Utilizing the lateral buds of known high rubber producing plants as explants in culture medium specifically designed to engender shoot development and to prevent callus formation, unlimited numbers of replicate plants can be produced. Each has the same genotype as the parent. This procedure has long been used to rid plants of virus, the latter generally does not occur in the embryonic tissues of the bud; it also, by virtue of its axenic nature, eliminates all microorganisms characteristic of the parent plant. Auxins were found essential to callus formation, but since the latter is known to bring about chromosomal aberrations, it was avoided. The cytokinin benzylaminopurine strongly stimulated shoot growth, and the number of regenerated buds on the inoculum was proportional to its concentration. These buds produced shoots several centimeters in length which were caused to root on medium containing indolebutyric acid. Transferred to the septic condition of soil, the plantlets were gradually brought into full sunlight where they showed a brief vegetative growth with production of mature leaves, and flowered

Ball, E. A.

English

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TITIE PAGE
1. Title of Report: Development of Guayule ( Perthenium ar¢entatum)
Research in Cell Culture.
2. Contractor's Report Number: UCI 59913.
3. Final Report.
4. Author: Ernest A. Ball.
5. Publication date: January 2, 1981.
6. JPL Contract No. 954955.
7. Name and location of contractor: Dr. Ernest A. Ball (work performed in
Department of Developmental and Cell Biology, University of California,
Irvine, Calif. 92717), present address: Biology Board of Studies,
Dnivercity of California, Santa Cruz, Calif. 95064.
This vcrk vas performed for the Jet Propulsion Laboratory, California Insitute
of Technology, sponsored by the National Aeronautics and Space Administration
under Contract NAS7-100.
(NASA-CH-163945) DEVELOPMENT OF GUAYUAE
	 Ndl-17oy1
(PA$THENIUM AnGENTATUM) 4it,SEAitCH IN Cbi.L
CULTURE Final Ht port (c:alif oraid Univ.)
14 p HC A02/MF AO1
	 CSCL OoC	 Ulicias
63/51 41358
This report contains information prepared by Dr. Ernest A. Ball under JPL
sub-contract. Its content is not necessarily endorsed by the Jet Propulsion
laboratory, California Institute of Technology, or the National Aeronautics
and Space Administration.
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ABSTRACT
Utilizing the lateral buds of known high rubber producing plants as
explants in culture medium specifically designed to engender shoot develop-
ment and to prevent callus formation, anlimi r . numbers of replicate plants
can be product-1. Each has the same genotype as the parent. This procedure
has long been used to rid plants of virus, since the latter generally does
not occur in the embryonic tissues of the bud; it also, by virtue of its
axenic nature, eliminates all microorganisms characteristic of the parent
plant. Auxins had been found essential to callus formation, but since the
latter is known to bring about chromosomal aberrations, it was avoided.
The cytokinin benzylaminopurine strongly stimulated shoot growth, and
the number of regenerated buds on the inoculum was proportional to its
concentration. -These buds produced shoots several centimeters in length
which were caused to root on medium containing indolebutyric acid. Trans-
ferred to the septic condition of soil, the plantlets were gradually brought
Into lull sunlight Where they showed a brief vegetative growth with produc-
tion of mature type leaves, and flowered. In contrast, seedlings of the
same age remained vegetative. Chromosome studies of root smears from the
tissue cultured olantlets showed that 2n = 36, the normal number for sex-
ually reproducing guayules.
3
INTRODUCTION.-- The importance of maintaining the genotypes of plants that
produce a high amrant of rubber was early recognised in this work, and pro-
cedures were established for the cloning of such plants by tissue culture.
The advantages of this technique are that unlimited numbers of plants of
the original genotype can be produced from a single high yielding individual.
The axenic procedure of tissue culture necessarily rids the explant of all its
surface-living microorganisms, and the use of the semi-microscopic shoot tip
as inoculum is a well-known method of ridding higher plants of viruses.
Since it is known that the precursory formation of callus in tissue culture
cloning of flowering plants often leads to chromosomal aberrations, the
present work utilized culture media that presented the development of such
undifferentiated tissue from which regeneration of buds occurred. Since
auxin, both natural as well as synthetic, engenders formation of callus in
