PlantCeli Reports

Plant Cell Reports (1990) 9:139-142

9 Springer-Verlag1990

Callus formation from root protoplasts of Quercus rubra L. (red oak) M. Brison and A. Lamant Laboratoire de Physiologie Cellulaire V+gttale, Universit6 de Bordeaux I, Avenue des Facultts, F-33405 Talence Cedex, France Received November 8, 1989/Revised version received March 15, 1990 - Communicated by A. M Boudet

ABSTRACT

Root protoplasts of Qu~Acu6 rubra L. were isolated from 12 day old seedlings with an enzyme mixture containing Cellulase RI0 + Rhozyme HPI50 + Macerozyme RI0, supplemented with cysteine and bovine serum albumin. Protoplasts were purified by a Ficoll density gradient centrifugation and cultured at low density in a liquid medium. The modified woody plant medium, containing 2.2 ~ M benzyladenine + 1.8 ~ M zeatin + 5.3)]M a-naphthaleneacetic acid + 2.2124 dichlorophenoxyacetic acid, allowed sustained divisions and formation of

article of Lang and Kohlenbach (1988), or that of Merkle and Sommer (1987) which give good surveys of these works). In some species successful regeneration of whole plants occurred. In order to reach the same result for red oak, we must decide which material would be the most valuable for regeneration studies. We chose to begin with roots because this tree readily throws up suckers. We report here on conditions allowing the induction of sustained cell division and callus formation from root

protoplasts of Qu~eu6 rubra i.

microcalluses.

MATERIAL AND METHODS

Protoplast - derived microcallus developed into green and compact callus when transferred to an agarose solidified medium, supplemented with casein hydrolysate and indole 3-acetic acid (devoid of 2,4 dichlorophenoxyacetic acid) and placed under low illumination.

Plant material : Seeds were germinated as described previously (Devilder 1988). Apical root segments (2 cm long) were excised from 12 day old seedlings, their surface was sterilised with 0.1% w/v mercuric chloride in distilled water for 2 min, and they were washed three times with sterile water.

ABBREVIATIONS

IAA : indole-3-acetic acid ; IBA : indole-3-butyrie acid ; BA : benzyladenine ; BSA : bovine serum albumin ; 2,4-D : 2,4-dichlorophenoxyacetic acid ; FW : fresh weight ; GA 3 : gibberellic acid ; MES : 2-(Nmorpholino) ethanesulfonic acid ; MS medium Murashige and Skoog medium (1962) ; NAA : a-naphthaleneacetic acid ; WPM : woody plant medium (Lloyd and Mc Cown, 1981).

Protoplast isolation : The root segments, excluding the two tips (3 mm), were chopped with a razor blade into 1-2 mm thick pieces and incubated in a sterile medium (pH adjusted to 5.6 with KOH) consisting of 0.6 M mannitol, 3 mM CaCI~ i0 mM cysteine 0.5 mM MES 0.3% BSA, supplemented wi~h different concentrations ' of cellulase (Onozuka RI0, Yakult Honsha, Japan), hemicellulase (Rhozyme HP 150, Rohm and Hass, Philadelphia USA), pectinase (Macerozyme RI0, Yakult Honsha, Japan) and with or without driselase (Fluka Chemie AG - Buchs, Switzerland)(Table i).

INTRODUCTION

~sgrcu6 ~ubra {red oak) has many appreciated qualities, such as resistance to frost and diseases, low nutrient requirements and hard wood. It is thought to be a promising species for referestration pregrammes in many parts of France. Micropropagation from oak stem cuttings is difficult (Juncker and Favre 1989) and, to our knowledge, all attempts using red oak have been unsuccessful. For these reasons, protoplast culture may be a useful tool, not only for plant propagation, but also for red oak improvement through genetic transformation or somatic hybridization. Isolation and culture of woody plant protoDlasts issuing from leaf mesophyll, callus or cell cultures has often been reported (for more details refer to the

Offprint requests to: M. Brison

The incubation lasted 5h on a gyratory shaker (40 rpm) at 23 ~ I~ in the dark. The larger cell debris and undigested tissues were then removed by filtration through a nylon sieve (60 ~ m pore size). Protoplast purification : The protoplasts in the enzyme solution were purified as described by Attree and Sheffield (1986), using a Ficoll (400-Pharmacia) density gradient centrifugation. The only modification made to this method was changing the Ficoll concentration (from 12% to 10%) in the middle layer of the gradient. The protoplasts were collected at the interface 10% - 0% (w/v) of the gradient, washed twice in 0.6 M mannitol, 5 mM CaCI (pH 5.6) and centrifuged at 800 rpm for 5 min. 2

