually resulted in their domestication (Sharma et al. 2002; Saklani and Rao 2002). Parkia timoriana (DC.) Merr., popularly known as ‘tree bean’, belongs to the family Leguminosae (nom.alt. Fabaceae) and sub-family Mimosoiodeae. Tree bean is distributed from northeast India to Irian Jaya and is the most widely distributed species of the Indo-Pacific region (Hopkins 1994). It is a highly branched and medium height (10–12 m) multipurpose tree having potential commercial and ecological significance in the region (Kanjilal et al. 1982). The pod is consumed as vegetable, salad and chutney in all its developmental stages starting from the green tender pods to the matured black seeds either fresh or sun-dried for future use during off-season. The branches and woods are used as firewood or as timber. The tree is well adapted to grow in different agro-climatic regions from colder hilly regions to the hotter plains. It is commonly grown wild in the Jhums, forests and backyard of houses throughout northeast India without any special care. However, there are a few reports on the scientific research on this tree species. Suvachittanont et al. (1996) reported the presence of thiazolidine-4-carboxylic acid (thioproline), a cyclic sulphur-containing amino acid, contributing for the pungent smell in the seeds. Thioproline is known to be anti-carcinogenic and inhibits the formation of squamous cell carcinomas in the fore-stomach of rats (Tahira et al. 1984, 1988). Longvah and Deosthale (1998) reported the high nutritional values in the pods of tree bean. Tree bean is conventionally propagated through seeds and vegetative cuttings. However, vegetative cuttings are not feasible in view of low rooting percentage. Propagation using seeds is bestowed with severe fungal and pest problem during storage. During the last decade, many cases of mass-death of tree bean trees have been observed in the northeast Indian region due to the die-back symptoms seriously affecting socio-economic situation of the growers. Infestation of Cadra cautella on the pods collected from field and storage have been well documented (Thangjam et al. 2003a). The problems of die-back symptoms in this tree have also been found to be associated with the infestation of Anoplophora glabripennis (Motchulsky) commonly known as Asian longhorned beetle (manuscript under preparation). For a sustainable commercial cultivation of this tree, it would require large amount of superior quality and genetically improved planting materials that may be difficult to obtain by conventional methods of propagation. Thus, there is an urgent need for biotechnological interventions using tissue culture techniques for mass production of quality planting materials and genetic modification using gene(s) coding for insect resistance or proteinase inhibitors. The genetic improvement of any plant species requires the development of an efficient transformation and regeneration procedure for further transfer of gene of interest. However, there are no reports for such studies on this tree except for earlier report on the development somatic embryos (Thangjam and Maibam 2006) and also in another species P. biglobosa (Amoo and Ayisire 2005). The conditions for establishing an effective in vitro regeneration and Agrobacterium tumefaciens-based transformation through direct multiple shoot organogenesis from cotyledonary-node explants without cotyledons and the establishment of an optimal selection system has been reported here for the first time in this multipurpose tree legume.

Materials and methods

Plant material and explant preparation

Mature black pods were collected from an 18-year-old elite plus tree selected from a population of Narankonjin cultivar grown in Narankonjin area, Imphal (24.73°N latitude and 93.93°E longitude at an elevation of 780 m above sea level), India. The seeds were extracted and washed thoroughly with running tap water, pretreated with 0.1% aqueous solution of Bavistin (a systemic fungicide) for 15 min and washed three times with sterile water under laminar hood. After this, the seeds were treated with 0.1% mercuric chloride (HgCl2) solution for 15 min and rinsed five times with sterile water. The softened black coats were then removed with a sterile blade and the intact cotyledons were germinated on 1% water agar medium. Cotyledonary node explants of approximately 10 mm in size were excised from 10-day-old seedlings by removing the cotyledons and cutting both epicotyls and hypocotyls 20 mm above and below the nodal regions and use for the experiments.

