\
Pathology
ALTERED RUST RESISTANCE AND YIELD COMPONENTS ARISING
FROM LEAF TISSUE AND BUD CULTURE IN SUGARCANE
J.P. Peros*, E. Bonnel*, L. Fererol** a n d J.C. Mauboussin***
**
* CIRADARAT, St Denis, Reunion, France
CIRADARAT, Petit Bourg, Guadeloupe, France
***
~
CIRADARAT, Montpellier, France
Key words: somaclonal variation, b u d culture, rust, y i e l d components,
flowering, isozymes
ABSTRACT
I
Sugarcane plants (cv B 4362) regenerated from calii (S) were compared with plants derived from cuttings (C) or micropropagated from axillary buds (B) for susceptibility to
Puccinia melanocephala and yield components. On overage, rust symptoms oppeared
to be more severe in the S and B plants, even after further propagation by cuttings of
the S plants. The S and B plants showed a decline in stalk diameter. The S plants had
reduced vegetative vigour, while tillering increased in the plant stage but decreased
in ratoons. None of the S plants was found to be resistant to rust, but in Reunion two
of them exhibited a high proportion of flowering stalks although cv B 4362 is considered
to be non-flowering there. One of the two S plants was characterized by the lack of an
isozyme common to the leucine and valine aminopeptidase systems.
I
INTRODUCTION
Since the early seventies, i n vitro methods have aroused great interest for sugarcane
improvement (MaretzkiI5). Especially, it has been suggested that plants regenerated
from calli (somaclones) differ from the original clone in their genetic characters. Heinz6,
Krishnamurthi and Tlaska19, Larkin and Scrowcroft" and Liu12 succeeded in obtaining
somaclones resistant to diseases through i n vitro culture of leaf tissues from susceptible
cultivars. Yield components can also be different in the somaclones, eg Bonnel et aP
a n d Kresovitch et a18 reported increased tillering and reduced stalk diameter, while Liu
et all3 obtained an improved somaclone from the cultivar H37-1933.
However, the usefulness of somaclonal variation for sugarcane improvement depends
on its rate, and Irvine7 showed that it has very likely been overestimated in the past.
In addition, Bonnel et aI2 and Kresovitch et a18 demonstrated that ranges of variations
in somaclonal and clonal populations were similar.
Regeneration has been proposed as another useful method for propagation (Anderlini
and Kostkal). However, as modified somaclones may be propagated it would be better to use meristems or axillary buds.
In the work presented here, plants of cultivar B 4362, derived from tissue culture, were
compared with their cutting-derived counterparts for rust susceptibility and yield components in two locations. In addition, plants derived from bud culture were included
in the experiment in one locarion.
MATERIALS AND METHODS
Somaclone production
Leaf explants were removed from B 4362 plants grown in the greenhouse at Montpelli-
I
II
J.F! PEROS, E. BONNEL, L. FEREROL AND J.C. MAUBOUSSIN
I
Experiments in Reunion
er, France. Calli were initiated on MI1 medium (Chagvardieff et a13) and subcultured
on either the same medium or a CS3-3-medium (Larkinlo). Somaclone (S) regeneration was achieved by transferr~ngthe calli to MOO medium (Chagvardieff et UP). Those
plantlets regenerated from calli, which had been grown only on M11, were subsequently designated S1, and the others S2. The somaclones were propagated on a MOO medium supplemented with kinetin (0.5 mg/f?), then transplanted into a MOO medium with
high sucrose content (80 g/!) for rooting. They were subsequently sent to Guadeloupe
and Reunion.
The somaclones (S plants) and pre-germinated one-eye cuttings (C plants) were simultaneously moved to polyethene bags containing 0.3 !of a v:v mixture of soil and pozzolana. Both populations were grown in a greenhouse made safe against fungus spore
penetration, with mist irrigation and weekly complete fertilization.
When the plants were 2 to 3 months old, they were inoculated by settling uredospores.
from Puccinia rnelanocephala on the lower blade surface of the youngest leaf with a
visible dewlap (P6ros17). The ten plants simultaneously inoculated constituted a group
of two C plants randomly distributed among eight S plants. After 15 days, the number
of sorl on a 10-cm2 surface area of the inoculated leaf were counted.
The groups of plants, inoculated or not, were moved to the field from October 1984 to
September 1985 according to the design presented in Fig 1. Each individual plant transferred to the field in 1984, provided it produced at least 18 three-eye cuttings, was replanted in December 1985 as a single-row plot 3-m long, with S and C plots alternating both
In a row and between different rows.
