Indian Journal of Experimental Biology Vol. 39, July 2001 , pp. 705-709

Regeneration from leaf protoplasts of Arabidopsis thaliana ecotype estland

Rita Gandhi & Paramjit Khurana*

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi II 0 021 , India

Received 10 July 2000; revised 26 March 2001

Protoplasts (2xl07/g fresh wt) were isolated from leaves of A. thaliana ecotype estland, with a viability of more than 90%. Protoplasts cultured in calcium alginate beads or layers showed division while culture in liquid or agarose beads failed to elicit any division. Effect of culture density showed highest frequency of division occurring at 5xl05 while no division was seen when cultured at a density of 5xl04. Culture in MS medium resulted in higher division frequency and better sustenance of microcolonies as compared to B5 medium. Under optimized conditions, macrocolonies were formed at a frequency of 1.8%. Shoot regeneration was seen in 50% of microcalli transferred to shoot induction medium for regeneration. Shoots were rooted and plantlets transferred to pots. The plants produced flowers and were fertile.

Over the years, Arabidopsis thaliana has been established as a model system for plant gene structure and function analysis and has been put to extensive experimentation in various fields of plant research. CelVtissue culture system and transformation technology using Agrobacterium and PEG-mediated gene transfer in protoplasts have also been worked out14

• We have been working on various aspects of Arabidopsis regeneration, transformation, EMS and T-DNA insertion mutagenesis5

•7 in ecotype estland.

Protoplast culture of Arabidopsis ecotype estland has been worked out in order to develop a system of PEG­mediated gene transfer7

• For mesophyll protoplast regeneration, various procedures have been developed for each ecotype as there is no protocol that is applicable to all ecotypes8

• We have reported here efficient isolation, reproducible division, and subsequent regeneration of fertile plants from mesophyll protoplasts of Arabidopsis thaliana ecotype estland.

For raising aseptic plants which served as a source of leaf material, seeds were surface sterilized by treating with sodium hypochlorite (2%) and Triton X-100 (0.01%) for 5 min and subsequently given 5-6 washes with sterile distilled water. Seeds were inoculated in 250 mL Erlenmeyer flasks containing 85 basal medium9 and incubated in light (16 hr light/8 hr dark cycle).

For isolation of protoplasts, leaves from 3-4 week old aseptically raised light-grown plants were incubated in a cell wall degrading enzyme solution

*Corresponding author. E-mail: paramjitkhurana@hotmail.com

(l% cellulase RlO, 0.5% macerozyme RlO, 0.5 M mannitol, 5 mM MES; prepared in CPW salts10

). The pH was adjusted to 5.7 with NaOH. Each leaf was cut into 2-3 parts in the enzyme solution (ca. 500 mg tissue in lO mL enzyme solution). Enzyme solution was filtered aseptically using 0.22 J..Lm nylon filter prior to use. Incubation was done overnight (-14 hr) at 24-26°C in dark with initial 30 min on a shaker at 40rpm.

Protoplasts were purified as described earlier' with minor modifications. The enzyme mixture containing the protoplasts was passed through a stainless steel mesh (40 J..Lm) to remove the coarse debris. The resulting filtrate containing protoplasts, isolated cells, and organelles from the damaged protoplasts was transferred to 15 mL centrifuge tubes and centrifoged at 100 g for 3 min. The supernatant was removed with the help of pasteur pipette and the pellet was suspended in washing solution I ( 15 rnM, MgCh; 0.5 M mannitol; 5 mM, MES; pH 5.7; washing solution 1: WS I) with the help of a pasteur pipette. The protoplasts were centrifuged again for 3 min at 100 g and the pellet was suspended in WS I. The protoplast suspension was then layered on to 6-7 mL of sucrose solution (21% sucrose; 15 rnM, MgCh; 5 mM, MES; pH 5.7) and centrifuged at 100 g for 3 min. The ring of floating protoplasts at the sucrose junction was carefully collected with the help of a pasteur pipette, added fresh WS I to the protoplast and centrifuged at 100 g for 2 min. The protoplast pellet was finally resuspended in 2 mL of WS I and the desired density was adjusted. Viability of protoplasts was tested by fluorescein diacetate (FDA) staining11

•

706 INDIAN J EXP BIOL. JULY 200 1

For liquid culture, protoplasts were pelleted from WS I at 100 g for 2 min, and resuspended in filter­sterilized culture medium (B5 or MS medium 12

containing 0.4 M glucose, I mg/L 2,4-D, 0.5 mg/L BAP, 0.1 mg/L NAA, pH 5.8). The density of the protoplasts was adjusted to 2.5x105 cells/mL and 1.5 mL of protoplast suspension was cultured in 35 mm Petri dishes (Nunc, Denmark) in dark.

