Plant regeneration after long term callus culture in clones of Asparagus officinalis L.*
A. C. Pontaroli and E. L. Camadro
Estación Experimental Agropecuaria (EEA) Balcarce, Instituto Nacional de Tecnología Agropecuaria (INTA) and Facultad de Ciencias Agrarias (FCA), Universidad Nacional de Mar del Plata (UNMdP); C. C. 276, (7620) Balcarce, Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina. Current Address: Dept.of Genetics, University of Georgia, 30602 Athens, Georgia, U.S.A.
Address correspondence to: Ing. Agr. Elsa L. Camadro. Casilla de Correo 276, (7620) Balcarce, ARGENTINA. FAX: (+54-2266) 439101. E-mail: ecamadro@balcarce.inta.gov.ar
*Part of the first author's doctoral thesis.
ABSTRACT: Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3% sucrose, 0.9% agar, 1 mg.l-1 kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg.l-1 2,4-D and 1 mg.l-1 kinetin. Shoot primordia (SP) induction from >18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89%) and average number of SP per callus (8.6) were obtained with clone ‘265' on MS medium with 5 mg.l-1 2iP, 1 mg.l-1 IAA, 3% sucrose and 0.9% agar. The highest percentage of root induction (100%) was achieved with clone ‘265' on MS medium with 0.1 mg.l-1 kinetin, 0.1 mg.l-1 NAA, 1.32 mg.l-1 ancymidol, 7% glucose and 0.8% agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.
Keywords: Callus. Shoot primordia induction. Rooting. Medium x genotype interactions.
Abbreviations: 2,4-D: 2,4-dichlorophenoxyacetic acid; 2iP: 2-isopentenyladenine; BA: 6-benzylaminopurine; FFW: final fresh weight; IAA: indoleacetic acid; IFW: initial fresh weight; MS: Murashige and Skoog (1962); NAA: 1-naphtaleneacetic acid; PGRs: plant growth regulators; PVP: polyvinylpyrrolidone; SP: shoot primordia.
Introduction
‘Crown and root rot', a disease caused by a fungal
complex of Fusarium species, is the main cause of asparagus
decline in the world (Dan and Stephens, 1995).
Conventional breeding of resistant cultivars is a slow
process that could be accelerated if more efficient selection
methods were available. In other pathosystems,
genetic materials with increased disease resistance were
generated through in vitro selection in presence of pathogen
toxins (in Jin et al., 1996). In asparagus, the feasibility
of in vitro selection for resistance to Fusarium toxins is scarcely documented. Selection methods exploit
the occurrence of somaclonal variation induced
during long-term callus cultures. However, a suitable
protocol for regenerating whole plants from surviving
long-term calluses must be available (Duncan, 1997).
In this regard, organogenesis has been suggested as the
most adequate pathway for preserving somaclonal variation
(Duncan, 1997).
There is a wide variety of published protocols for
asparagus callus culture and organogenesis from callus
(Harada, 1989; Reuther, 1990, Dan and Stephens, 1995;
among others). However, they were developed for particular
genetic materials, and are not broadly applicable,
as has been our experience with selected genotypes of
cv. Argenteuil (unpublished data). Thus, the objective of
this work was to develop an efficient protocol for (1) longterm
callus maintenance and growth and (2) plant regeneration
via organogenesis from callus, that could be utilized
in future in vitro selection in our breeding program.
Materials and Methods
Two staminate elite clones (genotypes) of cv.
Argenteuil, ‘265' and ‘357', were used for all experiments.
In all of them, the experimental unit was one
test tube, and a completely randomized design was used.
Callus induction was evaluated in three separate experiments
with ten replications each. Internodal spear sections
(2 mm long), excised from micropropagated
plants, were cultivated in the dark at 26ºC for 60 days in
110 x 15 mm test tubes with Murashige and Skoog's
(1962) basal medium (MS) plus 3% sucrose, 0.9% agar
(Sigma), and combinations of plant growth regulators
(PGRs; not shown). Using similar culture conditions,
the best three of these combinations, 1 mg.l-1 kinetin +
1.5, 5 or 10 mg.l-1 2,4-D, were tested in a callus growth
assay with 23 replications. The initial (IFW) and final
(FFW) callus fresh weights were recorded for each of
two subcultures. Callus relative growth was calculated
for each subculture as [(FFW-IFW)/IFW] and analyzed
through standard ANOVA and Tukey test for mean
comparison.
Five consecutive experiments were performed for
designing a suitable protocol for shoot primordia (SP)
induction from callus. The first was carried out with
18-months-old calluses, and each subsequent experiment
was performed with calluses 2 1/2 months older
than the used previously. The experiments consisted in
the culture of callus pieces in test tubes (experimental
unit=one test tube) with different SP induction media
(Table 1) in the dark at 26ºC. Sixty days after culture
initiation, the percentage of calluses with SP was determined
on 20-25 replications per treatment. In the second
experiment, the number of SP per callus was recorded
as well. Log-linear models and standard ANOVA
were used for analyzing the percentage of calluses with
SP and the number of SP per callus, respectively.
