Plant Cell, Tissueand Organ Culture 35: 99-106, 1993. © 1993 KluwerAcademicPublishers. Printedin the Netherlands.

Somaclonal variation within poplar P.L.E. Antonetti & J. Pinon*

Laboratoire de Pathologie ForestiOre, Centre de Recherches de Nancy, INRA, F-54280 Champenoux, France (*corresponding author) Received 2 February 1993; accepted in revised form 18 April 1993

Key words: Hypoxylon mammatum, Melampsora, Populus spp. ploidy, somaclonal variation, resistance Abstract

One thousand and ninety-two poplars were regenerated in vitro from callus of 13 poplar clones representing the Leuce, Aigeiros and Tacamahaca sections. At lest 44 of the regenerants differed in some way from the original clones. Somaclonal variation occurred more frequently in poplars of the Leuce section (8%) than in those of the Aigeiros or Tacamahaca sections (1%). Variation was noticed in growth habit, leaf shape or indentation but not in the reaction to four Melampsora races. However, after one growing season in the field, a few regenerants from calli of two clones ('Ogy' and 'Rap') differed in their susceptibility vis ~ vis the original clones. Cultivation of callus from Leuce poplars that had survived exposure to increasing concentrations of toxins from Hypoxylon mammatum gave rise to a toxin-tolerant line from which toxin tolerant plants were regenerated. Flow cytometry to measure the D N A content of nuclei showed that regenerants tended to be tetraploid.

Abbreviations: NAA-naphthaleneacetic acid, BAP-benzylaminopurine, T D Z - t h i d i a z u r o n , M S Murashige & Skoog medium

Introduction

Tissue culture is useful for the mass propagation of plants but the products are not always 'trueto-type' (Skirvin 1978; Mestre & Benbadis 1985): altered morphology and changed tolerance to fungal or bacterial toxins, herbicides or salts have been reported (e.g. Larkin & Scowcroft 1981). Such tolerance is potentially valuable and this has encouraged the assessment of somaclonal variation as a complement to classical hybridisation/selection breeding of food plants (e.g. Solanurn tuberosum; Behnke 1980, Triticum aestivum; Pauly et al. 1987 or Brassica napus; Sacristan 1982) and also poplar (Lester & Berbee 1977; Ostry & Skilling 1988; Prakash & Thielges 1989). In this paper we record the cytological and morphological attributes of Aigeiros,

Tacamahaca and Leuce poplars which regenerated from callus. We also described the reaction to foliar rust fungi (Melampsora larici-populina Kleb. or M. allii-populina Kleb) of Aigeiros and Tacamahaca poplars regenerated from callus. In addition, to extend knowledge implicating toxins from Hypoxylon mammatum (Wahl.) Miller in the aetiology of Hypoxylon canker (Pinon & Manion 1991) on Leuce poplars, we describe the in vitro selection of a callus line tolerant to toxins of this fungus followed by the regeneration of toxin tolerant plants.

Materials and methods

Poplar clones Eight

poplar

clones

from

Aigeiros

and

100 Table 1. Known behaviour to rusts (three races El, E2, E3 of M. larici-popufina and one race of M. allii-populina) of Populus x euramericana (=eur, Aigeiros section) and of P. x interamericana (=inter, the hybrids between Aigeiros and Tacamahaca sections), according to Pinon (1992).

Clone

Botanical type

M. larici-populina

M. allii-populina

Reaction to races

Robusta Guariento Ogy Rap L. Avanzo Carpaccio Barn Beaupr6

eur eur eur inter eur eur inter inter

E1

E2

E3

S S R R R R R R

S S S S R R R R

S S R R S S R R

Level of infection in nature

Level of infection in nature

H H V V V V N N

H M V V V V M H

s = susceptible, R = resistant, H = high, M = medium, N = null, V= variable (depending on race populations). T a c a m a h a c a sections listed in Table 1 and five L e u c e clones selected by L e m o i n e (1973) and

listed in Table 2 were included in this study. Foliage of the f o r m e r was previously challenged with M . allii-populina or three physiological races of M . larici-populina ( P i n o n 1992) whereas the latter were challenged in the past with H. m a m m a t u m (inoculated into stems of 4 - 5 yearold trees in the field) or its toxins (absorbed into petioles). Callus culture a n d regeneration

