Bulletin UASVM Horticulture, 67(1)/2010 Print ISSN 1843-5254; Electronic ISSN 1843-5394

In Vitro Propagation of Pinguicula vulgaris

Doina CLAPA 1) , Alexandru FIRA 1) , Ioan PACURAR 2)

1) Fruit Research Station Cluj, 5 Horticultorilor Street, 400457, Cluj-Napoca, Romania; www.scdpcluj.ro, [email protected] 2) University of Agricultural Sciences and Veterinary Medicine, 3-5 Manastur Street, 400372, Cluj-Napoca, Romania

Abstract . At the Fruit Research Station in vitro propagation and ex-vitro acclimation experiments were done for the species Pinguicula vulgaris . Rosettes and leaf cuttings were successfully used as explants. For maintaining the cultures, hormone-free Murashige & Skoog 1962 (MS) and Driver & Kunyuki Walnut (DKW) media were used, whereas for plantlet regeneration MS media with growth regulators were used, o.1 and 0.5 mg/l BAP or 2.5 mg/l kinetin. The gelling agents were Plant-agar at 6 g/l or Isubgol at 15 g/l. The highest multiplication rates (57.6-97.44) were obtained by using leaf cuttings as explants. Ex-vitro acclimation in liquid substrate was carried out successfully by using plantlets rooted on hormone-free MS as well as plantlets from the regeneration and multiplication media (MS containing growth regulators).

Keywords : Murashige&Skoog 1962, Driver&Kunyuki Walnut, Kinetin, 6-Benzylaminopurine

INTRODUCTION

Pinguicula vulgaris (fam. Lentibulariaceae , ord. Lamiales ) is a rare carnivorous species found in humid, swampy regions, especially in peat bogs. It has thick, fleshy leaves, with glandular hairs that produce a mucilaginous, sticky secretion which has the role of attracting, immobilizing and digesting small insects right on the spot. The species also has decorative value by its thick, fleshy leaves and flowers of various colours. In our country it can be found in the Bucegi Mountains. Pinguicula vulgaris can be efficiently propagated conventionally by leaf cuttings, which regenerate plants in their basal part. The in vitro propagation of this species, however, proved to be an extremely effective method. At the Fruit Research Station of Cluj experiments were done for the in vitro propagation of this species starting from plant material cultured in vitro , originating from the Vitosha Mountains in Bulgaria and some of the resulting plants will be tested in the perimetres of peat bogs studied in the framework of the project IDEI – 1478. Isubgol (Psyllium husk), used as geling agent, is a mucilage extracted from around the seeds of Plantago ovata and it was successfully used for the tissue culture of the orchid species Dendrobium chrysotoxum (Jain. and Babbar, 2005) and for the blueberry (Clapa et al . 2008). Pinguicula sp . was studied alongside with other rare plants, especially for the purpose of protection and conservation of the species. The reaction of Pinguicula sp . in tissue culture was also studied (Paunescu 2009, Davis 1993). Ex vitro acclimation in hydroculture, using tap water as substrate is a technique set up at the Fruit Research Station of Cluj and successfully used for various horticultural species (Fira and Clapa, 2009).

330 MATERIALS AND METHODS

The plant material used in the experiments provened from in vitro cultures on media without growth regulators. Whole leaves excised from the in vitro cultures were used as explants, as well as shoots (rosettes with excised leaves). Murashige & Skoog 1962 (MS) and Driver & Kunyuki Walnut (DKW) media were used in 4 experimental variants (Tab. 1). As gelling agents, Plant Agar from Duchefa (6 g/l) was used and, also, Psyllium husk (Isubgol) in the form of the product Colon Pure from the USA. Magenta GA7 vessels were used with about 60 ml of medium/vessel. All the components were added to the media before autoclavation, the pH of the media was adjusted to 5.8 with NaOH solution before adding the gelling agents. The media were sterilized by autoclavation for 20 minutes at the temperature of 121 °C. In the first experimental series the inoculi were cultured for 3.5 months on the media with agar and growth regulators, whereas in the second experimental series the inoculi were cultured for 3 months on the MS media with 15 g/l Isubgol and 0.5 mg/l BAP (Tab. 1). The third experimental series was done for the reason of keeping the plant material in vitro in order to maintain a permanent source of plant material (rosettes and especially leaves). For this, 820 ml jars with screw caps were used, the nutritive medium was Driver & Kunyuki Walnut (DKW) (Tab. 1), without growth regulators 100 ml medium/vessel). 10 rosettes were inoculated/jar and the cultures were kept for 5 months. Tab. 1. The variants of media used for the in vitro culture of Pinguicula vulgaris

