Plant Growth Regulation 12: 273-280, 1993.

273
0 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Factors affecting in vitro clonal propagation of Prosopis cineraria

N.S. Shekhawat, T.S. Rathore, R.P. Singh, N.S. Deora & S.R. Rao
Plant Biotechnology Laboratory, Department of Botany, University of Jodhpur, Jodhpur 342 001, India

Received 23 January 1992; accepted in revised form 20 May 1992

Key words: Prosopis cineraria, cloning, adult trees, tissue culture, repeated transferring, plantlets

Abstract

Genotype, age of tree, nature of explant and size (length and diameter), season of explant collection, explant
position on medium, plant growth regulators and certain additives (ascorbic and citric acids, adenine
sulphate, L-arginine, glutamine and ammonium citrate), incubation conditions, and subculturing period
greatly influenced the in vitro clonal propagation of P. cineraria. The maximum number of lo-12 shoots were
induced from the nodal shoot segment from pruned thorny adult trees on Murashige and Skoog’s (MS)
medium containing 0.1 mgl-’ indole- 3-acetic acid (IAA) + 2.5 mgl-’ benzylaminopurine (BAP) + ad-
ditives. Higher temperature (31 f 2 “C) and mixed (fluorescent and incandescent) light of 50 pmol me2 s-’
photon flux density for 12 h per day photoperiod favoured shoot induction and subsequent growth. Explants
from thornless trees produced 6-8 shoots per explant on MS medium containing 0.1 mgll’ IAA + 5.0 mg-
ll’ BAP + additives. Nodal shoot segments obtained from root and stump sprouts produced multiple
shoots. Root segments differentiated into multiple shoots on MS medium containing 0.5 mgll’ indolebutyric
acid (IBA) + 2.5mgll’ BAP.
Differentiated shoots multiplied best on MS medium containing 0.1 mgll’ naphthalene acetic acid
(NAA) + 1.Omgl-’ BAP + additives. To yield multiple shoots the original explant was transferred 6 times
on fresh medium after harvesting the differentiated shoots. Shoots were rooted by pulsing with
100 mgll’ IBA for 4 h and then culturing on hormone-free half strength MS medium. Initial dark incubation
for 5 days at high temperature (33 f 2OC) was found essential for root induction from shoots which was
63% within two weeks. The rooted plantlets contained a consistent number of chromosomes (2 n = 28). It
is suggested that the protocol developed could be useful for cloning of mature and tested trees of P. cineraria.

