MICROPROPAGATION OF ELITE
BIOTYPES OF FOURWING SALTBUSH
Swati Tripathy
J. R. Goodin
ABSTRACT
biotypes. The most important uses of saltbush (A.triplex)
species are for fuel wood and high-protein forage production (Northington and others 1979; Newton and others
1982). The species has a high range of polyploid levels
(Stutz and Sanderson 1979), and with the successful establishment of an efficient method for micropropagation,
it can be genetically manipulated under culture conditions
to produce even more economically useful genotypes.
Published information on tissue culture of saltbush
species is limited. A micropropagation method using
shoot tips for saltbush shoot multiplication and elongation
has been reported (Wochok and Sluir 1980). We have
developed another method for shoot multiplication and
elongation of this species from leaf segments. The process
varies with different genotypes.
Organogenesis in fourwing saltbush (Atriplex canescens)
(B3) was achieved in both semisolid callus medium and
single-cell suspension culture of 1/2-strength (MurashigeSkoog) medium supplemented with hormones. Maximum
shoot production was obtained by 112 MS + BA 0.4 mg/L
and NAA 0.05 mg/L in callus medium and by 1/2 MS + BA
0.1 mg/L, GA 3 0.1 mg/L,NAA0.05mg/LandKN03 450
mg/L in suspension cultures. A lower amount of auxin was
always an essential requirement in the shoot production as
well as shoot multiplication. Multiplication of 168 individual shoots and numerous shoot buds was also possible in a
1
12-strength MS medium in combination with Ki 0.4 mg/L,
GA 3 0.1 mg/L, and NAA 0. 05 mg/L. Elongation of the established shoots, up to a height of 1.2-1.5 em was possible
in 1/2 MS + BA 0.1 mg/L, Ki 0.25 mg/L, and GA3 0.1 mg/L
and KN0 3 450 mg/L, NHfi0 3 400 mg/L. Root induction
on these shoots is currently under study. For successful
root production a better shoot elongation medium needs
to be identified. Chromosome counts of different fourwing
saltbush population genotypes in the callus induction medium showed a wide range of ploidy level, as has been
established in naturally occurring populations. Among
the five different genotypes, B3 and B5 were dominated
by tetraploids, B4 and B6 by diploids, and B7 by hexaploids. A tendency toward mixploid production in all
phases of callus growth was remarkable. Aneuploid cells
around tetraploids, pentaploid and greater than octaploids
by 1-4 chromosome number were found. Based on these
studies, it can be suggested that a chromosome analysis of
callus culture is an important requirement in regeneration
studies to identify an optimum time period for subculture.
··,.·- ..·
MATERIALS AND METHODS
The five genotypes we selected for the study reported
here are designated as B3 and B5 (tetraploids), B4 and
B6 (diploids), and B7 (hexaploids). The shoot propagation
and multiplication procedure has been established with
B3 genotype.
The basal medium consisted of Murashige and Skoog
(1962) supplemented with organic source sucrose 30 mg/L
and the vitamins nicotinic acid 1.0 mg/L, pyridoxine
HC11.0 mg!L, thiamine HC110.0 mg!L, and myo-inositol
100.0 mg!L. The pH of the medium was adjusted to 5.8
with l.ON KOH or HCl prior to the addition of 1.6 mg/L
Gelrite in semisolid medium and autoclaved at 121 °C
with 15lb/in2 for 15 minutes. The amount of medium
used was 18 mL per 25- by 150-mm culture tube for semisolid medium, 50 mL per 250 mL Erlenmeyer flask for
suspension culture, 50 mL per 100- by 25-mm petri dish
and 30 mL per baby food jar. All petri dishes were sealei
with parafilm.
INTRODUCTION
Fourwing saltbush (Atriplex canescens) is a fast-growing,
widely distributed species in the United States, central
Mexico, and southern.Canada. Because of its adaptation
to diverse climatic and edaphic conditions, it is an excellent experimental material for micropropagation of elite
Explant Type
Young leaves offourwing saltbush were collected under
water from 1- to 2-year-old greenhouse plants. Mter surface sterilization, 4 to 5 explants of size 4-5 mm were inoculated per culture cube.
Sterilization Procedure
Paper contributed for the Proceedings of the Symposium on Cheatgrass
Invasion, Shrub Die-Off, and Other Aspects of Shrub Biology and Management, Las Vegas, NV, April 5-7, 1989.
