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Koroch, A., J. Kapteyn, H.R. Juliani, and J.E. Simon. 2002. In vitro regeneration and Agrobacterium transformation of Echinacea purpurea leaf explants. p. 522–526. In: J. Janick and A. Whipkey (eds.), Trends in new crops and new uses. ASHS Press, Alexandria, VA.


In Vitro Regeneration and Agrobacterium Transformation of Echinacea purpurea Leaf Explants*

A. Koroch, J. Kapteyn, H.R. Juliani, and J.E. Simon


*We would like to acknowledge the New Jersey Agricultural Experiment Station and the New Jersey Farm Bureau for their support of this research and for their support of the New Crops and New Use Agriculture program.

Echinacea has gained considerable attention because of its increasing economic value and use as a medicinal plant. The genus Echinacea, Asteraceae, is represented by eleven taxa found in the United States and in south central Canada. Echinacea purpurea is the most widespread species (McGregor 1968) and the most widely cultivated medicinal species of the genus (McKeown 1999). Echinacea species have long been recognized as important medicinal plants and were used by Native Americans for the treatment of many diseases, including colds, toothaches, snake bites, rabies, and wound infections (Bauer and Wagner 1991).

The pharmacological activity of Echinacea extracts has been widely studied with over 350 published studies to date (Briskin 2000) together providing strong evidence for an immune modulating activity of these extracts. However, little scientific research has been directed toward understanding the biosynthetic pathways of the several classes of compounds that are thought to be responsible for the biological activity of Echinacea extracts or the physiology regulating their accumulation.

The transfer of foreign genes into plants has provided new ways to study regulation of development and biosynthetic processes. Agrobacterium tumefaciens mediated transformation is preferred because of its simplicity and efficiency in providing stable integration of transferred DNA into the plant genome. In vitro shoot regeneration of E. purpurea has been reported from petiole explants (Choffe et al. 2000a) and leaf tissue (Koroch et al. 2001) while root organogenesis has been obtained from hypocotyl and cotyledon explants (Choffe et al. 2000b). The objective of this work was to develop an efficient in vitro regeneration method and an Agrobacterium tumefaciens transformation method for E. purpurea leaf explants.

EXPERIMENTAL PROCEDURES

Plant Material and Regeneration

Young leaves of E. purpurea were collected from 4 month-old plants grown at the Rutgers University, Cook College, Department of Plant Biology greenhouse. Leaves were surface sterilized by soaking for 17 min in a 1.05% (w/v) sodium hypochlorite solution (20% v/v commercial bleach) containing 0.1% Tween 20 and then they were washed three times with sterile water in a laminar flow hood.

The leaf margins were removed along with the tip and basal portions. Leaf sections (0.7 × 0.7 mm) were placed on callus and shoot induction media with the adaxial surface toward the media. The callus and shoot induction media was composed of MS (Murashige and Skoog 1962) basal medium (4.32 g L-1) containing myo-inositol (100 mg L-1), thiamine (0.4 mg L-1, and sucrose (2% w/v); this media was supplemented with different concentrations of 6-benzylaminopurine (BA) alone or in combination with naphthaleneacetic acid (NAA). The pH of the medium was adjusted to 5.8 with KOH before adding agar (7 g L-1). Medium without plant growth regulators was used as a control. Cultures were maintained in darkness at 28°C. Each treatment consisted of 12 explants per dish (100 × 15 mm) and was replicated 10 times. The rate of callus formation and the number of shoots/explant was determined after 4 weeks. After incubation in darkness, all treatments were moved to lighted conditions with a 16 hr photoperiod of 25 mmol m-2 s-1 at 25°C for 1 week.

For rooting isolated shoots (1.5 cm or longer) were transferred to basal medium without plant growth regulators (PGR), pH 5.8. After 4 weeks of culture, rooted plants were removed from culture, rinsed in water to remove media, and transferred to potting medium (Pro-Mix BX, Premier Company, Pennsylvania) in a mist chamber in the greenhouse. After two weeks, plants were transferred to the greenhouse supplemented with a 16 hr photoperiod of 85 mmol m-2 s-1. The percent survival of regenerated plantlets was recorded after a total of 4 weeks in potting medium and greenhouse conditions

The experimental design was fully randomized. Data were analyzed statistically by analysis of variance (ANOVA) followed by the Tukey test, with the level of significance set at 1%.

Transformation

Agrobacterium tumefaciens strains EHA105 (Hood et al. 1993) and GV3101 containing the binary vector pBISN1 (Narasimhulu et al. 1996) were used in preliminary transformation experiments. Leaf sections infected with EHA105 demonstrated a higher level of transient GUS expression than those infected with GV3101, and EHA105 was used in all subsequent experiments. pBISN1 contains the neomycin phosphotransferase II (nptII) coding region under the control of the nopaline synthase promoter and an intron containing gusA gene under the transcriptional control of the super promoter (Ni et al. 1995).

Agrobacterium were grown at 30°C in AB minimal medium (Lichtenstein and Draper 1986) in the presence of 50 mg L-1 kanamycin and 10 mg L-1 rifampicin. Agrobacterium cells were harvested in log phase, washed, and resuspended in liquid MS media at an OD600 of 0.6–0.8. Leaf sections were incubated with resuspended cells for 30 min and blotted dry before placing on shoot induction media. Leaf sections were co-cultivated with Agrobacterium for different periods of time ranging from 0 to 72 hr before transfer to shoot induction media containing Timentin for control of Agrobacterium and kanamycin for selection of transformed tissue. Leaf sections were assayed for GUS activity 4 days after the co-cultivation period.

Shoots were regenerated in the presence of 50 mg L-1 kanamycin and 300 mg L-1 Timentin using the method previously described and rooted with antibiotics of the same concentrations in MS media without PGR.