Callus was o b t a i n e d f r o m internodes of d o r m a n t twigs (diameter 1 - 1 . 5 cm) cut f r o m field-grown trees ( 1 - 3 years old). T h e twigs were surface sterilized in 70% alcohol (20 min), rinsed twice in sterile water, i m m e r s e d in a q u e o u s mercuric chloride ( 0 . 5 g 1 - 1 ; 6 0 m i n ) and finally rinsed asseptically (4 x 10min) in sterile water. T h e b a r k was r e m o v e d to expose c a m b i u m and twig slices ( 0 . 5 - 1 . 0 cm) were placed on Linsmaier &

S k o o g m e d i u m a u g m e n t e d with 10 I~M N A A and 4.5 IxM B A P ( C h a l u p a 1974). In all instances, g r o u p s of seven twig sections were incubated in Petri dishes at 25°C u n d e r fluorescent light (16-h p h o t o p e r i o d , 25 ixmol m -2 s-a). Later, all tissue cultures were achieved u n d e r these conditions of light and t e m p e r a t u r e . T h r e e to four weeks later, calli were cut into small pieces and subcultured o n the same m e d i u m . A t intervals of six weeks thereafter, half of these calli were subcultured on the same m e d i u m while the other half was transferred to the m e d i u m s u p p l e m e n t e d with plant g r o w t h regulators to facilitate bud formation (see Table 3). M i c r o p r o p a g a t i o n o f original clones

T h e original clones were m i c r o p r o p a g a t e d to p r o v i d e material for c o m p a r i s o n with putative s o m a c l o n a l variants. Lateral buds f r o m twigs of 1 - 3 y e a r old glasshouse-grown trees were established in vitro f r o m nodes that had b e e n steril-

Table 2. Botanical type and reaction of Leuce clones to Hypoxylon mammatum (recovery after inoculation) and leaf sensitivity to the toxins of the culture filtrate (foliar test: 0 = insensitivity, 100 = maximum sensitivity) according to Pinon (unpublished).

Clone

Botanical type

717-1-B4 5972-$38 802-36-7 706-8 333-4

P. tremula x P. alba P. alba P. alba × P. tremuloides P. tremula x P. alba P. tremula x P. tremuloides

*percentage of trees which heal after inoculation.

Recovery after inoculation %* 94 88 75 38 6

Sensitivity of leaves to culture filtrate 0 2 92 64 46

101

Table 3. Concentrations of NAA, BAP and TDZ (thidiazuron) (added after autoclaving) used to initiate bud formation on calli of the different clones according to Antonetti (1990). Concentrationsused (IxM) Clones

Ogy Robusta Carpaccio L. Avanzo Guariento Beaupre Barn Rap 333-4 5972-$38 706-8 717-1-B4 802-36-7

NAA

BAP

5 10 10

5 2.5-5 2.5-5 2.5 2.5 5 10 10 0.01

25 10 25

0.01 1

TDZ

0.1 0.01 0.1 0.01-0.1 0.1

ized in aqueous mercuric chloride (1 g 1-1) for 30 min before two rinses (each of ten minute duration) in sterile aqueous calcium chloride (2.5 g 1-1) and four ten minute rinses in sterile water. The mercuric chloride caused some necrosis at cut surfaces and such dead tissue was excised before the nodes were placed on medium. For Leuce clones, a modified MS medium without plant growth regulators was used and, for Aigeiros or Tacamahaca clones, MS medium with 0.1mM NAA and 0.5txM BAP was used (Antonetti 1990). After one month, the shoots which developed were excised and subcultured on the relevant media.

(5 g 1-1). Calli resisting the highest toxin concentration were regenerated.

Culture of regenerated plants Regenerated shoots (1 cm long) were transferred into tubes allowing elongation and rooting. Leuce clones were rooted on the MS modified medium without plant growth regulator (Antonetti 1990). Aigeiros and Tacamahaca clones were first transferred to an elongation medium (MS with 0.1 p~M NAA and 0.5 p.M BAP) and later rooted on the same medium as Leuce clones plus 0.5 ~M NAA (Antonetti 1990). Rooted plantlets (including those regenerated from calli and those derived directly by micropropagation of the original clones) were subsequently planted in containers (5 cm diameter) in equal parts of sphagnum peat and vermiculite at a high humidity under light (16-h photoperiod, 70 Ixmol m -2 s-l). One month later, the plants were transferred into one liter containers containing a mixture of half sphagnum peat and half sand (v/v) complemented with 2.5g 1 1 of dolomite and fertilized with 5 g -Osmocote Plus [N, P, K, MgO: 15, 10, 12, 2% and microelements: Fe (0.15%), Mn (0.06%), Cu (0.05%), Mo (0.02%), B (0.02%) and Zn (0.015%)] and grown at 21°C (continuous fluorescent light, 701~m -2 s -l and 70% relative humidity). Observations of morphology and phytopathological tests were initiated when the plants were established (after 2 weeks minimum) and thereafter the poplars were transplanted in the field.