TABLE 1.
Clones and media components evaluated in five consecutive experiments
for adjusting shoot primordia induction from callus.
All calluses in which SP were induced in the first experiment were cultivated for 60 days on ‘85' medium (Daorden, pers. comm), to promote shoot and minicrown growth. This medium was composed of MS basal salts, ‘85' vitamins (1 mg.l-1 thyamine, 1 mg.l-1 nicotinic acid, 1 mg.l-1 pyridoxin, 1 mg.l-1 calcium panthotenate, 1 mg.l-1 glycine, 100 mg.l-1 myoinositol and 0.01 mg.l-1 biotin), 0.2 mg.l-1 kinetin, 0.2 mg.l-1 NAA, 0.1 mg.l-1 BA, 0.1 mg.l-1 ancymidol, 3% sucrose and 0.7% agar. The derived plantlets were randomly assigned to eight rooting media, constituted by combining two carbon sources -7% sucrose or glucose-, with two gelling agents -0.8% agar or gellan gum-, and two basal rooting media, (1) ‘102' (Daorden, pers. comm.), composed of MS salts, ‘85' vitamins, 0.1 mg.l-1 kinetin, 0.5 mg.l-1 ancymidol and 0.2 mg.l-1 NAA, and (2) ‘DES' (Desjardins et al., 1987, with modifications), composed of MS salts, 0.1 mg.l-1 kinetin, 1.32 mg.l-1 ancymidol and 0.1 mg.l-1 NAA. After 45 days, the percentage of plantlets with healthy roots was determined on 20 replications; this variable was statistically analyzed using log-linear models.
Results
Statistical analysis of callus relative growth detected
significant contributions of clones and 2,4-D levels.
Calluses of ‘357' showed, on average, a higher relative
growth than calluses of ‘265' in both subcultures
(p<0.05). Callus relative growth diminished (p<0.05)
with increased levels of 2,4-D, mainly in subculture 1.
In both clones, a high proportion of calluses was of desirable
appearance at all three 2,4-D levels, except for
calluses of ‘357' on medium with 10 mg.l-1 of this auxin,
which were compact and exhibited browning. Based
on these results, the medium supplemented with 1.5
mg.l-1 2,4-D and 1 mg.l-1 kinetin was selected for further
inducing callus initiation and growth.
For adjusting SP induction from callus, five consecutive
experiments were performed because poor results
had been previously obtained after following several
published protocols (Harada, 1989; Reuther and
Becker, 1987 in Reuther, 1990; Reuther, 1990; Dan and
Stephens, 1995). In experiment 1, SP induction from calluses
varied between 0 and 62.5% in ‘265', depending
on the culture medium; in contrast, no SP induction was
observed in calluses of ‘357', that exhibited a compact
structure and browning. Statistical analyses, performed
only for data of ‘265', detected a significant contribution
of the second order interaction; thus, the effects of each
factor were evaluated for each of the levels of the remaining.
The highest percentages of SP induction were
found, in general, in media with 2iP; no clear trends were
observed in relation to cytokinin concentration (Fig. 1).
The effect of the addition of IAA depended on the type
and concentration of cytokinin. When 1 mg.l-1 IAA was
used in combination with any of the three levels of 2iP
or with 5 mg.l-1 kinetin, SP induction was increased
(p<0.05) in comparison with the observed in the same
media without IAA, while the opposite was found when
IAA was used in combination with 1 or 10 mg.l-1 kinetin
or any of the three levels of BA. The best three out
of the 18 PGR combinations evaluated in experiment 1
were selected for performing experiment 2.
FIGURE 1. Average shoot primordia induction from callus of ‘265' in experiment 1. Cytokinins: a) 2iP; b) kinetin; c) BA. Cytokinin concentrations: 1 mg.l-1 (grey); 5 mg.l-1 (white); 10 mg.l-1 (black).
Shoot primordia induction from callus was observed
in almost all treatments of experiment 2. However,
there were important differences between genotypes:
depending on the culture medium, the percentage
of SP induction varied between 39 and 89% in ‘265' and between 0 and 43.8% in ‘357', and the average number of SP per callus ranged between 2.4 and 8.6 in ‘265' and between 1 and 6.5 in ‘357'.
Significant contributions (p<0.05) to the statistical
model of the percentage of SP induction were given
only by main effects. On average, ‘265' showed a
higher percentage of SP induction than ‘357'; furthermore,
calluses of ‘357' exhibited the same appearance
as in the previous experiment. The reduction in the
concentrations of MS salts from 100 to 50%, and of
sucrose from 3 to 2%, negatively affected SP induction.
Conversely, the increase in both the concentration
of 2iP from 1 to 5 mg.l-1 and of IAA from 0 to 1
mg.l-1 enhanced SP induction. The only significant
contribution (p<0.05) to the statistical model of the
number of SP per callus was given by the MS salts
concentration; the highest value was observed when
the full strength (100%) was used.