(m) : observed (non o b s e r v e d ) C p l a n t s
o: S p l a n t s
A:
B
plants
Figure 1. Diagram of two neighbouring sets of the single-stool trial in Reunion (1)
and in Guadeloupe (2)
After natural infection, uredosori numbers were counted in 4 month-old plants as previously described. For the single-stool trial, counts were made on the third unfolded leaf
PATH0LOGY
of the main stalk of all individuals in plant cane, and of the individuals transplanted
in 1984 in first ratoon. In plant cane of the single-row trial, counts were averaged for
five leaves sampled along each plot.
For the individuals transplanted in 1984, yield components were measured at the time
of plant cane harvest (December 1985), then at the age of 5.5 months for the first ratoon
stage and the plant cane of the single-row trial.. Data included:
i ) stalk number in each stool (single-stool trial) or in each row (single-row trial) plot;
i i ) height of the tallest stalk (single-stool plant cane), height and diamater of the tallest
two stalks (single-stool first ratoon) or three stalks (single-row plant cane); and
iii) stalk weight in each stool (single-stool cane).
Experiments in Guadeloupe
Bud culture was carried out using the procedure of Sauvaire and ~ a l z ~However,
' ~ .
only
very young buds were excised and they were not subjected to disinfection. The B plantlets
thus obtained were propagated on MOO medium. S and B plantlets were simultaneously transferred to small peat pots kept damp on benches maintained in the shade.
Disease evaluation was done only under field conditions and only in the single-stool
trial. Groups composed of two B plants distributed at random among eight S plants, were
planted in the field from August 1985 to October 1986, and were bordered with C plants
obtained from two-eye cuttings directly planted in the field (Fig 1).
Uredosori counts were carried out during the plant and first ratoon stages, in plants three
and two months old respectively. Numbers of stalks per stool and means calculated from
height and diameter of the highest two stalks were determined for the second ratoon
crop at the age of six months.
Enzyme electrophoresis
Amylases, esterases, glutamate oxalotransaminases, peroxidases, valine and leucine
aminopeptidases were studied using the methods-defined by Feldmann5.
RESULTS
Susceptibility to rust
All the somaclones evaluated in the greenhouse exhibited at least one uredosorus, as
did those which were only examined in the field in Reunion and Guadeloupe.
As somaclone susceptibility to rust did not seem to depend on the medium in which
it was grown, the results are presented for the whole somaclonal population (Tables
1 and 2). In Reunion, all means obtained for S were higher than those for C. In
Guadeloupe, S and B were found to be more susceptible than C in plant cane in the
single-stool trial, but there were no differences in the first ratoon. Neither the coeficients of variation nor the standard errors differed significantly or repeatedly depending on the plant origin. In addition, no correlation was observed between the reponses
obtained for the same individuqls in successive evaluations.
Yield components
In Reunion, the rate of survival in the C population transplanted to the field proved higher
in both plant and ratoon stages than that in the S1 population, which in turn was higher
.than that in the 52 population. The S plants sbowed poorer vegetative vigour than the
C plants, and their leaf blades were shorter and narrower. Results of the measurements
J.P. PEROS, E. BONNEL, L. FEREROL AND J.C. MAUBOUSSIN
Table 1
- Comparison of sugarcane plants (cv 84362) derived from cuttings (C) and
tissue culture (S) for susceptibility to P melanocephala in Reunion.
C
S
Treatment
n
m
CV
SE
n
m
CV
SE
Potted plants
Single-stool plant
Single-stoolratoon
Single-row plant
170
188
88
109
30.1
70.8
48.2
20.9
133
74
45
61
f3.1
k3.8
f2.3
f 1.2
393
350
97
109
62.6
95.3
66.1
39.3
73
82
42
61
f 2.3
f 4.2
f2.8
f2.3
: number of observated individuals
n
m : mean uredosori on a 10 cm2 leaf area
CV : coefficient of variation (%):
SE : standard error
Table 2
- Comparison at the single-stool stage of sugarcane plants (cv 84362) derived from cuttings (C), bud culture (B) and tissue culture (S) for susceptibility to /? melanocephala in Guadeloupe.
C
Crop
Plant
Ratoon
n
m
1 1 1 64.5
102 54.4
B
S
CV
SE
n
m
CV
83
56
f5.1
f3.0
87
74
94.5
49.5
75
60
SE
n
m
f 7 . 6 328 85.0
f 3 . 6 181 51.1
CV
SE
77
60
f3.6
f2.3
Legend as in Table 1
performed are presented in Table 3. In plant cane of the single-stool trial, the somaclones
were characterized by increased tillering and considerably reduced we~ght,but primarily stalk length was comparable with that of the C plants. In the first ratoon, they appeared to.be inferior to the control in stalk diameter, equivalent or inferior in tillering,
and inferior in stalk height. In the single-row trial, the only remainig differences between S and C plants were in leaf aspect and stalk diameter. These morphological differences were more prominent in the S2 population. Coefficients of variation and standard errors were higher in S than in C populations (Table 3).