Selection with toxins A hundred calli from Leuce clones (706-8, 3334 and 802-36-7) were cultivated on Chalupa's liquid medium supplemented with progressively increasing concentrations of H. mamrnatum culture filtrates. At the beginning of the experiments, the H. mammatum culture filtrate was diluted (1:32) with sterile water. Calli that survived the toxin at this concentration were then exposed to a 1:16 dilution and subsequent increasing concentrations until a dilution of 1:2 was reached. To prepare culture filtrate containing toxins, H. mammatum was grown at 26°C on wort medium (MacWilliam 1968) diluted (1:10) and complemented with paper pulp

Morphological observations and pathological tests The general growth habit, number of leaves developed within a month and leaf morphology (size, shape , hairiness and surface appearance) were noted. Resistance tests of Aigeiros and Tacamahaca poplars to the three races of M. larici-populina (Pinon et al. 1987; Pinon & Peulon 1989) and to M. allii-populina were done. Altogether sixteen leaf disks (30 mm in diameter) were taken from each plant for further inoculation which was achieved by spraying spore suspensions (adjusted to 5000 spores/ml of water). Each isolate (one of

102 M. allii-populina and the races El, E2 and E3 of M. larici-populina) was applied to four disks out of the sixteen disks per plant. These disks were then incubated in Petri dishes, foating on water under fluorescent light (50~mol m -2 s-l). Flecking time, sporulation time and sore number of each disk were recorded. After on year in the nursery, natural infection of 'Rap' and 'Ogy' with M. larici-populina was measured using the methods described by Pinon (1991). Statistical analysis for calculation of confident limits of means were performed according to Sokal & Rohlf (1973). The reaction to culture filtrates from H. mammatum was assessed on four leaves from each Leuce by estimating the time of appearance of leaf necrosis as earlier described by Pinon (1984) and Pinon & Manion (1991). To determine the mass of nuclear DNA, labelled with fluorochrome, flow cytometry was applied to four sets of tissue: calli, putative somaclones, regenerant plantlets lacking variant characteristics and the 'original' clones. Pieces (1 mm 2) of poplar leaf were assayed against a Petunia hybrida leaf standard (2C = 3.2 pg DNA) in the medium described by Galbraith et al. (1983) supplemented with Bmercaptoethanol (1 Ixl m1-1) and 2 txg m1-1 Hoechst 33342 fluorochrome. Poplar callus and P. hybrida leaves were cut in the solution described by Bergounioux et al. (1986) supplemented with Bmercaptoethanol (1 ~1 m1-1) and 2.5 Ixg m1-1) of the fluorochrom. Each analysis included approximatively 10,000 nuclei and was conducted at 4°C on EPICS V (Coulter Electronics Hialeah, FI.) equipped with a 100 ~ nozzle and a UV krypton laser (346 nm). Fluorescence was logarithmically amplified. Taking account of the fluorochrom used and the possibility that Petunia and poplar have different proportions of A and T in their DNA, only relative amounts of nuclear DNA were recorded.

Fig. I. Modificationsin foliar morphology and growth habit in P. alba cv. '5972-S-38' (Leuce section). Left: variant, right: original clone.

unusual leaf shape or modified growth habit (Figs 1, 2). Four of eight Aigeiros and Tacamahaca clones had distinguishable leaf morphology (Table 4); in 'Rap' this variation was obvious after only two cycles of subculturing. Variants were recovered in 'Robusta', 'Ogy' and 'Barn' during four to six callus subcultures. Inoculation of leaf disks (of all the regenerated plants and of original clones of Aigeiros and Tacamahaca sections) showed clearly that susceptibility or resistance of the regenerated plants to the four Melampsora isolates were indistinguishable from that of the original clones indicating that genes for racespecific resistance had not been lost or altered. No differences appeared for the duration of

Results and discussion

Detectable variation was noted in 44 of the 1092 regenerated poplars. The most obvious changes were short internodes, early branching, deeper indentations on leaf margin, loss of hairiness on the abaxial side of the leaf blade (Leuce section),

Fig. 2. Modifications in the shape of leaf blade in P. × euramericana cv. 'Ogy' (Aigeiros section). Left and right: variants, center: original clone.