Swati Tripathy is Research Associate, Department of Biological
Sciences, Texas Tech University, Lubbock, TX 79409; present address
is 2219 Carriage Hill, Denton, TX 76201; J. R. Goodin is Dean, College
of Arts and Sciences, Texas Tech University, Lubbock, TX 76201.
Surface sterilization of the entire leaves was carried
out through washing with distilled water and 5 minutes
soaking in 10 percent NaOCl and a few drops of Tween
336
This file was created by scanning the printed publication.
Errors identified by the software have been corrected;
however, some errors may remain.
20 with a prerinse in 70 percent alcohol for 2-3 minutes,
and a postrinse in three passes of sterile distilled water.
This gave almost 95 percent infection-free callus. Cultures were incubated at 20 °C ± 1°, 40E cm2/s fluorescent
light with a 16-hour photoperiod. The length of incubation in each medi urn is discussed in the individual stages
to shoot formation.
capacity. They are: BA 0.5 mg/L, NAA 0.05 mg/L;
BA 0.1 mg/L, NAA 0.1 mg/L; and BA 0.45 mg/L, NAA
0.1 mg/L. The hormones BA and NAA were found to
operate more efficiently in combination rather than in
isolation toward morphogenic variation.
CALLUS INITIATION AND GROWTH
The regeneration capacity of the competent cells was
tested through both suspension culture and a semisolid
medium. Both the distinct pathways proved to be successful; the suspension route was more efficient in mass
production.
SHOOT PRODUCTION
A wide range of different 2,4-D and kinetin concentration and combinations were examined for callus induction and differentiation in basal MS medium. Calli
were easily formed from the cut edges of the leaf explant
on MS medium. For our study, a combination of kinetin
0.25 mg/L with 2,4-D either at 0.25 mg/L or 0.50 mg/L
was favorable for further regeneration studies. The callus
produced was very light gre~n to green, friable and globular with normal callus growth, and had more than 40 percent green spots all over it. Callus from higher concentration and combination of 2,4-D and kinetin produced
abnormally developed neomorphs.
SHOOT PRODUCTION ON
SEMISOLID MEDIUM
The cytokinins tested were butyric acid (BA) and kinetin (Ki), and auxins were naphthalene acetic acid (NAA),
isopentenyl adenine (2-ip), and gibberellic acid CGAa>· A
little higher concentration of cytokinins accompanied by
a very low concentration of auxin was always favorable
(table 1). A combination ofBA and Ki with GAa was more
effective than BAalong with GAa and NAA when mass
production was of concern. Kinetin 0.25 mg/L with BA
and GAa 0.1 mg/L in 112-strength MS medium greatly enhanced shoot production to 80 percent rather than in any
other combination. A further increase of BA concentration to 0.25 mg/L along with kinetin and GAa reduced
shoot production to only 20 percent. The combination
that produced 80 percent shoot was tried at least 2-3
times and, depending on the previous incubation time,
it has consistently produced 60-80 percent shoots. BA
0.1 mg/L along with GAa 0.1 mg/L and NAA 0.05 mg/L
produced 40 percent shoots and 2-ip 1.0 mg/L with GAa
0.1 mg/L gave 20 percent shoots under similar conditions.
Shoot production was always followed by less than normal amounts of callus production and these calli also gave
rise to new shoot buds. Shoots produced this way had
attained a height to a maximum of 3-5 mm with four to
six elongated leaves within 30-35 days and had a rosette
kind of appearance (fig. 1). GAa was always found to be
essential for shoot bud production as has been reported
by a number of workers (Ripley and Preece 1986; Geier
1986; Noh and Minocha 1986). The 112-strength MS salts
were just optimal for shoot growth, because in our study
full strength MS has never produced shoots or shoot buds
either through suspension or on semisolid medium.
COMPETENCE CELL PRODUCTION
After an initial callus differentiation process of 40-45
days, approximately 1.0-1.2 gm of the light-green, friable
callus mass with a few green spots was subcultured to a
1
12-strength MS salt medium with vitamins and sucrose
and a factorial combination of nine and four levels of BA
and NAA, respectively. There was no organ formation;
instead the original callus gradually turned brown with
or without a reddish-brown secretion, which presumably
inhibited further growth. New, green to dark green, par.tially friable and partially compact, globular callus was
formed above the old brown callus within 15-20 days and
this callus was found to be determined or fated for further
shoot production.