1. Introduction the Indian Thar Desert. The ecosystem in this

Prosopis (Mesquite) has proven value in arid and
semi-arid lands for production of forage (leaves
and pods), human food and firewood [16]. These
trees enrich soil by fixing atmospheric nitrogen and
provide shelter to life. Their CO,-fixation rates
among the highest known for trees render them
capable of rapid growth and enable them to thrive
even under most adverse conditions.
At least 14 Prosopis species are potentially im-
portant for afforestation and fodder production [9,
231. Prosopis cineraria (Khejari) is highly valued in
region is based on this life-supporting tree in the
stressed and densely populated desert environment.
It is the most accepted tree species for agroforestry
since it provides maximum productivity with
minimum input. P. cineraria profoundly influences
both the economy and the environment of the
region. The importance of this plant can be judged
from the 1730 incident where hundreds of men and
women of a desert community reportedly sacrificed
their lives to save Khejari trees from being felled by
the order of the contemporary ruler of the Marwar
State (Jodhpur).
274
Marked genetic variability exists in P. cineraria,
in mean biomass productivity per tree, quality and
quantity of fodder, pod production, and bole
length and diameter. This variability can be
attributed to the self-incompatibility of Prosopis
species [36] thus making it obligatory out-crossing
and preventing true-to-type propagation.
Therefore the only method of multiplication of
selected germplasm is by clonal propagation; varia-
tions can be exploited by clonal propagation.
During a field survey of the Thar desert it has been
recorded that P. cineraria propagates through root
suckers in the fields (Shekhawat et al. unpublished
data). Vegetative propagation was reported in P.
cineraria and P. julifIora [3 13. Air layering has been
demonstrated and suggested but the results are
preliminary [34, 371. Tissue culture is finding
applications in horticulture, forestry and economic
agriculture [l, 6, 8, 14, 29, 38, 391 particularly for
clonal and mass propagation of desired genotypes.
Several attempts have been made to demonstrate
applicability of tissue culture technology for Pro-
sopis species. This has been a subject for review and
discussions [18, 27, 301 and has centered on ex-
plants taken from young plants. This technology as
yet has no relevance to cloning of adult, tested and
selected plants of Prosopis species. The explants
taken from mature and aged trees behave entirely
differently in culture and require new tissue culture
approaches. Tissue culture of P. cineraria has been
established and success in organogenic differentia-
tion was reported [3, 4, 12, 131. Kackar et al. [20]
reported 5-7 fold multiplication of P. cineraria
through axillary branching by culturing of nodal
shoot segments of four and half-year-old plants.
However, there is no report on cloning of adult
trees of P. cineraria, selected for fuel, fodder and
fruit production and other desirable attributes. The
rate of clonal multiplication reported even for
young trees was limited. The management and cul-
ture of source tree, tree age, type of explant and the
season affect the results significantly e.g. extreme
pruning of the trees in a particular season is
important for obtaining treatment-responsive
explants. Rooting of in vitro produced shoots
from explants of mature trees has been extremely
difficult and unpredictable. We here report a
protocol developed for cloning of adult trees of
P. cineraria.
2. Materials and methods
2.1 Selection of source trees
Explants were taken from ‘Plus’ (tree/s in native
stands showing superior phenotype/s) trees of Pro-
sopis cineraria selected as follows:
1. Rapidly growing (5year-old), at Indira Gandhi
Canal Command Area, Mohangarh (Jaisalmer,
Rajasthan) by the State Forest Department
2. Thorny/thornless/sparsely thorny (40-year-old)
for good quality fodder yield by farmers in their
fields
3. Sparsely thorny (125 to 150-year-old) produc-
ing highly acceptable, sugar-rich fruit selected
by us with the help of farmers/villagers
2.2 Explants and their culture
Some of the trees were severely pruned during the
months of November-January and May-July.
Root and stump sprouts of (50-year-old) adult trees
were watered regularly to maintain a continuous
source of explants. Explants were taken from: (1)
pruned and non-pruned trees, (2) root and stump
sprouts, and (3) roots. These were thoroughly
washed with autoclaved water containing Tween-
80. The explants were submerged for 30min in an
aqueous solution containing ascorbic (1OOmgl~‘)
and citric (50mgl-‘) acids and polyvinylpyr-
rolidone (PVP, IOOmgl-‘). These were then surface
sterilized with 0.1% mercuric chloride for 3-5 min
and washed 8-10 times with autoclaved water con-
taining lOOmgl- each of ascorbic acid, citric acid
and PVP. Explants of various sizes (0.5-3.0cm
long; 0.2-0.5 cm thick) were cultured aseptically on
various nutrient media; namely Broad Leaf Tree
Medium (BTM), B5, MS and Woody Plant (WP)
medium [7, 11, 24, 261, respectively containing dif-
ferent concentrations and combinations of plant
growth regulators. The explants were positioned
vertically or horizontally on the culture medium.
For each experiment 15 replicates were taken and
each experiment was repeated three times. Ad-
ditives, namely ascorbic acid (50mgl-‘), citric acid,
adenine sulphate, arginine (25 mgl-’ each),
glutamine (148 mgl-‘) and ammonium citrate
(100 mgl-‘) were incorporated together in the fin-
ally selected culture medium. 3.0% sucrose was
used as carbohydrate source and the medium was
 215