For agarose bead culture, protoplast (5x105/mL) suspended in the WS I were mixed with equal volume of 1.2% of low melting point agarose (Sigma, USA) prepared in WS I and poured as drops in Petri dish (35 mm). After they solidified, liquid culture medium was poured over them and the petridishes were sealed with parafilm and incubated in dark.

Culture in alginate layers requires the purification steps to be free of any calcium which might result in premature formation of calcium alginate from sodium alginate. Thus protoplasts suspended in WS I were mixed with equal volume of sodium alginate (1.2%) prepared in WS I (devoid of Ca2+) and 1 mL of this mixture was poured over solidified medium (30 mM CaCI2.2H20, 0.5 M mannitol, 0.8% agarose, 5 mM MES, pH 5.7) in Petri dish (35 mm). Gradual uptake of calcium from the lower layer results in the formation of calcium alginate which is solid and thereby immobilizes the protoplasts. After 1 hr, poured 1 mL of washing solution II (15 mM, CaCh.2H20, 0.5 M mannitol, 5 mM MES, pH 5.7) and .incubated at 4 °C for 24 hr. After 24 hr, removed calcium alginate layer contammg immobilized protoplast from the lower agarose medium layer with the help of a spatula and transferred to a 55 mm petridish containing 2 mL of culture medium. Made four parts of the circular layer with the help of a spatula and floated in the fresh culture medium (2-3 mL) and incubated in dark. Cultures were periodically observed under the microscope and the division frequency was assessed after 15 days.

Cell wall formation was assessed after 24 and 48 hr of culture by using calcofluor white. A stock of 5 mg/mL cilcofluor white was prepar~d in water and 10 J.!L of this stock was added to 5 ml of WSI. The protoplasts suspension was incubated with diluted solution of calcofluor white in equal volume~ for 2-5 min in dark. The protoplasts were examined under Leitz Orthoplan fluorescence microscope (Leitz, Germany) using BP450-490 excitation filter and LP515 suppression filter.

After an interval of 10 days, fresh culture medium was added to the dividing/proliferating protoplasts till

the colonies reached a size of 1-2 mm. Macrocolonies were released from the calcium alginate layer by

' incubating the layers with a solution of sodium citrate (20 rnM, sodium citrate; 0.3 M, mannitol; 5 mM, MES; pH 5.8) with shaking at 50 rpm. The macrocolonies were transferred on B5 solid medium containing 5 mg/L 2iP and 0.15 mg/L IAA for further growth and differentiation in light/dark cycle of 16/8 hr. Shoots regenerated from protoplast-derived calli were transferred toMS medium with 1 mg/L NAA for root formation. Regenerated plantlets were transferred to pots filled with Soilrite mix (Kelperlite, Bangalore, India) and grown to maturity. The plants were watered with mineral medium 13

•

Although protoplast isolation has been reported from varied parts of Arabidopsis such as leaf, root, callus tissue 14, callus and cell suspension cultures15, plantlet regeneration has been reported from protoplast isolated from regenerable callus lines 16, fast growing suspension cells 17-18, auxin conditioned root cultures 19, and mesophy II tissues 1.3.

20-22. In the present investigation, mesophyll protoplast was employed for culture as they could be isolated in high numbers -2x107/g fresh wt (Fig. 1a) with a viability of more than 90% as assessed by FDA staining. To optimize culture conditions for mesophyll protoplasts, they were cultured under various conditions. Protoplasts cultured in liquid medium (MS or B5) or embedded in the agarose beads did not show any division. However, the protoplasts embedded in calcium alginate layers started dividing after 5-7 days of culture and after I 5 days, 8-16 celled microcolonies were formed (Fig. 1 b).

Density at which protoplasts were cultured had a profound effect on the division frequency of protoplasts. The culture of protoplasts was carried out at different densities in alginate layers in B5 r:nedium. A culture density of 5x105/mL showed the highest frequency of division (6%) while no division occurred when protoplasts were cultured . at a density of 5x104/mL. A culture density of 5x105/mL has also been used by Damm and Willmitzen 1 and Karesch et aP0

.