plant tissue cultures, its use was avoided. In contrast, cytokinins, which
are entirely synthetic, and consist of various chemical substitutions of
such nucleic acid components as adenine, (e.g., kinetin or N 6
 furfuryl-
adenine), stimulate the formation of buds and no callus is developed.
Our preliminary work with experimentally farmed callus tissue shared no
microchemically detectable rubber. It was decided therefore to clone plants
that were known, by biochemical tests, to be high producers of rubber.
Since the guayule plant at maturity shows prolific formation of lateral
buds, the majority of which can be removed without affecting the growth
of the parent plant, they were excised as explants for tissue culture
cloning. By variations in the culture medium, each such bud can be caused
to develop into one to many (up to fifty) new plants. Since it is known
that cytokinin inhibits formation of roots, it was necessary to place the
regenerated shoots upon medium containing auxin in order to regenerate the
former.
4
MATERIALS AND METHODS.-- The most regenerative explants from mature
plants of Parthenium areentat-m (0593 from the University of Arizona,
Tucson) were small shoot tips. These explants were excised according
to the procedure described by Ball (1946) and measured 1 x 1 x 1 mm.
and contained the shoot apex, the three youngest foliar primordia and a
small extent of basal pith parenchyma. The buds of mature plants prior
to dissection were surface-sterilized by immersion in 3 per cent sodium
hypochlorite, rinsed in 50 per cent ethanol and lastly in sterile distilled
water. The inocula were excised by microscalpels made according to the
technique of Ball (1946) and placed upon slants of agar culture medium
consisting of the minerals of Murashige and Skoog (1962) and 2 per cent
sucrose (W/V); the medium was solidified by 1.3 per cent agar. All com--
stituents of the medium, except, the agar, were filter-sterilized by being
forced through a Millipore filter (pore-size 0.4 micrometers). The inocula
on the agar, in culture tubes 25 x 200 mm., were grown wader light of 300
f.c. derived from an equal-wattage combination of white fluorescent tubes
and 25-watt incandescent bulbs.
Auxins, e.g., indoleacetic acid, 1-naphthaleneacetic acid, at concen-
trations of 0.1 milligram per liter, were inhibitory to growth of the ex-
plants. Cytoxinins, however, were stimulatory to growth by cell division.
The most satisfactory cytokinin was N6-benzyladenine, or benzylaminopurine,
BAP. On culture medium consisting of minerals, sugar and water, growth con-
sisted of the limited development of foliar primordia already present on
the inoculum, and there was no development of basal parenchyma. The leaves
that showed limited maturation were without the lobes characteristic of the
species, thus constituting the juvenile type. (Fig. 1).
RESULTS. The inocula responded quickly to the presence of BAP at 0.1
mg/l, produced additional leaves which became lobed in subsequent development,
thus constituting the mature type. The basal parenchyma bore adventitious
buds (Fig. 2).
5
Higher concentrations of the cytokinin accelerated these developments.
At 1 mg/Y new lobed foliar primordia were quickly produced, along with ad-
ditional adventitious buds on the basal parenchyma (Fig. 3). At 2 MA
the regenerative process was temporarily slowed (Fig. 4), but later showed
the same development. When these cultures on higher concentrations of cyto-
kinin were allowed to grow for about a month, they became messes of adven-
titious shoots on medium with 1 mg/1 BAP (fig. 5), and mostly buds on med-
ium with 2 no BAP (Fig. 6). The latter high concentration of cytokinin
inhibited shoot elongation.
After a month's growth upon the cytokinin medium, the cultures were
either transferred intact, or as.neparate excised shoots, to rooting medium.
The latter contained no cytokinin, but included 3 per cent B-indolybutyric
~ - acid. The numerous shoots from original growth upon medium containing 1 m9/1
BAP were separated and their bases inserted individually - into tubes of root-
ing medium where they produced adventitious roots from the basal parenchyma.
(Fig. 7). Such plantlets were always short and thick-stemmed, and the basal
parenchyma large. Plantlets produced upon medium containing 0.1 mg/1 BAP
were always slender and had small basal parenchyma (Fig. 8). Within thirty
days most of the plantlets had produced numerous adventitious roots (Fig. 9).
Although these plants had developed in axenic conditions, transfer to
non-i;terile soil brought about few losses. Microorganisms of soil had no
deleterious effect. The plantlets were transferred to plastic tubes of soil