Induction of multiple shoots, rooting and acclimatization

The explants were cultured in a vertically upright position with the hypocotyl cut ends slightly embedded in the MS basal medium (Murashige and Skoog 1962) and Gamborg’s B-5 basal medium (Gamborg et al. 1968) supplemented with NAA and BA. All the culture media were supplemented with 3% (w/v) sucrose and 0.7% (w/v) agar (Hi-media, Mumbai, India). The pH of the medium was adjusted to 5.8 prior to autoclaving at 15 psi and 121°C for 20 min and was maintained at 25 ± 2°C under a 16 h photoperiod with a photosynthetic photon flux density of 35 μmol/m2/s provided by cool white fluorescent tubes (Philips, India). They were sub-cultured every 2 weeks into a fresh media containing the same composition. After 4 weeks of culture, the efficacy of each treatment on shoot induction were determined by recording the percentage of explants forming multiple shoots, and the average number of shoots per explants. After 8 weeks of culture, individual shoots were separated from the explants and transferred in medium containing full and half-strength MS basal medium (MSB) supplemented with various concentrations of IBA and NAA. Plantlets with well-developed roots were removed from the culture medium, washed gently under running tap water, and transferred to plastic pots containing soil, vermiculite, and vermicompost (1:1:1). Plants were covered with transparent polyethylene bags to maintain adequate moisture for a week and transferred to the greenhouse (28°C day, 20°C night, 16 h day-length, and 70% relative humidity). After a week, the plastic covering was removed and the plantlets were maintained in the greenhouse in plastic pots containing normal garden soil until they were transplanted to the nursery. Each treatment in the experiment had three replicates of five explants each and repeated thrice.

Genetic transformation

Prior to the transformation experiment, an effective concentration of kanamycin for the selection of transformed shoots was determined by culturing non-transformed (control) nodal explants on shoot regeneration (SRM) medium (MSB + 2.7 μM NAA + 11 μM BA + 500 mg/l cefotaxime) containing different concentrations of kanamycin (0, 50, 75 and 100 mg/l). The cultures were transferred to a fresh medium containing the same level of antibiotic every 2 weeks and then scored for the frequency of regeneration and number of shoots per explant. Similarly, a kanamycin concentration that suppressed root induction was determined by transferring shoots (3–5 cm in length) regenerated from non-transformed (control) explants to rooting medium (RM) containing MSB supplemented with 9.8 μM IBA, 3% sucrose, and 500 mg/l cefotaxime supplemented with different concentrations of kanamycin (0, 1, 5, 7.5 and 10 mg/l).

For the transformation experiments Agrobacterium tumefaciens strain EHA105 harbouring a binary vector pCAMBIA2301 which contains β-glucuronidase (gus) with an intron in the coding region and neomycin phosphotransferase (nptII) genes, both driven by CaMV 35S promoter was used. Single colony of the bacterial strain was inoculated in 25 ml of liquid AB minimal medium (Chilton et al. 1974) containing 10 mg/l rifampicin and 50 mg/l kanamycin, and grown overnight at 28°C until OD600 reached to 0.8. The cells were collected by centrifuging at 5000 rpm for 5 min and the pellet was resuspended in liquid co-cultivation medium (LCM) containing MSB supplemented with 2.7 μM NAA + 11 μM BA, pH 5.8. The cotyledonary-node explants excised from 10-day-old seedlings were gently stabbed four to five times using a sterile needle (261/2 G) at the nodal region before being immersed in bacterial suspension for 25 min with occasional shaking. Inoculated explants were blotted on sterile filter paper and co-cultured in Petri dishes lined with filter paper moistened with LCM for 3 days 25 ± 2°C and maintained under a 16/8 h photoperiod under a cool white fluorescent light at an intensity of 35 μmol/m2/s provided by cool white fluorescent tubes. After co-cultivation, the explants were washed three to four times with LCM with vigorous stirring and blotted dry on sterile filter paper. Different experiments were conducted independently to evaluate the factors influencing the transformation efficiency, as follows: (1) cotyledonary nodal explants were co-cultivated with Agrobacterium for 0, 1, 2, 3 and 4 days to determine the optimum duration for co-culture; (2) explants were inoculated for 0, 15, 30, 45 and 60 min with Agrobacterium suspension to determine the optimum duration of inoculation period; (3) co-cultivation medium was supplemented with 0, 50, 100 and 200 μM acetosyringone to determine the optimum concentration of acetosyringone for transformation.