Componenta Concentration Variant 1 Variant 2 Variant 3 Variant 4 Salts MS * MS * MS * DKW ** Myo-inositol 100 mg/l 100 mg/l 100 mg/l 100 mg/l Vitamin B1 1 mg/l 1 mg/l 1 mg/l 2 mg/l Vitamin B6 0.5 mg/l 0.5 mg/l 0.5 mg/l - Nicotinic acid 0.5 mg/l 0.5 mg/l 0.5 mg/l 1 mg/l Glycine - - - 2 mg/l Sugar 30 g/l 30 g/l 30 g/l 30 g/l Agar 6 g/l 6 g/l - 6 g/l Isubgol - - 15g/l - BAP 0,1 mg/l - 0,5g/l - Kinetin - 2,5 mg/l - - pH adjusted to 5.8 * Murashige & Skoog 1962 ** Driver & Kunyuki Walnut

Some of the plantlets (whole rosettes, with non-excised leaves and rooted in vitro ) from the regeneration media containing 0.1 mg/l BAP were transferred to hormone-free MS medium (16 inoculi/vessel). The plantlets that were regenerated on the media presented above were transferred ex- vitro into trays containing tap water with neutral pH and cultured for 2 months for being acclimated. The acclimated plantlets were transplanted into trays and pots containing peat and perlite and studied in the greenhouse in the period March-May.

331 RESULTS AND DISCUSSION

On all the tested media, the rosettes regenerated plantlets especially from the wounded surfaces., whereas the leaves and leaf fragments regenerated plantlets from the basal parts. After 3 months of in vitro culture, the plantlets regenerated from the leaves rooted by themselves, on the same medium. Such, hundreds of rooted plantlets resulted on the regeneration media from each Magenta vessel. These plantlets were long and thin. From the rosettes, plantlets were regenerated which grew in a non-uniform way, some of them vigorous, others long and thin. The multiplication rates were much higher in the leaf explants compared to the rosette explants. The highest multiplication rate (97.44) was obtained on MS medium+0.1 mg/l BAP using leaf explants (Tab. 2). On the media with kinetin multiplication rates were lower than those on the media with BAP, but vigorous plantlets resulted. Tab. 2 The multiplication of species Pinguicula vulgaris on agar-gelled media, using different explants

Variant Nutritive medium Gelling agent Type and no. of No. of plantlets Multiplication inoculi/vessel resulted/explant rate 1 MS+0.1 mg/l BAP 6 g/l Plant-Agar 1 shoot (rosette) 54 54 2 MS+0.1 mg/l BAP 6 g/l Plant-Agar 9 shoots (rosettes) 237 26.33 3 MS+0.1 mg/l BAP 6 g/l Plant-Agar 9 leaf fragments 877 97.44 4 MS+2.5 mg/l kinetin 6 g/l Plant-Agar 9 leaf fragments 396 44

In the second experimental series (MS + 0.5 mg/l BAP + 15 g/l Isubgol) only leaves were used, having in view the results from the first series. High multiplication rates were obtained (Table 3) and the plantlets were relatively vigorous, in spite of the high concentration of BAP. Isubgol proved to be a very effective gelling agent. The plantlets could be easily extracted from the nutritive medium, in spite of the sticky nature of the gelling agent. Keeping the Pinguicula vulgaris plantlets in large jars on hormone-free DKW medium ensured 100 % viability of the cultures. Taking into account the rosettes regenerated as propagation material, the multiplication rate was of 13.8, whereas for the leaves it was of 31.05 times (Tab. 4). Because for multiplication the rosettes as well as the leaves can be used, the multiplication rate in this case is of 44.85. These results show that leaves are the most adequate explant source for the in vitro propagation of Pinguicula vulgaris , ensuring multiplication rates superior to those provided by rosettes. The leaf explants abundantly generated plantlets on all the nutritive media we tested and the plantlets rooted directly on the regeneration media, becoming suitable for ex- vitro acclimation in hydroculture. The medium with kinetin ensured the regeneration of fewer ut more vigorous plantlets than that with BAP (Fig. 1). Tab. 3 The multiplication of species Pinguicula vulgaris on MS medium with 0.5 mg/l BAP and 15 g/l Isubgol, using leaf cuttings as explants

Vessel Nutritive medium Gelling agent No. of No. of plantlets Multiplication rate initial resulted/vessel inoculi 1 MS+0.5 mg/l BAP 15 g/l Isubgol 5 352 70.4 2 MS+0.5 mg/l BAP 15 g/l Isubgol 5 224 44.8 Average 288 57.6