We believe that this medium is an intermediate stage
to organogenic differentiation, where few cells having
organogenic potential survive and grow with the aging
and changing of hormonal balance in the old tissue and
under the influence of suitable phytohormone combination. In field bindweed (Convolvulus arvennis) genotypes,
root-competent cells are formed in the shoot induction
medium and vice versa, suggesting the fact that they
are two independent and not alternate processes
(Christianson and Warnick 1985).
Of all the combinations ofBA x NAA growth regulators,
only three of them had the ability to sustain regeneration
337
Table 1-5hoot production of fourwing saltbush (83) through callus (semisolid) media
Previous
culture
condition
mg/L
112
MS +
BA0.4,
NAA0.05
Subcultured
condition
Shoot
produced/
callus
mg/L
Percent
Number
of
shoots1
40
16+
BA 0.25,
NAA0.05,
GA3 0.1
1
/2
MS +
BA0.1,
NAA 0.1
"::~"
Callus
growth2
'12 MS +
BA0.1 NAA
0.05, GA3
0.1
'12MS +
BA 0.45,
NAA0.05
Number
of
roots
++
+++
112
MS +
BA0.1, NAA
0.05
+++
BA0.25,
NAA0.05
+++
'12 MS +
BA 0.1, Ki
0.1, GA3
0.1
+++
BA 0.1 Ki
0.25, GA3
0.1
BA 0.25,
Ki 0.25,
GA3 0.1
1
12 MS +
BA0.5,
NAA 0.05
'hMS+
2ip 1.0,
GA3 0.1
80
54++
++
20
2
+
20
8
++
2ip 1.0,
GA3 0.1,
NAA
0.05
++
2ip 1.0,
GA3 0.1
BA0.1
++
'The number listed is number of shoots. The +'s refer to relative number of uncounted shoot buds
2'fhe +'s refer to relative amounts of callus growth.
·
numerous single cells to few chlorophyllus cell clusters that
varied greatly in size and shape. The latter phase of suspension consisted mostly of small-to-large cell clusters and
shoot buds and very elongated cells of different shapes and
sizes. The shoot primordia developed from any point on a
chlorophyll us cell cluster with the appearance of a dark
green spot made up of a large number of very small cells
(which might be dividing continuously). A striking variation in cell size common in the gigas plant and to some
extent in normal has been reported (Stutz and others
1975).
Three types of suspension produced shoots. BA 0.4 mg!L
with NAA 0.05 mg/L produced vitreous shoots on plating
the suspension. The multiplication of these vitreous shoots
SHOOT BUD PRODUCTION AND
MORPHOLOGY OF SUSPENSION
CULTURE
Approximately 0. 75 mg of green globular callus from
the competent cell production medium was subcultured
to 112-strength MS liquid medium supplemented with BA
0.1 mg/L, GA3 0.1 mg/L, NAA 0.05 mg/L, and KN03
450 mg/L (KN03 amount was 3/4 strength of the original
MS medium). Shoot buds appeared in a dark green suspension within 30 days of first culture and within 10-15
days in subsequent subcultures, which was used as the
regular subculture time. Suspension culture was fast
growing. The initial phase of suspension was full with
338
Figure 1-Photograph of shoot development and shoot bud initiation.
was normal, but the growth in length was restricted. BA
0.25 mg/1 with GAa and NAA 0.1 mg/L and KN03 and
NH 4N03 450 mg!L and 400 mg!L, respectively, produced
some shoot buds. The regeneration capacity was lost
completely on subsequent culture. Only BA 0.1 mg/1
with GAa 0.1 mg/L, NAA 0.05 mg/L, and KN03 450 mg/L
have been producing shoots to some amount successfully
in every subculture.
A little higher level ofKN03 was essential to shoot bud
emergence, because when 450 mg/L KN03 was added to
BA 0.1 mg!L, GAa 0.1 mg/L and NAA 0.05 mg/L shoot buds
appeared within a few days. Thus, KN03 alone might
have helped in breaking the dormancy. The beneficial
effect oflow ammonium/nitrate ratio on growth and shoot
production has been reported (Geier 1986; Zens and
Zimmer, in press). The combination ofNH+ 4 and N0-3
ions in our study did produce a green suspension, but it
is the N0-3 ion ~lone that accelerated the shoot bud
production.
and NAA 0.05 mg!L. This particular medium produced
maximum shoot height between 2-3 mm and 4-6 mm in
25-30 days. Two other types of medium were also found
to multiply shoots to some extent but with more time. In
general, higher concentration of kinetin, (0.4 mg/L with
GAa) and NAA in lower concentration helped in maximum
multiplication (table 2). Complete elimination ofNAA
from the medium led to the production of only green
callus.