Regeneration and histochemical GUS assay of putatively transformed plants

The transient gus expression in cotyledonary explants was scored after 3-day-cocultivation. Histochemical GUS assays (Jefferson 1987) were used to assess transient expression of the gus gene, and the number of explants showing transient gus expression at their edges were scored by immersing the tissue materials in GUS substrate solution for 24 h at 37°C. Following incubation, tissues were bleached with 100% ethanol, and examined under microscope. The efficiency of transformation was calculated by taking percentage of the GUS+ explants for evaluating the optimal condition of transformation.

Under the optimized transformation conditions, 3 independent experiments were conducted separately with 25, 30 and 40 explants, respectively, for the transformation assay. For the shoot regeneration, the co-cultured explants were cultured on semi-solid SRM containing 100 mg/l kanamycin and 500 mg/l cefotaxime. The explants were transferred onto fresh medium containing the same levels of antibiotics every 2 weeks for a total of 8–10 weeks, until the shoots attained a height of 3–4 cm. After 8 weeks of culture, the regenerated shoots were separated from the explants as individual shoots and transferred in the medium containing MSB supplemented with 9.8 μM IBA, 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime for rooting (RM, rooting medium). Histochemical GUS activity was carried out on the regenerated shoots by cutting 2 mm basal sections. The frequencies of putatively transformed plants (T0) were calculated based on the numbers and percentages of GUS positive (GUS+) shoots, which showed any GUS activity.

Molecular analysis of the putative transformants

For the PCR screening of the putative transformants (T0) total genomic DNA was extracted from fresh leaves of putatively transformed and non-transformed (control) plants by modified CTAB method (Thangjam et al. 2003b) and screened by the polymerase chain reaction (PCR) for the presence of the nptII gene. The 540-bp coding region of nptII was amplified using 20-bp oligonucleotide primers (5′-CCACCATGATATTCGGCAAC-3′ and 5′-GTGGAGAGGCTATTCGGCTA-3′). The amplification reaction was carried out using a thermal cycler (Perkin Elmer, Foster City, CA, USA) under the following conditions: one cycle of 94°C for 1 min; 35 cycles of 94°C for 1 min (denaturation), 58°C for 1 min (annealing), 72°C for 2 min (extension); a final extension at 72°C for 7 min (1 cycle). The PCR was performed in a 25 μl reaction using approximately 100 ng of purified genomic DNA, 2 μl of 25 mM MgCl2, 0.2 mM dNTP mix, 2.5 U/100 μl Taq DNA polymerase, 50 pmol primers specific for nptII gene. All the PCR reagents are of Bangalore Genei, India make. To ensure that reagents were not contaminated, DNA from non-transformed (control) plants was included in the experiments. The amplified products were separated by electrophoresis on a 1% agarose gel and visualized with ethidium bromide (Sambrook et al. 1989). For Southern analysis 20 μg of DNA samples from transformed and non-transformed (control) plants were digested with KpnI (Fermentas, USA) and separated on 0.9% agarose gel, blotted on positively charged nylon membrane (Hybond-N, Amersham Life Science) and probed with DIG-labelled probe (540 bp fragment of nptII) following supplier’s instructions (Boehringer Mannheim, Germany).

Statistical analysis

All experiments had three replicates per treatment. Each replicate consisted of a Petri plate with 5 explants, in a total of 15 explants per treatment. The experiments were repeated at least twice. The percentage data represented in the tables were arcsine transformed before being analyzed for significance using ANOVA (analysis of variance, P < 0.05). Further, the differences in means were contrasted using Duncan’s new multiple range test following ANOVA. All statistical analysis was carried out using SPSS statistical software package version 16.0.