103 Table 4. Variations occurring within Aigeiros and Tacamahaca clones in regenerated plants. Clones

Robusta Ogy Guariento Carpaccio L. Avanzo

No. of regener ated plants

Variants among regenerated plants No. (%)

No. of subcultures (No. of variants analysed)

Variant characteristics

Ploidy

81 257

1( 1.23) 5( 1.94)

6( 1) 4(2) 5(1)

p 2LS LI

4n 4n 4n

18 12 17

Non variant regenerated plants

Analysed calli

Analy(No.)

No. of subcullures

Ploidy

37

1 to 5

2n

0 0 0

Rap Barn Beaupre

183 55 61

2(1.09) 1(1.81) 0

Total

674

9(1,33)

2(2) 6(1)

2LI L]

4n 4n

35

lto5

2n

No.

No, of sub cultures

Ploidy

2

10

4n

1 1

10 20

4n 4n

1

10

4n

Foliar morphology: LS = shape of leaf blade modified. LI = leaf indentation modified, p = shorter petiole.

incubation and latency between the original clones and the regenerated plants. To obtain a quantitative estimate of susceptibility to natural infection by M. larici-populina, rust was assessed on regenerated plants of 'Ogy' and 'Rap' cultivated in the field. Figure 3 presents, for those two clones the population of the regenerated plants in comparison with the original clones propagated in vitro. For both clones F tests achieved to compare variances indicated that OGY

.

.

I0

.

15

.

20

.

.

25

3O m

.

.

.

.

.

35 4O 4G Go INFECTION (X) ~ge~roted I~3 orSglnat

im GG

60

85

70

7G

RAP

: 15

0 20

:

0 25

:

0 50

2

:

•

: 35

0 4O

M

2

. • 0 : : : . . 50 55 80 65 70 INFECTION (X) reQentr~tod ~ original :

0

,15

:

0

i, . . . 75 80

. 85

,3 90

95

Fig. 3. Natural infection by rust (M. larici-populina) in the nursery of the original clones ('Ogy' and 'Rap') and of the populations of plants regenerated from these clones. (Number associated with each class of infection represents its upper limit).

there was no significant differences between the mean infection of regenerated and original clones. For the clone 'Ogy', the means of percentage of infection of the two populations of plants were not significantly different at p < 0.05: 29.3 - 65.04 (--+ standard deviation) for the original clone and 30.1-+2.03 for the regenerated plants. Taking into account the standard deviation of the regenerated population, we calculated the confidence limits assigned to individuals from this population at different levels of risks (p < 0.05; p < 0 . 0 1 ; p <0.001). This revealed that respectively 9, 5 and 0 individuals were out of the acceptance region of standard deviation, which indicates that they may be variants characterized by a higher susceptibility. For " R a p " , the means did not differ significantly at p <0.05: 76 +- 5.5 for the original clone and 73.8 -+ 1.9 for the regenerated plants. At p < 0.05, one regenerated plant with high infection and three with small infection were out of the acceptance region. At p < 0.001, those three were still outliers suggesting that they may be variants with reduced infection. Therefore, plants presumed to be variants for their infection by rust have been multiplied for further examination. Our results suggest that qualitative reaction to rusts may be less subject to variation than quantitative characteristics. Quantitative variations have been also described for the susceptibility of poplar to Septoria musiva (Ostry & Skilling 1988) and to M. medusae (Prakash & Thielges 1989). In the Leuce section, all clones except one gave rise to a higher frequency of variants than clones of Aigeiros and Tacamahaca sections

104 Table 5. Variations occuring within Leuce clones in regenerated plants.

Clones

N u m b e r of

regenerated plants

333-4 5972 $38 706-8

92 23 57

717-1-B4

231

802-36-7

15

Total

418

Variants among regenerated plants Number (%)

0 5(21,7) 8(14,03)

20(8,65)

2(13,33)

Analysed calli

N u m b e r of subcultures (number of variants analysed)

Variant characteristics

Ploidy

4(2) 2(1) 4(1)

LS, LS + A LI LS

2n 4n 2n + 4n

4(2) 5(1) 6(1) 5(1)

LH, LI i LI LS

2n,4n 2n 4n 2n

Number

N u m b e r of subcultures

Ploidy

1 1 1

5 10 10

4n 2n + 4n 4n

35(8,37)

A = abnormal growth habit, i = shorter internodes, L H = loss of hairiness, LI = leaf indentation modified, LS = shape of leaf blade modified.