gelled by adding 0.7% agar. pH of the medium was Table 1. Effect of explant harvest period (season) on multiple
shoot induction in P. cineraria on MS + IAA0.1 mgl-’ +
adjusted to 5.8 before autoclaving at 15 lb for
BAP2Smgl-’ + additives, after three weeks
15 min. The cultures were incubated in a plant
growth chamber under controlled conditions of Explant Explants Shoot number/ Shoot
temperature, humidity, and light (mixed light pro- harvest period responding responsive length (cm)
(months) (%) &SD. explant f S.D. f SD.
vided by fluorescent tubes and incandescent
lamps). Jan-Feb 66.0 f 5.5 4.12 + 0.78 3.17 + 0.26
Mar-Apr 84.0 * 4.9 10.07 * 1.10 5.19 + 0.52
May-Jun 54.0 + 4.9 6.16 k 1.06 4.20 5 0.31
2.3 Subculturinglrepeated transferring
Jul-Aug 60.0 + 6.3 5.00 + 0.57 4.40 + 0.33
Sep-Ott 55.0 + 5.0 3.40 + 0.48 3.77 f 0.32
The shoots differentiated in culture were cut into Nov-Dee 28.3 + 9.0 1.80 f 0.74 2.85 5 0.34
1.5-2.0 cm single node segments and trasferred to
shoot multiplication medium. We tried several
combinations of growth regulators in the culture region of the explants (Fig. 1). Explants derived
medium. The media were steam or filter sterilized. from non-pruned thorny trees showed delayed bud
The original shoot explants were repeatedly trans- breaking and only 4-5 shoots were regenerated
ferred to fresh medium after harvesting the shoots from each node. The explants from non-pruned
and removing the brown and darkened tissues. thornless trees manifested browning and produced
Repeated transferring was performed both on only 2-4 shoots per explant. Hard, fleshy, nodal
semi-solid and liquid media containing activated shoot segments (2.0-2.5 x 0.4-0.5cm) proved
charcoal (0.5-2.0 gl-‘) and IAA (0.1 mgl-‘) + better than tender and woody shoot explants. Bud
BAP (2.5 mgl-‘) + additives. A three week period breaking of vertically placed explants was earlier
elapsed between each subculturing/transferring than that of horizontally positioned ones. Bud
step. breaking occurred on 6 th to 8 th day of culture at
31 + 2°C under 12 h photoperiod and
2.4 Rooting of shoots 45 pmol m-* s-’ photon flux density. Incubation at
lower temperatures (20-27 “C) resulted in very slow
The in vitro produced shoots were individually bud breaking. For multiple shoot induction MS
excised and cultured on several rooting media. Full, was found to be the best culture medium followed
l/2, and l/4 strengths of MS medium, Heller’s [15] by B5, BTM and WP media (Table 2). The explants
and White’s [40] media were tried for culture of derived from thornless trees required
shoots for root induction. Several treatments of 5.0mglI’ BAP for maximum (6-8) shoot induc-
IAA, naphthalene acetic acid (NAA) and naph- tion. Root and stump sprouts (Fig. 2), showed
thoxy acetic acid (NOA) and their mixture were proliferation of shoots from nodal region on
applied under different environmental conditions MS + 0.1 mgll’ IAA + 2.5mgll’ BAP -I- ad-
in liquid and semi-solid media. ditives, respectively within two weeks of culture.
Explants from thornless trees showed bud breaking
on media containing 1 .O-50.0 mgll’ BAP. Con-
3. Results centrations of BAP exceeding 1Omgll inhibited
shoot growth but enhanced the number of shoots
3.1 Bud breaking and multiple shoot induction per explant. However, the explants of thorny trees
did not tolerate more than lO.Omgl- BAP.
The nodal shoot segments harvested from pruned Kinetin proved to be less effective than BAP (Table
trees during the months of March/April and July- 4). Addition of IAA or NAA exceeding 0.1 mgl-’
September were found to be the best explants both induced callusing from the surfaces of the explants
for thorny and thornless trees (Table 1). The and also inhibited shoot induction. About 20% of
maximum bud breaking and shoot proliferation the root segments differentiated to produce multi-
occurred on MS medium containing ple shoots (Fig. 3) on MS + 0.5mgll’IBA +
0.1 mgll’ IAA + 2Smgll’ BAP plus additives. 2.5mgll’ BAP plus additives (Table 3). 2, 4-D
About 8-10 shoots differentiated from the nodal prevented bud breaking from all the types of ex-
276