The number of shoots produced in each subculture is
shown (table 3); the second subculture has a maximum
of 168 countable shoots and many shoot buds.
Table 2-Shoot multiplication media for fourwing
saltbush (83)
Plating media with shoots (mg/L)
Shoot mass1
112
MS + Ki 0.4, GA3 0.1, NAA 0.05
MS + Ki 0.25, GA 3 0.1, BA 0.1 +
KN03 450, NH 4N03 400
112 MS + BA 0.1, GA 0.1, NAA 0.1 +
3
KN03 450
112
SHOOT MULTIPLICATION
Organized growth and multiplication of the shoot buds
and cell clusters were obtained when 0.75 gm of the shoot
buds were spread out evenly on a petri dish containing
50 mL of 112-strength MS and Ki 0.4 mg/L, GA3 0.1 mg/L,
1The
339
+'s refer to relative amounts of shoot mass.
+++
++
+
Table 3-Shoot multiplication with time in fourwing saltbush (83)
Suspension medium: 112 MS + BA 0.1 mg/L, GA3 0.1 mg/L, NAA 0.05 mg/L,
KN03 450 mg/L
Number
or
subculture
Subculture
time
Plating media
(mg/L)
30
0
10
Shoot production h1
days lnterval1
20
30
Ki 0.4, GA3 0.1,
BA0.1
40
50
1+
4+
9+
11+
75+++
90+++
129++
168++
Ki 0.4, GA3 0.1,
NAAO.OS
BA 0.1, GA3 0.1
NAA 0.05 KN03 450
2
13
Ki 0.4, GA3 0.1
NAA0.05
2+
17++
1
Number of shoots followed by (+'s) relative number of shoot buds.
latter phase of callus induction, these dominant ploidy
levels gradually decreased accompanied by an increase
of other ploidy levels, finally giving ri·se to a complete
mixploid culture. A higher percentage of octaploid and
higher ploidy level in B3 and B 7 culture suggested the
occurrence of chromosome doubling through endoreduplication (D'Amato 1977). Genotypes B4 and B6 had maximum diploid chromosomes after 38 days of inoculation,
which became dominated by tetraploids after 48 days,
suggesting the same process of endoreduplication. Few
aneuploid cells were always present in all phases of callus
differentiation and were associated with tetraploids,
pentaploids, and hexaploids with 1-4 extra chromosomes.
The odd-ploidy chromosome numbers (triploid, pentaploid,
heptaploid) were found to be of common occurrence in
fourwing saltbush tissue culture. These usually appear
from nuclear fusion (D'Amato 1985) and tripolar spindle
formation; the latter was observed in this study.
The occurrence of a wide range of ploidy level, especially in the early phase of callus induction (28 days),
might be partly due to the preexisting cell condition in
the explant and partly due to the result of nuclear processes such as endoreduplication and nuclear fragmentation occurring at the time of callus induction. Naturally
occurring polyploid populations of fourwing saltbush have
been reported from sand dunes in central Utah (2n = 18),
New Mexico and western Texas (2n = 36, 54), and the
Mojave desert (12 ploid) (Stutz and Sanderson 1979;
Dunford 1984). Our genotypes have been collected from
different localities of New Mexico and Texas.
Thus, this investigation concludes that subculture
time can be different in genotypes of the same species
and long-term callus condition can sometimes reestablish
the original ploidy level and may help in the regeneration
by gradual elimination of other polyploids and aneuploids.
Genotype B7 had maximum hexaploids again after 48
days and much fewer >octaploids and no aneuploids.
SHOOT ELONGATION
The only medium that successfully elongated the shoots
to a maximum height of 1.2 to 1.5 em is 112-strength MS
with Ki 0.25 mg/L, BA 0.1 mg/L, GAa 0.1 mg/L, and KN0 3
450 mg!L, NH 4N03 400 mg/L.