140 Protoplast culture : Purified protoplasts were resuspended in a sterile cultur~ medium, an~ plated at densities varying from 3 x i0- to i0 x i0 in 60 mm diameter Petri dishes. Three methods were tried : 2 ml liquid medium, 1 ml liquid medium on 3 ml gelled medium and droplets (Fa~e and David 1983). The dishes were incubated at 23 - I~ for two weeks in the dark and thereafter under a 16h21ight/Sh dark cycle (light intensity : 5 to 6 ~ o l . m - s ~ ) . With regard to the macronutrlentS, the plating medium was either the modified woody plant medium (WPM) developed for POp~s protoplast culture by Russell and Mc Cow~ (1986) or half-strength Murashige and Skoog (1962) medium (MS/2). both devoid of ammonium. Micronutrients (Heller 1953), vitamins (except vitamins A-D~ -B.~), organic acids and amino acids (Kao and Mich~ylu~Z1975) were added. Sugar components consisted of 55 mM sucrose, 450 mM glucose, 20 mM sorbitol, 20 mM xylose and 20 mM mannitol. This medium was supplemented with growth regulators at different concentrations : BA (4.4 or 2.2)~M), NAA (16.1, 10.7 or 5.32/M) and zeatin (I.8~M) with or without 2,4-D (i.i or 2.2)JM) and adjusted to pH 5.6.

enzymes by phenolics. We can also suppose that roots, which grow without any cuticle protection in hard soil conditions,are resistant to chemical attack.

Table 1 : Effect of different enzyme combinations and concentrations on protoplast release. The results (means of 3 e• were obtained after a maceration time of 5h at 23 ~ 1 ~ C, pH 5.5 - 5.6. Cellulase Rhozyme Macerozyme Driselase Protoplastyield RiO %

HP 150%

0.5 0.6 o. 7 0.7 1 1.2

o.25 0.25 o.3 0.4 o.3 0.3

Table After 7 days in culture and, every ten days thereafter, 1 ml of fresh medium with reduced osmoticum (in 0.i M decrements) was added, until the overall concentration had reached approximately 0.3 M. Callus culture : Callus was cultured in a plating medium supplemented with 150 mg/1 casein hydrolysate, vitamin free (Merck), 2 mM NH 4 N03 and various growth regulators : BA (4.4, 3.3 or 2.2 ~M), IAA (5 or 0.5 ~M), NAA (8 or 5.3)/M), zeatin (0.4)/M). Its sugar content was reduced to 55 mM glucose and 30 mM sucrose. It was gelled with either 0.7~ agarose (type VII, low gelling temperature agarose ; Sigma) or 0.7% D i f c o Bacto agar. The microcalluses in Petri dishes were placed under a 16~ light/8h d ~ k cycle (light intensity : 10-12A~nol m-~s - ) at 23 - I~ When the microcalluses were 5 mm in diameter, they were transferred to an MS/2 differentiation medium gelled with Difco Bacto agar.

RESULTS AND DISCUSSION

Prot0Plast isolation an_d purification Isolation Several mixtures and digestion media were { e s t ~ ? - T h e best result was obtained with i~ Cellulase RI0 + 0.3~ RhozyUne HP 150 + 0.3% Macerozyme RI0 (Table i). The hemicellulase Rhozyme HP 150 has been reported to be particularly effective on the digestion of root cell walls (Xu e~t as 1982a ; Ochatt 6~ ~s 1987). The use of cysteine as a pretreatment (Senn and Pilet 1980 ; Faye and David 1983) or directly in the digestion medium, as we did, was decisive for the release of protoplasts. Without cysteine, root tissue turned dark brown and delivered few protoplasts (Table 2). The yield of ~eleased protopla~ts before purification (i0 - ii x i0 /g FW or 16 x l0 per i0 mm root segment) appears to ~ low compared to that of leaf protoplasts: from 9 x i0 to 3 x i0 /g FW when very young leaf tissue of Popus was used (~ussell and Mc Cown 1986). However, enzymatic isolation of root protoplasts has rarely been reported (Xu 6s ~s 1982a ; Xu Zs ~s 1982b; Senn and Pilet 1980) especially for woody species (Faye and David 1983) and the yield of protoplasts was low : ii x i0 per 20 mm root P ~ 6 pina6t~A (Faye and David 1983}. This can be attributed, in addition to a lower number of cells in root tissues, to a low activity of cellulolytic enzymes on root cell walls (Ruesink and Thimann 1966) or to the inhibition of the

%

%

o.25 0.25 o.3 0.25 o.3 0.3

0.125 0.125

ig FW

6 7-8 9-i0 9-10 lO-ll lO-ll

x x x x x x

~05 i0~ lo_ ~ i0~ lO~ i0m

2 : Effect of various modificationsof digestion medium and of preplasmolysis on protoplast release (mean values of 3 experiments).