Figs. I-8. (1) Multiple shoot induction from a stem explant derived from a severely pruned adult thorny tree of P. cinerariu on MS
medium + 2.5 mgl-’ BAP + 0.1 mgl-’ IAA + additives at 31 f 2 T; (2) A root-sprout-derived explant of thornless P. cineraria; (3)
Regeneration of multiple shoots from a root segment on MS + 0.5mgl-’ IBA + 2.5mglF’ BAP + additives; (4) Multiple shoots
differentiated from a repeatedly subcultured root-sprout-derived explant of thornless P. cinerariu; (5) Multiple shoots produced from
a subcultured shoot segment on MS medium + 0.1 mgl-’ IAA + 1.0 mgl-’ BAP + additives; (6) A rooted shoot of P. cineraria; (7)
A tissue culture-derived plant of P. cineruria; (8) Chromosomes (2 n = 28) of a root tip cell of a plant of P. cinerariu raised by tissue
culture.
 277

Table 2. Effect of various media on multiple shoot induction Table 4. Effect of growth regulators on multiple shoot induction
from nodal shoot segments of P. cineraria,* after three weeks from nodal shoot segments of P. cineraria on MS medium
f0.1 mgl-’ IAA + additives
Media Explants Number of Shoot
used sprouting (%) shoots/explant length (cm) Treatment Explants Shoot number Shoot
f S.D. f SD. f SD. mgl-’ responding (%) per explant length (cm)
+ SD. f S.D. f S.D.
White’s 26 ) 5 1.2 f 0.4 1.2 * 0.2
Heller’s 38 + 7 1.8 + 0.7 1.4 + 0.3 CONTROL 68.0 f 1.9 1.2 * 0.4 2.3 & 0.1
h 60 + 6 3.4 + 0.5 2.5 + 0.3
KINETIN
WP 68 + 4 2.6 f 0.5 2.2 f 0.3
0.1 68.8 f 1.6 1.4 * 0.5 2.7 i 0.7
BTM 14 * 5 2.3 + 0.4 4.0 & 1.0
0.5 72.0 + 2.4 1.6 f 0.5 2.8 + 0.3
MS/2 52 & 4 4.8 k 0.8 3.6 + 0.3
1.0 75.2 k 3.1 2.1 & 0.4 2.9 + 0.3
MS 86 + 5 10.8 + 1.9 5.2 k 0.6
2.5 82.7 k 2.1 4.8 + 0.7 3.7 f 0.4
*Media supplemented with IAA 0.1 mgl-’ + BAP2.5mgl-’ 5.0 85.2 f 2.2 6.5 & 1.1 4.1 + 1.0
+ additives. 10.0 74.6 + 2.4 7.1 f 1.1 3.1 & 0.4
25.0 56.6 k 2.3 5.9 + 1.2 1.8 + 0.2
50.0 27.5 f 1.9 3.9 k 0.8 0.9 + 0.1
plants evaluated. The additives incorporated in the
BAP
MS medium also caused multiple shoot induction,
0.1 69.8 + 1.7 1.2 f 0.4 2.1 * 0.3
branching and growth of shoots. Activated char- 0.5 79.2 f 2.9 2.6 & 0.8 2.7 _+ 0.3
coal (500-1000mglI’) added to the culture 1.0 82.3 f 2.1 5.1 f 1.1 3.0 * 0.3
medium reduced the number of shoots per explant. 2.5 81.6 * 2.7 10.6 i 1.0 5.0 k 0.6
5.0 85.4 f 4.0 8.7 f 0.8 3.5 * 0.5
10.0 17.1 * 1.9 7.9 f 0.9 2.1 f 0.5
3.2 Repeated transferring of original explant
25.0 46.1 + 4.2 4.5 * 0.7 7.0 k 0.1