Results and discussion

Multiple shoot induction and regeneration

The present study provides a method for in vitro plantlet regeneration from cotyledonary nodes of P. timoriana. The data on the effect of MS basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of NAA and BA on cotyledonary node explants of P. timoriana is given in Table 1. Explants cultured on hormone-free media resulted in the formation of single shoot (Fig. 1a) while the media supplemented with different concentrations of NAA and BA either singly or in combinations showed varied morphogenetic response within 16–21 days of culture. In both the media tested, supplementation of NAA and BA favours multiple shoot development with varying response accompanying with calli and root development. In the present study, callusing and rooting at the base increases with advancement in subculturing. Initially, tiny protuberances emerged from the cotyledonary nodes and then shoot buds developed. The shoots got elongated into slender shoots. Explants raised on MS medium produced larger amount of callus than those raised on Gamborg’s B-5 basal medium. However, the presence or absence of callus does not seem to have any effect on multiplication of shoots of tree beans as this callus is of non-regenerative type. This finding is in agreement with those of Acacia auriculiformis (Mittal et al. 1989). The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoot (96.66%) formation and number of shoots per explants (6.60), respectively (Fig. 1b). Whereas, in Gamborg’s B-5 medium the maximum frequency of multiple shoot formation (86.66%) and number of shoots per explant (6.20) were observed with 2.7 μM NAA supplemented culture (Fig. 1c). In both the media, supplementation of low concentration of NAA favours in the production of higher number of shoots per explant while the number of shoots per explant gets reduced with the increasing concentrations of NAA. On the other hand, multiple shoot induction was favourable with the increase concentrations of BA in both the media. Frequency of shoot formation and further development were greatly influenced by the presence of auxin and cytokinin in the medium. BA-induced axillary shoot proliferation from the cotyledonary nodes of seedlings was also reported in several tree species including Aegle marmelos (Arumugam and Rao 1996), Sterculia urens (Purohit and Dave 1996) and Dalbergia sissoo (Pradhan et al. 1998). The requirement of growth hormones as a supplement for obtaining optimal response for sprouting and further shoot differentiation is well documented in a number of species (Datta and Datta 1983). Hossain et al. (1994) observed similar response in hypocotyl explants of A. marmelos. Ajithkumar and Seeni (1998) obtained enhanced shoot production from nodal segments of A. marmelos when they were cultured in the medium augmented with 11.0 μM BA + 5.7 μM Indole acetic acid (IAA), rather than when the medium was supplemented with BA or Kinetin (KIN) alone. Al-Wasel (2000) reported that multiple shoot formation was better in Acacia seyal shoot tips than when the culture medium was supplemented with BA and NAA, but not when either of them was used separately. Overall, in the present study, the percentage of explants showing multiple shoot induction was higher as well as number of shoots per explants was more in MS medium compared to B-5 medium. This indicates that for culture establishment and multiple shoot induction MS medium is better than B-5 medium. This is in agreement with the findings of Dewan et al. (1992) and Joshi et al. (2003) that MS medium performs better than other media compositions.
Table 1

Effect of MS basal medium (Murashige and Skoog 1962) and Gamborg’s B-5 basal medium (Gamborg et al. 1968) media supplemented with plant growth regulators on shoot induction from cotyledonary node explants of P. timoriana after 4 weeks of culture

NAA (μM)

BA (μM)

No. of explants cultured

MS basal medium

Gamborg’s B-5 basal medium

No. of shoots/explant

Explants showing multiple shoots (%)

No. of shoots/explant

Explants showing multiple shoots (%)