(Table 5). Morphological variations appeared after the 4th subculture and affected a large range of characters. Among calli treated with H. mammatum toxins one revealed an acquired tolerance. It was derived from the clone 706-8 and six plants were regenerated from this callus line variant. Leaves from these plants were also more toxin-tolerant than those of the original clone (Table 6). While most of the regenerated plants did not significantly differ from the original type in the appearance time of leaf necrosis,

Table 6. Sensitivity of leaves from regenerated plantlets of P. tremula × P. alba cv. '706-8' to Hypoxylon mammatum

culture filtrate (foliar test). Regenerated plants all (57) individual variants: ***

Delay to necrosis (days)*

A B&C D E F

1 1.25 2.5 2.75 3.25

- 0 . 1 3 7 -+ 0.38**

*Toxin-tolerance is expressed by a delay in the development of necrosis compared to the original clone (whose value is zero) on a scale of 0-5 (Pinon 1984) ** -+ standard deviation ***These six variants have been regenerated from callus line which revealed an acquired tolerance to Hypoxylon maremature culture filtrate.

these six plants had a delayed reaction indicating a higher level of toxin tolerance. For all poplar genotypes, flow cytometry of leaves or young twig tissue indicated that most variants were tetraploid or heteroploid whereas the original clones were all diploid (Table 4, 5). Some of the calli were heteroploid but tetraploidy was more frequently observed. As shown in Fig. 4, flow cytometric analysis on 'Rap' revealed that, in the apical tissue of the original clone, a peak corresponding to 2C (most nuclei at G1) was accompanied by a smaller peak corresponding to 4C (reflecting the presence of cell nuclei at G2). By contrast, the fully expanded leaf gave only the peak equivalent to 2C. When the variants were tested, only the peak corresponding to 4C was visible indicating tetraploidy. Flow cytometry was done also on some regenerated plants which doesn't show variations. All tested plants were diploid (Table 4). After a growing season, flow cytometry analysis have been done again for many variants and the results are identical indicating a stability of the ploidy variations. A similar tendency towards tetraploidy was described by Lutz (1985). Somaclonal variation was not frequent (1.33%) in plants with Aigeiros or Tacamahaca parentage and in these, morphological differences vis ~ vis the original clones were not readily seen. By contrast, regenerants with

105

2C

Leuce poplar or perhaps may result from the use of thidiazuron to achieve the regeneration of those poplars. This phenylurea has a strong cytokinin activity and present results on Leuce poplars raise the question of an eventual mutagenic activity of this compound. However, only changes in plant metabolism due to thidiazuron have been recorded in the literature in the last decade. To clarify this point, it would be necessary to check if some variations obtained in Leuce poplars are heritable and to compare the effects of thidiazuron and BAP on variation levels on the same clones (including Aigeiros and Tacamahaca poplars).

4CP

True-to-type

(apex)

Conclusion

2C

P

True-to-type (leaf)

•

•

,

•

, , ; , ,

•

•

,

t •

, , , , ,

,

,

•

, , , , , o

~P

V a r i a n t V1 Fig. 4. Flow cytometry analysis of the clone 'Rap': fre-

quencies of nuclei with different relative nuclear D N A (logarithmic scale for DNA). Spectra of original clones (apex: top and leaf: middle) and of a variant (bottom). P = Petunia hybrida, 2C = nuclei at stage 2C, 4C = nuclei at stage 4C.