The multiple shoots which proliferated in culture

were harvested and the original explants could be caused elongation of shoots. In the case of root and
repeatedly transferred up to 6 times. In the three stump sprout-derived explants, transfer to fresh
initial culture transfers, an enhanced number of medium increased the number of shoots as much as
shoots were produced from the nodal regions. 30-35 per explant (Fig. 4).
Transferring to fresh medium after three weeks was
3.3 Subculturing of in vitro produced shoots
found to be essential to prevent defoliation and
culture deterioration and for sustained shoot
growth. Washing the original explant with 50 mgl-’ Shoots produced in vitro were excised and cultured
each of ascorbic acid and citric acid after each away from the mother explant, their growth being
very poor. We tried several media for multiplica-
transfer considerably improved growth and
number of shoots/explant. Repeated transfer of tion and culture of these shoots. Only those shoots
explants into a thin layer of liquid medium contain- which produced some callus at the cut ends could
ing activated charcoal enhanced shoot number and survive and multiply. Each of the segments of cul-
tured shoots proliferated and produced 4-5 shoots
per node on MS medium + 0.1 mgl-’ NAA and
Table 3. Effect of explant types on multiple shoot induction in
P. cineraria on MS medium containing IAA 0.1 mgl-’ + l.Omgl-’ BAP + additives (Fig. 5).
BAP 2.5mgl-’ + additives, after three weeks
3.4 Rooting of shoots
Explant Explants Number of shoots Shoot
type responding (%) per explant length (cm)
_+ S.D. k S.D. _+ S.D.
The shoots obtained in culture were induced to root
by pulse treatment with IBA. The maximum
Nodal shoot 86.0 f 4.9 10.8 + 1.9 5.5 f 0.6 number of shoots (about 65%) rooted (Table 5)
segment
Apical shoot 70.0 + 7.5 3.0 k 0.8 2.1 f 0.9
when treated with 100mgll’ IBA in half-strength
segment MS medium for 4 h followed by transfer to half-
Stump sprout 62.0 f 4.9 8.9 k 0.9 3.9 f 0.5 strength hormone-free agar-gelled medium con-
Root sprout 57.0 & 4.0 11.9 + 2.5 3.2 + 0.3 taining 500mgll’ activated charcoal (Fig. 6). In-
Root segment 16.0 f 4.9 3.8 f 0.8 3.3 f 0.2 cubation in the dark for 5 days at 33 + 2°C
278