The highest percentages of SP induction from calluses
of both ‘265' (62.5% in experiment 1 and 89% in
experiment 2) and ‘357' (43.8% in experiment 2) were
achieved with full strength MS basal salts supplemented
with 5 mg.l-1 2iP, 1 mg.l-1 IAA, 3% sucrose and 0.9%
agar. Thus, this culture medium was utilized as a control
in further experiments, carried out with ‘357'.
In experiment 3, the percentage of SP induction
from callus varied between 0 and 31.25% in ‘357', depending
on the culture medium, and was 52% in ‘265' (control). Only agar concentrations significantly contributed
to the statistical model; the lowest concentration,
0.5%, enhanced SP induction in comparison with
the remaining. However, generalized callus browning
was observed in ‘357', as described earlier. In experiments
4 and 5, neither was SP induction from calluses
of ‘357' attained, nor callus browning avoided, even with
the use of antioxidants. On the other hand, SP induction
from calluses of ‘265' was 41% in experiment 4 and
30% in experiment 5.
The percentage of root induction in ‘265' ranged
between 0 and 100%, depending on the culture medium,
but was not evaluated in ‘357' because few plantlets were
available. The utilization of gellan gum resulted in a significantly
lower percentage of root induction than the
achieved with agar (p<0.05). The remaining main effects
were analyzed jointly because a significant interaction
between them was detected. When ‘102' was the basal
rooting medium, the percentage of root induction was
higher with sucrose than with glucose: the opposite response
was obtained in ‘DES' basal rooting medium. On
the other hand, the average percentage of root induction
was higher in ‘DES' (67.25%) than in ‘102' (59.25%).
The best percentage of root induction (100%) was obtained
with ‘DES' basal rooting medium, 7% glucose and
0.8% agar. This medium was preliminarly selected and
tested in >100 plantlets of each of the two clones; on
average, 45% and 65% root induction were obtained with ‘357' and ‘265' respectively.
Discussion
The aim of this study was to establish and characterize
an in vitro culture system in asparagus for using
in prospective long-term in vitro selection programs for
resistance to Fusarium toxins. The medium selected for
callus induction and growth is now routinely used for
maintaining fast growing, healthy calluses of these and
other clones of cv. Argenteuil in our laboratory.
Important interactions between IAA and the type
and level of cytokinin occurred in SP induction from
calluses of ‘265' (see experiment 1). The subsequent
experiments showed conspicuous differences between
clones concerning percentage of SP induction, number
of SP per callus and callus appearance. Additionally, a
loss of organogenic capacity was observed in both clones
after the second SP induction experiments. Considering
SP induction from calluses in the control medium,
the only difference between experiments was callus age:
as previously stated, experiment 1 was carried out with
18-months-old calluses, and each subsequent experiment
was performed with calluses 2 1/2 months older
than the used previously. The adjustment of SP induction
accompanied the development of a long-term in
vitro selection program for callus resistance to Fusarium toxins in our laboratory (unpublished data), and the utilization
of calluses of similar age to the ones under selection
was deemed convenient. According to our results,
plant regeneration should be attempted in calluses
approximately 20 months old.
In conclusion, an in vitro culture system for plant
regeneration after long term callus culture is now available
for two elite clones of Asparagus officinalis cv.
Argenteuil. Although the medium selected for callus
induction and growth is of broad application, the conditions
for plant regeneration would have to be tested
and, likely, adjusted for other genetic materials due to
the presence of genotype x medium interactions.
Acknowledgements
This study was partially financed by CONICET and UNMdP.
References
1. Dan Y, Stephens CT (1995). The development of asparagus
somaclones with high levels of resistance to Fusarium
oxysporum f. sp. asparagi and Fusarium proliferatum. Plant
Dis 79: 923-927.
2. Desjardins Y, Tiessen H, Harney PM (1987). The effect of sucrose
and ancymidol on the in vitro rooting of nodal sections of
asparagus. HortScience 22: 131-133.
3. Duncan RR (1997). Tissue culture: induced variation and crop improvement.
Adv Agron 58: 201-240.
4. Harada T (1989). Studies on organogenesis of asparagus (Asparagus
officinalis L.) tissues and its utilization. Memoirs of the
Faculty of Agriculture, Hokkaido University 16: 301-346.
5. Jin H, Hartman GL, Huang YH, Nickell CD, Widholm JM (1996).
Regeneration of soybean plants from embryogenic suspension
cultures treated with toxic culture filtrate of Fusarium
solani and screening of regenerants for resistance. Phytopathology
86: 714-718.
6. Murashige T, Skoog F (1962). A revised medium for rapid growth
and bioassays with tobacco tissues cultures. Physiol Plant 15:
473-497.
7. Pontaroli AC, Camadro EL (2001). Selección de callos de genotipos
selectos de Asparagus officinalis cv. Argenteuil por
comportamiento frente al filtrado tóxico de Fusarium (Abst).
J Basic Appl Genet 14: 112.
8. Reuther G (1990). Histological and cytological selection of
somaclonal and induced variants of asparagus derived from
callus cultures. Acta Hort 280: 385-393.
Received on September 28, 2004.
Accepted on May 11, 2005.