To facilitate high rate of shoot production and multiplication, 112-strength MS media containing GAa and cytokinin are essential. Our study provides a double pathway
for shoot production and the rooting of these shoots; further establishment on soil can be achieved with time and
effort. Further research will be focused on these aspects
of Atriplex micropropagation.
PLOIDY LEVEL IN CALLUS
INDUCTION MEDIUM
The ploidy levels offourwing saltbush genotypes selected
for regeneration studies were evaluated in the callus induction medium (MS + 2,4-D 0.5 mg/L, Ki 0.25 mg!L) with
a purpose of establishing an optimum period for subculture
and successful plant production. Chromosomal changes
often occur in plant cells grown in vitro (D'Amato 1977,
1978), and this chromosomal analysis of cultured plant
tissues is an essential requirement in the in vitro regeneration study of any plant species.
Leaf explants induced callus proliferation within 25-28
days and cells were sal}lpled after 28, 38, and 48 days of
inoculation for chromosome analyses.
Among the five different genotypes, B4 and B6 were
found to be diploids (2n = 18), B3 and B5 were tetraploids
(2n = 4x = 36), and B7 was probably a hexaploid variety.
The early phase of callus induction was already influenced
by a wide range of polyploid levels (fig. 2) in all the genotypes. B3 and B5 were both dominated by tetraploids
(4x = 11.8 and 7.7 percent, respectively), B4 and B6 by
diploids (6.2 and 4.9 percent, respectively), and B7 by hexaploids (4.3 percent) and tetraploids (3.7 percent). In the
340
Genotype 83
Genotype 84
I
Ploidy ._..,.I
ISSJ
~
38 aays
1ZZJ
.o~e aaya
Ploidy
ISS!
:18 aaya
Genotype 85
Genotype 86
Ploidy LAvel
ISSI
1ZZJ
38 aays
Ploidy LAvel
ISS!
:18 aaya
Genotype 87
~
5
l
~
~I
38 aaya
"'
~
~
~
~
L
.1
Plofdy LAvel
38 daya
!SSI
~
.o~e
oays;
Figure 2-Pioidy levels in various genotypes over time. The horizontal axis lists ploidy levels of 2x, 3x, 4x,
5x, 7x, Bx, and Bx+ as x2, x3, ... x8+, respectively.
341
38 aaya
~ "'' aaya
ACKNOWLEDGMENTS
Newton, R. J.; Puryear, J.D.; Goodin, J. R.; Magar, D. L.
1982. Biomass from unconventional sources in semiarid west Texas. In: Klass, D., ed. Energy from biomass
and waste. VI. Chicago: Institute of Gas Technology:
167-219.
Noh, E. W.; Minocha, S.C. 1986. High efficiency shoot
regeneration from callus of quaking aspen (Populus
tremuloides Michx). Plant Cell Reports. 5:464-467.
Northington, D. K.; Goodin, J. R.; Wangberg, J.D. 1979.
Atriplex canescens as a potential forage crop introduction into the middle east. In: Goodin, J. R.; Northington,
D. K., eds. Arid land plant resources. Lubbock, TX:
Texas Tech University; International Center for Arid
and Semi-arid Land Studies: 425-429.
Ripley, K. P.; Preece, J. E. 1986. Micropropagation
Euphorbia lathyris L. Plant Cell, Tissue and Organ
Culture. 5(3): 213-218.
Stutz, H. C.; Melby, J. M.; Lingston, G. K. 1975. Evolutionary studies of Atriplex: a relic gigas diploid population of Atriplex canescens. American Journal of Botany.
62(3): 236-245.
Stutz, H. C.; Sanderson, H. C. 1979. The role of polyploidy
in the evolution of Atriplex canescens in arid land and
plant resources. In: Goodin, J. R.; Northington, D. K.,
eds. Lubbock, TX: Texas Tech University; International
Center for Arid and Semi-arid Land Studies: 615-621.
Wochok, C. S.; Sluir, C. J. 1980. Gibberellic acid promotes
Atriplex shoot multiplication and elongation. Plant Science Letters. 17: 363-369.
Zens, A.; Zimmer, K. [In press]. Untersuchungen zur
in vitro-Vermehrung von Anthurium scherzenrianum
Gartenbauwissenschaft.
This research was supported by a grant from the Rocky
Mountain Forest and Range Experiment Station, Forest
Service, U.S. Department of Agriculture, "New Biotechnologies for the Propagation of Elite Biotypes ofWoody
Plants."
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