2.7

0

45

6.50 ± 0.30a

82.30 ± 3.22a

6.20 ± 0.20a

86.66 ± 4.30a

5.4

0

45

1.60 ± 0.16b

53.33 ± 3.83b

2.10 ± 0.17bj

66.66 ± 4.07b

10.8

0

45

1.50 ± 0.16bc

50.00 ± 1.90bc

1.40 ± 0.51c

43.33 ± 1.93c

0

2.2

45

3.00 ± 0.14di

83.33 ± 2.30ad

2.20 ± 0.13djk

86.66 ± 4.30a

0

4.4

45

3.40 ± 0.22eij

86.66 ± 0.58ade

2.70 ± 0.21el

83.33 ± 1.07a

0

8.8

45

5.80 ± 0.20f

93.33 ± 0.20adf

3.40 ± 0.16f

80.00 ± 3.22a

2.7

2.2

45

2.10 ± 0.10bg

83.33 ± 0.10adg

1.90 ± 0.10gjk

69.99 ± 2.08bd

2.7

6.6

45

3.70 ± 0.26hj

90.00 ± 0.26adh

2.70 ± 0.15hl

81.22 ± 1.05a

2.7

11.0

45

6.60 ± 0.30a

96.66 ± 0.30adi

4.70 ± 0.30i

90.00 ± 3.22a

Mean (±) followed by the same letter(s) in each column were not significantly different at P < 0.05 using Duncan’s new multiple range test

https://static-content.springer.com/image/art%3A10.1007%2Fs11738-011-0917-3/MediaObjects/11738_2011_917_Fig1_HTML.gif
Fig. 1

a Single shoot induction on MS medium without hormones. b Multiple shoot formation on MS medium + 2.7 μM NAA + 11 μM BA. c Multiple shoot formation in B5 medium + 2.7 μM NAA. d Regenerated plantlet successfully hardened in pot. e PCR analysis of T0 plants using nptII primers (lane M marker, 1 control plasmid, 2 untransformed plant, 3–9 putatively transformed plants). f Southern blot analysis of genomic DNA of transformed and non-transformed (control) plants (lane 1HindIII digested Lamda DNA, 2 DNA from untransformed plant, 3, 4 transformed plants, 5 plasmid DNA)

For rooting, 8-week-old regenerated shoots (3–5 cm) were excised and transferred individually to half and full-strength MS medium supplemented with various single concentrations of NAA and IBA. The initiation of roots induction started between 16 and 26 days after culture in half-strength MS media while in full strength MS media it took 16–24 days after culture. Depending on the MS media strength, auxin type and concentration, root numbers ranging from a mean of 2.00 to 4.70 were observed in the auxin supplemented half-strength MS medium and a mean of 2.70–8.70 in full strength MS medium respectively (Table 2). None of the shoots cultured on an auxin-free medium (control) showed rooting. Concentration of auxin played a significant role in root formation. Higher numbers of roots were induced with increasing concentration of auxins and IBA showed higher performance than NAA in both the media composition tested. Full strength MS showed much better rooting performance as compared to half strength. Highest number of root formation was observed in full strength MS supplemented with 9.8 μM IBA. The fully rooted plantlets were taken out from the culture tubes, washed thoroughly to remove any remains of medium, and planted in small plastic pots containing pre-soaked containing soil, vermiculite, and vermicompost (1:1:1). Plants were covered with transparent polyethylene bags to maintain adequate moisture for a week and transferred to the greenhouse (28°C day, 20°C night, 16 h day-length, and 70% relative humidity). After a week, the plastic covering was removed and the plantlets were maintained in the greenhouse in plastic pots containing normal garden soil (Fig. 1d) until they were transplanted to the nursery. About 75% of the hardened plants survived in the nursery.
Table 2

Effect of half and full strength MS media (Murashige and Skoog 1962) supplemented with plant growth regulators on root induction in P. timoriana after 8 weeks of culture

NAA (μM)

IBA (μM)

No. of explants cultured

Half strength MS

Full strength MS

No. of days taken

No. of roots/shoot

No. of days taken

No. of roots/shoot

5.4

0

45

21–24

1.60 ± 0.16ag

20–24

1.70 ± 0.16a

10.8

0

45

21–26

2.00 ± 0.00bgi

21–24

2.60 ± 0.15bg

16.2

0

45

18–22

2.90 ± 0.17ch

19–22

2.70 ± 0.15cg

0

2.5

45

18–24

2.50 ± 0.16dhi

16–22

2.50 ± 0.16dgh

0

4.9

45

16–22

3.10 ± 0.31ei

16–22

3.10 ± 0.13egh

0

9.8

45

16–19

4.70 ± 0.21f

16–21

8.18 ± 0.33f

Mean (±) followed by the same letter(s) in each column were not significantly different at P < 0.05 using Duncan’s new multiple range test