Treatment

Protoplast yield Ig FW

Observation

.without cysteine

low

browning of the maceration solution and of root pieces

.Preplasmolysis with 10mM cysteine (lh)

7-8 X 105

no browning

.With 10mM cysteine in the medium -10mM cysteine alone -10mM cysteine + 3% BSA

no browning 8-9 X 105 i0-ii x 105

Purification procedures Red oak protoplasts are very {~giI~-~-a I~-~Sunt of debris was always present in the filtered maceration solution. Therefore, all experiments which attempted to purify the protoplasts by spinning them down in a ionic solution failed. A gradient procedure appears to be gentler for the protoplasts and more effective in removing the debris. Several Ficoll density gradient methods were tested, including those of Gronwald and Leonard (1982) and Attree and Sheffield {1986). The latter one was t~e most reliable. It delivered approximately 4 x i0 highly pure protoplasts/g FW. Prot0plast culture and first divisions (Fig. A-B) Two of the three culture methods tested, culture in droplets and culture in liquid medium on agar, did not succeed. The first one always led to clumping of a great nunlber of protoplasts even if some of them developed into clusters. With the second one, the protoplasts did not divide and died. Protoplasts were then cultured in thin layer (2 ml of liquid culture medium in 60 mm diameter Petri dishes), and the following results were obtained with this method. Plating density (Table 3). In our experiments, the ~ s { - ? e s u ~ s - w e r e obtained with a culture density of 5 - 7 x l0 protoplasts per ml. At a lower or higher density, the first divisions o c c u r e d b u t microcalluses did not develop. Dilution within the ~irst days with

141 fresh medium did not improve this result contrary to the observation of Lain4 s af. (1988). Moreover, we noted that sustained divisions were obtained only when a large part of the initial culture medium remained in the dishes. First divisions

(Figure B).

- 3 days after plating the healthiest protoplasts had reformed a wall and the first divisions occurred. The dividing protoplasts were the small ones. They had a dense cytoplasm and appeared more refringent. - 7 days after plating (Table 3), at most 30% of the protoplasts were alive (circular form, with clear regular cytoplasm). The estimated division rate (number of divisions/number of plated protoplasts) was low : 0.7 to 0.8%, possibly due to damage caused by the enzymatic maceration (Ishii 1988) or to the inability of the most woody plant cells to dedifferentiate (Russell and Mc Cown 1986). - After 7 days, the division rate, which remained low, became impossible to determine with precision because the cells issuing from the first divisions often separated, according to the observations of Tremblay (1988) and divided again. Culture medium : two culture media were tested : MS/2 and modified WPM. The latter enhanced not only the number of first divisions, but also the number of microcalluses formed. Therefore it was routinely used for plating. Sustained divisions were obtained only when the culture medium was sterilised by filtration (0.22 ~m Millipore filter), not by autoclaving, and when 2,4-D was added.

p i p e t t e d g e n t l y into the dishes containing the cell clusters. These continued to grow on a semi solid medium and later were transferred easily to solid agarose medium. This method was proved to be effective to recover a higher yield of viable microcalluses. Under the light conditions described in material and methods the calluses became compact and dark green (Figure D).

Table 3 : Effect of protoplast density on division rate and cluster formation in modified WPM medium. Protoplast density Viability Division rate protoplast (ml)(in 2ml culture medium) (%)a (%)a at 0 day

at 7 days

at 7 days

Cluster rate (%)a at 21 days

3 x 103

i0

low

--

5 x 103

24

0.3

0.4

7 x 103

30

0,75

0.4

(%) = in % of plated protoplasts

-

Optimal and sustained division was obtained with the following combination : 2.2 ~ M BA + 1.8 ~M zeatin + 5.3 ~ M NAA + 2.2 9 M 2,4-D. Callus formation and culture A small number of dividing cells developed directly into clusters (from 10 to 20 cells.) and finally into microcalluses. Others, which had separated soon after completion of the division, gave rise to two cells again or generated microcalluses so that numerous microcalluses could be successively obtained in the same culture dish (Lain6 ~ as 1988). This observation may explain why the number of clusters formed at 21 days was higher than the division rate at 7 days for a protoplast density of 5 x i 0 (Table 3).

Shoot regeneration

attempts

The callus was subcultured on a differentiation medium supplied with different growth regulator combinations and concentrations (0.4 , 13 ~M BA ; 1.4 - 8.6)JM GA3; I)~M NAA ; 0.2 - 2.8 ~M IBA ; i.i - 4.4)/M zeatin) in which BA was always present. The dishes wer~ p%aced under higher illumination (12 to 20 ~mol m--s-~). When the calluses remained for more than 1 month in the presence of a high overall hormone concentration (i0 - 159/M), they became brown and died. Their transfer to a medium containing a low hormone concentration was beneficial and allowed a longer survival. Until now, in spite of many attempts, bud formation did not occur, although some calluses grew up to i0 mm and remained green and compact, with sometimes a nodular zone, during four to five months before turning brown and gradually dying.

The size of the microcalluses reached 0.5 mm two months after protoplast isolation (Figure C). They did not grow further in liquid culture medium, but turned brown and died. However, transfer of microcalluses to a solid medium allowed their rapid growth, up to 5 mm within 2 weeks under the most favourable conditions. These required conditions were : - removal of all surrounding liquid after transfer of the calluses to the solid medium, - supply of the culture medium with casein hydrolysate, replacement of 2,4-D by IAA. Rapid growth was obtained with a combination of 5.711M IAA + 3.3 ~ M BA + 0.4 ~M zeatin. A small number of calluses continued to grow slowly when cultured on media gelled with 0 . ~ Difco Bacto agar. The use of 0.7% agarose type VII allowed rapid growth of a larger number of calluses (up to 25 calluses of 5 mm per dish). We observed that the earlier the mic