Tub/e 5. Effect of various concentrations of IBA for 4 h on root November-January of the preceding year. Only
induction from subcultured shoots of P. &m-aria* 2-4 shoots per explant were obtained if source
Treatment Shoots Number of Root plants were not pruned. Similarly, if root and
IBA rooting (%) roots per shoot length (cm) stump sprouts were used as explants, the number of
(wm’) _+ S.D. f S.D. f SD. shoots produced was higher. These observations
0.0 - - suggest that juvenile cuttings (rejuvenated and
10.0 16.6 f 4.7 2.2 + 0.7 1.1 f 0.2 reiterated) are suitable explants. Root and stump
25.0 28.3 i 6.9 2.5 k 0.8 2.0 f 0.5 sprouts are condensed and compact in P. cineraria
50.0 48.5 f 8.3 3.0 * 1.1 2.6 k 0.6 and have more nodes (and therefore several meris-
100.0 63.3 f 4.7 4.4 f 0.9 3.4 f 0.5
200.0 51.4 + 40 2.2 + 0.6 2.9 + 0.4 terns per unit length) per explant. Root sprouts are
persistently available for longer duration in com-
* Data were scored after three weeks. parison with stem shoots and are least influenced
by environmental changes. The explants derived
proved to be critical for early root induction. Tem- from thornless trees require more cytokinin and
peratures ranging from 28 + 2°C to 36 + 2°C produced fewer shoots than thorny explants, indi-
were tested. Root initiation started when the cul- cating genotype specific responses in culture.
tures were maintained under 12h per day Maximum bud breaking and sprouting occurred
photoperiod. In all responsive cultures rooting oc- at 31 + 2’C. At lower temperatures, response was
curred within 15 days of pulse treatment. Only poor and highly unpredictable. This reflects the
30-40% of shoots rooted when pulse treated with nature of P. cineraria which thrives in the hot and
lOOmgIl’ IAA/NAA or NOA. After root initia- dry environment of the Indian desert. BAP was
tion the plantlets grew rapidly but root growth was found to be more efficient than kinetin. Superiority
more rapid than the shoot growth. In a series of of BAP over kinetin has been found for
experiments ca 300 shoots rooted. Thirty plants micropropagation of other trees [17, 32, 331. The
were transferred to pots (Fig. 7) containing ver- mother explants could repeatedly be transferred to
miculite and sand (1 : 4, V/V). Cloned plants were obtain several crops of multiple shoots. Repeated
cytologically analysed and the root tip cells of transferring of explants has been suggested as an
cloned plants showed the normal chromosome efficient method of micropropagation in woody
number (2n = 28) (Fig. 8) and no chromosomal plants [2, 351. Hedging of the source plant and
aberration was observed. repeated transferring of woody explants in media
containing cytokinin (BAP) cause rejuvenation of
4. Discussion explant tissues taken from adult plants. These
treatments probably release the clone from the
Micropropagation methodologies were optimized developmental effects of past adult operating cycle
as a prerequisite for applying tissue culture biotech- and also reduce/remove accumulated inhibitory
nology for clonal propagation of P. cineraria. factors [lo, 281. These manipulations cause rein-
Woody explants taken from selected adult trees vigoration and rooting ability, vigor and resulting
could be cultured to obtain multiple shoots. shoot multiplication rates are improved. Repeated
Management of source plant, genotype, season, transferring causes activation [5] and conditioning
medium, incubation conditions and subculturing, of meristems. The browning of explants and cul-
influenced the efficiency of shoot induction, tures could be checked in P. cineraria by treating
multiplication and growth in culture. In earlier the explants and cultures with ascorbic acid, citric
attempts at cloning of this tree, explants were either acid and PVP. These compounds are known to
taken from seedlings or from very young plants [4, posses antibrowning and antioxidant activity [22]
12, 13, 201. in tissue cultures. Ascorbic acid also stimulates
During the course of present study, it was found growth and shoot differentiation in culture 1191.
that bud breaking and sprouting occurred in the Shoots regenerated in vitro from adult tree-
maximum number of shoot explants if these were derived explants could be rooted by a two step
taken during the months of March and April from method. IBA proved to be the best root inducing
source trees which were pruned back during auxin. Initial incubation in the dark for 5 days at
 279

33 “C promoted rooting. Thus P. cineraria requires of tree species in the Thar Desert through tissue culture.
elevated temperature for root initiation. Klass et al. Forest Ecol and Managem 16: 201-208
4. Batchelor CA, Yao D, Koehler MJ and Harris PJC (1989)
[21] reported that a temperature of 35°C and a In vitro propagation of Prosopis species (P. chilensis, P.
photoperiod of 12h per day were necessary for cineraria and P. jul$ora). Ann Sci For 46 (Supp): llOS-
optimum rooting of cuttings of P. alba clone B2 112s
V5Q. 5. Boulay M (1984) Some practical aspects and applications of
the micropropagation of forest trees. In: “Proc of the Inter-
Root tips of in vitro raised plants were cytologic-
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271
cloned plants. However this does not rule out the 8. Cheliak WM and Rogers DL (1991) Integrating biotechnol-
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Acknowledgements
Prosopis cineraria through tissue culture. Curr Sci 50: 468-
469
We are thankful to the Department of Biotechnol- 13. Goyal Y and Arya HC (1984) Tissue culture of desert trees:
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Government of India for financial support for this .I Plant Physiol 115: 183-189
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research (Project No. BT/TF/03/029/017/88) and
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the State Forest Department, Rajasthan and Pro- Veg 11 th Series 14: l-223
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