Optimization of kanamycin selection, co-culture duration, inoculation period and acetosyringone concentration for transformation

Prior to transformation, an effective concentration of antibiotic for the selection of transformants was determined by culturing cotyledonary node explants on SRM (MSB + 2.7 μM NAA + 11 μM BA + 500 mg/l cefotaxime) containing various concentrations of kanamycin (0, 50, 75 and 100 mg/l). Kanamycin concentration of 100 mg/l caused complete necrosis and inhibition of regeneration and growth of the explants leading to death by the 8 weeks of culture (Table 3). On the kanamycin free media, all the explants showed multiple shoot regeneration with an average of 6.07 shoots/explants after 8 weeks of culture. The results showed that kanamycin is an effective selection marker for P. timoriana. Similar results were also obtained in Vigna radiata (Jaiwal et al. 2001) and V. mungo (Karthikeyan et al. 1996). The supplementation of 500 mg/l cefotaxime in the SRM showed no adverse effect on the shoot induction and subsequent growth, but effectively controlled the Agrobacterium growth. Similarly, for rooting, supplementation of RM with kanamycin concentration between 7.5 and 10 mg/l was found to be ideal for selection with no sign of root induction in the cultured shoots even after 8 weeks of culture (data not shown). Rooting started within 15 days of culture in RM without kanamycin.
Table 3

Kanamycin sensitivity assay of cotyledonary node explants of P. timoriana cultured on SRM (MS + 2.7 μM NAA + 11 μM BA + 500 mg/l cefotaxime) after 8 weeks of culture

Concentration (mg/l)

No. of explants cultured

Survival (%)

No. of shoots/explant

0

45

100.00 ± 0.00a

6.07 ± 0.35a

50

45

59.99 ± 7.91b

1.40 ± 0.29b

75

45

11.11 ± 3.72c

0.40 ± 0.12ce

100

45

0.00 ± 0.00d

0.00 ± 0.00de

Mean (±) followed by the same letter(s) in each column were not significantly different at P < 0.05 using Duncan’s new multiple range test

In this study, extension of co-culture period of the explants with the Agrobacterium upto 3 days increased the transient transformation frequencies further extension in co-cultivation time decreased the transformation frequency resulting in bacterial over growth (Table 4). Similar observation was made in V. radiata (Jaiwal et al. 2001).
Table 4

Efficiency of A. tumefaciens strain EHA105 harbouring pCAMBIA2301 mediated transformation conditions on cotyledonary node explants of P. timoriana

Transformation parameters

No. of explants inoculated

Putative transformation (%)a

Co-culture duration (days)b

 0

45

0.00 ± 0.00a

 1

45

3.56 ± 0.29b

 2

45

6.27 ± 0.44c

 3

45

16.10 ± 0.50d

 4

45

11.00 ± 0.57e

Inoculation period (min)c

 0

45

0.00 ± 0.00a

 15

45

13.07 ± 0.63b

 30

45

39.03 ± 1.15c

 45

45

33.33 ± 0.88d

 60

45

30.00 ± 0.57e

Acetosyringone concentration (μM)d

 0

45

4.00 ± 0.57a

 50

45

14.66 ± 0.33b

 100

45

31.66 ± 0.33ce

 200

45

33.00 ± 1.73de

Mean (±) followed by the same letter(s) in each column of the same parameter were not significantly different at P < 0.05 using Duncan’s new multiple range test

aPercent of explants showing GUS+ activity

bCo-culture in LCM (MS + 2.7 μM NAA + 11 μM BA) without acetosyringone

cInoculation in LCM with 100 μM acetosyringone

dInoculation for 30 min and co-culture for 3 days in LCM

The explant inoculation efficiency also varies from placing the drops of bacterial suspension on explant surface (Moore et al. 1992) to different times of incubation, varying from 15 (Cervera et al. 2000) to 45 min (Perez-Molph-Balch and Ochoa-Alejo 1998). In this study, the longer the time of inoculation the percentage of GUS+ plants decreases. It can be concluded that time of inoculation between 30 and 45 min is the optimum for getting a good percentage of GUS+ plants (Table 4).