Histochemical GUS assays were performed on leaf tissue from regenerating kanamycin-resistant shoots by incubating with 5-bromo-4-chloro-3-indolylglucuronide (X-gluc) according to the method of Jefferson (1987).

DNA was isolated from leaf tissue of the same plantlets using DNeasy Plant Mini kits (Qiagen, California). Detection of nptII sequences in kanamycin-resistant plantlets was carried out by PCR analysis using the primer sequences and method of Lucas et al. (2000) to amplify a 320 bp region of the nptII gene.

RESULTS AND DISCUSSION

Regeneration

Leaf explants incubated on basal medium with different combinations of auxin/cytokinin demonstrated callus formation after 4 weeks of incubation (Table 1). BA alone produced green callus for each concentration tested, however only the lower concentrations (0.44–8.88 mM) showed adventitious shoot formation after 4 weeks.

Table 1. Effect of different combinations of NAA and BA on shoot regeneration from leaf explants of E. purpurea after 4 weeks of culture.

Growth regulator (mM) % explants
producing shoots
No. shoots/
explantz
NAA BA
0 0 0 0
  0.44 17 0.2 j
  2.22 97 4.2 cd
  4.44 97 2.3 efg
  8.88 59 1.9 de
0.054 0 0 0
  0.44 65 1.3 ghij
  2.22 75 3.3 de
  4.44 100 7.7 a
  8.88 0 0
0.54 0 0 0
  0.44 63 1.2 ghij
  2.22 89 1.8 fgh
  4.44 78 2.1 efg
  8.88 73 2.7 ef
2.69 0 0 0
  0.44 30 0.5 ij
  2.22 70 1.3 ghij
  4.44 91 5.7 b
  8.88 82 1.9 fgh

zMeans followed by the same letter do not differ statistically at p< 0.001 different according to Tukey test.

Of the combinations, MS medium supplemented with BAP (4.44 mM) and NAA (0.054 mM) was the most effective, providing shoot regeneration for 100% of explants associated with a high number of shoots per explant (7.7 mean shoots per explant). Explants grown in this medium for two weeks formed callus at the cut surface, and after 3 weeks the callus began to produce multiple shoot primordia, which developed into adventitious shoots.

Increasing NAA concentration resulted in increased callus production and low shoot initiation (Table 1). NAA alone (0.54 mM, 2.69 mM) induced direct and indirect root formation (data not shown). However, a low concentration of BA (0.44 mM) added to the medium resulted in a stimulation of shoot induction. The balance of auxin to cytokinin is a determining morphogenic factor. A combination of a high amount of NAA (2.69 mM) and a small amount of BA (0.44 mM) induced shoot proliferation and some adventitious roots. However, these roots formed independently of the shoots (data not shown).

In contrast, regeneration was slow or absent for explants grown on medium containing high levels of BA alone or with NAA. The callus observed with higher BA and NAA concentrations were brown and exhibited excessive necrosis, indicating toxic effects.

In previous reports, plant regeneration from petiole explants of E. purpurea was achieved by using only a small amount of BA (Choffe et al. 2000a), whereas, in the present study, BA in combination with NAA was most effective in inducing adventitious shoot regeneration from leaf explants. This difference between petiole and leaf explant response to BA and NAA concentrations in the media could be a reflection of probable differences of endogenous growth regulator levels in the explant sources or different tissue sensitivities to these plant growth regulators (Lisowska and Wysonkinska 2000).

All shoots longer than 1.5 cm were transferred MS media without growth regulators and began rooting. The survival rate of regenerated plantlets transferred to soil was 95%.

These results demonstrate that leaves of E. purpurea have a great organogenic potential for shoot formation, however the response is highly sensitive and directly related to the combinations of exogenous growth regulators in the culture medium.

Transformation

E. purpurea leaf sections incubated with Agrobacterium showed a high frequency of GUS expression after co-cultivation periods of 48 or 72 hr (95% for both treatments). For selection and regeneration of transformed plantlets, 640 leaf sections were co-cultivated with Agrobacterium for 48 hr and transferred to shoot induction media containing kanamycin and Timentin. After 6 weeks of selection/shoot induction, the first kanamycin-resistant shoots were transferred to MS media without PGR and with antibiotics for rooting. The first group of rooted kanamycin-resistant plantlet was transferred to the greenhouse approximately 19 weeks after infection. Leaf material for GUS assays and DNA extraction was harvested at this time.

Several morphologically normal appearing kanamycin-resistant plantlets (Fig. 1) did not exhibit GUS staining in histochemical GUS assays (Fig. 2 left), while most regenerated plantlets were GUS positive. PCR analysis for the presence of the nptII transgene (Fig. 2 right) correlated positively with GUS assay results. Thus, the relatively high concentration of kanamycin, which completely inhibited both callus and shoot formation in control (untransformed) leaf sections, was not adequate to completely prevent the occurrence of escapes. From the 640 infected leaf sections, 12 individual transgenic plants were produced, providing an initial efficiency of 1.9%.

Fig 1. Kanamycin-resistant E. purpurea plantlets 4 weeks after transfer to the greenhouse. The plantlets are the same as those used for GUS assays and nptII gene detection in Fig. 2.
Fig. 2. Leaves from kanamycin-resistant plantlets showing GUS staining, except rg2 and rg3, which were determined to be escapes (left). Amplification products of nptII transgene, present in GUS positive plantlets and absent from rg2 and rg3 (right).

CONCLUSION

The method developed here provides an efficient system of adventitious shoot regeneration from leaf explants of E. purpurea that will be useful for micropropagation of elite ornamental or chemotype selections of this medicinal plant species. In addition, this regeneration system, combined with Agrobacterium transformation, provides a method for routine genetic transformation of this important medicinal species.

REFERENCES