332 Tab. 4 Multiplication rates on the maintenance medium Driver & Kunyuki Walnut in Pinguicula vulgaris

No. of Rata de No. of leaves Regeneration No. of initial Vessel no. resulted regenerare- usable as rate-no. of inoculi plants nr. de plante inoculi leaves 1 10 124 12.4 364 36.4 2 10 152 15.2 257 25.7 Average 10 138 13.8 310.5 31.05

a.

b. Fig. 1. Pinguicula on the regeneration media: a) MS with 0.1 mg/l BAP; b) MS with 2.5 mg/l kinetin

Isubgol proved to be a very effective gelling agent in the Pinguicula vulgaris in vitro cultures because on the medium gelled with Isubgol vigorous plantlets regenerated, in spite of the high BAP concentration. This fact can also be due to the lower density of the inoculi, 5 inoculi/vessel being used instead of 9 unlike in the former experiment (Fig. 2).

Fig. 2. Pinguicula vulgaris on the MS medium with 0.5 mg/l BAP and 15 g/l Isubgol

333 The DKW medium proved to be very effective for the prolonged maintenance of the Pinguicula plantlets as a supply of plant material and a source of inoculi (Fig. 3).

a. b. Fig. 3. Pinguicula vulgaris kept on hormone-free DKW medium: a) for 2 months, b) for 5 months

The Pinguicula plantlets transferred ex-vitro directly from the regeneration media into the trays containing tap water survived in a proportion of about 80 % during acclimation, and the ones cultured on hormone-free MS medium had a survival rate of 100 % (Fig. 4). The plantlets transferred into the greenhouse and planted into trays and pots had a survival rate of about 75 % during March and April but they were severely affected by low temperatures, humidity and sunlight and many small plants as well as the leaves at the base of the big ones died. After putting the planlets into a shaded zone, these grew and developed very well an some of them flowered (Fig. 5). It is necessary to study thoroughly the effect of diverse concentrations of various growth regulators and that of various gelling agents and numbers of inoculi/vessel in order to obtain more conclusive results for the in vitro propagation of Pinguicula vulgaris . The ex-vitro acclimation of this species proved to be very efficient even when cultured for a long time, greatly exceeding normal acclimation time (2 months instead of 1). The peat + perlite mixture in 1:1 volume to volume ratio proved to be an adequate substrate for culturing this species in the greenhouse.

a. b. Fig. 4. Pinguicula plants acclimated in hydroculture a) from the regeneration media b) from the rooting medium (hormone-free MS)

334

Fig. 5. Pinguicula planted into peat+perlite mixture

CONCLUSIONS

The species Pinguicula vulgaris is wonderfully suitable for in vitro micropropagation. The basal nutritive media Murashige & Skoog 1962 (MS) and Driver & Kunyuki (DKW) and the two cytokinins (BAP and kinetin) can be successfully used for the in vitro propagation of this species. As gelling agents in the nutritive media, the product Plant-Agar as well as Isubgol pruved to be effective for the in vitro culture of Pinguicula vulgaris . The optimal explant type was represented by leaf explants. The plantlets obtained by direct organogenesis from leaves were successfully acclimated ex-vitro in hydroculture and then cultured in the greenhouse in peat and perlite mixture. It is recommended that they should be kept in a shaded place.

Acknowledgements. This work was supported by CNCSIS –UEFISCSU, Romania, project number 1089 PNII – IDEI , code CNCSIS 1478/2008.

REFERENCES

1. Clapa, D., Al. Fira and T. Rusu (2008). The use of Isubgol and Sequestrene 138 for the in vitro propagation of the highbush blueberry ( Vaccinium corymbosum L .). Journal of Food, Agriculture & Environment 6(1):132-134 2. Davis, G. (1993). Pinguiculas in tissue cultura. The International Pinguicula Study Group 3:5-6. 3. Fira, Al. and D. Clapa (2009). Ex-Vitro Acclimation of some Horticultural Species in Hydroculture. Bulletin UASVM 66(1):44-51. 4. Jain R. and S. B. Babbar (2005). Guar gum and isubgol as cost-effective alternative gelling agents for in vitro multiplication of an orchid, Dendrobium chrysotoxum . Current Science 88(2):292- 295. 5. Murashige T. and F. Skoog (1962). A revised medium for rapid growth and bioassays with tabacco tissue culture. Physiol. Plant 15:473-497. 6. Paunescu, A. (2009). Biotechnology for Endangered Plant Conservation: A Critical Overview. Romanian Biotechnological Letters 14(1):4095-4103. 7. ***http://www.carnivorousplants.org/IPSG/index.php 8. ***http://www.pinguicula.org/A_world_of_Pinguicula_2/Pages/my_experiences_with_tiss ue_culture.htm

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