In Guadeloupe, rate of survival in the S population was lower than in the B population.
A considerable percentage of the S2 individuals did not regrow following harvest. In
the second ratoon, S1 and S2 plants showed reduced tillering, stalk height and stalk
diameter in comparision with B plants. B responses seemed intermediate between those
of S and C. The statistical parameters (CV and SE) were highest in S and intermediate
In B populations (Table 4).
Flowering and electrophoresis
Though clone B 4362 is observed to be non-flowering in Reunion, two of the somaclones
exhibited a high proportion of flowering stalks in the first and second ratoon crops of
the single-row trial. One of the flowering somaclones, regenerated from a callus grown
on CS3-3 medium, showed poor vigour in the field. An electrophoresis analysis showed
that its enzyme systems were sim~larto those of the control and the other flowering
somaclone, except for the lack of one band common to the leucine and valine
aminopeptidases.
'
PATHOLOGY
Table 3
- Comparison of sugarcane plants (cv B4362) derived from cuttings (C) and
tissue culture (S) for yield components in Reunion.
Item
C
Single-stool plant n = 92
Height (cm)
Stalk number
Weight (kg)
n = 127
n = 78
m
CV
SE
m
CV
23
50
74
f4
k0.4
k0.4
154
9.5
3.5
32
62
101
m
18
45
16
SE
m
f4
143
k0.5 10.4
k0.3 3.0
SE
m
+3
112
f0.6 10.8
k0.05 1.88
Single-row plant n = 126
CV
SE
29
62
33
m
m
CV
10
15
10
SE
m
k1
139
k0.7 52.0
f0.02 2.28
CV
11
25
11
SE
30
59
99
+5
f0.7
k0.3
CV
f4
90
k0.9 9.1
f0.08 1.50
53
80
54
SE
+9
k1.4
k0.15
n = 43
n = 65
135
52.5
2.53
CV
n = 27
n = 59
CV
126
10.6
2.58
Height (cm)
Stalknumber
Diameter (cm)
S2
155
8.1
5.3
Single-stool ratoon n = 65
Height (cm)
Stalknumber
Diameter (cm)
S1
SE
m
CV
+2
134 22
k1.3 53.3 28
k0.03 2.19 16
SE
+4
k2.3
f0.05
S1 : somaclones derived from calli cultured on C11 medium solely
S2 : somaclones derived from calli also cultured on CS3-3 medium
Other symbols as in legend of Table 1
Table 4
- Comparison at the single-stool second ratoon stage of sugarcane plants (cv
84362) derived from cuttings (C), bud culture (B) and tissue culture (S) for
yield components in Guadeloupe.
B (n = 63)
C (n = 95)
Parameter
n
CV
Height (cm)
172 13
Stalknumber 10.6 46
Diameter (cm) 2.06 13
SE
n
CV
SE
S1 (n -57)
n
CV
52
166 19 +4
154 21
k0.5 7.6 54 f0.5 5.9 66
k0.03 1.91 22 f 0.05 1.75 29
SE
S2 (n = 25)
n
CV
SE
f4
157 23 f8
f0.5 5.7 63 f0.7
rt0.07 1.65 26 k0.09
Legend as in Table 3
DISCUSSION AND CONCLUSIONS
Nome of the somaclones exhibited complete resistance to P melanocephala. From the
simultaneous examination of plants derived from cuttings and from bud culture, it i s
apparent that somaclonal variation for rust severity may be considered similar to the
random variation of a quantitative variable. Bonnel et aI2 and White and lrvineI9 came
1
J.P. PEROS, E. BONNEL, L. FEREROL AND J.C. MAUBOUSSIN
to the same conclusion for somaclone fesistance to sugarcane borer and gumming,
respectively. In contrast, the variability for yield components was higher in somaclones
than in cutting-derived plants; this also applied, though to a smaller extent, in budderived plants. Unlike observations made by Lourens and l art in'^, comparative morphological variability in the vifroplants remained higher in the first ratoon crop and after the first replanting. Lack of synthesis of an isozyme by one of the somaclones might
be explained by either a chromosome deletion (1rvine7)or an alteration of the regulation mechanisms involved in protein synthesis.