Different workers have reported the use of various media including MS medium20, B5 medium1 and KM based medium 18. In the present study, protoplasts were cultured in B5 or MS medium at a density of 5x I 05 protoplast/mL and embedded in calcium alginate layers. Use of MS medium resulted in higher division frequencies, upto 10% as compared to -6% in B5 medium. Difference was reflected even during

GANDHI & KHURANA: REGENERATION FROM LEAF PROTOPLASTS 707

Fig. !- Regenerati on of plant le ts fro m mesophyll protoplast cultu re of Arabidopsis tlwliana ecotype ·est land ', (a)-Freshl y purified protoplasts at the time of c ulture; (b)-8- 16 ce lled microcolonies (marked by arrows) afte r 15 days of cu lture in alginate layers: (c)-A proliferating microcolony after 3 week s of culture: (d)- A macrocolony formed after 4-5 weeks of culture : (e)-Microca llus as seen after 2 weeks of growth o f a macrocolony on shoot induction medium (S IM ): (f)-Regenera ti on of multipk shoots from protoplast-derived callus afte r 2-3 weeks of culture o n S IM ; (g)-A regenerated shoot cultured fo r root induct ion on MS basal medi um ; (h & i)­Developing pl antlcts wit h nora! buds and fruit development (marked by arrows), respecti ve ly.

708 INDI AN J EXP BIOL, JU LY 200 1

further growth as the microcolonies formed in BS medi um never proliferated to form macrocolonies as compared to those formed on MS medium. Macrocolonies visible to the naked eye were formed at a frequency of 1.8% (Fig. I c, d). This plating effic iency of 1.8% is hi gher than that obtained by Damm and Willmitzer1 for 'estl and' ecotype. In fact, in their study involving regeneration of mesophyll protoplasts from 4 ecotypes, about 10-20 fold lower plating efficiency was obtained from estland and Iandsberg as compared to columbia (0.6%) and wassi lewsk ij a (0.4%). Genotypic differences are known to play a crucial role in affect ing the culture response, and 'estland' ecotype has not been worked out extensively and is reported to regenerate at a low frequency in studies publi shed so far 1

·15·n.26

•

Bigger macrocolonies/microcalli were subsequentl y transferred to BS medium solidifi ed with 0.8% of agar (supplemented with 3% sucrose, 5 mg/L 2iP and 0.15 mg/L IAA) for further growth and regeneration . After 2 weeks of incubation on the above medium, the microcalli increased in size and turned green (Fig. I e), and 50% of these regenerated shoots (Fig. I f) . These shoots were separated from the calli and transferred to MS medium contain ing 1 mg/L of NAA for root induction (Fig. 1 g). Some shoots, even without the format ion of roots, elongated and formed flowers and fru its inside the cu lture tube (Fig. 1 h,i). Valvekens et a/. 27 have also reported the formation of seeds in petri dishes without rooting of the shoots. Some of the plantlets were transferred to pots to complete their life cycle and seeds were set which were fert ile.

Protocol for protoplast isolat ion and culture will continue to play an important ro le in transient gene expression studies and for regeneration of transgenic plants. [n the present study, the protocol for isolation and regeneration of protoplasts has been employed for transient gene expression and for analysing

f . 7 ?8 trans ormatton parameters ·- .

The author thank Professor S C Maheshwari for his interest in thi s work . This work was financially supported by the Department of Biotechnology of Govt. of India and University Grants Commission, New Delhi. RG acknowledges the award of Junior and Sen ior Research Fellowships from CSIR, New Delhi.

References I Damm B & Willrnitzer L, Regeneration of fertile plants from

protoplasts of different Arabidopsis Ilwliana genotypes. Mol Gen Gene!, 213 ( 1988) 15.

2 Damm B, Schmidt R & Willmi tzer L, Efficient transformati on of Arabidopsis Ihaliana us ing direct ge ne transfer to protoplasts. Mol Gen Gene!, 217 ( 1989) 6.

3 Masson J & Paszkowsk i J, The cul ture response of Arabidopsis Ihaliana protoplasts is determined by the growth condition of the donor plants. Plant J, 2 ( 1992) 829.

4 Morris P & Altmann T, in Arabidopsis edited by EM Meyerowitz & CR Somerville (Cold Spring Harbor Laboratory Press, New York) 1994, 173.

5 Khurana JP, Kochhar A & Jain P K, Genetic and molecu lar analysis of light-regulated plant development. Genelica , 97 ( 1996) 349.

6 Gandhi R, Mahcshwari SC, Khurana JP & Khurana P, Isolation and characteriza ti on of aT-DNA tagged polyrosel/a mutant of Arabidopsis di splaying altered body pl an, in Abstracts-International symposi um on "Development, Growth and Differentiation", Mahabaleshwar, India, Dec 17-20, 1997,29.

7 Gandhi R, Mahcshwari SC & Khurana P, Transient gene cxprc sian and inOuencc of promoters on forei gn gene express ion in Arabidopsis Ihaliana, in Vilro Cell Dev Bioi -Plan! , 35 ( 1999) 232.