Leuce parentage varied more frequently (8.37%). Interestingly, although tetraploidy was frequent, the most pronounced variants were diploid. Such deep modifications and high frequency of variation may be characteristic of

Our results confirm that callus culture of poplar followed by regeneration can lead to plants variant for different characters: foliar morphology, growth habit, behaviour to rust or to a fungal toxin and ploidy. These characteristics were stable: after one growing season variation for rust behaviour was proved and tetraploidy was confirmed. The readiness with which tetraploidy was induced suggests new opportunities for poplar breeders. No variation was observed for qualitative resistance to rusts within Aigeiros and Tacamahaca poplars. Such resistance is generally controlled by a few major genes (Van Der Plank 1968) which apparently have not been lost or altered. This lack of qualitative trait differences may be just a matter of probability: fewer genes influence such traits and are less likely to be affected if somaclonal variation is random. After one growing season in the nursery exposed to natural inoculum, the levels of infection (and their variability) are similar for original clones and most of the regenerated plants. Nevertheless, a few regenerated plants were out of the acceptance region of the standard deviation which may indicate some variation for this quantitative trait [generally controlled by several minor genes (Van Der Plank 1968)]. Recovery of plants tolerant to H. mammatum toxins is encouraging. However, we must be sure that this trait will continue to be expressed in older plants and also determine the possible

106 relationship between toxin resistance and canker resistance. Lastly, it will be interesting in future to test if all these variations can be transmitted from the parent to its progeny. This point would permit to see if these variations are or not epigenetic.

Acknowledgements Authors are indebted to D. Chriqui, S. Brown & D. Marie for flow cytometry analysis. We thank A. Schipfer and M. Perochon for their technical assistance, respectively, in rust-resistance tests and plant cultivation in the nursery. Research was founded by INRA biotechnology grant. Thidiazuron was kindly supplied by B. Liche (Schering AG). Grateful thanks are due to Dr JI Cooper for reviewing the manuscript.

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among black poplar trees derived from callus culture. For. Sci. 23:122-131 Lemoine M (1973) Am61ioration des peupliers de la section Leuce sur sols hydromorphes. Th6se, Univ. Nancy I, no 8205 Lutz A (1985) L'expression de la variabilit6 morphologique apr6s r6g6n6ration dans les cultures de tissus de cellules, In: La Variabilit6 des Cellules V6g6tales Cultiv6es In Vitro. Bull. Soc. Bot. Fr, Actual Bot 132. 3 / 4 : 3 5 - 5 0 MacWilliam IC (1968) Wort composition - a review. J. Inst. Brew. 74:38-53 Mestre JC & Benbadis A (1985) La variabilit6 des cellules v6g6tales cultiv6es in vitro. Bull. Soc. Bot. Fr. 132, Actual Bot 185 (170 p) Ostry ME & Skilling DD (1988) Somtic variation in resistance of Populus to Septoria musiva. Plant Disease 72: 724-726 Pauly MH, Shane WW & Gengenbach BG (1987) Selection for bacterial blight phytotoxin resistance in wheat tissue culture. Crop. Sci. 27:340-344 Pinon J (1984) Propri6t6s biologiques de la toxine d'Hypoxylon mammatum, parasite des peupliers de la section Leuce. Rev. Cytol. Biol. V6g6t. Bot. 7:271-277 Pinon J, van Dam B, Genetet I & De Kam M (1987) Two pathogenic races of Melampsora larici-populina in NorthWestern Europe. Eur. J. For. Path. 17:47-53 Pinon J & Peulon V (1989) Mise en 6vidence d'une troisi~me race physiologique de Melampsora larici-populina Kleb. en Europe. Cryptogam Mycol. 10:95-106 Pinon J (1991) Comportement des principaux clones de peuplier fi l'6gard des rouilles et plus praticuli~rement de Melarnpsora larici-populina. Rev. For. Fr. 43:301-308 Pinon J & Manion PD (1991) Hypoxylon mamrnaturn and its toxins-recent advances in understanding their relationships with canker disease of poplar. Eur. J. For. Path. 21: 202-209 Pinon J (1992) Poplars rusts: variability and populations in France. Proc. IPC, Saragosse, Spain, 22-25 Sept 1992: 211-222 Prakash CS & Thielges BA (1989) Somaclonal variation in eastern cottonwood for race specific partial resistance to leaf rust disease. Phytopathology 79:805-808 Sacristan MD (1982) Resistance responses to Phoma lingam of plants regenerated from selected cell and embryogenic cultures of haploid Brassica napus. Theor. Appl. Genet. 61:193-200 Sokal & Rohlf (1973) Introduction to biostatistics (pp 103107). Freeman, San Francisco Skirvin RM (1978) Natural and induced variation in tissue culture. Euphytica 27:241-266 Van Der Plank JE (1968) Disease resitance in plants. Academic Press (London) (206 p)