Besides somaclonal variation, evidence is given in this study of overall effects related
to in vitro culture, since frequent recurrence of differences was observed through successive stages and between sites. Plants derived from calli are characterized by higher
susceptibility to rust, increased tillering in the plant crop, reduced stalk diameter in all
stages examined, and poor vegetative vigour, especially in ratoons. In Guadeloupe,
differences were also noticed between B and C populations, but it is possible that the
mode of planting and the position of C plants may have caused such differences, especially for vigour and tillering.
Comstock and Maretzki4 showed that rust-susceptible somaclones obta~nedfrom a resistant cultivar retained their increased susceptibility even after a second vegetative propagation. It was observed here that a somaclone population of the susceptible cultivar
B4362 kept its increased susceptibility after one vegetative propagation. This altered resistance to rust might partially account for declining vigour, but morphological modifications arising from vitroculture have been noted previously in sugarcane (Bonnel et aI2,
Kresovitch et aI8). The amount of variation was clearly related to the methods used,
since bud-derived vitroplants seemed to differ less from control plants than somaclones,
and since somaclone characteristics were influenced by the callus culture medium.
It has been suggested that i n vitro propagation induces a return to a juvenile condition
( ~ o r e z a n ' ~This
) . suggestion might be of interest for sugarcane, a vegetatively propagated plant, in which iuvenile characteristics are still incompletely elucidated. Original seedlings are known to favour rust expression and to produce great numbers of thin stalks.
In sugarcane, clonal specificity in regard to response to vitromethods (Lourens and
art in'^) makes the conditions of their use difficult to define. It therefore seems necessary to examine, on a clone by clone basis, the stability of the modifications induced
and their positive or negative effects on yield prior to any commercial application. In
view of the results obtained here, it is also Important to note that these effects are not
peculiar to somaclones but may also concern plants micropropagated from bud cultures.
ACKNOWLEDGEMENTS
The authors thank Miss Paulet, Miss Boisne-Noc, Mr Lombard, Mr Gelabale and Mr Telismart for technical assistance. They also are indebted to Dr Chagvardieff and Mr Girard
for having contributed to the initiation of this study, and to Dr Glaszmann for elecirophoresis analyses.
REFERENCES
1. Anderlini, TA and Kotska, SJ (1986). Initial yield responses of Kleentek tissue culture
produced seed cane in Louisiana. Proc int Soc Sug Cane Technol 19: 391-401.
2. Bonnel, E, PBros, JP and Girard, JC (1988). Evaluation of sugarcane somaclones for
gumming disease resistance and agronomic performances. Sug Cane (4): 15-17.
3. Chagvardieff, P, Bonnel, E and Demarly, Y (1981). La culture in vitro de tissus somatiques de canne a sucre (Saccharum sp). Agron Trop 36: 266-278.
PATHOLOGY
4. Comstock, JC and Maretski, A (1987). Selection of somaclonal variapts to the sugarcane rust pathogen, Puccinia rnelanocephala. Phytophatology 77: 1776 (Abstract).
5. Feldmann, P (1985). Identification varietale de la canne b sucre (Saccharurn sp) par
I'electrophor6se d'isozymes. Agron Trop 40: 124-128.
6. Heinz, DJ (1973). Sugarcane improvement through induced mutations using vegetative propagules and cell culture techniques. l,n: Induced mutations in vegetatively
propagated plants. Int Atom Energy Agen, Vienne 1972, pp 53-59.
7. Irvine, JE (1984). THe frequency of marker changes in sugarcane plants regenerated
from callus culture. Plant Cell Tissue Organ Cult 3: 201-209.
8. Kresovitch, S, McGee, RE, Drawe, HJ and Rivera, JL (1986). Variability of agronomic
characters in populations of tissue culture-derived and vegetatively-propagated sugarcane. Proc int Soc Sug Cane Technol 19: 528-532.
9. Krisnamurthi, M and Tlaskal, J (1974). Fiji disease resistant Saccharurn officinarurn
var. Pindar sub-clones from tissue cultures. Proc int Soc Sug Cane Technol 15: 130-137.
10. Larkin, PJ (1982). Sugarcane tissue and protoplast culture. Plant Cell Tissue Organ
Cult 1: 149-164.
11. Larkin, PJ and Scowcroft, WR (1983). Somaclonal variation and eyespot toxin tolerance in sugarcane. Plant Cell Tissue Organ Cult 2: 111-121.
12. Liu, MC (1981). In vitro methods applied to sugarcane improvements, pp 299-323.
In: Thorpe, TH (Ed) Plant tissue culture methods and applications in agriculture. Academic Press, London.