8 Sando S & Goto N, A novel method for regenerating plants from mcsophyl l protoplasts of Arabidopsis Ihaliana line WS. Plan/ Cell Rep, 14 ( 1994) 75.

9 Gamborg OL, Miller RA & Ojima K, Nutrient requirements of suspension cultures of soybean root cel ls. Exp Cell Res, 50 ( 1968) 151.

I 0 Frearson EM, Power JB & Cocking EC, The isolat ion, culture and regeneration of Pelunia leaf protoplasts . Dev Bioi, 33 ( 1973) 130.

II Widhalm JM, The usc of Ouorescene cliacctatc :md phenosafran ine for determining viabili ty of cultured plant cells. Stain Techno/, 47 ( 1972) 189.

12 Murashi gc T & Skoog F, A revi sed med ium for rapid growth and bioassays with tobacco tissue cu ltures. Physiol Plan/, 15 ( 1962) 473.

13 Somerville CR & Ogren WL, Isolat ion of photoresp iration mutants of Arabidopsis Ihaliana. In Methods in Chloroplast Molecular Biology ed ited by M Edelman, R B Hallick & N­H Chua (Elsevier Biomedical Press, New York) 1982, 129.

14 Bhalla-Sarin N, Sopory S K & Gulla-Mukherjee S, Studies on the isolat ion and fusion of protoplasts of Arabidopsis. Arabidopsis lnf Serv, 13 ( 1976) 200.

15 Ncgrutiu I, Bccftin k F & Jacobs M, Arabidopsis Iha/iana as a model system in somatic cell genetics I. Cell and ti ss ue culture. Plan/ Sci Leu, 5 ( 1975) 293.

16 Gresshoff PM, Protoplast and callus regeneration of Arabidopsis Ihaliana. Arabidopsis lnf Serv. 13 ( 1976) 211.

17 Xu an L T & Mcnczcl L, I mprovcd protoplast culture and plant rcgcnctation from protoplast-derived callus in Arabidopsis thaliana. Z Pjlanzenphysiol, 96 ( 1980) 77 .

18 Ford KG, Plant regeneration from Arabidopsis Ihaliana protoplasts. Plmll Cell Rep, 8 (1990) 534.

19 Mathur J, Koncz C & Szabados L, A si mple method for isola ti on. liquid culture, transformation and regeneration of Arabidopsis Ihaliana protoplasts. !'/ant Cell Rep , 14 ( 1995) 221.

20 Karesch H, Silang R & Potrykus I, Arabidopsis thaliana: protocol for plant regeneration from protoplasts. Plant Cell Rep, 9 ( 1991) 575.

GANDHI & KHURANA: REG ENERATION FROM LEAF PROTOPLASTS 709

21 O' Ne il CM & Mathi as RJ , Regeneration of plants fro m protoplasts of Ambidopsis thaliana L cv . Columbia (C24), via di rec t embryogenesis . 1 Exp Bot, 44 ( 1993) 1579.

22 Siemens J, Torres M, Morgner M & Sac ri stan MD, Pl ant regeneration from mesophyll -protoplasts of fo ur diffe rent ecoty pes abel two marker lines from Arabidopsis thaliana using a uni que protocol. Plant Cell Rep , 12 ( 1993): 569.

23 Negrutiu I & Jacobs M, Factors which enhance in vit ro morphogenesis of Arabidopsis thaliana. Z Pflanzenphysiol, 90 ( 1978) 423.

24 egrutiu I & Jacobs M, Restorati on of the morphogenetic capacity in long- term ca ll us cu ltures of Arabidopsis thaliana. Z Pflanzenphysiol, 90 ( 1978) 43 1.

25 Negrutiu 1. Jacobs M & Cachita D, Some fac tors controll ing in vitro morphogenesis of Arabidopsis tlwliww. Z Pflanzenphysiol, 86 ( 1978) 11 3.

26 Negrut iu I, Jacobs M & De Greff W, In vitro morphogenesis of Arabidopsis thaliana: The ori gin of the explant. Z Pjlanzenphysiol, 90 ( 1978) 363 .

27 Valvekens D, Van Montagu M & Van Lij sebettens M, Agrobacterium tumefaciens- mediated transformation of Arabidopsis thaliana root explants by using kanamyc in selection. Proc Nat/ Acad Sci USA , 85 ( 1988) 5536.

28 Gandhi R, Maheshwari SC & Khurana P, Genetic and molecular analys is of Arabidopsis thaliana (ecotype Estland) transformed with Agrobacteri11 111. in Vitro Cell Dev Bioi­Plant, (200 1) (In Press).