(Berkeley mix of peat, sand and loam) and protected for the first week in the
greenhouse by an encompassing glass culture tube (40 mm dia.). They were sub-
sequently conditioned gradually to higher light intensity in the lath house,
transferred to pots and grown in full sunlight out of doors.
Along with the potted' plantleta, equal-aged seedlings were grown in full
sunlight for thirteen months. During the first six months a few of the plantlets
6
1
flowered, but none of the seedlings did so. At thirteen months, all (30 of 30)
plantlets flowered (Fig. 10, 11), but only one (1 of 20) of the seedlings did
so. Most of the latter had continued vegetative growth, with no signs of inflor-
escence stalks (Fig. 12). The morphology of the flowers in the inflorescences on
the plantlets was normal (Fig. 13).
The chromosome counts of the plantlets and - of the seedlings were 2n = 36.
This chromosome number, according to the NAS report (1977) is the normal one
for sexually reproducing plants of guayule. The finding of the normal tr mber
of chromosomes in the tissue culture derived plantlets is evidence that the
procedures utilized in their derivation had not induced abnormal cytology.
DISCUSSION.— A tissue culture method has been described that successfully
clones an individual plant of Parthenium arsentatum. This procedure can now be
used to make an indefinite number of replicates of a high-rubber yielding plant
for commercial purposes or for breeding. Depending upon the concentration o
the cytokinin, e.g., benzylaminopurine, a single explant can be caused to
produce either a few shoots, or up to fifty. Each shoot can then be rooted,
transplanted to soil where it will grow into a mature rubber yielding plant.
With the successful achievement of this in vitro propagation procedure,
it is now possible to institute an improvement program for rubber production
by Parthenium areentatum. It will be important, both practically and theoret-
ically, to ascertain whether plants derived by tissue culture under the influence
of a cytokinin, will carry on the biochemical capacity for rubber production of
and original plant that contributed the explants. While the present work demon-
strated that there was no change in the chromosome number in plantlets produced
under the influence of a cytokinin, it will be necessary to test the biochemical
competence of resulting mature plants.
The donor plants were mature and bore numerous infloresences. This mature
state was perpetuated in the plantlets produced by tissue culture, for most of
7
them flowered during their growth in soil by thirteen months. Exceptional
ones that remained vegetative had been previously defoliated by insects.
In contrast, seedlings of the same age rarely flowered. This perpetuation
of the mature 'state in the tissue cultured plantlets Mill be an important
time-saver for breeding. 	 .
REFERENCES
Ball, 8.-A. 1946. Development in sterile culture of stem tips and subjacent
regions of Trovaeolum ma us L. and of Lupins albus L. Amer. Jour. Bot.
33: 301-318.
Hwashige, T. and F. Skoog. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue cultures. ?lhysiologis Planterum
15: 473-498.
National Academy of Sciences, Washington, D.C. Guayule, an alternative
sourse of natural rubber. Washington, D. C., Librr_ry of Congress
Catalog No. 72-62525. ix + 79 pp.
8
OL
+.
SMA.6"Aa . .joa .N.-A. _
Figure 1. Explant gram 23 days without cytokinin. Leaves are unlobed,
and no basal parenchyma occurred. No Further development was noted
after prolonged culture. 6Z.
Figure 2. Explant grown 14 days on medium containing 0.1 mg/l HAP. New lobed
leaves were produced and the basal parenchyma bore new adventitious buds. 61.
9
mar
Figure 3. Inoculum grown on medium containing 1 mg/1 Bk? for 14 days. In
this brief interval new lobed leaves had been produced, and adventitiou_r
buds formed upon the basal parenchyma. 6a.
Figure 4. Ihoculum grown on medium containing 2 mg/1 BAP for 14 days. A Alight
inhibition due to the high concentration of cytokinin slowed development
which later resumed. U.
10
fFigure 5. Gro.Ah of an inoculum for one month on medium containing 1 mg/1
BU. So many adventitious shoots were produced that the culture had
become a mass of teem. Most buds showed development of elongated stems. 6X.
Figuro 6,. Growth of an inoculum for one month on medium containing 2 mg/l
SAP. Numerous adventitious buds arose, but their stems usually elongated
only slightly, apparently due to the high concentration of cytokinin. 6x.
11
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)-	 1


Development of guayule (Parthenium argentatum) research in cell culture

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