The supplementation of the co-culture medium with acetosyringone stimulates the infection with Agrobacterium. In the present study, co-culture with increasing concentration increases the transformation frequency (Table 4). Thus, acetosyringone concentration of 100 μM was selected for subsequent experiments.

Regeneration and confirmation of putatively transformed plants

Following the optimized conditions of co-culture, the explants were cultured on semi-solid SRM containing 100 mg/l kanamycin and 500 mg/l cefotaxime for shoot regeneration an average of 11.67 kanamycin resistant shoots were obtained (Table 5). The explants were transferred onto fresh medium containing the same levels of antibiotics every 2 weeks for a total of 8–10 weeks, until the shoots attained a height of 3–4 cm. Initiation of shoot breaks from the sides of the wounded cotyledonary node explants started after 15–20 days of culture in the selection medium. After 25–30 days of culture, the multiple shoots were induced. The shoots were separated from the explants when they gave rise to 2–3 branches with leaves and then sub-cultured after every 2 weeks. The growth of kanamycin resistant shoots was rapid and during 3–4 weeks of culture, it developed into multiple branches and leaves. A total of 95 explants, in 3 different experiments, produced 35 resistant shoots (Table 5). Of the 35 shoots developed, only 22 regenerated into whole plantlets with rooting in the RM. Histochemical GUS activity in the regenerated shoots were detected with the appearance of blue colour at the excised basal section. Overall, a total of 12 GUS+ shoots were obtained from the 3 different independent transformation experiments (Table 5). The PCR analyses of the regenerated kanamycin resistant GUS+ plantlets showed amplification of the 540 bp fragments corresponding to the nptII gene, indicating the presence of transgenes but no amplification in the control non-transformed plant (Fig. 1e). However, not all of the 22 regenerated plantlets showed the presence of the fragment. This is probably due to the genomic position effects, deletion of the promoter or part of the coding region or gene silencing due to DNA methylation (Baulcombe 2004). The frequency of transformation revealed by the percentage (%) PCR + plantlets of the total number of explants varied from 12.50 to 16.00%. Southern analysis of putative transgenic PCR + plants revealed different patterns of junction fragments between the T-DNA and the plant genome, depending on the integration site (Fig. 1f). This indicates that these plants were derived from independent transformation events. The T-DNA of pCAMBIA2301 (5.3 kb) contains a single KpnI site at the multiple cloning site located in the lacZ alpha region. The sizes of the bands detected were greater than that of the size of DNA fragment (nptII gene) from KpnI site to the left border (2.1 kb) confirming the integration of T-DNA into the plant genome. The number of hybridization signals indicated the single copy insertion of T-DNA into the genome of the transgenic plants. DNA isolated from non-transformed plants did not hybridize with the nptII probe.
Table 5

Summary of the transformation of cotyledonary node explants of P. timoriana cocultured with Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA 2301

Experiment

No. of explants inoculated

No. of explants showing resistant shootsa

No. of resistant plantsb

Gus+/analysed shoots (%)

cPCR+ (%)

1

25

9

6

4/9 (44.44)

4/25 (16.00)

2

30

11

7

3/12 (33.33)

4/30 (13.33)

3

40

15

9

5/15 (33.33)

5/40 (12.50)

Total

95

35

22

12

13

Values represent three independent experiments conducted seperately each with 25, 30 and 40 explants, respectively

aMedia used: MS + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime

bOnly one shoot from one explant was scored

cResistant plants were checked by nptII-PCR for its presence (+)

To our knowledge this is perhaps the first report of successful in vitro regeneration and establishment in the field for P. timoriana besides our earlier report on the induction of somatic embryos (Thangjam and Maibam 2006), which fail to regenerate into shoots. This protocol offers itself not only as a highly efficient method for mass propagation of this species but also for its conservation. Further, in conclusion, the Agrobacterium-mediated transformation protocol presented here opens up an avenue for future genetic improvement programs for this multipurpose tree legume.

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2011