13. Liu, MC, Yeh, HS and Chen, WH (1984). Tissue and cell culture as aids to sugarcane
breeding a high-sucrose and vigorously growing calliclone 71-4829. Taiwan Sug 31:
(3): 77-83.
14. Lourens, AG and Martin, FA (1987). Evaluation of i n vitro propagated hybrids for
somaclonal variation. Crop Sc 27: 793-796.
15. Maretzki, A (1987). Tissue culture: its prospects and problems, pp 343-384. In: Heinz,
DJ (Ed). Sugarcane improvement through breeding. Elsevier, Amsterdam.
16. Nozeran, R (1978). Polymorphisme des individus issus de la multiplication vegetat ~ v edes vegetaux superieurs, avec conservation du potentiel genetique. Physiol V6gBt
16: 177-194.
17. PBros, JP (1989). Etude quantitative de la resistance de la canne tr sucre b Puccinia
rnelanocephala. Agron Trop (in press).
18. Sauvaire, D and Galzy, R (1978). Multiplication vegetative de la canne a sucre (Saccharurn sp) par bouturage i n vitro. CR Acad Sc Paris (D) 287: 467-470.
19. White, WH and Irvine, JE (1987). Evaluation of variation in resistance to sugarcane
borer (Lepidoptera: Pyral~dae)in a population of sugarcane der~vedfrom tissue culture. J Econ Entornol 80: 182-184.
MODIFICATIONS DE LA SENSlBlLlTE A LA ROUILLE ET DE
PARAMETRES DU RENDEMENT APRES CULTURE DE TlSSUS
FOLlAlRES OU CULTURE DE BOURGEONS DE CANNE A SUCRE
JP PBros*, E Bonnel*, L Fereol** et JC Mauboussin***
**
* CIRADARAT, St Denis, Reunion, France
CIRADARAT, Petit Bourg, Guadeloupe, France
* * * CIRADARAT, Montpellier, France
Des plants de canne a sucre (cvB43-62) issus de neoformation-de-cals (S), de boutures
(C) ou de culture de bourgeons (B) ont Bt6 compares pour leur sensibilite b Puccinia
melanocephala et pour certains parametres du rendement. En moyenne, les symptBmes
de rouille s'averent plus severes sur les plantes S et B, m8me apres une replantation '
I'\
des plantes S Les plantes S et B presentent une diminution du diametre des tiges. La
vigueur des.plantes S est reduite alors que leur tallage est augment6 en vierge et diminui.
1
en sepousses. Aucune plante resistante a la rouille n'a kt6 trouvee alors que deux d'entre
elles ont fleuri. Le clone B43-62 est connu pour ne pas fleurir a la Reunion. Un de ces
deux somaclones se caracterise por ['absence d'une bande disozyme commune aux
leucine aminopeptidases et valine aminopeptidases.
MODlFlCAClONES DE LA RESISTENCIA A ROYA Y COMPONENTES
DEL RENDlMlENTO SURGIDAS DE CULTIVO BE TEJIDOS DE HOJAS Y
YEMAS EN CANA DE AZUCAR
JP Peros*, E Bonnel*, L FBreol** et JC Mauboussin***
* CIRADARAT, St Denis, RBunion, France
* * CIRADARAT, Petit Bourg, Guadeloupe, France
* * * CIRADARAT, Montpellier, France
Palabras claves: variaci6n somaclonal, cultivo d e yema, roya, componentes
d e rendimiento, floracibn, isoenzimas.
RESUMEN
Se compararon plantas de catia de az6car (cv 843-62) regenerados a partir de callos
(S) con plantas provenientes de trozos de tallos (C) 6 micropropagadas a partir de yemas axilares (8) para susceptibilidad a Puccinia melclnocephala y componentes del rendimiento. En promedio 10s sintomas de roya parecieron ser mas severos en las plantas
S y 8. Este efecto permaneci6 a6n despues de la propagacibn por trozos de tallos de
las plantas S. Las plantas S y B mostraron una disminuci6n en el diametro del tallo. Las
plantas S tuvieron una reduccibn en el vlgor vegetativo e increment0 en macollaje en
el estado de planta per0 reducido en socas. Ninguna de las plantas S se encontr6 resistente a roya sin embargo en Reunion dos de ellas presentaron una alta proporci6n de
tallos florecidos a pesar de que se conoce que el cultivar B 43-62 no florece en ese
lugar. Una de las dos plantas S se caracteriz6 por la ausencia de una isoenzima comun
al sistema de leucina y valina aminopeptidasa.